Journal of Surgical Research

Increased Intracellular Creatine is not Harmful to the Mouse Heart

2012-02-01

Creatine Supplementation Ameliorates Doxorubicin Cardiac Cytotoxicity

M.D. Darrabie, J. Mantilla Arango, R. Mishra, D.O. Jacobs, L. Santacruz — 2012-02-01

Calcineurin Inhibition During Zebrafish Heart Development Blocks Ventricular Cardiomyocyte Hypertrophy and Leads to a Spectrum of Heart Defects

N.D. Andersen, N.C. Lent, M.R. Hutson, M.L. Kirby — 2012-02-01

Baseline Macro and Microhemodynamics in Anesthetized Landrace Large White Swine: Reference Values for Cardiovascular Surgical Research Studies

2012-02-01

Circumferential Coronary Artery Dissection on the Beating Heart for Perivascular Flow Probe Placement Using Ball Burnisher: A New Use for an Old Instrument and Refined Surgical Technique

2012-02-01

Sex Disparities in the Myocardial Response Following LAD Ligation

M. Wang, H. Gu, C. Huang, M. Wang — 2012-02-01

Mechanism for Reduced Pericardial Adhesion Formation in High-cholesterol Fed Pigs Supplemented With Vodka

A.D. Lassaletta, L.M. Chu, N.Y. Elmadhun, Z.G. Hoffman, D.J. Kim, F.W. Sellke — 2012-02-01

Ablation of Toll-like Receptor 4 Enhances Murine Mesenchymal Stem Cell Survival After Hypoxic Injury Via Increased AKT Salvage Signal

J.D. Rouch, B.D. Brewster, M. Wang, D.R. Meldrum — 2012-02-01

Withdrawn

2012-02-01

Therapies Targeting the Tumor Microenvironment After Surgery Can Prevent Cancer Relapses

J.D. Predina, B. Judy, V. Kapoor, G. Chen, S. Singhal — 2012-02-01

The Natural History of Moderate Aortic Stenosis in a Veteran Population

2012-02-01

Is Aortic Valve Replacement in Elderly Patients Justified?

C.M. Bhamidipati, R.R. Gopaldas, J.G. Markley, C.J. Lutz — 2012-02-01

Contemporary Results of Acute Type a Dissection Repair at a High Volume Aortic Center

N.D. Andersen, J.B. Williams, J.G. Gaca, G.C. Hughes — 2012-02-01

A Population-Based Analysis of Isolated Single Versus Bilateral Internal Mammary Artery Revascularization

R.R. Gopaldas, C.M. Bhamidipati, C.J. Lutz — 2012-02-01

The Impact of Glycoprotein IIB/IIIA Antiplatelet Infusion on the Choice of Revascularization Conduits in Patients Undergoing Coronary Artery Bypass Grafting

J.C. Hardaway, C.M. Bhamidipati, J.D. Markley, R.R. Gopaldas — 2012-02-01

Significant Predictors of Complications Post Sternal Wound Reconstruction: A 21-Year Experience

2012-02-01

Video-assisted Thoracoscopic Management of Primary Spontaneous Pneumothorax

G.S. Schwartz, S.S. Razi, H. Guend, D. Lee, C.P. Connery, F.Y. Bhora — 2012-02-01

Perioperative Outcomes of Patients With Less Than Clinical N2 NSCLC Receiving Neoadjuvant Vs. Adjuvant Therapy

2012-02-01

Stage I Non-small Cell Lung Cancer Treated by Lobectomy: Does Tumor Anatomic Location Matter?

B.A. Whitson, S.S. Groth, R.S. Andrade, E.B. Habermann, M.A. Maddaus, J. D'Cunha — 2012-02-01

Spontaneous Closure of the Ventricular Septal Defect in Fetuses and Neonates With Complete Atrioventricular Septal Defects (AVSD)

D. Adebo, S. Sivanandam, J. St. Louis — 2012-02-01

Routine Chest X-ray Prior to Thyroid Surgery? Is it Always Necessary?

H. Mazeh, B.W. Hong, H. Chen, R.S. Sippel — 2012-02-01

Optimizing Ambulatory Thyroidectomy: Predicting Success in the Outpatient Population

J. Hill, O.D. Guillamondegui, J.M. Ehrenfeld, K.W. Sharp — 2012-02-01

Operate Another Day? Increasing Utilization of Cholecystostomy Tube Drainage for Acute Cholecystitis Nationwide

2012-02-01

Prophylactic Mastectomy in High Risk Patients: Is There a Role for Sentinel Lymph Node Biopsy?

V. Murthy, R.S. Chamberlain — 2012-02-01

Association Between Patient Age, Volume of Tissue Excised During Breast Conserving Surgery, and Local Recurrence

H. Mazeh, I. Sagiv, D. Katz, H.R. Freund, T. Peretz, T.M. Allweis — 2012-02-01

Reliability of Fine Needle Aspiration for Thyroid Nodules Greater Than or Equal to 4 Cm

M.B. Albuja-Cruz, M. Goldfarb, S. Gondek, J.I. Lew — 2012-02-01

Automated Detection of Wound Margins Using a Multilayer Feed-Forward Artificial Neural Network in a Stented Excisional Model of Murine Wound Healing

M. Januszyk, G.C. Gurtner — 2012-02-01

The Incidence of Thyroid Cancer by FNA Varies by Age and Gender

L.J. Bessey, N.K. Lai, N.E. Coorough, H. Chen, R.S. Sippel — 2012-02-01

Outcome of Surgery for Toxic Goitres in Maiduguri: A Single Teaching Hospital's Perspective

N.-. Ali, M.A. Gamace, D.-. Dilli, A.A. Gadzama, M.B. Gali — 2012-02-01

Influence of Operative Approach and Conduit Type on Short-Term Outcomes After Esophagectomy

R.P. Merkow, K.Y. Bilimoria, M.D. McCarter, C.Y. Ko, D.J. Bentrem — 2012-02-01

Surgical Therapy for Epidermoid Carcinoma of the Anal Canal: A NSQIP Assessment of Short-term Outcomes

M.W. Causey, S.R. Steele, J. Maykel, M.J. Martin, B. Champagne, E. Johnson — 2012-02-01

Outcomes for Interval Appendectomy After Non-Operative Management of Perforated Appendicitis: What Are the Operative Risks and Luminal Patency Rates?

2012-02-01

The Impact of Resident Involvement on Patient Outcomes During Elective Inguinal Hernia Repair and Laparoscopic Cholecystectomy: An Analysis of Over 50,000 Cases from the ACS-NSQIP

C. Gonczy, V. Advani, S. Ahad, S. Markwell, I. Hassan — 2012-02-01

Complications, Mortality and Failure to Rescue After Abdominal Gunshot Wounds: A Nationwide Study

B.L. Zarzaur, M.A. Croce, T.C. Fabian — 2012-02-01

Comparison of Pediatric Appendectomy Outcomes Between Pediatric Surgeons and General Surgery Residents

2012-02-01

PROMIS for Laparoscopy: A Pilot Study

J. Bingener-Casey, P. Novotny, A. McConico, J.M. Swain, D.R. Farley, J. Sloan — 2012-02-01

Goal-Directed Fluid Therapy in Major Elective Rectal Surgery: A Prospective Study

S. Srinivasa, P.P. Singh, D.P. Lemanu, M.H. Taylor, A.G. Hill — 2012-02-01

Increasing BMI Portends Abbreviated Survival Following Pancreatoduodenectomy for Pancreatic Adenocarcinoma

A. Mathur, K. Luberice, H. Paul, F. Co, J. Hernandez, A.S. Rosemurgy — 2012-02-01

Budget Impact Analysis of Evidence Based Thyroidectomy

A. Sanabria, X. Gomez, C. Osorio, V. Vega, L.C. Dominguez — 2012-02-01

Exposing Inefficiencies in the Operating Room Case Flow

R. Phillips, T. Yonce, C. Sievers, V. Tsirline, P. Montero, D. Stefanidis — 2012-02-01

Do Racial Disparities Exist for Gastric Bypass Surgery?

N. Lodhia, A. Eltorai, L. Almario, J. Kattan, H. Rivas, J. Morton — 2012-02-01

State by State Variations in Emergent Versus Elective Colon Resections: Room for Improvement

2012-02-01

Are Surgeons with More Years in Practice Able to Adapt to Pay-for-performance (P4P)?

D.Y. Lee, M.J. Latif, R.E. Ross, J. Fisch, C. Jimenez, S.J. Belsley, F.Y. Bhora — 2012-02-01

Medical Malpractice and Hernia Repairs: An Analysis of Case Law

A.L. Walters, K.T. Dacey, A.Y. Zemlyak, A.E. Lincourt, B.T. Heniford — 2012-02-01

What Does It Cost to Treat A Recurrent Rectal Cancer?

S.S. Patel, S. Mahanti, J. Choi, G. Ault, A. Kaiser, A.J. Senagore — 2012-02-01

Patient Deconditioning and Failure to Rescue from Surgical Complications

A.A. Ghaferi, C.J. Sonnenday, J.D. Birkmeyer, J.B. Dimick — 2012-02-01

Measuring Quality of Decision-Making for Advance Care Planning and Surgery

2012-02-01

Defining HIV Risk in the Surgical Population

A. Adewole, A.B. Chagpar — 2012-02-01

Risk Factors for Interhospital Transfer After Emergency Abdominal Surgery

D.N. Holena, C.E. Reinke, B.G. Carr, A.M. Mills, R.R. Kelz — 2012-02-01

Epidemiology of Clostridium Difficile Colitis in Hospitalized Patients in the United States

C.V. Mehta, S. Vaid, A. Adedeji, D. Vibhakar, T. Bell, R. Grim, V. Ahuja — 2012-02-01

Maximizing the Utility of the Morbidity and Mortality Conference for Quality Improvement and Safety Initiatives

H.L. Chang, R. Thielke, L.D. Cassidy, M.J. Arca — 2012-02-01

The Cost-effectiveness of Elective Ventral Hernia Repair

A. Stey, M. Danzig, S. Yin, C. Divino — 2012-02-01

Day of Surgery not Associated with Length of Hospital Stay in Patients Undergoing Esophagectomy

C. Merkhofer, A. Kothari, S. Kolonko, T. Bretl, T.L. Weigel — 2012-02-01

Repair of Femoral Hernias Improves Quality of Life (QOL)

I. Belyansky, V.B. Tsirline, A. Walters, A.E. Lincourt, B. Heniford — 2012-02-01

The Use of a Research Registry Improves Patient Recruitment in Surgical Research

R.C. Wright, C.M. Mueller — 2012-02-01

Variability of NSQIP Assessed Surgical Quality Based on Age and Disease Process

2012-02-01

Teaching Single Incision Laparoscopic Appendectomy in Pediatric Patients to Mid Level Surgical Residents: An Early Experience

H.S. Nerkar, S.C. Burjonrappa — 2012-02-01

Competency Documentation in Residency Training: A Challenging Pathway

E. Desrosiers, M. Lacasse, F. Douville, M. Rousseau, L. Ct, F. Legare — 2012-02-01

Validity and Reliability of a Sensor Enabled Intubation Trainer

N. Issa, L. Salud, K. Woods, C. Pugh — 2012-02-01

Get on Your Boots: Preparing Fourth Year Medical Students for a Career in Surgery, Using a Competency-Based Curriculum to Teach Professionalism

2012-02-01

The Effect of Timely Completion of an Objective Assessment Tool

K. Laeeq, D. A. Diaz Voss Varela, M.U. Malik, H.W. Francis, N.I. Bhatti — 2012-02-01

Value of Fundamentals of Laparoscopic Surgery (FLS) Training in a Fourth-Year Medical School Advanced Surgical Skills Elective

D.A. Edelman, M.A. Mattos, D.L. Bouwman — 2012-02-01

Prospective Analysis of the 2-Year Outcomes from a Competency-Based Preparatory Course for the PGY-1 Resident: Modeling Standards for the Future

M.B. Antonoff, C.A. Green, J.A. Swanson, M.A. Maddaus, J. D'Cunha — 2012-02-01

New Resident Clinical Simulations to Improve Communication and Team Performance

L.C. Sakata, C.I. Anderson, L. Laczynski, J.A. Ghiardi, B.D. Mosher — 2012-02-01

Use of Mobile Learning Module Improves Skills in Chest Tube Insertion

2012-02-01

Laparoscopic Simulation Training? Does Timing Impact the Quality of Skills Acquisition?

E.M. Bonrath, E. Rijcken, M. Fritz, B. Weber, N. Senninger, T.P. Grantcharov — 2012-02-01

Who Teaches the Medical Student and in What Setting: Comparison of Student Reported Experience and Faculty Opinion for Teaching Basic Procedural Skills

J. Carr, J.J. Dehmer, K.D. Amos, T.M. Farrell, A.A. Meyer, M.O. Meyers — 2012-02-01

Assessing the 3rd Year General Surgery Clerkship Trauma Rotation: Are we Meeting the Learners? Needs?

H. Copeland, J.L. Wynne, E. Ong, W.J. Adamas Rappaport, A. Alseidi — 2012-02-01

Dimensions of Culture: Faculty and Student Characteristics at an Urban Public Medical School

A.O. Sanni, J. Reilly — 2012-02-01

Evaluation of a Clinical Campus Model for Surgical Clerkships

D.A. Edelman, R. Page, P. Bridge — 2012-02-01

The Educational Impact of a 2-Week Surgical Oncology Rotation for Third-Year Medical Students: A Prospective Study

2012-02-01

Videoconference Interviews as an Adjunct to Traditional Resident Recruitment Interviews

J.D. Alder, C. Howard, C. Ruiz — 2012-02-01

Innovative Information Delivery for the Modern Trainee: Objective Impact of Audio Podcasts to Augment Trainee Knowledge Base

2012-02-01

Are we Satisfying Our Patients?

I. Kwok, I. Basu — 2012-02-01

Creating a Healthy Culture for Women in Surgery

E.A. Berdan, J.I. Asghar, C.E. Stewart, J.M. Lewis, T.B. Dunn, R.F. Kelly — 2012-02-01

Cost-Comparison Analysis of Cholecystectomy During Bariatric Surgery

J. Benarroch-Gampel, V. Ho, C.A. Boyd, K.M. Sheffield, D.R. Merrell, T.S. Riall — 2012-02-01

Volume-Outcome Association in Bariatric Surgery: A Systematic Review

B. Zevin, R. Aggarwal, T.P. Grantcharov — 2012-02-01

Outcomes of Antireflux Surgery in Lung Transplant Recipients

A. Kilic, C.G. Gourin, A.O. Lidor — 2012-02-01

Evaluation of the International Study Group of Pancreatic Surgery (Isgps) Definition of Post-Pancreatectomy Haemorrhage (Pph) in a High-Volume Centre

F. Rckert, N. Hippe-Davies, H. Saeger, R. Grtzmann — 2012-02-01

Lymph Node Evaluation by Tumor Location in Colon Cancer Elderly Patients: A SEER-Medicare Study

J.J. Tucker, F. Yanagawa, N. Ahuja, T. Bell, R. Grim, V. Ahuja — 2012-02-01

High-Volume Hospitals are Associated With Improved Recovery Following Colorectal Surgery

2012-02-01

Perioperative use of Statins in Elective Colectomy

2012-02-01

Distribution of Feeding Styles After Pyloromyotomy Among Pediatric Surgical Training Programs in North America

D. Juang, O.O. Adibe, C.A. Laituri, D.J. Ostlie, G.W. Holcomb, S.D. St. Peter — 2012-02-01

Effect of Immunosuppression on Mortality Following Enteral Access Using the Enterprise Data Warehouse: A Novel Tool for Quality Improvement and Research

2012-02-01

Recurrence Patterns and Predictors of Recurrence After Neoadjuvant Chemoradiation (CRT) Followed by Surgery for Esophageal Cancer

A. Rosenbluth, R. Anne, J. Andrel, E.P. Mitchell, E.L. Rosato, A.C. Berger — 2012-02-01

The Influence of age on Postoperative Morbidity and Mortality After Pancreatoduodenectomy. An Analysis of 6293 Cases from the Acs-Nsqip

V. Advani, C.R. Gonczy, S.J. Markwell, S. Ahad, I. Hassan — 2012-02-01

Effects of Intestinal Resection on Growth Parameters in Children with Crohn's Disease

J. Pettiford-Cunningham, E.M. Knott, A. Gasior, S.D. St. Peter, D.J. Ostlie — 2012-02-01

A Statewide, Community-Based Assessment of Alvimopan's Effect on Surgical Outcomes

2012-02-01

Mk2206, an Akt Inhibitor, Attenuates Growth and Neuroendocrine Tumor Markers Expression in Human Gastrointestinal Carcinoid BON Cells

K.M. Simon, S. Kunnimalaiyaan, H. Chen, M. Kunnimalaiyaan — 2012-02-01

Appendectomy and Pregnancy: Gestational age may not Affect Position of Incision

2012-02-01

Perioperative Glucose Control in the Gastric Bypass Population: How Well Do We Do, How Well Do We Think We Do, and is It Predictable

M.J. Perna, T.K. Byrne, K.A. Morgan, M.K. Baker — 2012-02-01

Safety and Efficacy of Laparoscopic Sleeve Gastrectomy in the Super-obese

D.P. Lemanu, S. Srinivasa, P.P. Singh, A.D. MacCormick, A.G. Hill — 2012-02-01

Outcome Analysis of Laparoscopic Sleeve Gastrectomy for Morbid Obesity. Initial Experience of 100 Patients With BMI>45

I. Sucandy, G. Antanavicius, F. Bonanni — 2012-02-01

Robotically-assisted Laparoscopic Biliopancreatic Diversion With Duodenal Switch. Initial Experience and Outcomes from a Teaching Hospital

I. Sucandy, G. Antanavicius — 2012-02-01

Predictors of Marginal Ulcers After Laparoscopic Roux-En-Y Gastric Bypass

2012-02-01

Minimally Invasive Surgery for Rectal Cancer Results in Improved Short-Term Outcomes Without Compromising Oncological Results Compared to Open Surgery

S.T. Orcutt, L.T. Li, D.A. Anaya, A. Artinyan, S.S. Awad, D.H. Berger, D. Albo — 2012-02-01

Cancer Cell-Derived Interleukin-1alpha Promotes HGF Secretion by Stromal Cells and Enhances Metastatic Potential in Colon Cancer Cells

Y. Matsuo, H. Takahashi, N. Ochi, K. Tsuboi, H. Funahashi, Y. Okada, H. Takeyama — 2012-02-01

Utility of MiRNA Profile for Predicting Recurrence of Rectal Cancer

2012-02-01

Dual FAK-Hsp70 Inhibition Synergistically Inhibits Colon Cancer Cell Viability and Improves the Efficacy of Chemotherapy

M.D. Heffler, V.M. Golubovskaya, W.G. Cance, K.M. Bullard Dunn — 2012-02-01

Slug Expression Inhibits Vitamin D-mediated Sensitivity to Radiation in Colorectal Cancer

2012-02-01

The Association Between Insurance Coverage and Increasing Age Leads to a Lower Risk of Metastatic Colon Cancer in the Elderly

2012-02-01

Decreasing Mortality in Young Colorectal Cancer Patients: Are We Doing Better?

M.L. Campbell, J.E. Sanchez, D.M. Davis, S.H. Rasheid, J.E. Marcet — 2012-02-01

Aggressive Resection of Colorectal Liver Metastases After Approval of Molecular Target-Based Drugs

T. Ochiai, K. Ohta, Y. Kumagai, M. Iida, S. Yamazaki, S. Tanaka, S. Arii — 2012-02-01

Surgical Treatment of Heptocellular Carcinoma With Inferior Vena Cava Tumor Thrombus: A New Classification Guiding for Surgery

A. Li — 2012-02-01

Age-Related Outcomes Following Hepatectomy and Liver Ablation: An Analysis of 8,734 Patients from the National Inpatient Sample (2004? 2008)

R. Tran, S. Patil, R.S. Chamberlain — 2012-02-01

Zerumbone Inhibits Tumor Angiogenesis in Gastric Cancer

2012-02-01

Overexpression of SCCRO2 in Hepatocellular Carcinoma Correlates With Increased Neddylation Activity

V.B. Weeda, H. Sun, J. Au, Y. Ramanathan, Y. Fong, B. Singh — 2012-02-01

Establishing a Colon Cancer Stem Cell Model to Study Tumor Chemoresistance

A.M. Jarrar, F. Lotti, K. Sukhedo, J. Rich, M. Kalady — 2012-02-01

Partial Stomach-Partitioning Gastrojejunostomy (PSPG) and the Success of This Procedure in Terms of Palliation

2012-02-01

The Timing of Chemotherapy Correlates With Survival in Patients With Resectable Gastric Adenocarcinoma

R. Wiatrek, R. Nelson, M. Le, W. Lee, J. Garcia-Aguilar, J. Kim — 2012-02-01

Generation of an RNA Aptamer Against Glucose-Related Protein 78 (GRP78)

A.S. Barbas, K.L. Rialon, R. Ray, A.J. Jiang, G.G. DeRidder, R.R. White — 2012-02-01

Human Mesenchymal Stem Cell and Epithelial Hepatic Carcinoma Cell Lines in Admixture: A Novel Method Demonstrating Concurrent Stimulation of CAF and EMT Markers

S.D. Bhattacharya, Z. Mi, L.J. Talbot, H. Guo, P.C. Kuo — 2012-02-01

ALCAM (CD166) Expression is a Positive Predictor for Overall Survival in Colorectal Cancer Patients

2012-02-01

Fluorescence-Guided Surgery of Human Colon Cancer Increases Disease-Free Survival in an Orthotopic Nude Mouse Model

C.A. Metildi, S. Kaushal, C.S. Snyder, R.M. Hoffman, M. Bouvet — 2012-02-01

The Loss of Thymosin Beta 10 Increases Cell Migration of Cholangiocarcinoma Cells

S. Sribenja, S. Wongkham, C. Wongkham, K. Vaeteewoottacharn, Q. Yao, C. Chen — 2012-02-01

Impact of Lymph Node Disease in Soft Tissue Sarcomas

D.C. Johannesmeyer, V. Smith, D.J. Cole, N.F. Esnaola, L. Leddy, E.R. Camp — 2012-02-01

The Prognostic Significance of Lymph Node Ratio in Melanoma

2012-02-01

Merkel Cell Carcinoma: High Recurrence Rate Despite Aggressive Treatment

M.C. Soult, E.C. Feliberti, M.L. Silverberg, R.R. Perry — 2012-02-01

Expression Profiles of Metalloproteinases in Cutaneous Squamous Cell Carcinoma

A.P. Tufaro, N. Prasad, A. Chuang, J. Wright, N. Liegeois, A.C. Fischer — 2012-02-01

Oral Cavity Squamous Carcinoma; Effects of Primary Site on Survival

M.S. Wachtel, T. Nguyen, J. Cordero — 2012-02-01

Molecular Mechanism of SFRP2 in Angiosarcoma and Development of SFRP2 Targeted Therapeutic

2012-02-01

Fertility Preservation in Young Cancer Patients: Transplantation of Isolated Ovarian Follicles in Biomaterials in a Mouse Model of Infertility

A.N. Hardy, S.A. Boukaidi, L.D. Shea, J.S. Jeruss — 2012-02-01

Imatinib-Resistant GIST Metastases: Predicting Outcomes of Surgical Resection

K.T. Chen, J. Farma, M. Von Mehren, J.C. Watson — 2012-02-01

Does Complete Pathologic Response to Neoadjuvant Radiotherapy Predict Oncologic Outcome in Patients With Soft Tissue Sarcoma?

D.R. Shah, D. Borys, S.R. Martinez, C. Li, R.J. Tamurian, R.J. Bold, R.J. Canter — 2012-02-01

Importance of Connective Tissue Growth Factor in Sox2-Induced Oncogenesis

J.M. Hanna, M. Onaitis — 2012-02-01

Mutations in SMAD4 Lead to Increased Invasive Potential

J.C. Carr, F.S. Dahdaleh, D. Wang, J.R. Howe — 2012-02-01

One-step Nucleic Acid Amplification (OSNA) Assay for Lymph Node Metastases in Lung Cancer Patients

2012-02-01

Radioguided Detection of Lymph Node Metastasis in Non-Small Cell Lung Cancer

2012-02-01

Pre-Operative Core Muscle Cross-Sectional Area is Associated With Long-Term Survival Following Tri-Modality Management of Esophageal Carcinoma

2012-02-01

Withdrawn

2012-02-01

Validation of Quantitative Histopathology in Identifying Aggressive Cutaneous Squamous Cell Carcinoma

2012-02-01

Regulatory T-cell Subpopulations in Patients With Pulmonary Adenocarcinoma

2012-02-01

Evaluation of TNFAIP3 Gene Polymorphisms as Prognostic Markers in Complete Resected Only Surgically Treated Esophageal Cancer

2012-02-01

Integrated Genomic Analysis of Esophageal Adenocarcinoma Identifies DNA Copy Number Changes and Related Gene Expression Alterations That are Associated With Survival

2012-02-01

EMT-related Brachyury Gene Expression is a Significant Prognostic Factor for Primary Lung Carcinoma

A. Haro, T. Yano, M. Kohno, T. Yoshida, T. Okamoto, Y. Maehara — 2012-02-01

Clinical Significance of Negative Ultrasound and Positive Sestamibi Scan in Patients With Primary Hyperparathyroidism

C. Cen, L.A. Dultz, J.B. Ogilvie, K.S. Heller, K.N. Patel — 2012-02-01

Metastatic Carcinoid Tumors-Are we Making the Cut?

K.E. Coan, R.J. Gray, R.T. Schlinkert, B.A. Pockaj, N. Wasif — 2012-02-01

Double Adenoma? Assessment of True, Incidence in Terms of Pathology, Pre-Operative Imaging and Clinical Characteristics

P.K. Shah, K.K. Shah, G.C. Karakousis, C. Reinke, R. Kelz, D.L. Fraker — 2012-02-01

Is BRAF a Cost-Effective Marker for Indeterminate Thyroid Lesions?

N.H. Patel, L.A. Dultz, J.B. Ogilvie, K.S. Heller, K.N. Patel — 2012-02-01

MicroRNA Profiles Distinguish Papillary Thyroid Cancers of Varying Aggressiveness

J.C. Lee, J. Zhao, A. Gill, M.S. Sywak, L.W. Delbridge, S.B. Sidhu — 2012-02-01

Aggressive Surgical Management Best Serves Patients With Small Intestinal Neuroendocrine Tumors

L.R. Imhoff, J. Keiser, E. Bergsland, E.K. Nakakura — 2012-02-01

Role of Adrenal Vein Sampling (Avs) in Patients With Primary Hyperaldosteronism and Unilateral Adrenal Mass by Imaging

2012-02-01

Impact of Surgical Mentorship on Retroperitoneoscopic Adrenalectomy With Comparison to Transperitoneal Laparoscopic Adrenalectomy

J.T. Broome, C.C. Solorzano — 2012-02-01

Additional Imaging may Benefit Patients With Primary Hyperparathyroidism and Discordant Preoperative Localization Studies

C. Cen, L.A. Dultz, J.B. Ogilvie, K.S. Heller, K.N. Patel — 2012-02-01

The Disparity in Thyroid Cancer Incidence Between Native South Korean and Korean-American Women

A.K. Arrington, R. Nelson, L. Uyeno, S.L. Chen, J. Kim, J.H. Yim — 2012-02-01

Hereditary Medullary Thyroid Cancer: Age-Appropriate Thyroidectomy Improves Disease-Free Survival

K.H. Shepet, A.Y. Alhefdhi, N. Lai, H. Mazeh, R. Sippel, H. Chen — 2012-02-01

Disparate Gland Weight in Multigland Primary Hyperparathyroidism? A Surgical Caveat

L.A. Dultz, J.B. Ogilvie, K.S. Heller, K.N. Patel — 2012-02-01

Radioactive Iodine: An Unlikely Cause of Primary Hyperparathyroidism

A.L. Amin, S. Wu, T.W. Yen, K. Doffek, T.S. Wang — 2012-02-01

Factors That Influence Parathyroid Hormone Half-life: Are New Intraoperative Criteria Needed?

A.J. Leiker, T.W. Yen, D.C. Eastwood, K. Doffek, A. Szabo, D.B. Evans, T.S. Wang — 2012-02-01

Routine Thyroid Lobectomy for Thyroid Nodules on the Same Side of a Localized Parathyroid Adenoma During Directed Parathyroidectomy May Reduce Reoperative Thyroid Surgery

L. Potdevin, T. Davidov, S.Z. Trooskin — 2012-02-01

Is Intra-operative Parathyroid Hormone Monitoring Necessary in Patients With Two Concordant Preoperative Localization Studies?

C. Cen, L.A. Dultz, J.B. Ogilvie, K.S. Heller, K.N. Patel — 2012-02-01

Is Thyroidectomy in Patients With Hashimoto's Thyroiditis More Risky?

C.M. McManus, J. Luo, R. Sippel, H. Chen — 2012-02-01

The Natural Flavinoid, Chrysin, in the Treatment of Medullary Thyroid Cancer

B. Zarebczan, M. Kunnimalaiyaan, H. Chen — 2012-02-01

Evidence of a Stabilizing Mutation of-Catenin Encoded by CTNNB1 Exon 3 in a Large Series of Sporadic Parathyroid Adenomas

L.F. Starker, A. Fonseca, G. Akerstrom, P. Bjorklund, G. Westin, T. Carling — 2012-02-01

Down-Regulation of Notch1 Expression in Papillary Thyroid Carcinoma Correlates With Advanced Age and Tumor Aggressiveness

X. Yu, H. Chen, R.S. Sippel — 2012-02-01

Inhibition of Focal Adhesion Kinase (FAK) Leads to Decreased Cell Survival in Malignant Renal Rhabdoid Tumor Cells

M. Megison, L. Gillory, J.E. Stewart, H. Nabers, E.A. Beierle — 2012-02-01

Genetically Engineered Herpes Simplex Virus Expressing IL-12 Suppresses Murine Neuroblastoma Tumor Growth

2012-02-01

Notch1 Signaling in Neuroblastoma

J.F. Burke, Q. Khan, S. Kunnimalaiyaan, M. Roy, M. Kunnimalaiyaan — 2012-02-01

Single Lgr5+ Intestinal Stem Cells Co-Cultured With Intestinal Subepithelial Myofibroblasts (ISEMF) Differentiate into Absorptive and Secretory Cell Lineages Without Exogenous Growth Factors

E.R. Barthel, X. Hou, J.A. Matthews, A.L. Speer, F.G. Sala, T.C. Grikscheit — 2012-02-01

Withdrawn

2012-02-01

Formation of Intestinal Atresias in the Fgfr2IIIb-/- Mouse Model is not Associated with Defects in Notochord Development or Alterations in SHH Expression

A.L. Reeder, R.A. Botham, M. Franco, K. Zaremba, P.F. Nichol — 2012-02-01

Selective Laser Photocoagulation Manipulates Blood Flow Dynamics in Microcirculation

2012-02-01

Reduction of Bioavailable Vascular Endothelial Growth Factor (VEGF) Alters Terminal Differentiation of the Absorptive and Secretory Lineages in Neonatal Mouse Intestine

2012-02-01

Pharmacokinetics and End Organ Effect of PDE5 Inhibitors in the Fetal Lamb Model of Congenital Diaphragmatic Hernia

E.H. Shue, S. Schecter, W. Ferrier, D.N. Miniati — 2012-02-01

Human Cholangiocyte Susceptibility to Infection With Rotavirus - Parallels to the Murine Model of Biliary Atresia

A.C. Coots, B.M. Donnelly, S.K. Mohanty, M. McNeal, K. Sestak, G.M. Tiao — 2012-02-01

A Multi-scale Agent-based Model of Necrotizing Enterocolitis Integrating Oxidative Stress, Inflammation, and Microvascular Thrombosis

M. Kim, S. Christley, J. Alverdy, D. Liu, G. An — 2012-02-01

Site-specific Impaired Mucosal Immunity in the Murine Neural Crest-Specific Deletion of Endothelin Receptor-B Model of Hirschsprung's Disease

A. Gosain, A. Heneghan, J. Pierre, A. Gosain — 2012-02-01

Diverse Early Intestinal Microbiota as a Contributing Factor in Necrotizing Enterocolitis

2012-02-01

The Gut Barrier Protective Effect of Low Dose Celebrex, a Selective Cox-2 Inhibitor, in Experimental Peritonitis

S.S. Short, S.L. Castle, J. Wang, A.V. Grishin, H. Ford — 2012-02-01

The Effect of Diabetes on the Biomechanical Properties of the Skin

2012-02-01

Angiotensin II Expression in Dermal Scar, Functional Activation in Dermal Fibroblasts, and Receptor Inhibition in Vivo, Validate Angiotensin II as a Target to Prevent Dermal Fibrosis

J.E. Bond, L. Ren, G. Kokosis, L. Chen, M. Selim, H. Levinson — 2012-02-01

Increased Expression of MicroRNA-29a During Diabetic Wound Healing

J. Xu, W. Wu, L. Zhang, W. Dorsett-Martin, M.E. Mitchell, K.W. Liechty — 2012-02-01

The Increased Diabetic Wound Healing Impairment Seen After Recurrent Injury is Associated with Increased Matrix Metalloproteinase-9 (MMP-9) and Decreased Heat Shock Protein-47 (HSP-47) Gene Expression

2012-02-01

Fetal Tendon Scar Formation is Associated With Increased Inflammation and Decreased MicroRNA-146a Production

2012-02-01

Notch1 Signaling is Critical for Keratinocyte Migration During Wound Healing

T.J. Jaraczewski, M. Roy, S.J. Schossler, B. Allen-Hoffmann, H. Chen, T.W. King — 2012-02-01

Gut Epithelial Gene Expression Is Altered In Hirschsprung’s Disease

A.C. Pieretti, C.E. Steiger, A.M. Goldstein — 2012-02-01

Functional Genomics of Hepatic Ischemia Reperfusion Injury in Mice

G.W. Nace, H. Huang, R.E. Eid, J.R. Klune, G.D. Leikauf, A. Tsung — 2012-02-01

Improved Allogeneic Liver Transplant Survival With Over-expression of IRF-2 to Block IRF-1 Mediated Hepatic Injury

S. Kimura, J.R. Klune, S. Ueki, R. Dhupar, S. Yokota, N. Murase, D.A. Geller — 2012-02-01

Circulating Gamma Delta T-Lymphocytes are Activated and Accumulate in the Lungs Following Hemorrhagic Shock? Resuscitation

2012-02-01

Genomic Characterization of Hepatic Stem Cells During Liver Regeneration

M. Manivannan, C.T. Barry — 2012-02-01

Determinants of Early Outcome in Pediatric Liver Transplantation

2012-02-01

Risk Factors and Prevention of Sepsis and Surgical Site Infection After Living Donor Liver Transplantation

2012-02-01

Combined Heart-Liver Transplantation in the United States

R.M. Cannon, M.G. Hughes, C.M. Jones, M. Eng, M.R. Marvin — 2012-02-01

Patients With Caroli's Disease After Orthotopic Liver Transplantation Have Improved Long-Term Survival

T.R. Harring, N.T. Nguyen, H. Liu, J.A. Goss, C.A. O'Mahony — 2012-02-01

Incidental Hepatocellular Carcinoma in Liver Transplant Recipients is Associated With Fatty Liver Disease and Conveys a Low Risk of Recurrent Malignancy After Transplant

2012-02-01

Early Post-Transplant Alanine Aminotransferase (ALT) Level Predicts the Outcome of Donation After Cardiac Death (DCD) Livers

2012-02-01

Surgical Complications in 275 Human Immunodeficiency Virus (HIV) Infected Liver and/or Kidney Transplant Recipients

2012-02-01

National Outcomes for Simultaneous Thoracic and Abdominal Transplantation

2012-02-01

Video-assisted Living Donor Lateral Segmentectomy and Left Hepatectomy Through a Reduced Upper Midline Incision for Liver Transplantation

2012-02-01

Increasing Organ Donation Outcomes Among High School Students

A. Salim, C. Berry, E.J. Ley, D. Schulman, S. Navarro, L. Zheng, L.S. Chan — 2012-02-01

Cumulative Incidence of Cancer in Solid Organ Transplant Recipients

E.C. Hall, R.M. Pfeiffer, E.A. Engels, D.L. Segev — 2012-02-01

In the Resuscitation of Hemorrhagic Shock, Transient Hemodynamic Responders Have a Higher Acute Mortality and an Altered Inflammatory Response Compared to Definitive Responders

2012-02-01

Efficacy of Combat Gauze Dressing in the Setting of Severe Acidosis and Coagulopathy

M.W. Causey, S. Miller, D. McVay, A. Beekley, M. Martin — 2012-02-01

Angiotensin II Expedites Scar Contracture Independent of Myofibroblasts

L. Chen, J.E. Bond, L.C. Ren, G. Kokosis, A.M. Selim, H. Levinson — 2012-02-01

The Identification of a Novel TLR4 Inhibitor With Potent Anti-inflammatory Effects in Vivo Using a Combined in Silico and in Vivo Approach

2012-02-01

Phosphatidylserine is a Critical Lipid Signaling Mediator in Hemorrhagic Shock

L.Y. Yeung, E.J. Miraflor, A. Garcia, B. Curran, G.P. Victorino — 2012-02-01

Dynamic Knowledge Representation of Surgical Wound Healing Using an Agent-based Model

V. Gopalakrishnan, M. Kim, J. Alverdy, G. An — 2012-02-01

BTLA Expression is Increased on Circulating CD4+ T-cells in Trauma Patients. a Potential Mechanism for Trauma Induced Immunosuppression

D.S. Heffernan, S.F. Monaghan, N. Shubin, L.R. Irwin, W.G. Cioffi, A. Ayala — 2012-02-01

TSLP Expression Negatively Impacts Survival and Bacterial Clearance in a Murine Model of Sepsis

P.S. Prakash, K.R. Kasten, C.C. Caldwell — 2012-02-01

Mice Subjected to Severe Peripheral Trauma Are Resistant to Pseudomonas Infection at 48 Hours Despite Suppression in Lymphocyte Responses

M.K. Hoffman, S.S. Darwiche, K.R. Zettel, T.R. Billiar — 2012-02-01

Creating a “Pro-Survival” Phenotype Through Epigenetic Modulation

2012-02-01

Effects of Traumatic Brain Injury on Intestinal Motility

A.B. Olsen, R. Hetz, K. Uray, C. Cox — 2012-02-01

Designing a Novel Focal Lung Injury Model

S.Y. Tan, M.A. Krasnow — 2012-02-01

DEL1 Protects Against Osteoarthritis by Preventing Chondrocyte Apoptosis

2012-02-01

The Fate of Internalized Alpha-5 Integrin is Regulated by Fibronectin Matrix Assembly

H.C. Hsia, M.R. Nair, S.A. Corbett — 2012-02-01

Interleukin-13 Induces Interferon Regulatory Factor-2 (IRF-2) Expression in Hepatocytes

J.R. Klune, L. Shao, C. Bartels, D.A. Geller — 2012-02-01

Anti-Inflammatory Effects of Valproic ACID in a Rodent Model of Hemorrhagic Shock

2012-02-01

Human Multipotent Adult Progenitor Cells: Effect of Osmolarity on Viability and Differentiation Capacity

R.A. Hetz, C.S. Cox — 2012-02-01

Sunitinib Reduces Recurrent Pelvic Adhesions in a Rabbit Model

2012-02-01

Prevalence of Hypoferremia and Iron-Deficient Erythropoiesis in Anemic Critically Ill Trauma Patients Early After Admission, and Correlation With Severity of Illness

A.R. Cappa, B.M. Jaouen, E.E. Moore, F.M. Pieracci — 2012-02-01

Injured Adolescents--Not Just Large Children: Differences in Care and Outcome Between Adult and Pediatric Trauma Centers

K. Matsushima, E.W. Schaefer, E.J. Won, H.L. Frankel — 2012-02-01

Does Payer Status Matter in Penetrating Trauma?

2012-02-01

Understanding Risk in Emergency Abdominal Surgery in Our Elders

2012-02-01

Helmet Use in Pediatric ATV Accident Victims and Associated Closed Head Injuries

K.H. Nagarsheth, S. Kurek — 2012-02-01

Fighting the Weekend Trend: the Alarming, Increased Mortality Among Elderly TBI Patients Admitted on Weekends

2012-02-01

Increasing Numbers of Rib Fractures Do not Worsen Outcome: An Analysis of the National Trauma Data Bank

B.A. Whitson, M.D. McGonigal, C.P. Anderson, D.J. Dries — 2012-02-01

Does Insurance Status Affect Outcome in Patients With Gunshot Wound to the Abdomen?

M. Smith, R.H. Kim, A. Youssef, B.D. Li, Q.D. Chu — 2012-02-01

Predicting the Daily Incidence of Major Traumatic Injury in Chicago

J. Kieltyka, D. Kyriacou, M. Crandall — 2012-02-01

Risk Factors for the Development of Intra-Abdominal Abscess Following Splenectomy for Trauma

2012-02-01

Splenic Conservation in Adolescent Blunt Abdominal Trauma: Variation Between Pediatric and Adult Centers Within the Same Region

2012-02-01

The Etiology of Pneumoperitoneum in the 21st Century

A. Kumar, M.T. Muir, M.A. Salhanick, D.B. Lankford, V.S. Katabathina, S.M. Cohn — 2012-02-01

Incidence and Outcomes of Venous Thromboembolism After Inferior Vena Cava Injury

2012-02-01

Trauma Center Level Impacts Survival for Cirrhotic Trauma Patients

2012-02-01

Contemporary Trends in the Incidence, Management, and Outcomes of Necrotizing Soft Tissue Infections in the United States

C.M. Psoinos, S. Ng, Y. Li, J. Shaw, J. Tseng, H. Santry — 2012-02-01

Disparities in Trauma Outcomes Between Patients With Different Types of Insurance: Analysis and Implications for Healthcare Reform Policy

2012-02-01

Gunshot Wound (GSW) Victims Arriving by Private Vehicle Have Better Survival Than Those Transported by Emergency Medical Services (EMS) or Police

2012-02-01

Race and Insurance Type as Predictors of Mortality After Trauma

M.B. Singer, M.A. Clond, M. Bukur, R. Chung, D.R. Margulies, A. Salim, E.J. Ley — 2012-02-01

Fall from a Ladder: Age Matters More Than Height

2012-02-01

Helmet use Among Pediatric Cyclists in Residential Areas

2012-02-01

Long-term Effect of Trauma Splenectomy on Blood Glucose: the Emerging Role of the Spleen as a Source of Pancreatic Stem Cells

M.B. Singer, M.A. Clond, M. Bukur, D.R. Margulies, A. Salim, E.J. Ley — 2012-02-01

Osteoclast-like Cells in Abdominal Aortic Aneurysm

D. Yamanouchi, C. Stair, S. Morgan, D. Yamanouchi — 2012-02-01

Inhalational Carbon Monoxide Protects from Abdominal Aortic Aneurysm Formation in Mice

R.M. McEnaney, C. Go, B. Liu, E. Tzeng — 2012-02-01

Unique Features of Smooth Muscle Cells Derived from Abdominal Aortic Aneurysms

2012-02-01

Withdrawn

2012-02-01

In Vivo Imaging of Apoptosis in Early Aneurysm Development in Marfan Syndrome

2012-02-01

Atherosclerosis in Sporadic Ascending Aortic Aneurysms

2012-02-01

Inhibition of Vein Graft Intimal Hyperplasia With a Blue Marking Dye in a Rabbit Carotid Interposition Model

2012-02-01

Analysis of Femoral Artery Intima-Media Thickness, Vessel Diameter and Intima Cross-Sectional Area During the Cardiac Cycle

2012-02-01

SiRna Transfection and Gene Silencing in Human Aortic Smooth Muscle Cells from Electrospun PET

2012-02-01

A Swine Model for the Evaluation of Glutaraldehyde-Treated Bovine Pericardium and Dacron Arterial Patches in a Topically Infected Environment

J.J. Johnson, M.A. Jacocks, T. Garwe, R.F. Wolf, J.S. Lees — 2012-02-01

Increased Monocyte-Endothelial Cell Dysfunction in Patients With Peripheral Vascular Disease (PVD) Versus Patients With Abdominal Aortic Aneurysms (AAA)

E.S. Lee, Q.S. Shen, R.L. Pitts, M.H. Wu, S.Y. Yuan — 2012-02-01

Genome-Wide Differences in Inflammatory Gene Expression Predict Success Versus Failure in Lower Extremity Angioplasty/Stenting

2012-02-01

Cytokine Prediction Modeling of Vein Bypass Failure

2012-02-01

A Non-Toxic Blue Dye Restores Functional Viability After Stretch-Induced Injury in Saphenous Veins

2012-02-01

A Non-Toxic Blue Dye Enhances Smooth Muscle and Endothelial Function of Human Saphenous Vein Bypass Grafts

2012-02-01

Vascular Surgery Patient use of the Internet

L.M. Harris — 2012-02-01

Apolipoprotein A-1 Reduces Clot Strength and Augments Fibrinolysis

2012-02-01

In Vivo Directed Differentiation of Pluripotent Stem Cells to Bone Lineage and Repair of a Skeletal Defect

2012-02-01

Introduction: There remains a pressing need for a suitable alternative to currently available techniques for tissue engineering. the use of pluripotent stem cells for tissue engineering has thus far required a period of ex vivo differentiation prior to in vivo implantation to avoid the formation of a teratoma. in this study, we set out to differentiate pluripotent cells down an osteogenic lineage in vivo without pre-differentiation through the creation of a novel osteogenic niche. Methods: Human induced pluripotent stem cells (h-iPS) were derived from human adipose-derived stromal cell using the four Yamanaka factors and characterized for pluripotency. Human embryonic stem cells (hESC) were obtained from existing federally mandated cell lines. The ability of h-iPS and hESC to differentiate down an osteogenic lineage was confirmed in vitro through q-PCR, western blotting, and immunocytochemistry. h-iPS and hESC cells were seeded onto an osteogenic micro-niche consisting of a hydroxyapatite-coated osteoinductive scaffold with BMP-2 and implanted into a 4mm critical-sized calvarial defect macro-niche without prior ex-vivo differentiation in athymic mice. Implanted cell viability was followed through bioluminescence and histological analysis using human nuclear antigen. in vivo calvarial defect healing was assessed by microCT and histology. Results: We demonstrate that the implanted h-iPS and hESC cells undergo teratoma formation when placed in vivo either subcutaneously or within the renal capsule. We show that these cells can undergo osteogenic differentiation in vitro, as revealed by an up-regulation of alkaline phosphatase activity, mineralization and numerous gene markers (RUNX2, OCN and ALP) when placed in osteogenic medium. Most importantly, we also demonstrate that pluripotent cells seeded on an osteoconductive scaffold survive and participate in de novo bone formation, contribute to the healing of skeletal defects, and do not form a teratoma as defined by calcification on Micro CT and histological analysis. Conclusions: These data support that an osteogenic micro-niche scaffold placed into a macro-environmental niche of a skeletal defect promotes in vivo-directed pluripotent cell osteogenic differentiation without the formation of a teratoma. We believe this is the first example of directed lineage differentiation of pluripotent cells without teratoma formation in vivo and without a period of ex-vivo pre-differentation. Development of an in vivo model for directed differentiation of pluripotent cells would be important for these cells as we move closer to clinical applications.

Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier Breakdown

2012-02-01

Introduction: Intestinal barrier failure may lead to a systemic inflammatory response and distant organ injury in patients following severe injury. Enteric Glia Cells (EGCs), part of the Enteric Nervous System, have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO). We have recently shown than Vagal Nerve Stimulation (VNS) increases EGC activation which was associated with improved gut barrier integrity in an animal model of severe burn injury. In these experiments, we utilized an in vitro co-culture model of human enterocytes and EGCs to understand a mechanism by which EGCs prevent gut barrier breakdown after injury. We hypothesized that EGCs would improve gut barrier function through improved expression and localization of intestinal tight junction proteins, and that the protective effects of EGCs could be reproduced through treatment with GSNO. Methods: Caco-2 human enterocytes were grown to confluent monolayers and incubated alone, or in co-culture with EGCs, and exposed to Cytomix (TNF-α, INF-γ, IL-1β) for 24 hours. To determine the effects of GSNO, confluent monolayers were also incubated with GSNO and GSNO + Cytomix for 24 hours. Enterocyte monolayer permeability to 4kDa FITC-Dextran was measured. Confocal microscopy was used to assess cytoplasmic localization of tight junction proteins Occludin and ZO-1. Changes in Occludin protein expression were analyzed by Western blot. Results: Cytomix stimulation of Caco-2 monolayers caused a 6-fold increase in paracellular permeability which was associated with altered localization of Occludin and ZO-1 away from areas of cell contact and an overall decrease in Occludin protein expression. the addition of EGCs to Cytomix-stimulated Caco-2 monolayers restored permeability to control levels (Figure). Improved barrier function in co-cultured cells was associated with a normal distribution of Occludin and ZO-1 at the cell periphery and restoration of Occludin protein expression. Treatment of Caco-2 monolayers with GSNO also prevented Cytomix-induced barrier dysfunction, with permeability similar to control. Stimulated cells treated with GSNO demonstrated a normal localization of ZO-1 and Occludin at areas of cell contact and Occludin protein expression which was similar to control. Conclusions: EGCs improve gut barrier function after injury through improved expression and localization of the tight junction proteins Occludin and ZO-1. The protective effects of EGCs were reproduced through treatment with GSNO. Therapies which increase EGC activation, such as VNS, may be a novel strategy for limiting intestinal barrier failure in patients following severe injury.

European Society for Surgical Research: Biological Properties & Regenerative Potential, in Vitro & in Vivo, of Human Cardiac Stem Cells Isolated from the Adult Human Heart

2012-02-01

Inhibition of Cold Ischemia and Reperfusion Injury Mediated by Matrix Metalloproteinases (MMPs) by NFkB Inhibitor PS-341

2012-02-01

Introduction: Steatotic livers are more susceptible to cold ischemia and reperfusion (CIR) injury. Our lab has previously shown that MMPs play a role in the increased susceptibility of steatotic allografts to CIR injury. Further, MMP inhibitors have been shown to be protective following liver injury. Since CIR injury is associated with increased NFkB activation, we hypothesized that PS-341 (bortezomib), a proteasome inhibitor, may decrease the MMP related injury. Methods: We evaluated the role of MMPs in steatotic liver injury using an orthotopic liver transplant (OLT) model in Zucker rats. Genetically obese to lean OLTs were performed with and without intravenous PS-341 treatment (0.25 mg/kg) immediately prior to graft procurement (n=3). Following 2-hour cold ischemia and 2- or 24-hour reperfusion, tissue and serum were collected. MMP-9 and MMP-2 expression/activity were examined using zymography and densitometry. MMP expression was further validated by Western Blots using specific antibodies. Cytokines were assayed from liver tissue extract by ELISA. Normalized data were analyzed by 2-tailed unpaired t-test. Results: MMP-9 and MMP-2 were identified in all liver and serum samples. When compared to controls, PS-341 treated samples demonstrated significantly decreased MMP-9 and MMP-2 activity in both liver extract and serum at 2-hours following reperfusion (MMP-9: 0.11±0.04 vs. 0.78±0.05, p=0.0005; and 0.12±0.03 vs. 0.66±0.04, p=0.0003. MMP-2: 0.04±0.01 vs. 0.49±0.04, p=0.0003; and 0.15±0.03 vs. 0.45±0.05, p=0.008). There were no differences in MMP-9 or MMP-2 expression in both liver extract and serum at 24-hours following reperfusion (MMP-9: 0.43±0.04 vs. 0.40±0.01, p=0.47; and 0.38 ±0.12 vs. 0.57±0.16, p=0.39. MMP-2: 0.13±0.05 vs. 0.08±0.01, p=0.40; and 0.42±0.03 vs. 0.41±0.06, p=0.96). PS-341 treatment also reduced the expression of hepatic pro-inflammatory cytokines TNF-α and IL-1β when compared to controls (TNF-α: 223.8±29.9 vs. 518.5±66.5 pg/2μg protein, p=0.003; IL-1β: 6.0±0.9 vs. 19.8±5.2 pg/10μg protein, p=0.02) at 2-hours following reperfusion. Conclusions: PS-341 effectively inhibits MMP-9 and MMP-2 expression along with pro-inflammatory cytokines at 2-hours following the reperfusion of steatotic allografts in an OLT model in Zucker rats. Therefore, proteosome inhibition using PS-341 may represent a novel therapeutic strategy to improve early post-transplant allograft dysfunction due to steatosis.

Surgical Research Society of Southern Africa: Triple Negative Breast Cancer, Incidence and Patterns of Presentation

T. Monareng — 2012-02-01

Resistance of KRAS Mutant Colon Cancers to Cetuximab May Be Overcome Through Antibody Engineering

2012-02-01

Introduction: Cetuximab is an antibody that is FDA approved for treatment of colorectal cancer (CRC). Unfortunately, half of all CRC patients cannot benefit from cetuximab because their tumors have KRAS mutations that confer resistance to the agent. However, cetuximab may induce potent antitumor immune responses in some patients with KRAS mutant tumors because of expression of high affinity FcγIII receptors (FcRIII) on natural killer (NK) immune cells. High affinity FcRIII can bind the constant (Fc) domains of tumor-associated antibodies, allowing NK cells to kill tumors directly via antibody dependent cellular cytotoxicity (ADCC). FcRIII affinity is dictated by the amino acid located at the 158 position of the receptor. If a valine (V) is present, binding affinity is high, while if a phenylalanine (F) is present, affinity is low. We hypothesized that the 6-15% of humans with the homozygous FcRIII-158-V/V genotype (who only express high affinity FcRIII) could kill KRAS mutant tumors through ADCC in response to cetuximab, while those with the FcRIII-158-F/F genotype (who express only low affinity FcRIII) would not. Furthermore, we hypothesized that novel, engineered versions of cetuximab with increased avidity for FcRIII would enhance ADCC killing of KRAS mutant tumors by FcRIII-F/F donors. Methods: NK cells were isolated from the blood of healthy donors (n=48) and genotyped (FcRIII-V/V, n=7 [14%], V/F= 21 [44%], and F/F=20 [42%]). Cetuximab-induced ADCC against KRAS mutant SW480 colon cancer cells was tested using a standard chromium release assay with appropriate isotype controls. We also tested if ADCC could be enhanced in FcRIII-F/F donors (healthy and colon cancer patients) by novel versions of cetuximab with increased affinity for FcRIII. These included a cetuximab variant with a defucosylated Fc domain (made by kifunensine culture), and a stradobody™ version with polyvalent Fc domains. Results: FcRIII-V/V and V/F donors mounted significantly greater ADCC against KRAS mutant tumors than FcRIII-F/F donors (Median lysis: 38%, 39%, and 18%, respectively, p<.001). Engineered cetuximab variants with increased avidity for FcRIII significantly enhanced ADCC by FcRIII-F/F donors, including colon cancer patients who might have tumor induced immune tolerance and impaired NK cell cytotoxity (Figure 1). Conclusions: FcRIII-V/V and V/F genotype patients who express high affinity FcRIII may benefit from cetuximab even if they have KRAS mutant tumors. Novel, Fc engineered versions of cetuximab with increased avidity for FcRIII may enhance ADCC and tumor regression in FcRIII-F/F genotype patients who express low affinity FcRIII. These Fc engineered antibodies may be a unique and practical form of tumoricidal immunotherapy for treating advanced CRC and a variety of other cancers.

Endothelial Progenitor Cells Dependent Post-Pneumonectomy Compensatory Lung Growth (PPCLG): Role of Proliferation and Apoptosis

2012-02-01

Introduction: We previously demonstrated an essential role of bone marrow (BM)-derived endothelial progenitor cells (EPCs) in PPCLG, but the mechanisms by which they enhance compensatory lung growth are unknown. We hypothesized that EPCs enhance PPCLG by modulating proliferation and apoptosis in the regenerating lung. to define the role of EPCs in PPCLG, we performed pneumonectomy (PNX) in matrix metalloproteinase-9 knockout (MMP9-/-) mice known to be defective in EPCs mobilization, as a loss-of-function and analyzed cellular proliferation and apoptosis in the lung. in a gain-of-function, we rescued the MMP9-/- phenotype using Stem Cell Factor (SCF), known to bypass the MMP-9 dependent step in mobilization of BM EPCs. Methods: Resection of left lung (PNX) was performed in 8 week old FVB/N mice grouped into: 1) Wild type mice (WT), 2) MMP9-/- mice, 3) MMP9-/- mice treated with recombinant mouse SCF, 4) in sham-operated controls, WT mice underwent left thoracotomy (n=5). Tail vein injection of SCF (0.2ug/gm/day) was done 2 days before pneumonectomy and then daily till harvest. the lungs were harvested at days 7 and 10, inflation fixed and paraffin embedded. Ki-67 and TUNEL immuno-staining were performed to determine cellular proliferation index (PI) and apoptotic index (AI) respectively. Data was analyzed using ANOVA and t test, and presented as mean ± SD. Results: Seven days post-op WT mice lungs showed significantly higher PI compared to sham controls (Fig. A) (WT 32.6±5.1 vs. Sham 16.3±2.6; p<0.001). In contrast, the MMP9-/- mice that are EPCs mobilization-deficient, had significantly lower PI compared to the WT mice (MMP9-/- 21.2±2.3 vs. WT 32.6±5.1; p=0.001). The addition of SCF to MMP9-/- mice resulted in a significant increase in PI(MMP9-/-+ SCF 28.3±3.8 vs. MMP9-/- 21.2±2.3;p=0.01) to level observed in WT (MMP9-/-+SCF 28.3±3.8 vs. WT 32.6±5.1; p=NS). at 7 days post-op there was no significant difference in AI in between groups (Fig. B). Ten days post-op PI decreased in all groups. WT mice still had significantly higher PI compared to the sham but with no significant difference compared to MMP9-/- mice (Fig.C). MMP9-/-mice lungs had 3-fold increase in the AI compared to the WT(MMP9-/- 30 ±15.2 vs. WT 7±9.4; p=0.02) and SCF treatment significantly rescued this phenotype attenuating the apoptosis (MMP9-/- 30 ±15.2 vs. MMP9-/-+SCF 0.05±0.06; p=0.002) (Fig.D). Conclusions: EPC recruitment appears to be an essential driver of compensatory lung growth through stimulation of cellular proliferation and inhibition of cellular apoptosis, in response to PPCLG. Augmentation of EPC recruitment could potentially be a potent therapeutic strategy to enhance compensatory lung growth in a range of clinical applications

Transforming Growth Factor- Beta and its Signaling Mediator Smad3 Enhance Cell Survival After Vascular Injury

S.M. Seedial, P.A. Suwanabol, X. Shi, B. Liu, K. Kent — 2012-02-01

Introduction: It is well established that Transforming Growth Factor-beta (TGF-β) promotes intimal hyperplasia (IH); levels of this cytokine are elevated after vascular injury and blocking TGF-β results in inhibition of the hyperplastic response. IH is usually accompanied by inhibition of apoptosis/increased cell survival, which contributes to the increased cellularity that accompanies hyperplastic plaque. Paradoxically, TGF-β traditionally is thought to be pro-apoptotic. We have previously shown injured smooth muscle cells (SMCs) contain elevated levels of Smad3 and may respond to TGF-β differently compared to normal SMCs. In the current study, we tested a hypothesis that TGF-β in the presence of elevated Smad3 may be anti-apoptotic. Methods: Cultured aortic SMCs were infected with adenovirus expressing Smad3 or GFP and then stimulated with TGF-β for 24 hours. Apoptosis was induced with 5 minutes of ultraviolet light. Cleaved Caspase-3 immunofluorescence and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to detect apoptosis. Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with adenovirus expressing Smad3; or GFP control and arteries harvested at 3 days were subject to TUNEL staining and immunohistochemistry for cleaved Caspase-3. Akt activation was measured with phospho-Ser473 Akt (p-Akt). SIS3 was used to inhibit Smad3 activation. Results: in vitro, stimulation of SMCs withTGF-β reduced the number of TUNEL positive cells (Control = 35.0 vs TGF-β = 22.6%, p < 0.05) and diminished activation of Caspase-3 (31.4 vs 21.8%, p < 0.01) suggesting that TGF-β has an anti-apoptotic or protective effect on vascular SMCs. These protective effects were reversed by knockdown (siRNA) of TGF-β’s primary signaling protein, Smad3 (cleaved Caspase-3, p < 0.05). Moreover, overexpression of Smad3 in SMCs further enhanced TGF-β’s antiapototic effect (cleaved Caspase-3, p < 0.05). TGF-β phosphorylation of Akt was time-dependent and significantly enhanced when Smad3 was overexpressed. Both chemical inhibition and knockdown of Smad3 prevented phosphorylation of Akt (n = 3, p < 0.01). in injured carotid arteries, overexpression of Smad3 resulted in a dramatic inhibition of apoptosis but increased phosphorylation of Akt in the medial layer of the arterial wall: cleaved Caspase-3 (GFP = 35.2 vs Smad3 = 4.9, p < 0.01), TUNEL (32.1 vs 8.5, p < 0.01) and p-Akt (6.2 vs 22.4 p < 0.05). Conclusions: Our data show that TGF-β promotes SMC survival both in vitro and in vivo. This anti-apoptotic effect may well be one of the primary mechanisms through which TGF-β enhances intimal hyperplasia. Knockout and overexpression studies suggest that TGF-β’s inhibitory effect on apoptosis is mediated through the signaling protein Smad3. the correlation with Akt activation suggests that this may be mediated by the Akt-cell survival pathway.

Upregulation of VEGF in Adult Leptin Deficient Diabetic Mouse Adipose Derived Stromal Cells Using Biodegradable Nanoparticulate Polymeric Vectors Can Be Used to Accelerate Wound Closure in a Diabetic Mouse Excisional Wound Healing Model

A.C. Nauta, L. Deveza, D. Montoro, J. Hyun, G.C. Gurtner, M.T. Longaker, F. Yang — 2012-02-01

Introduction: Angiogenesis is essential to wound repair, and adipose-derived stromal cells (ASCs) are attractive vehicles for affecting angiogenesis. However, diabetic ASCs are known to express VEGF to a lesser degree than wild type ASCs, and angiogenic growth factor expression in both cell types is inadequate to have an appreciable effect on wound healing. We have previously demonstrated both in vitro and in vivo efficacy of VEGF DNA plasmid delivery to wild type ASCs using a novel, non-viral biodegradable nanoparticulate polymeric vector. Diabetes is known to interfere with the stages of wound repair. Therefore, the focus of this study is to apply this novel technology to diabetic ASCs to enhance VEGF expression and improve wound healing. Methods: Mouse diabetic and wild type ASCs were transfected with DNA plasmid pEGFP-N1 or pBLAST49-hVEGF using biodegradable poly(β-amino ester). Control cells were transfected using Lipofectamine 2000, and transfection efficiency was determined by flow cytometry. VEGF concentration in conditioned media was measured by ELISA, and cell survival was measured using a viability assay. One million transfected diabetic cells were injected into excisional wounds in diabetic mice, and wound healing measurements were assessed every other day until closure. These measurements were compared to positive ASC-injected and vehicle-treated controls. Results: VEGF ELISA confirmed decreased VEGF concentration in conditioned media of diabetic ASCs compared to wild type ASCs. Polymer-mediated gene delivery to wild type and diabetic ASCs led to approximately two-fold increase in transfection efficiency compared to positive controls (9.91% and 8.70%, versus 3.55% and 3.61%, respectively, *p<0.05). VEGF ELISA showed the diabetic ASCs transfected with polymer/VEGF resulted in VEGF concentration almost three-fold that of positive controls. (*p<0.05). Cell viability on day 2 post-transfection was 60-70% and comparable to positive controls. Diabetic mice injected with VEGF/polymer transfected ASCs healed wounds in 10 days after the procedure and treatment, versus ASC-injected and vehicle controls that healed wounds at an average of 12-14 days (*p<0.05). Conclusions: the use of polymeric nanoparticles is a safe and effective means of upregulating VEGF in both wild type and diabetic ASCs, addressing the limitations of traditional gene delivery methods. Furthermore, VEGF transfected cells injected into excisional wounds of syngeneic mice accelerate wound closure. Therefore, this technology could be used in the development of novel gene-therapy based wound healing strategies for both healthy and diabetic subjects.

The Pro-Fibrotic Molecule, Connective Tissue Growth Factor (CTGF), is Regulated by MicroRNA-214 (miR-214) in Hepatic Stellate Cells

L. Chen, A. Charrier, Y. Zhou, D. Brigstock — 2012-02-01

Introduction: Connective tissue growth factor (CTGF) plays a central role in driving hepatic fibrosis in vivo and pathways of fibrogenesis in hepatic stellate cells (HSC), the principal fibrogenic cell in the liver. in response to fibrosing stimuli, CTGF is expressed by HSC and regulates their proliferation, myofibroblastic differentiation, and collagen production. in this study, we analyzed the regulation of CTGF production in HSC by microRNA-214 (miR-214). MiR-214 is located within the intron of the dynamin3 (DNM3) gene and produces a 7.9kb non-coding DNM3 opposite strand transcript (“DNM3os”). Methods: Mice were subjected to chronic intragastric ethanol infusion prior to assessment of hepatic CTGF or miR-214 expression. in situ hybridization (ISH) for miR-214 or DNM3os was performed using digoxygenin-labeled probes. Cultured HSC from normal mice were analyzed for expression of CTGF, collagen α1(I), or miR-214 either after exposure to 0-25mM ethanol or after transfection with miR-214 in the presence of a CTGF 3'-UTR target protector. Reporter assays were performed in HSC transfected with phCMV-CLUC into which CTGF 3'-UTR was cloned downstream of luciferase. Functional targeting of the CTGF UTR by miR-214 was assessed by HSC viability or luciferase production after transfection of the cells with a Fire-Ctx sensor reporter containing either wild type CTGF UTR or a mutant CTGF UTR lacking the miR-214 binding site. Results: MiR-214 was detected by ISH in day 3 cultured mouse HSC while DNM3os was localized by ISH to HSC and hepatocytes in normal liver. MiR-214 was decreasingly expressed as CTGF was increasingly expressed in steatotic liver or in primary mouse HSC in response to ethanol or TGF-ß, or during autonomous activation in culture. Activity of a CTGF 3'UTR luciferase reporter in HSC was reduced by co-transfection with pre-miR-214. Transfection of HSC with pre-miR-214 reduced expression of CTGF or collagen α1(I). Suppression of CTGF or collagen α1(I) expression or of CTGF 3'UTR luciferase reporter activity by miR-214 was prevented by a protector designed to block the miR-214 binding site whereas a control negative protector was ineffective. When miR-214-CTGF UTR was cloned into a Fire-Ctx sensor reporter and transfected into HSC, the cells exhibited high levels of Ctx drug-induced cell death except when the cells were co-transfected with pre-mir-214 which caused Ctx activity to be blocked and viability to increase; this rescue was absent in a deletion mutant lacking the miR-214 binding site. Conclusions: Elevated expression of CTGF during liver fibrosis or HSC activation is due to reduced expression of miR-214 which otherwise exerts suppressive effects on CTGF production via its direct binding to the CTGF 3'-UTR. the inhibition by miR-214 of expression or action of CTGF, a highly pro-fibrotic molecule that drives liver fibrosis in vivo, suggests that miR-214 may have therapeutic or diagnostic value in fibrosing liver injuries.

Induction of the Nrf2-Antioxidant Response Element Pathway (ARE) in Hepatic Stellate Cells Protects Hepatocytes from Oxidative Stress-Induced Injury

D.P. Foley, S.E. Nelson, D.A. Johnson, J.A. Johnson — 2012-02-01

Introduction: Oxidative stress (OS) plays a major role in the cellular damage sustained during hepatic ischemia reperfusion injury (IRI). the transcription factor, Nrf2, mediates the gene expression of several antioxidant proteins. Using transgenic reporter mice in a model of hepatic IRI, we have shown a greater induction of the Nrf2-ARE pathway in hepatic stellate cells (HSCs) compared to hepatocytes. the aim of these studies was to determine whether Nrf2 induction in HSCs could mitigate hepatocellular damage in in vitro models of OS. Methods: Primary hepatocytes were isolated from Nrf2 wild type (WT) mice using a standard in situ collagenase digestion technique and plated in 96-well, collagen-coated plates (20,000 cells per well) with 10% FBS, low glucose DMEM. HSCs were isolated from both Nrf2 WT and KO mice using both in situ and ex vivo pronase/collagenase digestion followed by density centrifugation in Percoll. HSCs were expanded on tissue culture plastic in the same media to obtain sufficient cells for experiments. to assess resistance to oxidative stress, we plated 5,000 HSCs per well. In co-culture experiments, HSCs were added to the hepatocytes at a 1:8 ratio. After 48 h in culture, 100 uM of tert-Butylhydroquinone (tBHQ), a known Nrf2-inducer, was added to cells. After stimulation for 24 h, cells were treated with incremental doses of tert-Butyl hydroperoxide (tBOOH), a known inducer of OS. Twenty-four hours later MTS assays were performed to assess cell survival. Alanine aminotransferase (ALT) assays were performed to evaluate hepatocellular death. Statistical analysis was performed using one-tailed Student's t-tests with p < 0.05 for significance. Results: tBHQ treatment of Nrf2-WT HSCs prior to OS resulted in significant increases in cell survival compared to vehicle controls (Fig. A). tBHQ pretreatment of Nrf2-KO HSCs provided no cellular protection from OS (data not shown). tBHQ pretreatment of hepatocytes did not prolong cell survival after OS compared to vehicle (Fig. B). in co-cultures with WT HSCs and hepatocytes, tBHQ pretreatment resulted in a significant reduction in ALT release with incremental doses of tBOOH compared to vehicle controls (Fig. C). in co-cultures containing Nrf2 KO HSCs, the reduction in ALT release with tBHQ pretreatment was minimal across multiple doses of tBOOH (Fig. D). Conclusions: Pretreatment of HSCs with tBHQ provides significant protection from OS-induced cellular injury. in contrast, tBHQ pretreatment of hepatocytes alone provides no protection from OS. However, Nrf2 induction with tBHQ in co-cultures of HSCs and hepatocytes provides significant hepatocellular protection from OS. Activating Nrf2 in HSCs may be an effective approach to mitigate the cellular injury seen in hepatic IRI.

Transplanted Fibroblast Cell Sheets Promote Migration of Hepatic Progenitor Cells in the Incised Host Liver in Allogeneic Rat Model

2012-02-01

Introduction: ‘Cell-sheet engineering’ using temperature-responsive dishes has been noted as a new approach in the tissue engineering field. This technique has been already used in clinical fields including autologous mucosal epithelial cell sheets for the treatment of esophageal ulceration after endoscopic submucosal dissection. Usefulness of cell-sheet engineering has been expected also in hepatic tissue engineering. on the other hand, it was reported that proliferation of smooth muscle cells in the site where skeletal cell sheets were transplanted successfully prevented the deterioration of the impaired myocardium. These data indicate that transplanted cell sheets may induce organ-specific progenitor cells. The objective of this study is to explore procedure to induce hepatic progenitor cells in the rat liver. Methods: SD rat dermal fibroblasts (DFs) were used as donor cells and male F344 nude rats as recipients. DFs were cultured on temperature-responsive dishes and DFs were then harvested as a cell sheet. In the DF group, DFs sheets were transplanted into the incised surface of the liver. In the control group, only a same incision was made. The rats were sacrificed on postoperative days (POD) 7, 14, and 28 (n=5). for histological examination, hematoxyline-eosin staining and immunohistochemistry for cytokeratin (CK)-8 (rat bile duct epithelial cell marker), OV-6 (liver progenitor cell marker) and α-fetoprotein (AFP) (immature hepatocytemarker) were performed. We also performed the transplantation of GFP-expressing DFs sheet into the incised surface of the liver. Results: In the control group, necrosis or inflammatory cell infiltration was observed on POD7, and those were absorbed eventually. in contrast, the DF sheet transplanted sites became thicker, and bile duct (BD)-like structures appeared on POD 7. On POD 14, the BD-like structures were more significant, and immature hepatocyte-like cells were observed. Histological assessment revealed that BD-like structures were CK8 positive, and hepatocyte-like cells induced around the BD were both OV-6 and AFP positive. Observing the GFP-expressing DFs sheet transplanted sites byimmunofluorescent microscope, the proliferation of GFP positive DF cells were found, but the cells forming new structures including BD were GFP negative. These findings may become suggest that engrafted DFs recruited hepatic progenitor cells and supported their differentiation to mature hepatic tissues. Conclusions: We observed engineering process of hepatic tissues after transplantation of DF sheets. We also demonstrated that the origin of newly formed structures was recipient liver, which means transplanted DFs sheets induced hepatic progenitor cells from the recipient liver. The present study described a novel aspect of hepatic differentiation process induced at liver injury site. These technologies may become a useful therapeutic method for liver diseases in the future.

Transforming Growth Factor- Beta and its Signaling Mediator Smad3 Enhance Cell Survival After Vascular Injury

S.M. Seedial, P.A. Suwanabol, X. Shi, B. Liu, K. Kent — 2012-02-01

Introduction: It is well established that Transforming Growth Factor-beta (TGF-β) promotes intimal hyperplasia (IH); levels of this cytokine are elevated after vascular injury and blocking TGF-β results in inhibition of the hyperplastic response. IH is usually accompanied by inhibition of apoptosis/increased cell survival, which contributes to the increased cellularity that accompanies hyperplastic plaque. Paradoxically, TGF-β traditionally is thought to be pro-apoptotic. We have previously shown injured smooth muscle cells (SMCs) contain elevated levels of Smad3 and may respond to TGF-β differently compared to normal SMCs. in the current study, we tested a hypothesis that TGF-β in the presence of elevated Smad3 may be anti-apoptotic. Methods: Cultured aortic SMCs were infected with adenovirus expressing Smad3 or GFP and then stimulated with TGF-β for 24 hours. Apoptosis was induced with 5 minutes of ultraviolet light. Cleaved Caspase-3 immunofluorescence and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to detect apoptosis. Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with adenovirus expressing Smad3; or GFP control and arteries harvested at 3 days were subject to TUNEL staining and immunohistochemistry for cleaved Caspase-3. Akt activation was measured with phospho-Ser473 Akt (p-Akt). SIS3 was used to inhibit Smad3 activation. Results: in vitro, stimulation of SMCs withTGF-β reduced the number of TUNEL positive cells (Control = 35.0 vs TGF-β = 22.6%, p < 0.05) and diminished activation of Caspase-3 (31.4 vs 21.8%, p < 0.01) suggesting that TGF-β has an anti-apoptotic or protective effect on vascular SMCs. These protective effects were reversed by knockdown (siRNA) of TGF-β’s primary signaling protein, Smad3 (cleaved Caspase-3, p < 0.05). Moreover, overexpression of Smad3 in SMCs further enhanced TGF-β’s antiapototic effect (cleaved Caspase-3, p < 0.05). TGF-β phosphorylation of Akt was time-dependent and significantly enhanced when Smad3 was overexpressed. Both chemical inhibition and knockdown of Smad3 prevented phosphorylation of Akt (n = 3, p < 0.01). in injured carotid arteries, overexpression of Smad3 resulted in a dramatic inhibition of apoptosis but increased phosphorylation of Akt in the medial layer of the arterial wall: cleaved Caspase-3 (GFP = 35.2 vs Smad3 = 4.9, p < 0.01), TUNEL (32.1 vs 8.5, p < 0.01) and p-Akt (6.2 vs 22.4 p < 0.05). Conclusions: Our data show that TGF-β promotes SMC survival both in vitro and in vivo. This anti-apoptotic effect may well be one of the primary mechanisms through which TGF-β enhances intimal hyperplasia. Knockout and overexpression studies suggest that TGF-β’s inhibitory effect on apoptosis is mediated through the signaling protein Smad3. the correlation with Akt activation suggests that this may be mediated by the Akt-cell survival pathway.

Assessment of the Implementation of a Surgical Preoperative Checklist

2012-02-01

Introduction: Peri-operative checklists are mandated by many hospitals based on the reduction in morbidity and mortality seen with utilization of the World Health Organization's (WHO) “Surgical Safety Checklist.” Although an adapted peri-operative checklist was implemented within our hospital system without formal system-wide training, compliance with the checklist is reported to be 100%. We hypothesize that compliance does not measure fidelity of implementation, in that all items on the checklist are not performed as intended. Methods: Over a 10 week period, a prospective study was performed evaluating the completion of the 12 pre-incision components of the surgical checklist. Pediatric surgical operations, occurring in the operating room or in neonatal and pediatric intensive care units, were randomly selected for direct observation. Emergent cases were excluded. the evaluated checkpoints include essential parties present, team members identified, patient name/procedure verified, incision site confirmed, team member concerns addressed, administration of appropriate antibiotics (if applicable), essential imaging displayed (if applicable), anticipated case length stated, anticipated risk of blood loss stated, and sterility indicator confirmed. Essential parties included anesthesiology attending or fellow, pediatric surgical attending or fellow, circulating nurse, and scrub technologist. Results: 142 pediatric surgical cases were observed. Hospital data demonstrated 100% compliance with the pre-incision phase of the checklist for these cases. Our observation revealed that in 3.5% of cases the checklist was not performed at all. None of the cases completely executed all items on the checklist, and the average number of checklist items performed in the observed cases was five. the most commonly performed checkpoints were the confirmation of patient name and procedure (99.3%) and administration of antibiotics (88.1%). the rest of the checkpoints were performed in less than 60% of cases (figure). Conclusions: These data show that despite the 100% documented completion of the pre-incision phase of the checklist, most of the individual checkpoints are not routinely performed. These findings demonstrate lack of fidelity in implementing the checklist, which may be a reflection of the poor strategy in disseminating and implementing this patient safety practice. Failure of the system to measure the appropriate implementation metrics and to fully adopt the evidence-based intervention could lead to failure to achieve the intended outcomes. the impact and etiology of non-compliance with all components of the surgical checklist requires further investigation.

Exploring Variation in Emergent Surgical Outcomes in the Michigan Surgical Quality Collaborative: a Fertile Area for Quality Improvement

2012-02-01

Introduction: Within a large, statewide collaborative, significant improvement in surgical quality have been appreciated (9.0% reduction in morbidity for elective general and vascular surgery). Our group has not noted such quality improvement in the care of patients who had emergent operations. With this work, we aim to describe the scope of emergency surgical care within the MSQC, variations in outcomes among hospitals, and variations in adherence to evidence based process measures. Overall, these data will form a basis for a broad based quality improvement initiative within Michigan. Methods: We report morbidity, mortality, and costs of emergency and elective general and vascular surgery cases (n= 211,903) within 34 hospitals participating in the MSQC from 2005 to 2010. Adjusted hospital specific outcomes were calculated using a step-wise multivariable logistic regression model. Adjustment covariates included patient specific co-morbidities, and case complexity. Hospitals were also compared based on their adherence to evidence based process measures (measures at the patient level for each case – SCIP 1 and 2 compliance). Results: Emergent procedures accounted for approximately 10% of total cases, yet they represented 32.0% of mortalities and 40.4% of surgical complications. the complication-specific costs to payers was $208 million for the emergent cases and $307 million for the elective cases. Adjusted patient outcomes varied widely within MSQC hospitals; morbidity and mortality rates ranged from 3.1% to 28.0% and 0% to 13.6%, respectively.The variation among hospitals was not correlated with volume of emergent cases and case complexity. for emergent colectomies, there was wide variation in compliance with SCIP 1 and 2 measures, and overall compliance (42.0%) was markedly lower than elective colon surgery (81.7%). Conclusions: Emergent surgical procedures are an important target for future quality improvement efforts within Michigan. Future work will identify best practices within high-performing hospitals and disseminate these practices within the collaborative.

Hesperetin Activates the Notch1 Signaling Cascade, Induces Cellular Differentiation, and Causes Apoptosis in Anaplastic Thyroid Cancer

P.N. Patel, Y. Xiao-Min, M. Kunnimalaiyaan, H. Chen — 2012-02-01

Introduction: Anaplastic thyroid cancer (ATC) is characterized by aggressive growth and extremely poor prognosis, for which there is no standardized therapy. in addition, the undifferentiated feature of ATC in which sodium iodide symporter (NIS) expression is reduced makes treatment such as radioactive iodine ineffective. Therefore, there is an urgent need for strategies that sensitize ATC cells to iodine treatment. It has been reported that the Notch1 signaling pathway, which affects cell proliferation and differentiation, is inactivated in ATC. However, it is unknown whether activation of Notch1 could yield therapeutic effects in ATC. Therefore, in this study we evaluated whether Hesperetin, a naturally occurring flavanone and potential Notch activator, effects ATC cell proliferation and possibly up-regulates thyroid-specific differentiation markers such as NIS. Methods: In-vitro studies were carried out to evaluate the drug effects using a human derived ATC cell line (HTH7). to determine whether Hesperetin activates Notch1 in ATC, expression of Notch1 mRNA and protein was evaluated upon treatment. in addition, mRNA levels of Notch1 response genes (Hes1 and Hey1) were measured, and the Notch1 activity was assessed using CBF-1-luc binding assay. Cellular proliferation was measured by viable cell counts every 24 hours post-treatment (0-200mM) for 72 hours, and the underlying mechanism was investigated by western blot. Expression of thyroid-specific differentiation markers including Thyroid Transcription Factor 1 (TTF-1), Thyroid Transcription Factor 2 (TTF-2), Paired Box Gene 8 (PAX-8), Thyroid Stimulating Hormone Receptor (TSHR), and NIS were also measured. Results: Hesperetin caused activation of the Notch1 signaling pathway. Treated ATC cells displayed elevated Notch1 protein and increased mRNA levels of Hes1 and Hey1, important Notch1 response genes. Treatment induced a dose-dependent increase in Notch1 activity as evidenced by increased luciferase activity. Hesperetin treatment also resulted in a time and dose-dependent decrease in cell proliferation: at 72 hours, 50mM and 100mM Hesperetin resulted in a 27% and 47% reduction respectively compared to control. Protein expression of apoptotic markers cleaved PARP, cleaved Caspase-3, and BAD increased with treatment. Importantly, Hesperetin up-regulated NIS mRNA levels, a thyroid-specific marker responsible for sensitivity to radioactive iodine. Other thyrocyte markers (TTF-1, TTF-2, PAX-8 and TSHR) were also increased, further evidence that Hesperetin induces differentiation in ATC. Conclusions: Hesperetin activates the Notch1 signaling cascade and has a significant antiproliferative effect on ATC that can be largely attributed to apoptosis. Hesperetin also induces differentiation in ATC, and by up-regulating markers such as NIS, could be useful in combination with radioactive iodine for treating ATC. Further investigation to elucidate the in-vivo effects of Hesperetin on ATC is warranted.

The Progressive Decline in Diabetic Skin Integrity is Associated With Decreased Collagen Protein Content and Dysregulation of MicroRNA-29a

2012-02-01

Introduction: We have shown that the biomechanical properties of murine and human diabetic skin are significantly inferior to non-diabetic skin at baseline. the major structural component of the skin responsible for skin integrity is type I collagen. MicroRNAs (miRNAs) are regulatory molecules that inhibit the translation of mRNAs at the post-transcriptional level, and have been shown to play an important role in the regulation of collagen content. Specifically, MiRNA-29a has been shown to inhibit the translation of Col1a2 mRNA. We hypothesized that the impaired biomechanical properties of murine diabetic skin at baseline is associated with decreased collagen protein and is due, in part, to increased miRNA-29a production. Methods: To test this hypothesis, we obtained samples from 4 and 20 week old diabetic and non-diabetic murine skin. the skin was processed for isolation of total protein and total cellular RNA. Western blot analysis was performed for type I collagen and real-time PCR performed for Col1a2 and miRNA-29a. Results: At 4 weeks of age diabetic and non-diabetic skin had similar type I collagen protein content, however, by 20 weeks the diabetic skin had significantly decreased type I collagen compared to non-diabetic (p<0.05) or 4 week old skin (p<0.05). at 4 weeks diabetic skin had significantly increased col1a2 and miRNA-29a expression compared to non-diabetic skin, and a col1a2/miRNA-29a ratio of 7.6:1 in diabetics. at 20 weeks the diabetic skin also had increased col1a2 and miRNA-29a expression compared to non-diabetic skin, but the col1a2/miRNA-29a ratio was significantly decreased to 2.7:1 in diabetics. Conclusions: These findings provide the first evidence that the impaired biomechanical properties of diabetic skin may be due, in part, to increased miRNA-29a expression relative to collagen gene expression, resulting in decreased collagen protein production. These results represent a novel potential therapeutic target to improve collagen content and subsequent biomechanical properties of diabetic skin to prevent injury, as well as to improve healing following injury.

Activated Mesenchymal Stem Cells Increase Wound Tensile Strength in Old Mouse Model Via Macrophages

S. Lee, E. Szilagyi, L. Chen, L.A. DiPietro, A.M. Bartholomew — 2012-02-01

Introduction: Tissue damage with a subsequent delay in wound regeneration affects morbidity, mortality, and increases healthcare costs. This is especially true for older population groups where the natural ability to regenerate tissue has been diminished. We have previously observed that a treatment of 50,000 dose of autologous MSC activated with interferon gamma (aMSC) increased tensile strength in a younger mouse model. Drawing from these observations, we tested the extent to which aMSC could improve wound tensile strength in an older mouse model. in addition, we also explored the possible role of macrophages in aMSC's augmentation of wound healing. Methods: A B6 mouse model testing 5 therapies was used to determine the extent to which activated MSCs augmented wound tensile strength in older animals (>12 months). Therapies were injections of aMSC, aMSC after macrophage depletion with clodranate, only clodranate treatment, fibroblasts (3T3 cell line), and vehicle. Cell doses of 50,000 were injected into 4cm linear incisions on the dorsum on each animal and then sutured closed. Macrophage depleted animals were treated with intraperitoneal injections of clodranate for 5 days prior to wounding. After 7 days, histological analysis and tensile strength measurements of the wounds were conducted. Results: In older mice, it was found that the tensile strength of healing wounds was significantly lower as compared to younger animals (<2 months) when only vehicle was administered (100% +/- 11.4 vs. 381% +/- 24.3, p = 3.67E-4). Older mice treated with aMSC showed significant and substantial increases in tensile strength of healing wounds when compared to older control mice treated with only vehicle (100% +/- 16.4 vs. 270% +/- 43.3, p = 4.61E-5). Macrophage depleted older mice treated with aMSC had tensile strength not significantly different compared to controls (104% vs. 100% +/- 21.8, p = n/s). Lastly, there was less pronounced increase in tensile strength in older mice treated with fibroblasts when compared to older controls (100% +/- 9.81 vs. 164% +/- 47.9, p = 4.44E-2). Conclusions: When compared to vehicle only controls, aMSC treatment had a significant statistical increase in wound tensile strength in older mice. the application of aMSC brought the augmented tensile strength close to that of younger control mice, suggesting that aMSC may be an effective therapy option for enhanced healing in older animals with a naturally diminished ability to repair wounds. Macrophage depleted older mice did not differ significantly in wound tensile strength, suggesting that these cells may act as a mediator for the aMSC in enhancing wound healing. Further study of this mechanism is planned. aMSC also enhanced wound healing better than fibroblasts, a current clinical treatment option, suggesting a potential alternative therapy option. Taken together, this data provides encouraging support for the development of aMSC therapies for enhanced tissue regeneration, especially for older population groups.

Reliability of Risk-Adjusted Outcomes as Hospital Quality Measures

A.E. Hozain, L.S. Kao, J.B. Dimick — 2012-02-01

Introduction: Risk-adjusted outcomes are increasingly used as indicators of hospital quality for major surgery. However, it is unknown whether outcomes are statistically reliable measures of hospital quality (i.e., whether high-performing hospitals can be distinguished from low-performing hospitals). Methods: We examined all patients who underwent several common procedures in the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) in 2008: colectomy, abdominal aortic aneurysm repair, pancreatectomy, lower extremity bypass, and ventral hernia repair (n = 44,830 patients, n = 211 hospitals). We first calculated the number of cases and the risk adjusted outcomes for each procedure. We then used hierarchical modeling to estimate the reliability of each measure at each hospital. for each procedure, we then calculated the proportion of hospitals that met the acceptable level of reliability (0.70). Results: Risk-adjusted morbidity was found to be the most reliable measure of hospital quality. for example, with colectomy, 55% of all hospitals surpassed the minimum reliability threshold. to achieve this level of reliability, 84 cases were required. for the other 4 procedures, however, a small fraction of hospitals met this threshold (0% for both abdominal aortic aneurysm repair and ventral hernia repair, only 2% and .5% for pancretectomy and lower extremity bypass, respectively). Risk-adjusted mortality was never a reliable measure of hospital quality. There were no hospitals which met the minimum reliability threshold for all 5 procedures. Conclusions: Except for rates of colon resection morbidity, risk-adjusted outcome measures are unreliable as indicators of hospital quality. to improve the reliability of outcome measures, quality measurement programs should increase sample size by collecting one hundred percent of cases for targeted procedures, and use statistical techniques to adjust for reliability.

Response of Type II Diabetes Mellitus to the Preoperative Liquid Diet as a Predictive Model for Diabetes Resolution in Bariatric Surgery Patients

S.M. Biro, D.L. Olson, M.J. Garren, J.C. Gould — 2012-02-01

Introduction: the prevalence of obesity and type II diabetes mellitus (T2DM) is rapidly increasing in the United States. Up to 90% of patients with T2DM are obese. in morbidly obese individuals with T2DM, bariatric surgery offers a novel potential therapeutic endpoint – complete disease remission. in our bariatric program, patients are placed on an 800 calorie/day, very low-calorie diet (VLCD) for 2 weeks prior to surgery. Some diabetic patients experience significant improvement in blood glucose control and reduced insulin requirements on this VLCD. We sought to define the relationship between diabetic response to the pre-op VLCD and T2DM resolution rates post-op in a select group of bariatric surgery patients with the most severe diabetes, those requiring injectable insulin. Methods: Our study group was comprised of 51 diabetic patients on injectable insulin who underwent bariatric surgery at the UW Health Bariatric Surgery Program from August 2006 to February 2011. Subjects' blood sugars and insulin requirements prior to initiating the VLCD were compared with those at 10 days on a VLCD. Patients with a ≥50% reduction in total insulin dosage to maintain appropriate blood glucose control were deemed ‘rapid responders’ to the pre-op VLCD. All others were deemed ‘non-rapid responders’. Subjects were followed up to 1-year post-op to determine T2DM resolution rates (complete cessation of all antidiabetic medication). Results: of the 51 subjects, 29 (57%) were ‘rapid responders’ to the pre-op VLCD and 22 were ‘non-rapid responders’. Rapid and non-rapid responders did not differ demographically based on age (56.8 yr. vs 54.4; p=.41), sex (female 52% vs 77; p=.08), pre-op body mass index (47 kg/m2 vs 50; p=.12), HbA1c (7.5 vs 7.7; p=.7), duration of T2DM (13.9 yr. vs 14.5; p=.75) or procedure (laparoscopic gastric bypass [LRYGB] 72% vs 64%; p=.55 or laparoscopic adjustable gastric band [LAGB] 10% vs 32%; p=.07). T2DM resolution rates were significantly greater in the ‘rapid responder’ group (Table 1). Evaluating LRYGB patients alone, rapid responders showed greater % excess weight loss (EWL) at 3 months (40% vs 28%; p<.01), 6 mo. (55% vs 40%; p<.01), and 12 mo. (65% vs 51%; p=.04). Conclusions: Insulin-dependent T2DM bariatric surgery patients who display a rapid response (≥50% reduction in insulin requirements) to the pre-op VLCD are more likely to experience early resolution of T2DM post-op, and ultimately greater weight loss. the diabetic response to the pre-op VLCD is an important predictive variable not routinely taken into account in the published literature pertaining to diabetes and bariatric surgery. Further study is necessary to clearly delineate the mechanisms for these observations.

The Severity of Disparity: Increasing ISS Accentuates Disparate Outcomes Following Injury

2012-02-01

Introduction: Multiple studies have demonstrated disparities in trauma mortality based on race and health insurance status. the underlying causes of these disparities are still largely unknown. Elucidating what groups are most at riskmay help in understanding these mechanisms. Given the predictable effect injury severity has on mortality, we sought to determine if injury severity modifies previously reported race and insurance status based disparities in survival after blunt traumatic injury. Methods: Adults (ages 18-64) in the 2003-2008 National Trauma Data Bank were stratified into six groups based on race and insurance status. Patients with Injury Severity Score (ISS) >=9 were included. Multivariable logistic regression compared the odds of death between the six race/insurance groups within different injury severity levels (ISS 9-15; ISS>15 & Systolic Blood Pressure (SBP)>90; ISS>15 & SBP<90). Age, gender, ISS, Glasgow Coma Scale (GCS) motor, presence of hypotension (SBP<90), admission year, and mechanism of injury were controlled for. Clustering was used to control for potential inter facility survival outcomes differences. Results: 760,598 patients met inclusion criteria. Crude mortality for moderately injured white insured patients was 0.75% and 1.52% (p<0.01) for uninsured blacks. for the hypotensive severely injured group, the difference in mortality increased to 34.5% for insured whites compared to 58.9% for uninsured black patients (p<0.01). (Figure) Adjusted analysis revealed an increase in disparities between race/insurance groups as the degree of injury worsened. (Table) Conclusions: Disparities in trauma mortality effecting minority and uninsured patients appear to worsen with increasing injury intensity. Understanding why the most severely injured suffer from even greater disparities may help us develop effective solutions to mitigate these inequities.

Is the Kampala Trauma Score an Effective Predictor of Mortality in Low-Resource Settings? A Comparison of Multiple Trauma Severity Scores

2012-02-01

Introduction: Injury results in an estimated 5.8 million deaths worldwide annually, with the majority occurring in developing countries. in the developed world, multiple injury severity scores have been developed and validated for trauma patients. Despite widespread use in resource-rich settings, few studies have supported effectiveness of these scores in the developing world. as a result, the Kampala Trauma Score (KTS) was developed for resource-limited settings and has been shown to be a robust predictor of death. However, the literature supporting its utility over other tools is limited to retrospective analyses and small patient cohorts. This study evaluates trauma scores as predictors of mortality in a large prospective patient cohort from the emergency ward of a teaching and referral hospital in Cameroon. Methods: This prospective study was conducted in the emergency ward of the Central Hospital of Yaounde, the largest trauma volume hospital in the capital of Cameroon. Collected information included patient demographics, clinical presentation and outcome. for each patient, Kampala Trauma Score (KTS), Injury Severity Score (ISS), Revised Trauma Score (RTS), Glasgow Coma Scale (GCS) and Trauma Injury Severity Score (TRISS) were calculated. Scores were evaluated as predictors of mortality using logistic regression models. Comparisons between areas under ROC curves were made using DeLong's nonparametric jackknife method. Results: A total of 2855 patients were enrolled. 73% of patients were male; 60% presented after a road traffic injury. the median age was 28 years. 60% of patients had injuries classified as mild (<9) on the ISS scale, 29% moderate (9-15), 8% severe (16-24) and 3% profound (> 24). the mortality rate was 0.6%. 2472 patients with non-missing values for KTS, ISS, RTS, GCS, and TRISS were included in logistic regression analysis which showed each score to be a statistically significant predictor of mortality. ROC curves are shown. the area under the ROC for KTS as a predictor of mortality was 0.7748 (95% CI: 0.6285 - 0.9212). No pairwise difference between ROC areas of KTS and other scores was statistically significant. Similar results were found when analysis was limited to severe injuries. Conclusions: This comparison analysis of KTS to trauma scores from the developed world supports the adoption of KTS for injury surveillance and triage in resource-limited settings. As severity scores are a necessary tool for evaluating patient care, it is important to develop and validate scoring systems for use in the developing world. We show that the Kampala Trauma Score is as effective as other scoring systems in predicting patient mortality. As KTS is simple to administer and record, it is therefore a valuable tool available for resource-limited settings.

Reliability of Risk-Adjusted Outcomes as Hospital Quality Measures

A.E. Hozain, L.S. Kao, J.B. Dimick — 2012-02-01

Quality Measures for Assessing Hospital Mortality: Evaluation Using Instrumental Variable Analysis

L.H. Nicholas, T.J. Iwashyna, J.B. Dimick — 2012-02-01

Introduction: Hospital quality indicators are often validated by assessing their relationship to risk-adjusted mortality. However, these evaluations may be biased due to unmeasured differences in patient severity. We sought to validate a composite measure of hospital quality using an instrumental variable analysis to account for potential unmeasured differences in patient severity. Methods: We studied patients undergoing 5 high-risk surgical procedures in the national Medicare population between 2005 and 2008. We measured hospital quality using a composite measure of hospital volume and risk-adjusted mortality for a two year time period (2005-06). We then examined the relationship between the composite score and future risk-adjusted mortality rates (2007 - 2008). to assess the impact of unobserved confounding variables, we performed this analysis with and without an instrumental variable approach. We used differential distance to the highest quality hospital as our instrumental variable. for these analyses, we compared mortality rates in the highest quality hospitals (top 20% on the composite measure) to all other hospitals. Results: Patients who lived closer (differential distance below the median) to highest quality hospitals were much more likely to have surgery at them for all 5 operations, varying from 3 times more likely (30.4% to 10.3%) for pancreatic resection to nearly 7 times more likely (36% vs. 5.3%) for aortic valve replacement. in instrumental variable analysis, patients undergoing surgery in 2007 - 2008 at hospitals with the highest 2005 - 2006 composite scores had significantly lower risk of mortality compared to patients in other hospitals: coronary artery bypass (OR 0.62; 95% CI, 0.57 to 0.68), aortic valve replacement (OR 0.59; 95% CI, 0.53 to 0.66), percutaneous coronary intervention (OR 0.69; 0.66 to 0.73), pancreatic cancer resection (OR 0.38; 95% CI, 0.27 to 0.53), esophageal cancer resection (OR, 0.60; 95% CI, 0.46 to 0.78). Conclusions: Composite measures forecast large differences in hospital mortality with surgery. Based on instrumental variable analysis, these differences in mortality are not due to unmeasured hospital differences in patient severity of illness, indicating that composite measures can be useful quality indicators for public reporting and pay-for-performance.

Racial Disparities in Readmission Rates Among Medicare Beneficiaries Undergoing Aortic Aneurysm Surgery

M. Girotti, P. Henke, J.B. Dimick — 2012-02-01

Introduction: Black patients are known to have increased rates of morbidity and mortality following vascular surgery when compared to non-black patients. We sought to examine whether this disparity extends to rates of post-operative readmission. Methods: National Medicare beneficiaries undergoing abdominal aortic aneurysm (AAA) repair in 2007-2008 were examined (n=58, 244). Hospitals were divided into quintiles based on the extent to which they served the black population during the study period. These were termed quintiles 1-5, quintile 1 contained the lowest 20% of black-serving hospitals and quintile 5 contained the highest 20%. the rate of risk-adjusted 30-day post-operative readmission was then compared between quintiles and logistic regression was used to examine the effect of race on this outcome. Risk adjustment included demographic information, patient co-morbidities, socioeconomic factors, and type of aneurysm repair (open versus endovascular). Results: the overall 30-day readmission rate for all patients following AAA repair was 11.8%. Across all hospitals, black patients had a 17% higher risk of 30-day readmission (odds ratio [OR], 1.17; 95% confidence interval [CI] 1.04-1.31) compared to non-blacks. When hospital-level analysis was performed, we found that quintile 1 hospitals treated 35% of the study population but actually treated zero black patients, compared to quintile 5 hospitals, which treated 20% of the study population but 67% of all black patients. We found a significant difference between 30-day readmission rates in non-blacks in quintile 1 compared to blacks in quintile 5 (11.3% versus 14.8%). Overall, blacks treated at the quintile 5 hospitals had a 30% higher risk-adjusted rate of 30-day readmission compared to non-blacks treated at quintile 1 hospitals (OR 1.30; 95% CI 1.12-1.50). Conclusions: Black patients experience a significantly higher risk-adjusted 30-day readmission rate following AAA repair compared to non-black patients. This disparity was even more pronounced at minority-serving hospitals.

Cognitive Impairment and Medicare Utilization Near the End-of-Life

L.H. Nicholas, D.M. Weir, T.J. Iwashyna, K.M. Langa — 2012-02-01

Introduction: Healthcare utilization near the end-of-life is a controversial topic; aggressive care for patients in the last six months of life are costly to the Medicare program, and it is unknown whether these treatments reflect patient preferences or improve quality of life. Although cognitive impairment may hinder patients' abilities to assess treatment options at the end-of-life, little is known about end-of-life care for cognitively impaired and unimpaired older adults. Methods: We use survey data from the Health and Retirement Study linked to Medicare claims for 3,302 respondents who died between 1998 and 2007. Respondent cognitive functioning is assessed using a Telephone Interview for Cognitive Status score and proxy reports of patient functioning. Inpatient hospitalization records are used to assess receipt of life-sustaining treatments including feeding tube placement, mechanical ventilation, hemodialysis, CPR, and intubation, in-hospital death and intensive care unit (ICU) stays. We used multivariate logistic regression to compare treatment of decedents with and without cognitive impairment. Results: 41% of Medicare decedents were unimpaired, 29% were borderline impaired and 30% were cognitively impaired. Cognitively impaired and unimpaired decednts were equallly likely to be hospitalized in the last six months of life, though only 40% of decedents with borderline or full cognitive impairment had written advance directives in place. Compared to unimpaired decedents, cognitively impaired patients were less likely to receive life-sustaining interventions (OR = 0.68, 95% CI [0.57,0.81]); equally likely to die in the hospital (OR = 0.98, 95% CI [0.82,1.16]), and less likely to have an ICU stay (OR = 0.82, 95% CI [0.67,0.99]). Conclusions: A large proportion of elderly decedents reach the end-of-life without documented treatment preferences or surrogate decision-maker and possible incapacity for informed consent. Although cogntive impairment is a terminal condition, impaired patients face similar odds of receiving many aggressive treatments near the end-of-life. Physician discussions earlier in life are potentially helpful to create awareness, facilitate decision-making while unimpaired.

Ranking Hospitals on Vascular Surgery Morbidity: Does the Type of Complication Matter?

M. Girotti, C. Ko, J.B. Dimick — 2012-02-01

Introduction: the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) reports rates of risk-adjusted morbidity for each participating hospital and subsequently ranks hospitals based on these rates. However, this approach does not consider the severity or number of complications that occurred. We sought to determine whether incorporating this consideration would alter hospital rankings. Methods: Patients undergoing vascular surgery in NSQIP hospitals during 2008-2009 were examined (n=39, 519). Post-operative complications were then categorized into minor or severe. We first evaluated the extent to which minor and severe complications increased a patient's risk of mortality and prolonged length of stay. We then re-ranked hospitals based on two alternative approaches, both by severity and number of complications. We determined the impact of these alternative methods by assessing the proportion of hospitals that moved out of the top and bottom 20% of the standard rankings when the alternative methods were applied. Results: Compared to patients with minor complications, patients with severe complications have a higher mortality rate (20.0% vs. 8.1%) and prolonged length of stay (66.2% vs. 53.3%). Patients with multiple complications (2 or more) had a higher mortality rate (23.7% vs. 15.5%) and prolonged length of stay (73.9% vs. 18.2%) than patients with only one complication. Compared to the current approach for assessing morbidity, ranking hospitals on severe complications resulted in 11 (26%) hospitals moving out of the top 20% and 18 (44%) hospitals moving out of the bottom 20%. A similar degree of reclassification was found when comparing the current morbidity rankings to an alternative approach that considered the number of different complications. Conclusions: Although the severity and number of postoperative complications affect mortality and length of stay, and subsequently hospital rankings, existing measurement systems do not account for this. Quality measurement platforms should consider weighting complications according to severity and number.

Hospital Characteristics and Participation in the National Surgical Quality Improvement Program

S.E. Regenbogen, N.H. Osborne, J.B. Dimick — 2012-02-01

Introduction: The National Surgical Quality Improvement Program (NSQIP) may not be nationally representative, as only a fraction of U.S. hospitals are enrolled. However, differences between NSQIP participants and non-participants have not been well described. We examined the structural characteristics, demographics, resources and surgical outcomes of high-risk surgery performing hospitals that do and do not participate. Methods: We linked 2008 inpatient Medicare claims for six high-risk general and vascular surgical procedures – colorectal resection, esophagectomy, pancreatectomy, abdominal aortic aneurysm repair, carotid endarterectomy or lower extremity arterial bypass – with the American Hospital Association Annual Survey, and classified hospitals according to their participation in NSQIP. We used chi-square tests to compare characteristics and outcomes between hospitals in each group. Results: of 3561 hospitals, 231 (6%) participated in NSQIP in 2008. NSQIP hospitals were far more likely to have more than 200 beds (89% vs. 34%), to be a teaching institution (71% vs. 17%), and to be among the two highest quintiles of procedure volumes (39% vs. 6% of non-participants). They were more likely to have high nurse-to-patient ratios (45% vs. 18%), and less likely to have highest-quintile surgical mortality rates (6% vs. 21%). NSQIP hospitals were less likely to be in the South (21% vs. 40%) or in non-urban settings (6% vs. 41%), with few sole community providers (3% vs. 12%) and only one critical access hospital (0.3% vs. 14%) included. NSQIP hospitals were equally likely, however, to have very high concentration of black patients (4% vs. 5%), Medicaid patients (22% vs. 20%), and high disproportionate share indices (19% vs. 16%). Conclusions: NSQIP participants do not represent the diversity of hospitals performing high-risk general and vascular surgery nationwide. to enable quality improvement efforts in small, rural, and critical access hospitals, which may have high surgical mortality rates and limited resources, other methods of outcome feedback are needed.

Exploring Variation in Emergent Surgical Outcomes in the Michigan Surgical Quality Collaborative: A Fertile Area for Quality Improvement

2012-02-01

Introduction: Within a large, statewide collaborative, significant improvement in surgical quality have been appreciated (9.0% reduction in morbidity for elective general and vascular surgery). Our group has not noted such quality improvement in the care of patients who had emergent operations. With this work, we aim to describe the scope of emergency surgical care within the MSQC, variations in outcomes among hospitals, and variations in adherence to evidence based process measures. Overall, these data will form a basis for a broad based quality improvement initiative within Michigan. Methods: We report morbidity, mortality, and costs of emergency and elective general and vascular surgery cases (n= 211,903) within 34 hospitals participating in the MSQC from 2005 to 2010. Adjusted hospital specific outcomes were calculated using a step-wise multivariable logistic regression model. Adjustment covariates included patient specific co-morbidities, and case complexity. Hospitals were also compared based on their adherence to evidence based process measures (measures at the patient level for each case – SCIP 1 and 2 compliance). Results: Emergent procedures accounted for approximately 10% of total cases, yet they represented 32.0% of mortalities and 40.4% of surgical complications. the complication-specific costs to payers was $208 million for the emergent cases and $307 million for the elective cases. Adjusted patient outcomes varied widely within MSQC hospitals; morbidity and mortality rates ranged from 3.1% to 28.0% and 0% to 13.6%, respectively. The variation among hospitals was not correlated with volume of emergent cases and case complexity. for emergent colectomies, there was wide variation in compliance with SCIP 1 and 2 measures, and overall compliance (42.0%) was markedly lower than elective colon surgery (81.7%). Conclusions: Emergent surgical procedures are an important target for future quality improvement efforts within Michigan. Future work will identify best practices within high-performing hospitals and disseminate these practices within the collaborative.

Using Simulation to Perform Root Cause Analysis of Adverse Surgical Outcomes

E.R. Simms, J.R. Korndorffer, M.E. Garstka, S.A. Tersigni, D.P. Slakey — 2012-02-01

Introduction: The Joint Commission on Accreditation of Healthcare Organizations (JCAHO) has published recommendations on conducting root cause analysis (RCA). Their recommendations include finding “root causes” rather than “proximate causes”, and identifying “common cause variation” rather than “special cause variation” in order to produce appropriate interventions. Simulation for root cause analysis of adverse medical outcomes is emerging as a novel technique for analyzing medical error, but has not yet been compared to traditional RCA methods. the purpose of this study was to compare simulation-based RCA of an adverse surgical outcome to traditional RCA methodology. Methods: 631 closed claims of a major medical malpractice insurance company were reviewed. Claims were categorized by comparing the predominant underlying cause documented in the case files to the major root causal categories described in the Eindhoven Classification Model. A single case of delayed recognition of a post-operative complication (hemorrhage) resulting in death was chosen. All records for the selected claim were analyzed using RCA methodology, and the root cause identified in the claim was congruent with the root cause identified by RCA techniques. A detailed scenario was developed and recreated in a simulation center. Confederates were trained to play key healthcare personnel, and six test subjects (general surgery residents) were presented with physical and laboratory findings identical to those in the closed claim case. Root causes identified by simulation analysis were compared to those identified by standard RCA technique. Results: Two of the six simulations performed duplicated the adverse event; in the other four, participants responded to the scenario in a way that avoided the adverse event. the root cause identified by standard RCA technique in the selected case was miscommunication of/inattention to physical signs and symptoms of bleeding in a postoperative patient (a “special-cause variation”). However, review of the simulation recordings revealed that the most significant root cause was not examining a key laboratory finding in the context of previous lab findings (a “common-cause variation”). the simulation-based RCA therefore identified a root cause more amenable to effective intervention. Conclusions: This study demonstrates that simulation-based RCA of adverse surgical outcomes is better at identifying root causes that are amenable to making changes in the healthcare setting than traditional RCA. Simulation-based RCA is therefore a more effective method of improving patient safety. the additional information simulation-based RCA provides can facilitate implementation of more appropriate corrective measures, decreasing the risk of error recurrence.

Does Hospital Transfer Predict Mortality in Very Low Birth Weight Infants Requiring Surgery for Necrotizing Enterocolitis?

L.I. Kelley-Quon, C. Tseng, A. Scott, H.C. Jen, K.L. Calkins, S.B. Shew — 2012-02-01

Introduction: Necrotizing enterocolitis (NEC) is one of the leading causes of infant mortality and the most common reason for emergent surgery in very low birth weight (VLBW, <1500g) infants. However, surgical capabilities are not available in all neonatal intensive care units (NICUs). the goal of this study was to investigate whether transfer for higher level of surgical care affects mortality for VLBW infants with surgical NEC. Methods: VLBW infants who underwent NEC surgery from 1999-2007 were retrospectively reviewed from the California Patient Discharge Linked Birth Cohort Database. Hospital admissions/transfers from birth to first discharge home were identified. Hospitals were stratified by NICU levels 2A-3C. NEC diagnosis and surgeries were identified by ICD9 codes. Transfer for emergent NEC surgery was defined as surgery ≤2d after transfer. Infants were categorized as either transferred for surgery or received surgery at their primary NICU. Mortality was analyzed with multivariate logistic regression using a fixed effects model at the individual hospital level. Covariates included transferring NICU level, peritoneal drainage, surgery <7d after birth (as a proxy for spontaneous intestinal perforation), birth weight (BW), maternal age, prenatal care, insurance, gender and major medical comorbidities. Results: Overall, 1,272 VLBW infants (BW: 860±260g) with surgical NEC were identified from 70 hospitals with a 39% mortality. the majority of the cohort underwent surgery at a 3C level NICU (63%). Overall, 406 (32%) infants were transferred for surgical care leaving 866 (68%) who had surgery at their primary NICU. Surgery <7d after birth occurred in 213 (17%) infants, most (73%) with a BW<1000g. Unadjusted mortality was not increased for infants transferred for surgery versus those not transferred for surgery, 37% vs. 40% (p=0.25). on multivariate analysis, adjusted mortality for infants transferred for surgery did not differ from those who received surgery at their primary NICU (OR 0.46, 95% CI 0.12-1.72). Lower BW, peritoneal drainage as sole surgical intervention, grade IV intraventricular hemorrhage, pulmonary interstitial emphysema, and pulmonary hemorrhage were associated with increased odds of mortality (p<0.05). Surgery <7d after birth was associated with decreased odds of mortality (OR 0.11, 95% CI 0.04-0.27). Exclusion of this subset on subsequent analysis did not impact the effect of transfer on mortality. Conclusions: VLBW infants with surgical NEC do not demonstrate increased risk of mortality when transferred emergently for higher level of surgical care. It is otherwise uncertain whether lack of surgically capable NICU's contributes to mortality prior to transfer or whether morbidity is affected for infants who survive transport. Future efforts must engage health professionals at all levels caring for this vulnerable population in order to truly maximize resource allocation and safety.

Using a Mixed-Methods Model to Improve Validity of Clinical Vignettes to Assess Variation in Surgeon Practice

2012-02-01

Introduction: Clinical vignettes have been used in multiple medical specialties to identify factors associated with variations in quality of healthcare delivery. However, developmental guidelines and validation of clinical vignettes for use in a surgical setting is lacking. Our objective was to develop a combined qualitative and quantitative approach to produce clinical vignettes appropriate for use while studying practice variation in the surgical setting. Methods: A four step mixed-method approach (Figure 1) was utilized to develop clinical vignettes for use in a study to investigate healthcare provider factors associated with disparities in trauma and emergency surgery care.The four steps were 1) literature review 2) expert panel review 3) cognitive interviewing 4) testing for heterogeneity of responses. at each step new iterations of problematic vignettes and questions were produced and re-tested to ensure the responses to each vignette were able to capture variations in practice. Clinical vignettes focused on the general care of surgical patients such as: treatment of traumatic brain injury and acute abdomen, the use of restraints, assessment of pain, substance abuse and domestic violence. Each vignette had four associated questions (mix of Likert scale responses and multiple choice) and were written and reviewed by an expert panel. Cognitive interviewing (CI) sessions of healthcare providers tested the appropriateness of each vignette in the four stages of the question-response process. Adjustments were made to vignettes based on these responses. Heterogeneity of response was determined graphically by comparing frequency of each answer choice among healthcare providers at the same level. Results: Nine clinical vignettes were tested on 30 healthcare providers. 7 out of the 9 clinical vignettes were substantively revised based on cognitive interview results. 25% of questions for the final vignettes were modified in order to increase heterogeneity of responses and improve the overall sensitivity of the survey instrument. Overall, vignettes and their associated questions underwent up to four different iterations. Conclusions: The four step model is applicable to the creation of a variety of clinical vignettes across a range of sensitive provider-level topics. Development and implementation of standardized approaches to the creation clinical vignettes is essential as we begin to use these tools for the evaluation of variations in quality of surgical care.

An Objective System for Measuring Surgical Complexity in Elderly Patients

W.B. Chow, K.Y. Bilmoria, B.L. Hall, C.Y. Ko — 2012-02-01

Introduction: The purpose of this study was to create a system to assess surgical complexity by developing an objective composite risk score (CRS) using postoperative adverse outcomes from high-quality ACS NSQIP clinical data on elderly patients. Methods: Patients age ≥65 years undergoing common or major general and vascular operations were selected from the ACS NSQIP 2005-2009 data. Procedures were clinically grouped into 67 categories. Hierarchical logistic modeling was used to calculate risk-adjusted rates for 23 postoperative adverse events, which included death, surgical site infections, and end-organ failure. Adverse events were assigned a weighted value from 1 to 5 according to severity. for each procedure group, the CRS was calculated as the weighted sum of rates for all postoperative events. the median operative time (OT) and length of stay (LOS) for operative cases without adverse events were also assessed. General surgeons were surveyed to rank the procedure groups in order of perceived complexity based operative risk, required technical skill and training, and surgeon effort for performing the operation and providing postoperative care. Comparisons were made between the CRS, OT, LOS, survey results, and work RVU. Results: 205,973 elderly patients met criteria for analysis. the CRS was rescaled from 0 for low risk to 100 for high risk. Operations with the highest scores were open repair of ruptured abdominal aortic aneurysm (100) and open repair of thoracic/thoracoabdominal aneurysm (80); the lowest scorers were open repair of reducible inguinal hernia (1.3) and laparoscopic repair of inguinal hernia (1.4). at the 50th percentile, open repair of non-reducible, recurrent ventral hernia and open cholecystectomy had scores of 16.4 and 18, respectively. Without adverse events, median OT ranged from 38 minutes (open repair of reducible umbilical hernia) to 342 minutes (proximal pancreatectomy); and median LOS ranged from 0 days (outpatient procedures) to 10 days (esophagectomy). the CRS had correlation coefficients of 0.46 with OT and 0.79 with LOS. Work RVU correlated at 0.59 with the CRS. the average rankings from the preliminary surgeon surveys had Spearman correlations of 0.89 with CRS and 0.57 with work RVU. Conclusions: This pilot study demonstrates that clinical data from ACS NSQIP can be used to assess surgical complexity. the CRS provides a single index value, reflecting both severity and likelihood of adverse outcomes that allows objective comparisons of the surgical risk across procedure groups. the work RVU does not strongly correlate with the CRS. the CRS more strongly correlated to the surgeons' rankings of procedures compared to the work RVU. in the next phase, the CRS will be refined based on surveys from a larger group of surgeons.

Emergency General Surgery: Does an Individual Surgeon?s Volume of Cases Matter?

2012-02-01

Introduction: Emergency General Surgery (EGS) has a higher risk of morbidity and mortality than non-emergent cases. the extent to which a surgeon's experience with EGS contributes to these outcomes is not well characterized. the purpose of this study is to examine the relationship of EGS case volume to mortality and morbidity. Methods: We performed a retrospective cohort study of surgical admissions using the Nationwide Inpatient Sample for calendar year 2006. Inclusion criteria were a Procedure Clinical Classification Software (PRCCS) code indicating a general surgery procedure; only cases performed by an individual surgeon with a volume of > 50 cases were included. Percentage of emergency general surgery (PctEGS) cases performed by an individual surgeon relative to that surgeon's overall volume was calculated and binned into low (LPct, <33%), moderate (MPct, 33.4-66.5%) and high (HPct ≥ 66%). Emergent cases were defined as those admitted through the emergency department that were operated on within 24 hours of presentation. Discharge weighting was used to recapitulate a nationally representative dataset. Univariate logistic regression was used to assess the relationship between patient factors (age, sex, and Elixhauser Comorbidity Index Score (ECIS)), and PctEGS and mortality and morbidity after emergent cases. Morbidity was defined as the presence of any of 11 ICD-9 defined post-operative complications: respiratory, postoperative myocardial infarction, dehiscence/wound disruption, postoperative ileus, postoperative shock, postoperative infection, hemorrhage, postoperative cerebrovascular accident, retained foreign body, postoperative fistula, and return to the operating room. Factors found to be significant at p ≤ .10 in univariate analysis were entered into multivariable models. Results: A total of 413 surgeons performed 29, 181 cases meeting inclusion criteria, of which 10,558 (36.2%) were emergent. Patients were 52% female, mean age 55 (SD 18) years, and a median ECIS of 1 (IQR 0-2). Overall, 18.7% of patients had at least 1 complication. in multivariable modeling, age, male sex, and EI were significantly associated with mortality but there was no association between mortality and the percentage of EGS performed by the individual surgeon. After adjustment for age, sex and ECIS, HPct EGS was found to be associated with lower morbidity after emergent general surgery (OR 0.89, 95% CI 0.83-0.95). Conclusions: Mortality and overall morbidity after EGS were strongly associated with patient comorbidities and age. Patients undergoing EGS by surgeons who performed ≥66% EGS had modest but statistically significant reductions in morbidity. the association between an individual surgeons experience and outcomes after EGS merits further investigation.

Assessment of Prophylactic Retention Suture in Reducing Dehiscince in Midline Laparotomy in High Risk Patients: A Randomized Clinical Trial

S. Shoar, B. Laghaie, A. Aminian, N. Hosseini Araghi, Z. Khorgami — 2012-02-01

Introduction: Abdominal wound dehiscence is a major postoperative complication with a high mortality and morbidity rate. Retention sutures are mostly used in repairing dehiscence to strengthen the wound. the objective of present study was to assess reducing dehiscence of midline laparatomy by prophylactic retention suture in high risk patients. Methods: 300 high risk patients with at least three risk factors for dehiscence, who were admitted to two referral affiliated hospitals between 2008 and 2010, were randomly divided into two equal groups. We used retention suture in patients who were included in case group. the fascia in control patients was repaired continuously with using a running looped #1 Nylon suture. in case group, we used retention suture in every 10 cm of the incision in addition to running Nylon suture which included skin and subcutaneous tissue and rectus muscle. We compared the rate of wound dehiscence, evisceration, wound infection, and postoperative pain (by visual analogue scale) in the two groups of patient with and without tension suture. Results: Our finding showed that the wound dehiscence occurred in 6 (4%) cases and 20 (13.3%) controls (P value = 0.003). Wound evisceration after surgery occurred in one case (0.7%) and 4 ones (2.7%) in controls (P value = 0.185).Wound infection occurred in 24 (16%) cases and in 19 (12.7%) controls (P value = 0.25). Mean postoperative pain (with scale of 0-10) was 7.4 in first day, 6.2 in second day, 4.7 in third day, and 3 in fourth day among cases and 7 in first day, 5.7 in second day, 4.1 in third day, and 1.2 in fourth day among controls, not statistically different. Conclusions: Prophylactic retention suture may reduce wound dehiscence in midline laparatomy in high risk subgroup of patients with multiple risk factors of impaired wound healing. It may reduce evisceration without causing more severe postoperative pain or higher wound infection rate but the latter findings need more studies.

Bayesian Meta-analyses of Cochrane Reviews for Reduction of Surgical Site Infections in Colorectal Surgery: are They More Informative Than Traditional Meta-Analyses?

U.R. Phatak, C. Pedroza, G.J. Chang, S.G. Millas, K.P. Lally, L.S. Kao — 2012-02-01

Introduction: Interventions other than appropriate administration of prophylactic antibiotics to prevent SSIs have not been widely adopted due to a lack of consensus regarding effectiveness. Estimates of treatment effect are often not statistically significant, even when all studies are combined in meta-analyses, using the traditional, yet arbitrary cut-off of a p-value less than 0.05. Bayesian methods, which produce probability distributions based on existing knowledge and new data, can provide probabilities of specific thresholds of benefit which may be more useful in guiding clinical decision-making. We hypothesized that ranking of evidence-based interventions would differ when using traditional versus Bayesian meta-analyses. Methods: We conducted a systematic search of the Cochrane database for reviews of interventions to reduce SSIs after colorectal surgery, other than prophylactic antibiotics. Traditional and Bayesian meta-analyses were performed using RevMan and WinBUGS (MRC Biostatistics Unit, Cambridge, UK). Bayesian posterior probabilities of relative risk reductions (RRRs) of greater than 0% and 10% were calculated using a neutral prior probability distribution. Interventions were ranked and compared using the frequentist point estimates for relative risks or odds ratios for SSIs and the Bayesian probabilities. Results: A total of 9 Cochrane reviews met the search criteria. Using traditional meta-analysis methods, only laparoscopic colorectal surgery resulted in a statistically significant reduction in SSIs and a recommendation for use of the intervention. the rankings differed when point estimates were used versus the Bayesian probability of any benefit in 6/9 (67%) analyses, with no more than a 2 point difference between rank numbers (Table). When a threshold benefit of more than 10% RRR was used, the rank order changed for 4/9 (44%) interventions. Conclusions: Bayesian probabilities and traditional point estimates of treatment effect yield similar information in terms of ranking interventions on potential effectiveness. However, Bayesian meta-analysis provides more clinically useful information about the probabilities and size of a beneficial effect, particularly when traditional meta-analyses fail to produce any treatment recommendations because they yield results that are interpreted as inconclusive or not statistically significant.

Composite Measures for Assessing Hospital Quality With Lower Extremity Bypass Surgery

N.H. Osborne, C.Y. Ko, J.B. Dimick — 2012-02-01

Introduction: Risk-adjusted morbidity rates are widely used as surgical quality indicators for vascular surgery. However, since many hospitals have low caseloads, morbidity rates can be unreliable. We evaluated whether a novel approach for creating composite quality measures can improve the reliability of hospital morbidity reporting. Methods: Using data from ACS-NSQIP, we studied all patients undergoing major general and vascular surgery in 2008. for lower extremity bypass surgery, we created a composite measure by combining quality indicators from several distinct domains of quality: morbidity, reoperation, length of stay, and hospital morbidity with other potentially related procedures. We empirically weighted each measure and filtered out measurement noise using multilevel modeling. Hospitals were then ranked based upon the composite and placed in quintiles (1- to 5-stars). to validate this approach, we assessed how well composite measures from 2008 forecasted risk-adjusted morbidity in the next year (2009) compared to the traditional approach of using risk-adjusted morbidity alone. Results: Composite measures were better at discriminating differences in the future risk of serious complications compared to the traditional approach. Hospitals rated as 1-star and 5-star had a 58% difference in future risk-adjusted morbidity (13.4% vs 8.2%) using the composite measure, compared to only a 36% difference (11.8% vs 10.0%) for the traditional NSQIP approach. the composite measure also explained a higher proportion of hospital-level variation in serious complication rates compared to the traditional NSQIP approach. Conclusions: Empirically-derived composite measures are more reliable measures of hospital morbidity for lower extremity vascular surgery than traditional approaches. These measures could be used to improve the accuracy of quality measurement programs such as ACS-NSQIP.

Payer Status is Associated With Use of Inferior Vena Cava Filters in High Risk Trauma Patients

D.M. Pickham — 2012-02-01

Introduction: Pulmonary embolism (PE) is a preventable and potentially lethal complication in trauma patients. It is controversial whether patients who are at high risk for PE and have a contra-indication to chemoprophylaxis should receive a prophylactic inferior vena cava (IVC) filter. This lack of clarity creates the potential for different levels of care based on socioeconomic factors. We hypothesized that un-insured patients would receive prophylactic IVC filters less frequently than those with insurance. Methods: This was a retrospective analysis of the National Trauma Databank (2002-2007). Patients were included if they had traumatic brain injury or spine injury (defined as high-risk), were >18 years of age, and admitted to the hospital. Exclusion criteria were abbreviated injury scale of 6, diagnosis of deep venous thrombosis or pulmonary embolism, or if the patient was admitted to a hospital that never placed an IVC filter. Logistic regression was performed to control for age, injury severity score (ISS), gender, race, and insurance status. Since payer status may cluster at particular centers, a hierarchical mixed effects model was also performed. Results: There were 129,454 patients available for analysis. of these, 6,220 (4.8%) received a prophylactic IVC filter. Those with IVC filters were slightly younger (46 vs. 47 years, p=0.000), more often male (73% vs. 69%, p=0.000), and had a higher mean ISS (30 v 19, p=0.000). Overall, 2.7% patients without insurance had an IVC filter placed compared to 4.8% of those with some form of insurance (p=0.000). After adjusting for age, gender, ISS>15, insurance status, and race, patients without insurance were significantly less likely to receive a prophylactic IVC filter (OR 0.63, p=0.000). This effect remained after controlling for center (OR 0.57, p=0.000). Conclusions: Prophylactic IVC filters are placed less frequently in patients without insurance. These findings suggest that when clinical guidelines lack clarity, unconscious bias has the potential to create a system with different levels of care based on socioeconomic disparities.

Pre-operative Risk Prediction: Does Experience Matter?

2012-02-01

Introduction: There are few methods for standardized risk prediction of post-operative complications, and most prediction is based on literature and surgeon experience. Predicting post-operative complications for patients undergoing elective operations would assist in identifying patients most at risk and potentially allow for interventions aimed at reducing risk. the objective of this study was to compare surgeons' assessment of mortality and complication risk with model-based estimates on a set of standardized cases. Methods: Ten standardized elective case scenarios of varying complexity were developed. Risk-adjusted point estimates for mortality and complications were calculated from models derived from the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) pre-operative risk data using regression analyses. A web-based survey was employed at two academic institutions to capture resident and faculty estimates for mortality and complications as well as risk category rankings (low, average, high) for the standardized cases. Surgeons' mean mortality estimates and risk category rankings were compared to the model results using t-tests and Spearman correlations. Comparisons were also stratified by experience level and institution. Results: A total of 53 respondents completed the survey, 41 residents and 12 faculty. Compared with the model estimates, surgeons significantly underestimated risk for the highest risk case (-44.7%, p<0.0001) and overestimated risk for the lowest risk case (+1.00%, p=0.0003), with no improvement by PGY year or attending and no difference by institution. the mean Spearman correlation between surgeon and model-based risk estimates was 0.70 (SD 0.12) with p<0.01 by randomization tests. Conclusions: Although the respondents' case rankings were somewhat consistent with model rankings, risk prediction estimates varied widely among respondents and underestimated the risk among high risk patients. Pre-operative risk assessment embedded in an electronic health record would assist with standardization of identifying high risk patients and allow for interventions directed at risk reduction.

Differences in Spousal and Lifestyle Choices Between Female and Male Physicians

R.L. Berger, C.J. Balentine, D.H. Berger — 2012-02-01

Introduction: Over the past several decades the number of female physicians has greatly increased. There is little data available examining the spousal and family choices female physicians make compared to their male counterparts. We sought to determine if there are differences in the spousal choices and family profiles of male and female physicians. Additionally we sought to determine how these choices relate to career measures such as income and hours worked. Methods: Subjects were identified based on their reported occupation of physician or surgeon from the five percent Public-Use Microdata Sample of the 2000 United States Census. We analyzed data concerning marital status, occupation, income, and hours worked for physicians and their spouses. Statistical analysis was performed using chi squared tests for categorical variables and the non parametric Mann-Whitney test for comparison of continuous variables. Results: A total of 36,622 subjects were identified with a total of 2,175 dual physician couples. of the total sample of physicians, 26,886 were male and 9,736 were female. Female physicians were more likely to be married to other doctors than were male doctors (22.3% versus 8.1% p<0.001). Spouses of male physicians were ten times more likely to report having no job than spouses of female physicians (29% versus 2.9%). Female physicians tend to work fewer hours per week (median usual hours worked per week for males is 50 versus 45 for females, p<0.001) and have a lower annual income than male physicians (median total personal income for males is $137,000, for females is $67,000, p<0.001). Within dual physician couples, male physicians were still more likely to work longer (55 versus 40 hours per week) and earn more than their female partners ($130,900 versus $75,000). Conclusions: Male physicians are more likely to be married to non-physicians compared to female physicians. Female physicians have a lower median income and usual hours worked per week than male physicians even within dual physician couples. Despite the significant advances made by women in the workplace over the last 50 years, physicians still seem to be more likely to follow traditional gender models of family and career. This has implications for the career advancement of female physicians.

Surgical Readmissions in the Obese Elderly Patient

2012-02-01

Introduction: Reducing readmissions has become a recent policy focus in an effort to improve healthcare quality and reduce costs. Obesity is one patient characteristic that may increase the risk of readmission, but is not captured in current risk-adjustment data. We hypothesized that obese patients would have an increased risk of readmission, even after controlling for their comorbidities, and that their most common reasons for readmission would be different than for the non-obese patients. Methods: A matched case-control study of Medicare patients who were readmitted following hip and knee surgery, colectomy, and thoracotomywas performed using claims (2002 through 2006) and abstracted charts from 47 participating hospitals throughout Illinois, New York and Texas. Patients were matched exactly for procedure, with near-fine balance for hospital, and balancing the means of age and sex. After matching, a conditional logistic regression was used to study the odds of readmission for obese patients while controlling for race, transfer status, and emergency status, both with and without controlling for preoperative comorbidities. the reference group was patients with a BMI between 20-30 kg/m2, and obesity was defined as BMI>35 kg/m2. ICD-9 codes for principal diagnosis for readmission were grouped into larger related categories and the most common causes of readmission were analyzed by BMI category. Results: We identified 1,380 readmitted patients and 2,760 controls. Obese patients had a significantly increased risk of readmission, both before and after controlling for comorbidities (OR 1.35, p=0.003; OR 1.30, p-value=0.013). the most common reasons for readmission varied by procedure type, and were not different by BMI category (Table). Almost a third of all readmissions after colectomy were due to GI complications, 39% of readmissions after thoracotomy were due to pulmonary or cardiac complications, and 22% of readmissions after orthopedic procedures were related to orthopedic complications. Infection was one of the top three reasons for readmission for all procedure types. Conclusions: Obese patients have an increased risk of readmission, independent of their age, sex, race, transfer status, emergency status, and comorbidities, yet their reasons for readmission appear to be related to the procedure performed, similar to the non-obese. As CMS plans to expand the list of procedures subject to penalties for readmissions over time, appropriate risk-adjustment that includes BMI will be essential to avoid disincentives to care for the obese patient.

Incidence of Venous Thromboembolism in Patients Undergoing Surgical Treatment for Malignancy: An Analysis of ACS NSQIP Data 2005-2008

C.E. Reinke, G.C. Karakousis, R.A. Hadler, J.A. Drebin, D.L. Fraker, R.R. Kelz — 2012-02-01

Introduction: Venous thromboembolism (VTE) events have been associated with surgical procedures and cancer status. Pulmonary embolism is a potentially fatal event, and long term consequences of deep venous thrombosis can result in significant disability. Data regarding VTE by tumor type for surgical patients are sparse. the current study was designed to define the incidence, relative risk, and adjusted odds ratio of VTE following surgical intervention by cancer site. Methods: We performed a retrospective cohort study of patients entered into the ACS-NSQIP database from 2005-2008 who underwent a surgical procedure and had a post-operative neoplasm diagnosis. the incidence of VTE was calculated by tumor type for malignant and benign/CIS neoplasms. the relative risk of post-operative VTE for cancer was calculated by tumor site. An odds ratio was calculated for each neoplasm site using logistic regression, and controlled for body mass index (BMI), age, operative time, and sex. BMI, age and operative time were divided into categorical variables in concordance with those indicated by VTE prophylaxis guidelines. Results: All patients with a neoplasm were identified (n=130,284); 4289 cases were excluded due the uncertain behavior of the neoplasm. We identified 92,072 malignant neoplasms and 33,923 benign/CIS neoplasms, which were used as controls. the presence of colon cancer, rectal cancer, pancreatic cancer, uterine cancer, ovarian cancer, and breast cancer had statistically significant higher relative risk for VTE in patients with malignant neoplasms before controlling for patient characteristics. After controlling for age, sex, BMI and operative time, only malignancies of the large intestine, uterus, ovaries, and adrenals remained as having statically significant elevated odds ratio of developing VTE after a surgical procedure. Conclusions: The incidence of VTE is higher in patients with malignant neoplasm undergoing surgical procedures when compared to their benign counterparts. the relative risk of VTE varies by tumor type and is not uniform across patients undergoing surgical procedures. After controlling for patient characteristics known to be associated with VTE, the odds ratio of VTE remained elevated for malignancies of specific sites. Recommendations for VTE prophylaxis for patients undergoing surgical procedures should be based on the overall risk profile of the patient including an adjustment tailored to the specific type of malignancy.

A Novel Floor Management Simulation Course for Medical Students: Providing Experience and Feedback in A Safe Environment

C. Reinke, C. Nelson, D.N. Holena, B. Kann, N. Williams, J. Bleier, R.R. Kelz — 2012-02-01

Introduction: the 2003 ACGME work rules changed resident and medical student (MS) education. Daytime floor management of surgical patients is often performed by midlevel providers. Knowledge previously acquired by experience must now be actively taught to avoid prolonging the training period. We report the feasibility of and MS opinions regarding a novelsimulated floor management course (SIMFLO) to teach three basic patient care concepts required on the surgical wards: documentation, communication and medical principles. Methods: A simulated hospital ward was populated with 3 SimMan3G simulators (Laerdal, Stavanger NOR) complete with physical exam findings and active vital signs. Surgical clerks gathered data during “morning rounds,” wrote notes and provided care for SimMan3G simulators throughout the duration of the course. the use of SimMan 3G also allowed students to participate in the active evaluation and treatment of a “change in patient condition” with nursing staff represented by participating housestaff officers. Findings and plans were communicated directly to their “chief resident”, a surgical attending, who gave them feedback on presentation style and appropriateness of plans. All patient encounters and oral presentations were recorded for future critical review. A 15-item survey was distributed to participating MS to determine attitudes and opinions about the course. Results: The course required five faculty, two medical educators, four surgical housestaff and 2.5 hours to accommodate 40-50 students per session. Faculty and surgical housestaff oversight provided guidance and feedback on clinical skills, including information gathering, oral presentation and organization of written content to fulfill documentation requirements. Fifty students completed the survey (56% response rate). Most medical students felt that SIMFLO improved their understanding of medical management of surgical issues (33/50 (66%, 95% CI 51-79%)) and their documentation skills (39/50 (78%, 95% CI 64-88%)). Medical students reported that attending surgeon involvement made the experience more valuable (42/47 (89%, 95% CI 77-96%)) and was not intimidating (31/47 (66%, 95% CI 51-79%). Most expressed an interest in participating in more clinical scenarios (34/47 (72%, 95%CI 57-84%)). Conclusions: A simulation course for teaching patient care concepts such as SIMFLO is feasible and regarded positively by medical student participants. It is an effective method to train medical students in basic ward management of non-procedural care issues without any concerns over patient safety or anxiety. Students felt that attending surgeon involvement was fundamental to making it meaningful. Further development and use of such simulated patient care exercise may be an effective adjunct for training future housestaff and hospital staff in patient care in a time of shifting paradigms on work hours.

Scientific Impact of AAS/SUS Plenary Session Abstracts Increases in the Era of the Joint Academic Surgical Congress, 2006-2010

2012-02-01

Introduction: A previous analysis of publications generated from plenary session abstracts from the separate Association for Academic Surgery (AAS) and Society of University Surgeons (SUS) annual meetings from 2002-4 revealed no significant difference in the publication rate, citation numbers, or average impact factor (IF) between the two societies. the aim of the current study is to conduct a similar analysis of the scientific impact of plenary meeting abstracts between the AAS and SUS since 2006, when both societies began hosting the combined annual Academic Surgical Congress (ASC), to determine if the merger has affected the scientific quality of the meeting. Methods: 76 abstracts from the AAS (n=40) and SUS (n=36) plenary sessions at ASC meetings (2006-10) were reviewed. Publication rate was determined using the PubMed database through 07/01/2011. Number of citations was determined using the Web of Science. the IF was obtained from the Journal Citation Reports. Statistical analysis was conducted using Fisher's exact test, and mean values were compared by two-tailed t-tests. Results: Overall, 60 (79%) of 76 ASC plenary abstracts presented from 2006-10 were published in peer-reviewed journals, with a mean time between presentation and publication of 11.9 months. Analysis revealed a higher publication rate for AAS (90%) compared to SUS (67%) plenary abstracts (p=0.02). Among papers published, the overall mean number of citations was 6.7. There was no difference in mean number of citations for AAS vs. SUS (5.9 vs. 7.8, p=0.42) publications. the mean annual citation rate for all papers was 2.5, with 2.2 citations/year for AAS and 3.0 citations/year for SUS (p=0.31) papers. the mean 5-year IF for all publications was 4.6 (AAS 4.3 vs. SUS 5.0, p=0.54). in comparison to the previously published analysis from the separate AAS and SUS meetings, the mean IF has increased for both AAS (0.56) and SUS (0.82) publications. (See Table). Conclusions: After initiation of the joint ASC meeting in 2006, the SUS and AAS plenary presentations continue to exhibit high-quality research as demonstrated by increasing journal IFs. A higher overall publication rate for the AAS plenary abstracts was observed; the numbers of citations and IFs were higher on average for SUS abstracts, but the differences were not significant. This study supports the benefit of a joint meeting for the AAS and SUS, as it has been associated with a high publication rate and an increasing overall scientific impact for plenary abstracts.

Procedural Skills of Medical Students: Opinion of Faculty Compared to Graduating Medical Students

J.J. Dehmer, K.D. Amos, T.M. Farrell, A.A. Meyer, M.O. Meyers — 2012-02-01

Introduction: Opportunities for medical students to learn and perform technical skills during their clinical years have decreased. What is not clear is the value that faculty place in teaching procedural skills and the level of competence faculty believe graduating students should have. We sought to evaluate the faculty perception of student competence in performing basic procedures and compare those to the perception and opinions of graduating students. Methods: Under IRB approval, a confidential, online cross-sectional survey was conducted in March and April 2011 of 4th year medical students and faculty at the University of North Carolina at Chapel Hill. Opinions were sought as to the experience, level of competence and desired level of competence for nine procedural skills (Table) using a 4-point Likert scale (1=unable to perform; 2=require major assistance; 3=require minor assistance; 4=independent). Responses were compared by Student's t-test and ANOVA. Results: 282 faculty (180 male/100 female) and 134 4th year students (62 male/69 female) responded. 63% of faculty reported having a procedure-based practice and 21% a surgical specialty; 54% of students planned to enter a procedural specialty. 92% of faculty thought teaching procedural skills was somewhat (38%) or very important (54%). for all skills, there was a significant difference in the faculty perception of actual competence vs. expected competence in graduating students (P<0.001 for all). Compared to 4th year students, faculty perception of actual competence was much lower than student perception (p<0.001 for all), with faculty suggesting that for most skills, students required major assistance to perform them. (Table) However, the faculty expected level of competence was exceeded by the student expectation of competence for all skills (P<0.001 for all) skills. (Table) A procedural practice was associated with greater expected competence of students for only Foley (p=0.02) and NGT (p=0.02). A surgical specialty was associated with greater expected competence only for Foley (p<0.001) and basic suturing (P=0.008) and non-surgical specialty favored competence only for LP (p< 0.001). Conclusions: 4th year medical students and faculty have differing opinions as to the level of competence students achieve by the time of graduation. They also differ in the level of desired competence, with students favoring a greater degree of proficiency. These results suggest that despite a change in culture of our medical training and the perception that students may not be as engaged in learning as they were in the past, they still desire to achieve independence in certain skills prior to entering residency. We should continue to explore novel ways to achieve these goals.

Experience and Opinions of 4th Year Medical Students in Acquiring Competence With Basic Procedural Skills

J.J. Dehmer, K.D. Amos, T.M. Farrell, A.A. Meyer, W.P. Newton, M.O. Meyers — 2012-02-01

Introduction: The Association of American Medical Colleges has identified key procedural skills that graduating medical students should acquire. Despite this educational goal, there is data to show that students are unprepared to perform even basic medical procedures upon graduation from medical school. Our aim was to characterize the experience and opinions of graduating students in acquiring these skills and elucidate predictors of competence. Methods: 156 graduating medical students were queried via online survey as to their experience, actual level of competence and desired level of competence for nine procedural skills (Foley catheter insertion, nasogastric tube insertion, venipuncture, intravenous catheter insertion, arterial puncture, basic suturing of lacerations, endotracheal intubation, lumbar puncture, and thoracentesis). Data were collected on a 4-point Likert scale (4 =independently performs skill to 1 = unable to perform skill) and analyzed by ANOVA and Student's t-test. Results: 134 (86%) students responded. 54% planned to enter a procedural specialty. Mean level of competence ranged from 3.13 ± 0.75 (Foley) to 1.7 ± 0.7 (thoracentesis). A gap in desired vs. actual level of competence existed for all procedures (p<0.0001). No relationship existed between experience prior to medical school and competence for any skill. Male gender favored competence for venipuncture (p=0.04) and IV insertion (p=0.05). A career plan to enter a procedural specialty was associated with competence only for intubation (p=0.03). There was a correlation between the number of times a procedure was performed and competence for all skills except arterial puncture and basic suturing. the only 2 skills that over 50% of students reported performing greater than 2 times were Foley catheter insertion and basic suturing. Conclusions: Senior medical students perform most skills infrequently and rate themselves as being unable to perform them without assistance. Strategies to improve student experience and competence of procedural skills need to evolve to improve the technical competency of graduating students. These findings may have implications for those entering surgical specialties and may inform future educational plans for training new residents as the actual competence of graduating students in performing technical skills varies widely.

Surgical Skills Training Restructured for the 21st Century

M.W. Morris, R.C. Caskey, M.E. Mitchell, D.E. Sawaya — 2012-02-01

Introduction: Few if any medical schools have a comprehensive surgical skills program taking medical students from learning basic knot tying and suturing skills to performing these skills at a level adequate for function during a primary care, surgical or subspecialty residency. We designed and continue to refine a skills program consisting of 5 workshops during the third year surgical rotation. Methods: During the first workshop students learn how to tie both one and two-handed surgical knots. the focus of the second workshop is teaching the differences in suture type and use, instrument handling and suturing techniques such as simple interrupted, running, running locking, subcuticular, and mattress stitches. the third workshop is a supplemental workshop to address problems and refine techniques previously learned. A final examination to evaluate suture and knot tying skills comprises the fourth workshop. to pass this skills exam students must be able to tie one and two-handed surgical knots and perform two of the previously mentioned suturing techniques. the fifth session is a voluntary knot tying and suturing competition evaluating three categories. the first category determined the speed and finesse of knot tying in thirty seconds. Knot-tying points were lost for different tying errors including improper form, dropping the suturing while tying, and throwing air knots. the second category determined suturing tightness, symmetry and aesthetics while closing a simulated laceration with a running suture. the third category determined the water-tightness of a repaired latex tube after a 75% transection. Photographs of all materials were obtained at the completion of the competition. Certificates are presented to the winners of each individual category. A “Top Gun” plaque is presented to the overall winner. Results: All third year students at our medical school participated in this program over the past 18 months, and each student demonstrated competency in basic knot tying and suturing for completion of the skills exam. the results of the knot-tying portion of the competition revealed the fastest student performing 16 throws every thirty seconds. 5% of students had excellent knot-tying technique losing no points for tying errors. Suturing evaluation revealed combined excellence in symmetry, tightness, and aesthetics in 10% of students. Results of the third category of the competition revealed water-tight competency of the vascular surgical repair during pressure testing in 10% of the students. Conclusions: This program produces medical students with a fundamental surgical skill set, appreciation of surgical technique, and the confidence to perform basic surgical skills when required during their internship. Additionally, identifying the most technically proficient students may assist with career counseling residency recruitment.

Results of a Leadership/Teamwork Program for Third-Year Medical Students

G.S. Cherr, K. Glaser, A. Panchal, E.A. Bowdish, J.M. Hassett — 2012-02-01

Introduction: Leadership and teamwork skills are an integral part of medical training and practice. Currently, medical students at our university do not receive leadership/teamwork training. No research exists to describe a model of medical student leadership/teamwork training. the purpose of this study is to assess the effectiveness of a novel leadership/teamwork program for 3rd-year medical students. Methods: We developed a leadership/teamwork skills curriculum. Surgery students rotating at one hospital participated in weekly sessions on leadership/teamwork, while those at others sites did not. to test the programmatic effectiveness, all surgery students completed a well-accepted, validated, reliable teamwork survey at the beginning and the end of the 3-month rotation between 9/1/2010-4/1/2011 (TeamSTEPPS Teamwork Attitudes Questionnaire). Scores were compared for students receiving leadership/teamwork training and those who did not (SPSS). Results: 64 students completed pre-test and post-test surveys. 22 participated in the leadership/teamwork curriculum. Compared to students not receiving leadership/teamwork training, program participants had significantly better scores for Team Structure (p<.001), Situation Monitoring (p=.045) and Communication (p=.046) but not Mutual Support (p=.249) or Team Leadership (p=.307). Using a Likert-scale evaluation (1=strongly disagree; 5=strongly agree), leadership/teamwork students ranked the following statements: “valuable educational experience” (Mean score=4.27), “…lead(s) to improvements in my patient care” (Mean Score=3.95), “…lead(s) to improved performance as a medical student” (Mean Score=4.36), and “…should be offered to medical students in the future” (Mean Score=4.41). Conclusions: We developed a leadership/teamwork skills program that was perceived as valuable and relevant by medical students. Compared to students without teamwork training, students participating in the program had improved teamwork knowledge and attitudes at the end of each rotation.

The Surgical Clerkship: A Contemporary Paradigm

E.L. Bradley, A.B. Littles, L.J. Romrell — 2012-02-01

Introduction: Traditional Surgical clerkships have been composed of a mixture of hospitalized patients, daily resident interaction, periodic attending rounds, and assigned texts, often augmented by lectures. Recently, academic research has challenged the underpinnings of this traditional curriculum. Moreover, traditional clerkships do not necessarily mirror today's surgical practice given its increasing short-stay and out-patient emphasis. Initiated in 2001, the allopathic Florida State University College of Medicine provided an opportunity to create a contemporary surgical curriculum, emphasizing learning rather than teaching. Methods: The eight week FSU surgical clerkship is a tutorial model. at each of our six regional campuses, students are assigned one-on-one to a carefully selected, board-certified community surgeon, responsible for daily in-patient and out-patient teaching. the academic spine for the rotation is provided by a central curriculum, containing defined objectives and competencies, administered during weekly meetings with the student by campus-specific surgical clerkship directors. Residents do not participate in student teaching, nor are lectures used. Students are free to choose from among suggested print materials or alternative learning sources. Patient contacts are entered into an electronic data collection system, capable of comparing both student and individual campus experiences. Evaluations of student performance are 360 degrees: by patients, staff, clerkship faculty, and clerkship directors. Results: to date, 456 students have graduated. No significant differences exist between campuses with respect to either the numbers or types of patients encountered. USMLE Step II CK and CS scores, or NBME Surgery Subject Examination scores (p>.05). Student satisfaction with the course is evidenced by exceptionally positive responses to the end-of-clerkship evaluations of both faculty and clerkship at each of the campuses, and by anonymous laudatory responses by graduating seniors to the AAMC GQ Survey. Overall, 23.3% of graduates have chosen some aspect of Surgery as a career. the community faculty retention rate exceeds 97%. Conclusions: 1) A quality clerkship experience in Surgery can be provided by a non-traditional community-based system, 2) the distributed campus model does not lead to inequality of learning opportunities or surgical experiences, 3) Both students and community faculty strongly support the tutorial model, 4) Student learning has been confirmed by national metrics.

Clinical Experience Correlates With Surgical Examination Scores

Y. Vigneswaran, B. Gabryszak, C. Golner, L. Fogg, S.D. Bines, J.A. Myers — 2012-02-01

Introduction: Medical schools worldwide continue to face the challenge to construct both a balanced curriculum between clinical experience, lectures, reading time and other activities and to find appropriate methods for clinical evaluation. Although most schools in the United States use the National Board of Medical Examiners (NBME) subject examinations as a method of at least partial assessment, there is still uncertainty of how well these examination scores correlate with clinical proficiency. Thus, we investigated which factors in a surgery clerkship curriculum have a positive impact on academic achievement on the NBME surgery subject examination. We hypothesized students with more free time for self-study outside of their clinical experience would achieve better scores on the objective NBME surgery subject examination. Methods: We performed a retrospective analysis of 83 third-year medical students at our institution that rotated on the 8 week core general surgery clerkship for the 2007-2008 academic year. During this academic year, reorganization of the clerkship allowed for analysis of four groups of students experiencing varying clinical volume: two groups with unequal clinical volumes and time for self-study during the first 6 months and two groups with equivalent clinical experiences during the last 6 months. Records of the United States Medical Licensing Examination (USMLE) Step 1 scores, NBME scores and essay examination scores in each set of groups were compared using single-factor analysis of variance testing. Additionally, to specifically characterize each service, student feedback for each surgical service was obtained evaluating the clinical course load, time allotted for self-study, time in lectures and teaching effectiveness. Results: Data from the first six months demonstrated NBME scores from the group with the heavier clinical loads and least time for self-study were statistically higher than the group with lighter clinical services and higher rated self-study time (p = 0.036). the last six months, when the clerkship was rearranged to create two groups of equal clinical loads, there was no statistical difference of NBME scores between the groups (p = 0.751). Baseline USMLE step 1 scores were not significantly different between the groups and no statistical difference was seen in the subjective essay examination scores. Conclusions: We found that students experiencing higher clinical volumes on surgical services, but less self-study time demonstrated statsically higher academic performance on objective evaluation, suggesting clinical experience may be of higher value than self-study and reading. Thus, as the structure of our training system adapts to abide by the duty hour restrictions for trainees, we must be conscientious that certain experiences on the floors and in the operating room are invaluable and cannot be substituted.

Attending Surgeons and Residents Underestimate Their Influence on Medical Students Entering Surgery

2012-02-01

Introduction: Positive surgical role models have been demonstrated to influence medical students to pursue a career in surgery. However, the perception by the role model of their effectiveness has yet to be examined. in this study, we sought to evaluate the influence of surgical role models on medical student career choice and how these role models perceive themselves. Methods: A voluntary and anonymous survey was distributed to the following: (1) third year medical students at the end of their surgery clerkship, (2) applicants for a residency in general surgery, (3) general surgery residents and (4) attending surgery staff at a tertiary care university hospital during the 2010-2011 academic year. Statistical analysis was preformed using the chi-squared and Cochran–Mantel–Haenszel tests with a p-value < 0.5 being significant. Results: Responses were obtained from 127 (84%) medical students, 62 (86%) resident applicants, 29 (53%) general surgery residents, and 22 (31.4%) attending surgery staff. Medical students and the resident applicants agreed that faculty and residents play an important role in shaping the career path of medical students (p=NS). However, the resident applicants were more likely to agree than medical students that clerkship role models (p=0.0049) and mentors (p=0.0035) affected their interest in surgery. Similar sentiments were also seen in those medical students expressing an interest in surgery (p=0.0281 and p=0.0068, respectively). at each level of responsibility on a surgical team, the resident applicants were more likely to agree than the medical students that attending surgeons (p=0.0004), senior surgery residents (p=0.0019), and junior surgery residents/interns (p=0.0028) served as positive role models. While the attending surgical staff and general surgery residents agreed with the medical students that each level of the surgical team serves as a positive role model (p=NS), they did not agree as strongly with the medical students that they play an important role in shaping their career path (p=<0.0001). Conclusions: Attending surgical staff, senior surgical residents, and junior surgical residents/interns serve as positive role models for medical students on their services. They play an essential role in shaping the career path of medical students interested in surgery and should be more cognizant of their influence, which may draw a student towards or lead them away from the field of surgery.

Motivations and Obstacles to a Career in Surgery in Developing Countries: An Association for Academic Surgery Survey of Medical Students in West Africa

S. Krishnaswami, A. Lamoshi, E. Ameh, P. Ekeh, J. Laryea, B. Nwomeh — 2012-02-01

Introduction: Surgical disease is an increasingly recognized burden in low and middle income countries. This situation is exacerbated by a critical shortage of surgical workforce in these regions. to address this problem, the Association for Academic Surgery (AAS) has developed surgical training, research and career development programs for physicians and medical students in several regions of the world. During a recent mentoring and career workshop in West Africa, we surveyed medical students for their specialty preferences and the factors influencing their choice of a surgical career. Methods: A survey was distributed to all medical students who attended the AAS mentoring and career workshop. Demographic information including gender and level of training was collected. Respondents were also asked questions to assess exposure to and level of interest in various specialties as well as factors affecting career choice. Results: Sixty-seven of 70 attendees (96%) completed the survey, of which 51% were female and 49% male. Although the workshop was open to medical students of all levels, all attendees were in their clinical years of training. the majority of respondents (97%) had experienced their Surgery and Medicine rotations, while most had completed their core rotations in Ob/Gyn (49%), Pediatrics (55%) and Public Health (70%). the pre-clinical subjects of greatest interest to the group were Physiology (64%) and Anatomy (46%) whereas the most interesting clinical subjects were Surgery (71%), Medicine (36%), and Ob/Gyn (31%). the top choices for a future career were Surgery (61%), Ob/Gyn (42%), Medicine (18%), and Public Health (13%). However, women were less likely than men to choose Surgery as a career option (47% vs 77%, p=.04). Among all students, a positive experienceduring the rotation (67%) and mentors in the specialty (57%) were the most frequent factors cited for specialty choices. These factors increased in importance when examined in relation to only those students stating Surgery as a top career choice (78% and 64% respectively). Other factors positively correlating with career choices included intellectual content of specialty (56%), financial reward (47%) and prestige of specialty (47%). Factors negatively affecting career choices were workload (40%), ability to cope with the rigors of training (39%) and on-call schedule (31%). Conclusions: Encouragingly, Surgery appears to be a top career choice among medical students in West Africa. Direct mentoring from local surgeons, as well as formal mentoring experiences like the AAS workshop, may be vital to sparking and nurturing this interest in Surgery. Additionally, improvement in work-life balance during residency, perhaps through duty hour regulations, may further this interest, increasing the number of surgical trainees and ultimately helping to bridge the existing gap in the surgical workforce.

Beta-Catenin is a Critical Regulator of Mitochondrial Function and Energy Balance in Liver Homeostasis and Disease

2012-02-01

Introduction: The liver is the central organ for regulating systemic energy balance and nutrient metabolism. the Wnt/ß-catenin signaling pathway is a critical molecular regulator of hepatic development, regeneration and carcinogenesis. Given these established roles of Wnt signaling, we hypothesized a likely as yet described role for Wnt signaling in regulating mitochondrial function and cellular energy balance in the liver. Methods: Mice with hepatocyte-specific ß-catenin deletion (KO) and wild-type littermate controls (WT) were fed either normal chow or administered ethanol intoxication using the binge drinking model. Energy balance was determined by ATP level, pyruvate and lactate concentration. Mitochondrial function was assessed by mitochondrial content, membrane potential as well as oxygen consumption and oxidative phosphorylation target genes. Hepatic steatosis was determined by ALT, triglycerides as well as tissue histology. Intracellular oxygen radicals were detected by DHE staining, MDA assay and NAD+/NADH ratio. Changes in fatty acid ß-oxidation were determined by qRT-PCR for changes in gene expression for three of the mitochondrial master regulator genes PPAR-a, Sirt1 and PGC-1a. Results: We report that ß-catenin deficient hepatocytes possess basal mitochondrial dysfunction and energy deficit as evidenced by decreased cellular respiration (O2 consumption), ATP production and reduced mitochondrial membrane potential along with increased overall lactate production. Despite these findings, there is no evidence for redox imbalance in the absence of nutrient stress. in ß-catenin deficient hepatocytes, nutrient stress in the form of ethanol intoxication leads to significant redox imbalance and further deterioration in mitochondrial function including compromised oxidative phosphorylation and ATP production. Ethanol feeding resulted in significantly increased steatosis and oxidative damage in ß-catenin KO mice compared to WT along with a disrupted NAD+/NADH ratio. We further demonstrate that KO mice show reduced fatty acid ß-oxidation with a disrupted Sirt1/PPAR-a signaling axis. Conclusions: Taken together, our results demonstrate that ß-catenin plays an important role in maintaining mitochondrial function and energy homeostasis in which a basal deficit in oxidative metabolism is further disrupted given nutrient stress resulting in significant oxidative cellular injury. These findings demonstrate an important link between mitochondrial function and regenerative capacity in the liver that may have significant implications for current therapeutic strategies targeting Wnt signaling for hepatic protection, regeneration and carcinogenesis.

Simultaneous Knock-down of Bcl-xL and Mcl-1 Induces Apoptosis in Gemcitabine-Sensitive and -Resistant Pancreatic Cancer Cells

2012-02-01

Introduction: Gemcitabine has been considered the standard chemotherapeutic agent in the treatment of pancreatic cancer (PaCa). However, gemcitabine have revealed only a marginal overall survival benefit. Several reports describe that expression of the anti-apoptotic Bcl-2 family proteins is correlated with cancer cell resistance to chemotherapy. However, the precise molecular mechanism and contribution of anti-apoptotic Bcl-2 family proteins to PaCa cell survival in general and the role of anti-apoptotic Bcl-2 family proteins on gemcitabine resistance in PaCa has not investigated. Our aim was to delineate the molecular mechanism of anti-apoptotic Bcl-2 family proteins in PaCa cell lines. Methods: Human PaCa cell lines BxPC-3, MIA PaCa-2, Panc-1, Capan-2, and AsPC-1 were obtained from the American Type Culture Collection (ATCC). Cell proliferation was measured by MTT assay. Whole cell lysates were analyzed for the expression of proteins by Western blot. Apoptosis was measured by cell death ELISA kit (Roche) and Hoechst 33258 staining. to knock-down Bcl-2, Bcl-xL, and Mcl-1, siGENOME SMARTpool siRNA (Dharmacon) were used. Results: Bcl-2 was expressed in MIA PaCa-2 and Panc-1 cells, but virtually undetectable in other cell lines. Bcl-xL was almost equally expressed in six cell lines. Mcl-1 was not detected in AsPc-1 cells. These proteins were mainly localized in mitochondria membrane fraction. to assess whether gemcitabine sensitivity is correlated with anti-apoptotic Bcl-2 family protein expression, cell proliferation assay was performed. BxPC-3 cells was sensitive to gemcitabine treatment (IC50 = 0.3μg/ml), but MIA PaCa-2 and Panc-1 cells were more resistant (IC50 = 39μg/ml and 91μg/ml, respectively), suggesting that gemcitabine sensitivity is not correlated with the expression of anti-apoptotic Bcl-2 family proteins. Mcl-1 knock-down by siRNA resulted in marked cleavage of PARP and induction of apoptosis. on the other hand, down-regulation of Bcl-2 or Bcl-xL protein expression had a much weaker effect on PaCa apoptosis. Surprisingly, simultaneous knock-down of Bcl-xL and Mcl-1 induced apoptosis strongly in both gemcitabine-sensitive BxPC-3 cells and gemcitabine-resistant MIA PaCa-2 or Panc-1 cells (Enrichment factor = 6.0, 8.1, and 10.2, respectively). However, simultaneous knock-down of Bcl-xL and Bcl-2 or Mcl-1 and Bcl-2 had no additive effect. We previously reported that Mcl-1 down-regulation reinforce gemcitabine treatment. But, the effect of Bcl-xL and Mcl-1 knock-down was much more robust than Mcl-1 knock-down plus gemcitabine treatment. the apoptosis inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 is associated with Bax activation, mitochondrial cytochrome c release, and caspase cascade. Conclusions: These data suggest Bcl-xL and Mcl-1 as a potential therapeutic target for PaCa treatment and may help to develop novel therapeutic strategies for chemo-resistant PaCa cells.

The Role of Dendritic Cell and Pancreatic Stellate Cell Cross-Talk in Chronic Pancreatitis

2012-02-01

Introduction: Chronic pancreatitis (CP) is characterized by fibro-inflammatory transformation of the pancreatic parenchyma. the precise cellular mediators in CP are incompletely understood. Dendritic cells (DC) have recently emerged as initiators of organ-specific inflammation. Pancreatic stellate cells (PSC) become potently pro-inflammatory in CP and are primarily responsible for deposition of extracellular matrix proteins and fibrillar collagen. We postulated that an intimate relationship between DC and PSC mediate the dramatic fibro-inflammatory changes in CP. Methods: DC were isolated from bone marrow aspirates and cultured for 8 days in complete RPMI supplemented with murine GM-CSF. PSC were isolated after mechanical and chemical digestion of pancreas, followed by density centrifugation and plastic adherence. DC-PSC co-culture was accomplished by culturing equal number of DC and PSC for 24 to 48 hours before washing off the non-adherent DC. Cell culture supernatant was assayed using cytometric bead or ELISA. for in vivo experiments, a three week caerulein model of chronic pancreatitis was employed in C57BL/6 mice with select mice receiving i.p. adoptive transfer of DC (1x106 thrice weekly). in vivo PSC activation was measured using monoclonal antibodies directed against Desmin and α-SMA. Results: in vitro DC-PSC co-culture resulted in activation of PSC surface phenotype and the production of higher levels of PDGF, as well as numerous chemokines and cytokines by PSC (Table). Effects were contingent on direct cellular interaction between DC and PSC as well as DC expression of ICAM-1 and MyD88. in vivo i.p. adoptive transfer of DC in murine caerulein-induced chronic pancreatitis resulted in markedly increased fibro-inflammatory changes in the pancreas as well as 4-fold increase in percent desmin-positive acini, and approximately 80% α-SMA-positive acini (Figure). However, whereas DC induced PSC to become pro-inflammatory, DC inhibited PSC proliferation, failed to stimulate PSC migration, and did not induce PSC to express higher levels of extra-cellular matrix proteins or Collagen I in co-culture experiments. Furthermore, our experimental data suggest bi-directional cross-talk as PSC prevented DC maturation but acted as a potent DC chemoattractant. Conclusions: DC and PSC induce robust bidirectional crosstalk that affects CP. DC induce PSC to become pro-inflammatory, but do cannot cause PSC to adopt fully mature myofibroblast-like properties.

Response of Type II Diabetes Mellitus to the Preoperative Liquid Diet as a Predictive Model for Diabetes Resolution in Bariatric Surgery Patients

S.M. Biro, D.L. Olson, M.J. Garren, J.C. Gould — 2012-02-01

Introduction: The prevalence of obesity and type II diabetes mellitus (T2DM) is rapidly increasing in the United States. Up to 90% of patients with T2DM are obese. in morbidly obese individuals with T2DM, bariatric surgery offers a novel potential therapeutic endpoint – complete disease remission. in our bariatric program, patients are placed on an 800 calorie/day, very low-calorie diet (VLCD) for 2 weeks prior to surgery. Some diabetic patients experience significant improvement in blood glucose control and reduced insulin requirements on this VLCD. We sought to define the relationship between diabetic response to the pre-op VLCD and T2DM resolution rates post-op in a select group of bariatric surgery patients with the most severe diabetes, those requiring injectable insulin. Methods: Our study group was comprised of 51 diabetic patients on injectable insulin who underwent bariatric surgery at the UW Health Bariatric Surgery Program from August 2006 to February 2011. Subjects' blood sugars and insulin requirements prior to initiating the VLCD were compared with those at 10 days on a VLCD. Patients with a ≥50% reduction in total insulin dosage to maintain appropriate blood glucose control were deemed ‘rapid responders’ to the pre-op VLCD. All others were deemed ‘non-rapid responders’. Subjects were followed up to 1-year post-op to determine T2DM resolution rates (complete cessation of all antidiabetic medication). Results: Of the 51 subjects, 29 (57%) were ‘rapid responders’ to the pre-op VLCD and 22 were ‘non-rapid responders’. Rapid and non-rapid responders did not differ demographically based on age (56.8 yr. vs 54.4; p=.41), sex (female 52% vs 77; p=.08), pre-op body mass index (47 kg/m2 vs 50; p=.12), HbA1c (7.5 vs 7.7; p=.7), duration of T2DM (13.9 yr. vs 14.5; p=.75) or procedure (laparoscopic gastric bypass [LRYGB] 72% vs 64%; p=.55 or laparoscopic adjustable gastric band [LAGB] 10% vs 32%; p=.07). T2DM resolution rates were significantly greater in the ‘rapid responder’ group (Table 1). Evaluating LRYGB patients alone, rapid responders showed greater % excess weight loss (EWL) at 3 months (40% vs 28%; p<.01), 6 mo. (55% vs 40%; p<.01), and 12 mo. (65% vs 51%; p=.04). Conclusions: Insulin-dependent T2DM bariatric surgery patients who display a rapid response (≥50% reduction in insulin requirements) to the pre-op VLCD are more likely to experience early resolution of T2DM post-op, and ultimately greater weight loss. the diabetic response to the pre-op VLCD is an important predictive variable not routinely taken into account in the published literature pertaining to diabetes and bariatric surgery. Further study is necessary to clearly delineate the mechanisms for these observations. Food consumption patterns during acclimation period, and Glucose and Insulin levels at harvest.

Dysregulated Intestinal Nutrient Sensing and Feeding Pattern in Obesity and Diabetes

H.Y. Bhutta, T. Deelman, D.B. Rhoads, A. Tavakkolizadeh — 2012-02-01

Introduction: Shift work, an increasingly prevalent work pattern, leads to misaligned circadian patterns and is associated with type 2 diabetes mellitus (T2DM) and obesity. Obese subjects also often report night eating, a behavior disruptive of circadian rhythms. We hypothesized that such deregulation of food intake and its subsequent effect on gut glucose sensing (via sweet taste receptors, T1R2) and transport (via glucose transporter SGLT1) leads to metabolic disease. We studied circadian patterns of feeding and gut glucose sensing and transport in a rat model of disease, and correlated them to glucose homeostasis and weight changes, to gain insight into mechanisms underlying the onset of metabolic disease in these conditions. Methods: 13-week old obese ZDF rats and lean littermates were acclimated for 12 days (n=24/group), with ad libitum high carbohydrate diet. Daily weights and food intake during light and dark hours were recorded. Animals were harvested at circadian time-points ZT3, 9, 15 (lights on 7am/ZT0). Proximal jejunal mRNA levels of SGLT1 and T1R2 were assessed and functional glucose uptake assayed using the everted sleeve technique. Homeostasis Model of Assessment-Insulin Resistance (HOMA-IR) was calculated. Statistical analyses included Student t-test, ANOVA and Cosinor. Results: As expected, obese rats ate more than lean littermates (Table 1). in both groups however, diurnal feeding patterns were diminished compared to other rat models (SD rats, data not shown). As a result, diurnal rhythmicity of SGLT1 expression and function were absent in both groups. Blood glucose levels were however higher in the obese rats with a significant increase in HOMA-IR (43.7 vs 21.9, obese vs. lean respectively; p<0.05; Table 1). Elevated serum glucose was also associated with a trend towards increased jejunal SGLT1 mRNA but a decrease in T1R2 mRNA in obese ZDF rats compared to lean littermates (p<0.05, Fig 1). Conclusions: In both obese ZDF and lean littermates, diurnal feeding pattern was dampened with resultant loss of rhythmicity in SGLT1 expression and activity. Since only ZDF rats developed the phenotype of obesity and T2DM, loss of intestinal rhythmicity per se (e.g. in shift work) does not appear to lead to disease. Elevated SGLT1 levels, despite low T1R2 levels in obese ZDF rats, indicate an inappropriate SGLT1 response to glucose-sensing in the proximal jejunum of these rats. Isolation of this segment of bowel in gastric bypass may in fact explain the anti-diabetic effect of surgery and highlight a new therapeutic pathway.

Formation of an Intestinal Epithelial Surface Using Collagen Gel

2012-02-01

Introduction: Intestinal stem cells can form spheroids when they are cultured within Matrigel supplemented with the Wnt agonist R-spondin that interacts with the Lgr5 receptor. A critical barrier to translate this methodology to cell-based therapy is the ability to generate a sheet of intestinal epithelial cells using well-defined extracellular matrix. We hypothesize that collagen gel will induce the formation of such epithelial cell sheets and eliminate the need for Matrigel. Methods: Intestinal crypts were isolated from transgenic GFP mice between 4-6 weeks of age using EDTA chelation. Intestinal subepithelial myofibroblasts were obtained from murine C57BL/6 pups. Crypts were mixed in collagen gel and were seeded on top of myofibroblasts. Crypts were also seeded on top of collagen gel without myofibroblasts in some experiments. Spheroids were counted by light microscopy, and quantitative PCR and immunohistochemical staining were performed on cells cultured for 7 days in media with R-spondin. Results: Crypts initial formed spheroids when they were cultured within collagen gel supported by myofibroblasts that expressed smooth muscle actine and vimentin. Cells within the spheroids expressed E-cadherin and Cdx2, confirming their intestinal epithelial origin. the cell mass increased in culture as confirmed by microscopy and GFP-DNA PCR. Epithelial cells were observed to migrate from these spheroids and formed a sheet of columnar cells at the liquid-gel interface. These cells possessed brush borders and basally located nuclei, and they also expressed E-cadherin and Cdx2. Goblet cells were observed in this newly formed epithelium. When directly seeded on top of collagen gel in the absence of myofibrolasts, crypts formed a sheet of cobblestone-patterned cells at the liquid-gel interface. Conclusions: Collagen gel can be used to support the in vitro growth of primary intestinal epithelial cells. the formation of an epithelial cell sheet instead of spheroids may allow scaling up of this methodology for clinical use. This technique eliminates the dependence of intestine epithelial cells on Matrigel and may facilitate the translation to cell-based therapy in the future.

Distribution of Growth Induced By Straight Versus Cuvilinear Distractive Enterogenesis

2012-02-01

Introduction: Length of remaining small bowel is an important prognostic factor in long term survival and ability to wean off parenteral nutrition in patients with short bowel syndrome(SBS). Distraction enterogenesis, using mechanical forces to lengthen intestinal tissue, is a novel, potential treatment option for SBS. Two newly developed intraluminal devices to achieve functional longitudinal small bowel growth in a pig model are described. the experimental aims were to test the characteristics of growth induced by both devices. Methods: Two engineered devices were implanted into live pigs. in one pig a straight-radio frequency controlled device driven using a shape memory alloy(SMA)-powered expansion mechanism(Figure 1A) was implanted and extended over an 8 day period. in a second pig, a curvilinear-shaped hydraulic-powered device was implanted and expanded over a 14 day period(Figure 1B). Devices were placed into roux-en-y segments to isolate them from enteric flow. Outcome measures were initial and final bowel length, epithelial cell proliferation using bromodeoxyuridine(BrdU), functionality using transepithelial resistance(TER) measured by an Ussing chamber, and calculating mesenteric microvessel growth using α-smooth muscle actin staining. Location of growth in the bowel relative to the devices was also examined. Results: Initial and final bowel lengths with the SMA device were 13 cm and 18.5 cm; 5.5 cm of lengthening. Initial and final lengths with the hydraulic device were: 15 cm and 24.9 cm; 9.9 cm of lengthening. Table 1 shows: percentage of BrdU+ epithelial cells; TER; and area of microvessels per 20x high powered field. Finally, because the degree and application of distractive forces varied between the two devices (curvilinear-shaped hydraulic device more even distribution vs. straight-shaped SMA device, force confined to both ends), the differences in growth between devices was examined. Growth, (BrdU+ epithelial cells) was higher in the mid-portion of the curvilinear case vs. concentrated near the ends for the straight device, correlating with higher microvessel growth distribution for each device. Conclusions: Longitudinal distractive forces appear to be an effective method for small bowel growth. Growth does not appear to occur evenly throughout the stretched segment, but is concentrated on the end for the SMA device and the middle for the hydraulic. These growth characteristics will prove useful in guiding future device development.

In Vitro Crypt Culture from Small Bowel Maintains Cephalo-caudal Gradients of Gene Expression

M.K. Fuller, D.M. Faulk, N. Sundaram, S.J. Henning, M.A. Helmrath — 2012-02-01

Introduction: Recent methodology to expand intestinal crypts to poly-cryptoid structures (enteroids) has been previously described by our group. We sought to determine whether enteroids in vitro maintained their in vivo characteristics by comparing regional variation in vitro. Methods: After IACUC approval, crypts were harvested from 5 cm of proximal and distal small bowel of C57BL/6J mice aged 6-8wk (n=3 animals). Crypts were cultured using a modification of the Sato technique. Proximal and distal small intestinal tissue, crypts, and enteroids (at 7, 14, and 28 days in culture) were collected. in addition, proximal and distal enteroids were co-cultured with both proximal and distal myofibroblasts and collected at day 14. mRNA was extracted via RNeasy Mini kit (Qiagen Biosciences), and reverse transcribed via High Capacity Reverse Transcription kit (Applied Biosystems). qRT- PCR with Taqman universal primer master mix (Applied Biosystems) for regionally expressed genes GIP, FABP1, GATA4, PYY, Asbt, and Cubulin was performed in triplicate. the data were analyzed using a relative standard curve method normalized for the expression of β-actin. *p < 0.05 was determined significant by two way ANOVA followed by Bonferroni post-test corrections. Results: Morphologically, proximal enteroids demonstrated significantly greater budding than distal at 7 days in culture (6.7 vs 5.1 buds per enteroid*). Gene expression of the proximal markers GIP*, FABP1*, and GATA4* was pronounced in proximal enteroids and modest in distal enteroids. Conversely, gene expression of PYY*, Asbt, and Cubulin* was elevated in distal enteroids and nearly absent in proximal enteroids. These patterns of expression were maintained through 3 passages and 28 days in culture. Upon co-culture with myofibroblasts, proximal and distal enteroids retained their original regional identity despite the influence of opposing myofibroblasts. (*p< 0.05) Conclusions: Intestinal crypt culture reproducibly maintains in vivo crypt identity. Regional identity is programmed within the crypt unit and this data is suggestive that it is regulated at the stem cell level. the combination of the ability to expand crypts in vitro and mimicry of in vivo behavior provides a promising assay toward examination of differences in regenerative capacity among small intestinal diseases and among potential therapies for short bowel syndrome.

Inducible Deletion of Rb in Enterocytes Enhances Adaptation After Small Bowel Resection

P.M. Choi, J. Guo, D. Wakeman, J. Leinicke, B. Warner — 2012-02-01

Introduction: Following massive small bowel resection (SBR), an endogenous compensatory process takes place in the remaining bowel termed adaptation. Adaptation induces villi growth, thereby increasing mucosal surface area for absorption and digestion. Previously, we have demonstrated that genetically disrupting retinoblastoma protein (Rb) expression in enterocytes results in taller villi, deeper crypts, and increased rates of enterocyte proliferation – all of which mimic resection induced adaption responses. When we performed SBR in these Rb null mice, no additional villus growth occurred. the purpose of the present study was to determine whether adaptation responses would be affected by inducibly disrupting Rb expression in enterocytes after the SBR procedure. Methods: A tamoxifen (TAM)-inducible villin (vil) promoter driving Cre recombinase in Rb floxed mice was employed to induce Rb deficiency specifically in enterocytes. Six to 8 week old male vil-Cre-ERT2(-)/Rb(flox/flox; wild-type -WT) and vil-Cre-ERT2(+)/Rb(flox/flox; induced Rb-null - i-Rb-null) mice underwent a 50% proximal SBR. One day later, Tamoxifen was injected intraperitoneally for 3 days, and the small intestine was harvested 28 days after surgery. Enterocytes were isolated, and absent Rb expression was confirmed by Western Blot. Small bowel samples were fixed for histology. Crypt depth and villus height were compared between samples obtained at the time of resection versus at the time of harvest 28 days later. Results: Small intestine morphology at the time of SBR was no different between WT and i-Rb-null mice: crypt depth (87.8 ±12.6vs 85.4±8.0 μm, p =0.73) and villus height (183.0±20.7 vs 159.2±25.2, p =0.14). at 28 days following SBR, WT mice demonstrated increased villus height (21.6%±13.9) and crypt depth (5.4%±5.2). However, a significantly greater adaptive increase in villus height (65.2%±16.2, p = .0002) and crypt depth (29.5%±14.4, p = .003) were observed after SBR in the i-Rb-null mice when compared with WT (Figure 1). Conclusions: Deletion of Rb after SBR, but not prior results in amplified intestinal adaptation. These findings implicate an important regulatory role for Rb in the genesis of this important response and underscore the significance of postoperative timing for interventions intended to enhance resection-induced adaptation in patients suffering from short gut syndrome.

RNA-Binding Protein TIA-1 Regulates Intestinal Epithelial Cell Apoptosis

2012-02-01

Introduction: Apoptosis plays an essential role in the maintenance of intestinal epithelial cell renewal and integrity. RNA-binding proteins (RNA-BPs) and microRNAs (miRNAs) jointly regulate numerous processes critical for cellular and organismal survival including apoptosis. RNA-binding protein TIA-1 is known to modulate the fate of its target mRNAs by binding to the AU-rich element (ARE) of their 3' untranslated region (UTR), thus resulting in regulation of translational efficiency, alternative pre-mRNA splicing, stress granule aggregation, and programmed cell death. Additionally, recent studies have identified a multitude of miRNAs as key contributors to regulation of apoptosis. MicroRNA-503 (miR-503) is one such miRNA that has been recently reported to regulate both apoptosis and RNA-BP activity. Prior literature suggests that TIA-1 may promote apoptosis in vitro in cancer cell lines. However, both the role of TIA-1 in normal physiologic IECs and the mechanisms that regulate TIA-1 abundance remain elusive. We hypothesized that TIA-1, in fact, acts in an anti-apoptotic manner in normal physiologic IECs and that miR-503 can directly regulate this effect. Methods: Utilizing the Cdx2/IEC-6 cell line derived from rat intestinal crypts, cells were transfected with siRNA specifically targeting TIA-1 (siTIA-1) or control siRNA (C-siRNA). Following 48 hours of transfection, apoptosis was induced by tumor necrosis factor-α (TNF-α)/cyclohexamide (CHX). Apoptosis was measured by both morphological analysis and annexin-V immunofluorescence staining. Expression of caspase-3 was measured by both Western blot analysis and ELISA. Results: TIA-1 silencing by transfection with siTIA-1 resulted in an increased susceptibility to apoptosis following exposure to TNF-α/CHX for 3 hours. the assessment of apoptosis was confirmed via Western blotting and ELISA by demonstrating an increase in levels of active caspase-3 in siTIA-1 cells as compared to controls. Immunofluorescence staining with Annexin V confirmed increased apoptotic cells in the TIA-1 silent population as compared to its controls. Therefore, our study demonstrates that TIA-1 functions in a protective, anti-apoptotic manner in physiologic intestinal cells. Additionally, in order to examine the role of miR-503 in the TIA-1-mediated down-regulation of apoptosis, Cdx-2 cells were transfected with miR-503 precursor (pre-miR-503) or its respective control. Transfection of pre-miR-503 not only resulted in a predictable increase in miR-503 expression and a significant decrease in TIA-1 expression, but also enhanced apoptosis after exposure to TNF-α/CHX. Conclusions: Therefore, our findings both identify miR-503 as novel regulator of TIA-1 and define a novel role for TIA-1 as a modulator of intestinal epithelial apoptosis.

The Impact of Neoadjuvant Chemotherapy on Wound Complication Risk After Breast Cancer Resection and Reconstruction - A Multi-Institutional Assessment

2012-02-01

Introduction: Neoadjuvant chemotherapy has been shown to be as effective as adjuvant therapy for the treatment of resectable breast cancer, with the advantages of monitoring the in situ tumor for response to therapy and the potential to convert from mastectomy to breast conserving surgery. As a result, the indications for neoadjuvant chemotherapy are expanding. However, limited data are available regarding the impact of neoadjuvant chemotherapy on the risk of post-operative wound complications. Our multi-institutional study addresses this gap. We hypothesized that neoadjuvant chemotherapy would not be associated with an increased risk of wound complication, regardless of type of breast procedure. Methods: Using the American College of Surgeons National Surgical Quality Improvement Program database, we selected patients with a diagnosis of malignant neoplasm of the breast, ductal carcinoma in situ, breast mass, or inflammatory breast disease who underwent 1) breast conserving surgery with lymph node biopsy or dissection 2) mastectomy or 3) mastectomy with immediate or delayed - immediate reconstruction from 2005 – 2009. We performed a stratified analysis of post-operative wound complication (surgical site infection and dehiscence) rates for each of the three surgical groups. Multivariable logistic regression was used to adjust for other risk factors for wound complication, including demographics and pre-operative characteristics. Our primary variable of interest was chemotherapy 30 days prior to surgery. Results: A cohort of 42,088 women with non-metastatic breast cancer who underwent surgery was identified. of these, 1550 (3.7%) received chemotherapy 30 days prior to surgery. the wound complication rate for recipients of neoadjuvant chemotherapy was 3.5%, compared to 2.9% for the remainder of the cohort (p = 0.19). After adjusting for other pre-operative and operative factors, neoadjuvant chemotherapy was not significantly associated with increased risk for wound complication for the cohort as a whole (OR = 1.03, 95% CI 0.76 - 1.38), or when the analysis was stratified by breast procedure. Risk factors that were associated with wound complication included smoking, dependent functional status, overweight and obesity, diabetes, hypertension, mastectomy (with or without reconstruction), and sentinel lymph node biopsy (Table). Conclusions: in this multi-institutional analysis, neoadjuvant chemotherapy was not associated with post-operative wound complication, regardless of the type of breast surgery performed. Patients and providers can be assured that receipt of neoadjuvant therapy does not increase the risk of post-operative wound complication.

Invasive Lobular Carcinoma Predicts Micrometastatic Disease in Stage I-III Breast Cancer

2012-02-01

Introduction: Invasive lobular carcinomas (ILC) represent 5-15% of all breast cancers, are usually well-differentiated, and almost all are estrogen receptor (ER) positive. Most delayed breast cancer recurrences (> 5 years) occur in ER positive patients. Disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) have been implicated in breast cancer recurrences. the purpose of this study was to determine if DTCs and CTCs are associated with ILCs in stage I-III breast cancer patients. Methods: Clinical stage I-III breast cancer patients seen at a single tertiary cancer center provided consent to participate in an IRB-approved study involving collection of bone marrow (5 ml x 2 tubes) and blood (7.5 ml x 2 tubes) at the time of surgery for their primary breast cancer. Both chemonaïve patients and those who underwent neoadjuvant chemotherapy (NACT) were included. DTCs were assessed by anti-CK antibody cocktail following cytospin. CTCs were detected using the CellSearch™ system. CTCs were defined as nucleated cells lacking CD45 but expressing cytokeratin (CK). A positive result was defined as the presence of one or more cells per 5 ml of bone marrow or 7.5 ml of blood since the threshold for positivity has not been established in non-metastatic breast cancer. Statistical analyses used chi-square and Fischer's exact tests. Results: Four hundred and twenty-two patients were prospectively enrolled. Mean age was 52 years. Median follow-up was 32 months. One hundred and twenty-six patients (30%) received NACT. Sixty four patients (15%) had ILC and 357 (85%) had invasive ductal carcinoma (IDC). ER positivity was much higher in ILCs (59/64, 92%) compared to IDCs (237/358, 66%). {P=0.0001} DTCs were identified in 109/366 patients (30%) and CTCs in 90/388 (23%). Either DTCs or CTCs were identified in 170/399 of all patients (43%). Thirty-four patients (54%) with ILC had DTCs or CTCs. DTCs were identified in a significantly higher percentage of ILC patients (21/53, 40%) compared to IDC patients (81/300, 27%). {P=0.03} Only 2/14 patients (14%) with ILC treated with NACT achieved pathologic complete response (pCR) compared to 34/122 patients (28%) with IDC. {P=0.09} No correlation was observed between the presence of DTCs and/or CTCs in ILC patients and tumor size, tumor grade, hormone receptor status, stage or administration of NACT. Conclusions: ILC was an independent predictor of the presence of DTCs and CTCs. Since most late recurrences are ER positive this raises the question of whether DTCs and CTCs are indeed responsible for late breast cancer recurrence. We are currently assessing DTCs and CTCs for ER status to determine discordance rates between these cells and the primary tumor.

Indications for Sentinel Lymph Node Biopsy in Multifocal and Multicentric Breast Cancer

L.C. Moody, C. Chao — 2012-02-01

Multifocal and multicentric breast cancer have been regarded as a relative contraindication for sentinel lymph node biopsy because initial validation studies noted an association between multifocal and multicentric breast cancer and higher false-negative results. Multifocal and multicentric breast cancer have a higher incidence of node-positive disease compared to unifocal cancers. Some single institution researchers have demonstrated false-negative rates comparable to that of unifocal tumors; however, these studies have been limited by low patient numbers. the purpose of this study is to perform a meta-analysis of the literature evaluating the feasibility and accuracy of sentinel lymph node biopsy in multifocal & multicentric breast cancer. Methods: A PubMed search was performed to retrieve original articles published between 2000 & 2010 evaluating the accuracy of sentinel lymph node biopsy in multifocal & multicentric breast cancer. Patients with multifocal & multicentric breast cancer who underwent sentinel lymph node biopsy followed by axillary lymph node dissection were included in the meta-analysis. Patients with unsuccessful lymph node mapping were excluded. Nineteen original articles were considered. Three of the articles were eliminated because there was insufficient data to perform validity calculations. the meta-analysis included 7 retrospective and 9 prospective studies. Results: A total of 932 patients with multifocal & multicentric breast cancer underwent sentinel lymph node biopsy followed by axillary lymph node dissection. 444 patients had a positive sentinel lymph node biopsy and 487 patients had a negative sentinel lymph node biopsy. There were 37 false-negative biopsies. 7 of the 37 false-negative patients had relative contraindications for sentinel lymph node biopsy including tumor size, palpable lymph nodes, prior breast surgery, and neoadjuvant chemotherapy. the overall accuracy rate, sensitivity, specificity, false-negative rate, and negative predictive value are 96%, 92.3%, 100%, 7.7%, and 92.4% respectively. If we exclude 7 of the 37 false-negative patients with at least one other known relative contraindication to sentinel lymph node biopsy, the accuracy rate, sensitivity, specificity, false-negative rate, and negative predictive value are 96.7%, 93.7%, 100%, 6.3%, and 93.5% respectively. Conclusions: in conclusion, in a select subset of patients with multifocal and multicentric breast cancer, sentinel lymph node biopsy can accurately predict lymph node status with false-negative rates comparable to that of patients with unifocal breast cancer. However, when a multifocal or multicentric breast cancer has an additional relative contraindication to performing sentinel lymph node biopsy, such as neoadjuvant chemotherapy or T>5cm, the false-negative rate increases.

Wound Complications After Inguinal Lymph Node Dissection for Melanoma: is ACS NSQIP Adequate?

C.E. Seaberg, D.Y. Greenblatt, R.J. Rettammel, H.B. Neuman, S.M. Weber — 2012-02-01

Introduction: in the treatment of melanoma, inguinal lymph node dissection (ILND) is the standard of care for palpable or biopsy-proven lymph node metastases. Wound complications after ILND are common, occurring in 77% of patients in one single-institution prospective clinical trial. in this trial's definition of wound complications, wound infections as well as seroma and skin necrosis were included. in the current study, we utilized the multi-center American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) to examine frequency of wound complications after ILND. Methods: Patients with cutaneous melanoma who underwent superficial and superficial with deep ILND (CPT codes 38760 and 38765, respectively) from 2005-2009 were selected from the ACS NSQIP database. Standard ACS NSQIP 30-day outcome variables for Wound Occurrence (superficial surgical site infection (SSI), deep SSI, organ space SSI, and dehiscence) were defined as wound complications. Seroma, hematoma, and skin necrosis were not included in the definition of wound complications as they are captured as “Other Wound Occurrence” by ACS NSQIP and not available as part of the Participant Use Data File. Rate of return to the operating room within 30 days was also noted. Univariate and multivariate analyses were performed to identify risk factors for wound complication after ILND. Results: Of 206 total patients,173 underwent superficial ILND and 33 underwent superficial with deep ILND. Only 30 patients (14.6%) had 34 wound occurrences: 24 superficial SSI, 3 deep SSI, 1 organ space SSI and 6 wound disruptions. in a multivariable model, superficial with deep ILND (OR 2.59, 90% CI 1.19-5.65), obesity (OR 2.61, 90% CI 1.30-5.21), and diabetes (OR 2.69, 90% CI 1.17-6.19) were significantly associated with wound complications. There was no difference in the rate of re-operation in patients with and without wound complications (p=0.243). Conclusions: ACS NSQIP appears to markedly under-report the actual incidence of wound complications after ILND. This may reflect the program's narrow definition of wound occurrence, as other relevant wound complications (i.e. seroma, hematoma, and skin necrosis) are not available as part of the Participant Use Date File. Additionally, while return to the operating room is captured, non-operative interventions such as seroma drainage are not. Future iterations of the ACS NSQIP for Oncology should expand the definition of Wound Occurrence to incorporate other clinically relevant post-operative wound outcomes, including seroma/hematoma, skin necrosis, and percutaneous drainage procedures. These variables would be relevant for outcomes measurement and quality improvement initiatives for soft tissue malignancies beyond melanoma, including breast cancer and sarcoma.

Predicting Disease Progression After Regional Therapy for In-Transit Melanoma

M.E. Lidsky, R.S. Turley, G.M. Beasley, K. Sharma, D.S. Tyler — 2012-02-01

Introduction: While approximately 30-50% of patients experience complete response after regional chemotherapy for in-transit melanoma, a subset of patients will develop rapidly progressive disease (PD). in the current era of an expanding armamentarium including both regional and systemic options for treating advanced melanoma, identifying perioperative factors that predict disease progression may obviate unnecessary morbidity associated with regional therapy and avoid delays in systemic therapy. Methods: A prospective database identified 215 patients who underwent 134 first-time isolated limb infusions (ILI) and 81 first-time hyperthermic isolated limb perfusions (HILP) for in-transit melanoma. Response was defined using RECIST modified criteria for cutaneous disease at 3 months after treatment. Survival analyses were performed using the method of Kaplan and Meier, with differences in survival curves compared using the log-ranks test. Potential preoperative and procedural predictors of in-field PD were analyzed using logistic regression. Results: 43/134 (32.1%) and 9/81 (11.1%) patients experienced in-field PD after ILI and HILP, respectively. Median survival for patients with in-field PD was 20.3 months and 15 months after ILI and HILP, respectively (Figure). in general, patients with PD were younger with advanced stage melanoma and increased tumor burden. Compared to complete responders (CR), in-field PD after ILI was associated with decreasing age (OR=0.94, 95% CI=0.90-0.98, p=0.002). for patients undergoing HILP, no clinically relevant preoperative predictors of in-field PD were identified. Procedural variables including chemotherapeutic dosing, the degree of acidosis or base deficit achieved, and peak temperature attained were not predictors of in-field PD after ILI or HILP. Conclusions: Patient, clinical, and procedural factors are unreliable predictors of in-field PD after regional therapy in patients with in-transit melanoma. Defining the potential utility of molecular markers in predicting response and failure in the regional therapy setting should be the focus of future research efforts.

GSTP1 Suppression Induces Apoptosis in Melanoma Cells Independent of BRAF Mutational Status

R.S. Turley, C.K. Augustine, M.E. Lidsky, D.S. Tyler, F. Ali-Osman — 2012-02-01

Introduction: Although specific inhibitors of the oncogenic mutant V600E BRAF kinase have improved rates of overall and progression-free survival for melanoma patients harboring the V600E mutation, durable responses are rarely achieved. There is, thus, the need for the development of novel treatment strategies for this disease. We have previously shown glutathione S-transferase P1 (GSTP1), a binding partner and negative regulator of MAP kinase signaling, to be important for survival of melanoma cells. the purpose of this study was to investigate the effect of GSTP1 knockdown on cell survival and MAP kinase signaling in both wild-type (WT) and mutated V600E BRAF melanoma cells. Methods: DNA was extracted from 24 melanoma cell lines and PCR amplified using specific BRAF primer sequences and the DNA products were purified and sequenced to identify the V600E (c.1799T>A) mutation. GSTP1 expression in these cells was determined by quantitative RT-PCR. the WT BRAF DM175 and the V600E mutated DM6 melanoma cell were transfected with GSTP1 siRNA or scrambled control. GSTP1 knockdown was confirmed by PCR and western blotting. Population growth kinetics of the GSTP1 knockdown and control cells was monitored in real time over 5 days by electronic cell sensing. Apoptosis was determined used flow cytometry of propidium iodide stained cells. MAP kinase signaling was assessed by determining the levels of JNK, ERK, and P38 by western blotting. Results: Of the 24 melanoma cell lines, 15 harbored the V600E BRAF mutation and 9 had the WT BRAF gene. GSTP1 was expressed in all cell lines at variable levels. Transfection of siRNA GSTP1 effectively decreased GSTP1 transcript and protein levels in both the BRAF V600E DM6 and the WT BRAF DM175. in both WT and V600E BRAF melanoma cells, transcriptional suppression of GSTP1 ceased cell population growth (Figure 1) and significantly increased basal levels of apoptosis. After GSTP1 KD, we observed a significant increase in activated ERK1/2 and JNK1/2 MAP kinases. Conclusions: The results allow the following Conclusions: 1) GSTP1 suppression induces apoptosis in GSTP1-expressing melanoma cells independent of BRAF mutational status; 2) GSTP1 suppression activates the JNK and ERK signaling pathways in V600E mutated melanoma cells and 3) targeted inhibition of GSTP1 may represent a clinically efficacious strategy for WT BRAF melanoma and melanoma tumors with the V600E mutation that have become resistant to BRAF inhibitors.

Impact of CDK4 Inhibition and Chemotherapy on Smad3 Action in Breast Cancer Cell Proliferation and Migration

E. Tarasewicz, A. Hardy, S. Zelivianski, J.S. Jeruss — 2012-02-01

Introduction: Several aspects of breast cancer onset and disease progression have been linked to members of the TGF-beta superfamily and their associated downstream signaling components, the Smads. Alterations in Smad signaling have been directly implicated in the dichotomous role of TGF-beta in malignancy, enacting both tumor suppressant and tumor promoting behaviors in breast carcinogenesis. Our previous work characterized Smad3 as a tumor suppressor and found that Smad3 action is inhibited upon phosphorylation by cyclin D/CDK4 in breast cancer cells. Others have shown that CDK phosphorylation of Smad3 can affect cell motility.Based on these findings, we hypothesized that activation of CDK4 leads to phosphorylation and inhibition of Smad3, thus releasing cell cycle arrest and promoting cell proliferation and metastasis. Methods: MCF7 parental and cyclin D overexpressing cells (MCF7 CD1) were treated with a CDK4 inhibitor alone or in combination with doxorubicin or paclitaxel and cell proliferation was determined using the MTS assay. A protein profile apoptosis array was also used to examine treated and control study cells. Transwell migration and Matrigel invasion assays were used to determine the effect that transfection with Smad3 CDK phosphorylation site mutants resistant to inhibitory cyclin D/CDK phosphorylation or treatment with a CDK4 inhibitor had on highly metastatic cyclin D expressing MDA-MB-231 cells. Study wells were fixed/stained and the number of migrated or invaded cells was counted. Results: Treatment with the CDK4 inhibitor alone resulted in a growth inhibitory effect on proliferation of MCF7 parental and CD1 cells. Treatment with the CDK4 inhibitor in combination with chemotherapy resulted in the greatest inhibition of cell proliferation. When 35 apoptosis related proteins were examined, treatment with the CDK4 inhibitor in combination with chemotherapy also resulted in maximal upregulation of apoptosis related proteins Fas and DIABLO and inhibition of Survivin and HSP70. Lastly, MDA-MB-231 cells transfected with the 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration and invasion, when compared with cells transfected with both vector control or WT Smad3. Treatment of the MDA-MB-231 cells with the CDK4 inhibitor also resulted in a significant decrease in cell migration and invasion. Conclusions: Mutation of CDK phosphorylation sites in the Smad3 construct or direct inhibition of CDK4 resulted in a decrease in breast cancer cell proliferation, migration and invasion. Treatment with the CDK4 inhibitor in combination with chemotherapy further augmented these results and potentiated early induction of apoptosis. Inhibition of CDK4 and restoration of Smad3 activity may have a contributory role in the treatment of breast cancers overexpressing cyclin D.

Preoperative Immune Manipulation of the Primary Site in Melanoma can Initiate Anti-Tumor Autoimmunity: A New Approach to Adjuvant Therapy

E.G. Elias — 2012-02-01

Introduction: Presently, systemic adjuvant therapy is being initiated in high-risk melanoma patients after complete resection of the disease, with the objective of prolonging survival. This has been met with limited success due to tumor heterogeneity and nonspecific immunotherapy. Therefore, new approaches are needed. Granulocyte-macrophage colony stimulating factor (GM-CSF) activates mainly dendritic cells, while interleukin-2 (IL-2) activates T cells. Each gave 60% response rate of long duration when administered intralesional in satellitosis and intransit metastases. Further, when GM-CSF was injected at the primary site, it increased the number and activation of CD8+ and dendretic cells in the paracortical zone of sentinel lymph nodes (LN). Hypothesis: the combined administration of GM-CSF and IL-2 at the primary site may yield higher tumor response, can have the same effect as intralesional therapy and initiate tumor-specific autologous immunotherapy. Methods: In a pilot study, newly diagnosed high-risk patients received a single short course of GM-CSF: 500 microgram at the primary (biopsy) site on day #1. This was followed by IL-2: 11 million IU per day, at the same site, on days 2 and 3, one week prior to sentinel LN biopsy (or LN dissection for palpable LNs) and wide excision of the primary site. Postoperatively, patients with LN metastases had both agents administered subcutaneously for variable periods of time depending on the extent of LN involvement. Results: This approach is safe and without systemic toxicity. However, there was moderate size local reaction at the injection site. Histological examination of the excised tissues showed complete tumor necrosis with massive histiocytosis at the primary site and the LNs. Immunohistochemistry studies revealed overexpression of CD8+, CD25+ and CD83+ cells at the primary site and LN, compared to controls, reflecting the effects of IL-2 and GM-CSF. Conclusions: The presence of GM-CSF and IL-2 and residual tumor cells or their debris forms a TRIAD at the primary site. This has the potential of inducing an autologous tumor-specific cytotoxic T cells in-vivo (patient specific) at the primary site that can migrate via the lymphatics to the regional LNs. This approach can initiate a specific and effective cytoxic cells and direct the specifity of postoperative systemic therapy. Postoperative administration of the same low doses of both agents subcutaneously could maintain and magnify the initial response. This hypothesis needs further confirmation in phase I clinical trials.

The Preclinical Development of FTY720, A Sphingosine-1-Phosphate Receptor Modulator, as a Novel Targeting Therapy Against Breast Cancer

2012-02-01

Introduction: Sphingosine-1-phosphate (S1P), is a bioactive lipid mediator and a ligand for five specific G-protein coupled receptors (S1P1-5), that regulates critical processes of cancer progression, such as cell proliferation, migration, and angiogenesis. As such, this molecule is expected to provide a good target against breast cancer. FTY720, a sphingosine analogue that antagonize the S1P1 receptor, is currently in clinical use for multiple sclerosis with FDA approval. Phase 3 clinical trials of FTY720 for the transplant population dosed the drug at 5mg po daily (equivalent to 0.8mg/kg po daily for mice) for up to 2 years with minimal side effects. Previously, FTY720 has been shown to inhibit breast cancer growth in a mouse model which required a notably higher dose (over 2mg/kg ip daily) above any other used in clinical trials. Thus, FTY720 has not been developed as an anti-cancer drug to date. Recently it has been shown that oral administration is more potent than intraperitoneal injection of FTY720. the purpose of this study was to evaluate the efficacy of FTY720 against breast cancer in an acceptable dosing under an appropriate route of administration, using oral instead of intaperitoneal injections. Methods: We used a syngeneic model with orthotopically implanted 4T1-luc2 murine breast cancer cells, which best mimics human breast cancer progression.FTY720 was then administered orally in either 1, 0.5 or 0.3mg/kg daily dosing. the overall tumor burden was quantified using bioluminescence technology over 19 days, and tumor weights were measured at the nineteenth day. Pathological analysis was performed to determine the anti-cancer effect of FTY720. Results: Overall tumor burden increased exponentially after 4T1-luc2 cell implantation, where lymph node metastases were confirmed from day 2, and lung metastasis from day 8 by bioluminescence. the groups treated with 1mg/kg and 0.5mg/kg FTY720 showed significantly reduced tumor burden compared with the control group and the 0.3mg/kg FTY720 group. at time of sacrifice, the tumor weights in the control groups were significantly heavier than in those groups treated with either the 1mg/kg or 0.5mg/kg FTY720 (p=0.005, p<0.001 respectively). the 0.3mg/kg FTY720 group, on the other hand, showed no difference in tumor weight or overall tumor burden compared with control group. Conclusions: They key role of S1P in breast cancer progression suggests that FTY720 would be an effective treatment against this disease. Previous studies required the dose of the drug in levels far exceeding tolerable dose for humans. in our study, we demonstrated that lower doses of FTY720 to 0.5mg/kg in mouse, which is equivalent to 3mg in human, are effective in inhibiting breast cancer progression when administered orally.

Vaccinia Virus GLV1h-153: Effective Novel Treatment Agent and Imaging Tool for the Detection of Positive Surgical Margins of Triple-Negative Breast Cancer

2012-02-01

Introduction: Triple-negative breast cancers (TNBC) have an aggressive biology and are associated with poor outcomes due to their lack of expression of targets, estrogen, progesterone, and HER-2 receptors, for hormonal therapy. the human sodium iodide symporter (hNIS) is a naturally occurring protein in human breast and thyroid tissue which enables cells to concentrate iodide, including radioiodide, as well as 99mTc-pertechnetate, which have been used to image and treat thyroid cancers. in this study, we investigated the therapeutic impact of a new oncolytic virus, vaccinia virus GLV1h-153, on a TNBC murine model and its potential for identifying positive surgical margins after tumor resection via PET imaging. Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against the TNBC cell lines MDA-MB-231, MDA-MB-468, HCC-38, HCC-1937, and HCC-1143 at various multiplicities of infections (MOIs, # of viral particles per cell). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice with MDA-MB-231 cells tagged with mCherry. Xenografts were treated with GLV-1h153 or PBS and followed for tumor growth. to determine the utility of GLV-1h153 in identifying positive surgical margins, uninfected xenografts were surgically resected and subsequently imaged after application of GLV-1h153 in the surgical wound. Serial Focus 120 microPET images were obtained from 3 to 72 hr post-tail vein injection of ∼150 mCi of 124I-iodide. Results: GLV-1h153 infected, replicated in, and killed all TNBC cell lines. Green fluorescent protein expression, a surrogate for viral infectivity, showed that infectivity was time and concentration dependent. Greater than 90% cell kill was achieved in all cell lines by day 5 at an MOI of 5.0. GLV-1h153 replicated efficiently in all cell lines and reached a peak titer of 26 million viral plaque forming units (>1300-fold increase from the initial viral dose) by day 4 in the cell line, MDA-MB-468. in vivo, mean volume of tumors after 2 weeks of treatment was 21 mm3 versus 273 mm3 in untreated controls (p=0.002). Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning (Figure). Conclusions: This proof-of-principle study is the first to demonstrate killing of TNBC by a novel vaccinia virus in vitro and in vivo. Our results suggest that GLV-1h153 is a promising therapeutic agent and imaging tool for identifying positive surgical margins.

The Effect of Depression on Diagnosis, Treatment, and Survival in Pancreatic Cancer

2012-02-01

Introduction: Depression has been associated with delayed presentation, inadequate treatment, and poor survival in patients with cancer. in particular, patients with pancreatic cancer have high rates of depression. Methods: Using Surveillance, Epidemiology, and End Results (SEER)-Medicare linked data, we identified patients diagnosed with pancreatic adenocarcinoma from 1992-2005 (N=23,745). ICD-9 codes were used to evaluate depression during the 3-27 months prior to cancer diagnosis. Patient and tumor characteristics were compared between the patients with and without depression. the effect of depression on survival was evaluated in univariate and multivariate models. Logistic regression was used to evaluate the effect of depression on receipt of therapy. Results: 7.8% of patients with pancreatic cancer had a diagnosis of depression (N=1,852). Depression was associated with increased age, female sex, white race, single or widowed status, and advanced stage disease (p<0.0001). Unadjusted median survival for patients with locoregional disease and depression was 5 months, compared to 8 months for patients without depression. Patients with locoregional disease and depression were less likely to undergo surgical resection, chemotherapy, or radiation, and less likely to see a surgeon or medical oncologist (p<0.0001). in a multivariate analysis, after adjusting for age, gender, race, marital status, SEER region, comorbidities, and physician visits, patients with locoregional disease and depression had 27.3% lower odds of undergoing surgical resection (OR=0.73, 95% CI 0.55 – 0.95). in a model without treatment, depression was associated with lower 2-year survival (HR=1.17, 95% CI 1.07-1.29). After adjusting for surgical resection and chemotherapy, depression was no longer a significant predictor of survival (HR=1.08, 95% CI 0.99 – 1.19). in patients who underwent surgical resection, depression was not a significant predictor of survival (HR=1.24, 95% CI 0.95 – 1.61). A similar pattern was seen in distant disease. Patients with distant disease and depression were less likely to receive chemotherapy and see a medical oncologist (p<0.0001) than those without depression. in addition, they had 31.5% lower odds of receiving chemotherapy (OR=0.685, 95% CI 0.57 – 0.82). Similarly, after adjusting for receipt of chemotherapy in patients with distant disease, depression was no longer a significant predictor of survival (p=0.3784). Conclusions: Patients with depression were less likely to receive appropriate treatment for pancreatic cancer. in multivariate models, depression was significantly associated with decreased survival, which appears to be mediated by a lower likelihood of appropriate treatment in patients with depression. Accurate recognition and treatment of pancreatic cancer patients with depression may improve treatment rates and survival.

Combination of TRAIL and JAK-2 Downregulation: Novel Therapeutic Strategy Against Pancreatic Cancer

2012-02-01

Introduction: TRAIL (Tumor Necrosis Factor-Related Apoptosis Inducing Ligand) is emerging as a promising anti-cancer therapy by virtue of its strong anti-tumor activity in wide range of cancer cells and minimal toxicity to normal cells and tissues. Unfortunately, pancreatic cancer is resistant to TRAIL. Elucidation of the mechanism of resistance to TRAIL-mediated apoptosis will help design strategies to overcome this resistance and thus develop TRAIL as an effective therapeutic strategy for this difficult to treat malignancy. JAK-2/STAT-3 is a pro-survival pathway which is up-regulated in a variety of cancer cells including pancreatic cancer. the aim of the current study was to evaluate the role of JAK-2/STAT-3 pathway in the resistance to TRAIL mediated apoptosis and to evaluate the combination of TRAIL and JAK-2 downregulation as a novel treatment for pancreatic cancer. Methods: Highly aggressive metastatic pancreatic cancer cell lines (S2013, S2VP10) were treated with JAK-2 inhibitors FLLL-31 (0-5μM) or WP1066 (0-5μM), TRAIL (0-40ng/ml) or a combination of JAK-2 inhibition and TRAIL. the effect on viability (MTT) and parameters of apoptosis (annexin V, caspase 3, 8 and 9 activation) was measured. the results were confirmed with specific inhibition of JAK-2 by siRNA. Results: Even though TRAIL in itself was ineffective in inducing cell death in pancreatic cancer cells, combination of TRAIL and JAK2 inhibitor FLLL-31 induced marked decreased in viability (Table). Combination of TRAIL and FLLL-31 also markedly increased annexin positivity as compared to TRAIL and FLLL-31 alone, suggesting that TRAIL and JAK-2 inhibition synergize in inducing apoptosis in pancreatic cancer cells (Table). Similarly, combination of TRAIL and JAK-2 inhibition induce significantly more caspase3, 9 and 8 activation when compared to TRAIL or FLLL-31 alone (Table). Similar results were observed with the combination of TRAIL and the other JAK-2 inhibitor WP1066 as well as with JAK-2 downregulation by siRNA (data not shown). Conclusions: Inhibition of JAK-2 pathway sensitizes pancreatic cancer cells to TRAIL induced apoptosis and cell death. Combination of JAK-2 silencing and TRAIL has immense potential to emerge as novel therapeutic strategy against pancreatic cancer.

Inhibition of Mammalian Target of Rapamycin (mTOR) Complex 1 and 2 by PP242 Induces G1 Growth Arrest in Pancreatic Ductal Adenocarcinoma and when Combined with Chloroquine Induces Cell Death

R. Daylami, S. Virudachalam, R.J. Bold — 2012-02-01

Introduction: Pancreatic adenocarcinoma shows high resistance to traditional apoptosis inducing chemotherapeutic agents due to activation of multiple proliferative pathways downstream of K-ras, including PI3K/AKT/mTOR as well as high expression of BCl-2. Recently mTOR inhibition has shown promise in the treatment of pancreatic neuroendocrine tumors, but trials of mTOR complex 1 (mTORC1) inhibitors in the treatment of pancreatic ductal adenocarcinoma have not demonstrated efficacy. We hypothesized that newer classes of dual mTOR inhibitors may be more effective in suppressing PDAC growth and that the combination of the mTOR inhibitors with the anti-autophagy compound chloroquine (ChQ) would enhance cell death induced by mTOR inhibition. Methods: the effect of mTOR inhibition by the dual mTORC1/2 inhibitor PP242 was tested on multiple PDAC primary cell lines by MTT. the effect of PP242 on growth arrest and cell cycle was assayed by trypan blue assay and flow cytometry. the effect of PP242 on signals upstream and downstream of mTOR was demonstrated by western blot. Autophagy was inhibited by addition of ChQ to PP242 and the effect of the combination treatment on apoptosis was measured by flow cytometry. the role of caspase dependent apoptosis in mediating this cell death was demonstrated by co-incubation with the pan-caspase inhibitor Zvad-fmk. Results: the PDAC cell line MIA-PaCa2, ASPC-1 showed similar sensitivities to PP242 with IC50 of 500nM, while PANC-1 showed a higher IC50 of 50uM. Trypan blue death growth/death assay in MIA-PaCa2 demonstrated that PP242 (1uM) significantly decreased growth, but did not induce significant cell death. PI cell cycle analysis demonstrated a strong G1 arrest in MIA-PaCa2 up to 48hours. Western blot demonstrated PP242 decreased insulin induced phosphorylation of AKT, mTOR, P70S6K and 4EBP-1. However this effect was not sustained at 24 hours, except in 4EBP-1. After 48 hours of co-incubation with PP242, there was an actual increase in pAKT and pP70S6K, but not 4EBP-1. the combination of ChQ (25uM) to PP242 increased cell death in both the sensitive cell line MIA PaCa2 as well as the more resistant cell line PANC-1. the enhanced cell death achieved by combining ChQ and PP242 was reversible with the pan-caspase inhibitor Zvad-fmk. Conclusions: Inhibition of mTORC1/2 by PP242 induces a G1 growth arrest in PDAC. the addition of ChQ to PP242 significantly inhibits autophagic flux and induces cell death. This cell death is reversible with the pan-caspase inhibitor Zvad-fmk suggesting that apoptosis is necessary for cell death induced by the combination.

Identification of Genes Associated with Histological Grade in Human Pancreatic Cancer Reveals Annexin A8 as a Therapeutic Target and Prognostic Marker for Pancreatic Cancer

J.M. Pimiento, D. Chen, B.A. Centeno, M.P. Malafa — 2012-02-01

Introduction: Histologic grade of pancreatic cancer defines morphologic subtypes, which are informative of outcomes after surgical resection. We hypothesized that deregulated genes that are associated with high grade, and thereby poor outcomes, might provide a potential target for the treatment of pancreatic cancer. Methods: We studied the gene expression profile of 61 resected primary pancreatic cancers through analysis of a custom Affymetrix microarray. Significance analysis of microarrays was used to select genes associated with grade. the effect of silencing one of the genes, Annexin A8(A-A8), which was induced with increasing grade was investigated. the relationship of protein expression of A-A8 and tumor grade was also studied with immunohistochemistry(IHC). Finally, the prognostic significance of A-A8 expression was investigated. Statistical analysis was performed with Fisher's exact test, T-test and ANOVA as indicated. Results: We initially profiled 44 tumors, and identified 60 probe sets corresponding to 49 genes. of the 49 genes, 34 were downregulated, and 15 were upregulated as tumor grade progressed from well to moderate to poorly differentiated. We identify A-A8 induction in this data set, then we profiled 17 more samples confirming this finding, with a 5.9 fold increased A-A8 expression from low to high grade tumors (r=0.31;p=0.019). Immunohistochemistry and western blotting confirmed increased expression of A-A8 in human pancreatic tumors and cell lines. the expression of A-A8 was correlated with tumor grade in a human pancreatic tumor tissue microarray (r=0.26;p=0.014). Knockdown of A-A8 in human pancreatic cancer BXPC-3 cells with siRNA resulted in significant decrease in cell viability (p<0.001), cell migration (2.5 vs. 0.9mm at 96h; p=0.048), and colony formation (p=0.007). Moreover, high expression of A-A8 is associated with a significant decrease in 5 year survival following resection of stage I and II human pancreatic cancer by Cox model (p=0.049). Conclusions: Studying the expression profiles of primary pancreatic cancer analyzed on Affymetrix microarrays, we identified A-A8 as a gene associated with pancreatic cancer differentiation. Downregulation of A-A8 by RNA interference in pancreatic cancer cells results in growth and migration inhibition. Finally we show that high A-A8 expression is associated with poor disease outcome in pancreatic cancer patients. These results support further investigation of A-A8 as a potential therapeutic target and prognostic marker for human pancreatic cancer.

Protein Phosphatase 4 (PP4) Is A Negative Regulator of Pancreatic and Duodenal Homeobox-1 (PDX-1) Via Enhancement of PDX-1 Ubiquitination

G. Zhou, S. Liu, K.M. Shahi, F.C. Brunicardi — 2012-02-01

Introduction: Our recent studies show that pancreatic and duodenal homeobox-1 (PDX-1), a pancreatic embryonic transcription factor, is an oncogene which is overexpressed in almost all solid cancers, including pancreatic cancer. Since phosphorylation plays a key role in cellular control of PDX-1 expression, the purpose of this study is to determine the role of protein phosphatase 4 (PP4) in regulation of PDX-1 expression. Methods: Murine Embryonic Fibroblasts (MEFs)were isolated from day 13.5 embryos (E13.5) of PP4 tamoxifen-inducible knockout mice (ER-Cre;pp4Flox/Flox) by treatment with trypsin/EDTA to produce single cell suspensions. MEFs, HEK293, HEK293 stably transfected with GFP-PDX-1, and human pancreas cancer cells, Panc-1 and Mia PaCa-2, were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin. MEFs were treated with 1 mM tamoxifen for 4 days to induce PP4 knockout. Effect of 100 ng/ml IGF-1 on AKT phosphorylation was examined in MEFs treated with or without 1 mM tamoxifen. Antibody array screen was performed by adding the HEK293-GFP-PDX-1 cell extracts to a membrane filter arrayed with 400 antibodies and preblocked in a buffer containing 5% nonfat milk. After overnight incubation, the presence of the antibody-antigen-GFP-PDX-1 complex was detected by horseradish peroxidase-conjugated anti-GFP antibody, followed by chemiluminescence. PP4-PDX-1 interaction was examined by immunoprecipitation and Western blotting. Transient transfections were performed using Lipofectamine 2000. Effects of 16.7 mM glucose on PP4-PDX-1 interaction in Mia Paca-2 cells and PDX-1 expression and AKT activation in Panc-1 cells were examined. Expression of PDX-1, PP4, PP4-RL, PP6, PP2A, AKT and phospho-AKT was measured by Western blotting using appropriate antibodies. PDX-1 ubiquitination was measured by immunoprecipitating PDX-1, followed by Western blotting of ubiquitin. Results: 1. PP4 was identified as a candidate PDX-1-interacting protein in an antibody array-based screen in HEK293-GFP-PDX-1 cells; 2. PP4-PDX-1 interaction was confirmed by immunoprecipitating PDX-1, followed by Western blotting against PP4 in the HEK293-GFP-PDX-1 cells; 3. Overexpressed PDX-1 was associated with overexpressed PP4-RL, a phosphatase-dead mutant, but not WT PP4, in HEK293 cells; 4. High glucose enhanced PP4-PDX-1 interaction in Mia PaCa-2 cells; 5. High glucose induced increased PDX-1 expression accompanied by AKT activation in PANC-1 cells; 5. PP4-knockout MEFsenhanced AKT activation by IGF-1; 6. Co-transfection of PP4 with PDX-1 inhibited PDX-1 expression, while PP4-RL, PP2A and PP6 did not have a down-regulating effect on PDX-1; 7. Co-transfection of PP4 with PDX-1 enhanced PDX-1 ubiquitination, while PP4-RL inhibited PDX-1 ubiquitination. Conclusions: PDX-1 expression is regulated by phosphorylation; PP4 is a negative regulator for PDX-1 via enhancement of PDX-1 ubiquitination.

MicroRNA-101 (miR-101) Enhances Chemosensitivity of Pancreatic Ductal Adenocarcinoma (PDAC) Cells By Inhibition of MTOR Signaling Via PRAS40

R.B. Batchu, O. Gruzdyn, A.M. Qazi, D. Bouwman, S.A. Gruber, D.W. Weaver — 2012-02-01

Introduction: Gemcitabine-resistant PDAC is traditionally managed with palliative care due to poor prognosis. in this study, we explored the possibility of employing the re-expression of miR-101, a tumor suppressor, to inhibit mTOR, which is responsible for gemcitabine-resistant PDAC. PRAS40 is a regulatory protein which binds and inhibits the mTOR enzymatic complex (mTORC1) in its active un-phosphorylated form. Methods: PANC-1 cell lines were propagated in DMEM medium. Standard protocols were followed for plotting growth curves, western blot analysis and immunoprecipitation assays. Gene profile analysis was conducted with Human Whole Genome OneArray v5. Results: PANC-1 cells that were treated either with 0.5?mM gemcitabine or transfected with pre-miR-101 alone produced 40.4% and 55.5% cell death, respectively; however, simultaneous treatment resulted in 81.5% cell death (Fig. 1A).While we observed unchanged mTOR and PRAS40 levels of miRNA in gene profile analysis and protein in western blot analysis, a significant reduction in the threonine-246 phosphorylation of PRAS40 (p-PRAS40thr-246) is evident after pre-miR-101 transfection, thereby increasing binding and inhibition of the mTORC1 signal pathway (Fig. 1B). Additionally, unbound, phosphorylated PRAS40 prevents cells from undergoing apoptosis. Co-immunoprecipitation assays revealed higher levels of PRAS40 interaction with mTOR in pre-miR-101 treated cells indicating lower levels of unbound PRAS40, which in turn increases apoptosis. Conclusions: Our results demonstrate that the re-expression of miR-101 not only enhances the chemosensitivity of gemcitabine but also results in the inhibition of mTOR through promoting its binding to PRAS40 which further increases apoptosis. This study provides insight into the mechanism underlying one of the anti-tumor effects of miR-101, which may pave the way for possible therapeutic intervention.

A Reciprocal Repression Between Tumor Suppressor MiR-198 and Mesothelin Regulates Proliferation and Metastasis in Pancreatic Cancer

C. Marin-Muller, U. Bharadwaj, M. Li, C. Chen, Q. Yao — 2012-02-01

Introduction: the reduced expression of microRNAs is a critical factor in the development of cancers. We have identified miR-198 as a key tumor suppressor in pancreatic cancer (PC). We have previously reported that the cell-surface glycoprotein mesothelin is overexpressed in a ∼90% of pancreatic cancers and increases the aggressiveness of tumor cells. in this study, we found that mesothelin overexpression results in downregulation of miR-198 in PC cells, and hypothesized that miR-198 plays a role in the pathogenesis of PC. We have dissected the mechanisms through which miR-198 repression contributes to tumor progression, and examined the anti-oncogenic effects of miR-198 reconstitution in vitro and in vivo. Methods: We examined miR-198 and mesothelin expression in PC patient tumors and cell lines. We confirmed the mechanism for miR-198 regulation using a combination of in silico analyses and functional assays using inhibitors and shRNAs against specific factors. We confirmed several targets of miR-198 using immunoblotting, luciferase assays and site-directed mutagenesis of predicted miR-198 binding sites. Lastly, we characterized the effects of miR-198 reconstitution in PC cells in vitro with functional assays including proliferation, migration, invasion, and colony formation, and in xenograft mouse models. Results: MiR-198 correlates negatively with mesothelin in a majority of PC cells and patient tumors. We identified an intricate mechanism through which miR-198 is repressed. Mesothelin overexpression leads to downregulation of miR-198 through a pathway involving constitutive NF-kB activation and induction of NF-kB-responsive negative transcription factors (TFs) which repress the miR-198 promoter, including ZEB1. Interestingly, miR-198 is involved in a reciprocal regulatory loop with mesothelin. MiR-198 directly targets mesothelin at multiple sites within its coding region, which contribute collectively to a near complete downregulation of mesothelin following miR-198 reconstitution. We also identified two additional targets for miR-198, the tumorigenic factors PBX-1 and VCP, and have determined that miR-198 repression leads to dysregulation of the PBX-1/VCP axis, contributing to PC progression. Most importantly, reconstitution of miR-198 reverses the tumorigenic properties and pro-survival effects conferred by mesothelin, PBX-1, and VCP, including proliferation, migration, and invasion in vitro and reduced tumor growth and metastasis in vivo. Conclusions: Our study reveals a novel role for miR-198 and highlights the involvement of several components of the mesothelin regulatory network in PC. the ability of miR-198 to target multiple transcripts indicates a broad applicability for miR-198 replacement therapy, with high potential for therapeutic benefit in pancreatic and other cancers.

Mesothelin Confers Resistance of Human Pancreatic Cancer Cells to Gefitinib By Activating ERK and Enhancing VEGF Production

Y. Yuan, C. Marin-Muller, C. Chen, Q. Yao — 2012-02-01

Introduction: Epidermal growth factor receptor (EGFR) is overexpressed in a significant portion of pancreatic cancers (PC), and correlates with disease progression and poor prognosis. Targeting the EGF/EGFR axis is therefore an attractive therapeutic strategy for PC treatment. However, the clinical response rate to EGFR tyrosine kinase inhibitor (TKI) gefitinib treatment has been disappointing. the mechanism of PC cell resistance to EGFR-TKI needs to be further explored. We have previously found that mesothelin (MSLN) is overexpressed in a majority of PC tissues and is a malignant factor that contributes of PC pathogenesis. the role of MSLN in PC drug resistance has never been explored. We hypothesized that MSLN plays a critical role in conferring EGFR-TKI resistance in PC cells through aberrant activation of several signaling pathways. Methods: Two MSLN-overexpressing stable cell lines (MIA-MSLN and PANC-1-MSLN) were generated from MIA-PaCa-2(MIA) and PANC-1 cells, which have low endogenous MSLN expression. the EGFR-TKI gefitinib was used for treatment at various concentrations ranging from 1 mM to 100 mM. Cell proliferation and survival were determined by MTT test and clonogenic assay. Cell migration and wound healing assays were also performed to determine the PC cell properties with or without gefitinib treatment. Cell culture supernatant was collected and Vascular Endothelial Growth Factor (VEGF) production was quantified in different PC cells by ELISA. Western blot was used to determine protein expression. Results: Gefitinib significantly inhibited low MSLN control cell growth, migration, and survival at clinically relevant drug concentrations, whereas it did not inhibit MIA-MSLN and PANC-1-MSLN cells. MSLN overexpression increased EGFR expression and activated EGFR signaling. Gefitinib inhibited baseline EGFR and extracellular signal-regulated kinase (ERK) phosphorylation in control cells, but had limited effects on baseline EGFR or ERK phosphorylation in MIA-MSLN and PANC-1-MSLN cells. SiRNA silencing of MSLN in MSLN overexpressing cells restored the sensitivity to gefitinib compared to control siRNA treatment. in addition, MSLN overexpression significantly enhanced the production of VEGF. Conditioned media from MSLN overexpressing cells promoted the phosphorylation of ERK1/2 in human vascular endothelial cells. Conclusions: We found that MSLN confers resistance of PC cells to gefitinib treatment by activating the Erk pathway and enhanced secretion of VEGF. the fact that silencing MSLN sensitized the gefitinib effect on PC cell growth furnishes exciting evidence for an effective combination therapy in PC. We believe that targeting of MSLN is necessary to sensitize PC cells for an effective EGFR-TKI treatment.

Tumor-Educated Macrophages Stimulate Tumor Progression in an Orthotopic Human Pancreatic Cancer Nude Mouse Model

2012-02-01

Introduction: There is a strong association between poor survival and increased macrophage density in many cancers including thyroid, lung, breast, and hepatocellular cancers. Macrophages promote tumor growth, stimulate tumor-associated angiogenesis, cancer-cell invasion, migration, and intravasation, as well as suppression of antitumor immune responses. Methods: Nude mice (n=15) underwent orthotopic implantation of the human pancreatic cancer cell line, BxPC3 stably expressing RFP. Tumors were allowed to grow on the pancreas until they were identifiable by fluorescence. Five transgenic nude mice ubiquitously-expressing GFP were injected subcutaneously with BxPC3 cells stably expressing RFP which subsequently formed subcutaneous tumors. GFP-expressing macrophages from these mice were harvested and defined as “tumor-educated macrophages”. Macrophages were also harvested from transgenic GFP mice (n=5) without subcutaneous tumors and identified as “naïve macrophages.” Three groups of mice with orthotopic BxPC3 pancreatic tumors were studied: 1) A control group without addition of macrophages (n=5); 2). A naïve group, with weekly intraperitoneal (ip) injection of 106 naïve macrophages per mouse (n=5); 3) A tumor-educated group, with weekly ip injection of 106 tumor-educated macrophages per mouse (n=5). the study ended with termination of the tumor-educated group after 8 weeks due to a pre-morbid state identified in all mice. All mice in each of the three study arms were terminated, imaging was performed, and total tumor weight was obtained. Results: Average body weight in the control group at the time of termination was 27.8 gm, with an average tumor weight of 514 mg; only one mouse developed peritoneal metastasis weighing 650 mg. Average body weight in the naïve-macrophage group was 27.9 gm (p=0.12), with an average tumor weight of 750 mg (p=0.16) and average peritoneal metastasis weight of 982 mg (p=0.034). Average body weight in the tumor-educated-macrophage group was 24.8 gm (p=0.14) with mice showing severe cachexia and ascites requiring termination. This group had an average tumor weight of 2235 mg (p=0.008), with two mice developing peritoneal metastasis weighting 830 mg and 3885 mg, respectively (p=0.15). Conclusions: Tumor-educated-macrophages, in contrast to naïve-macrophages, stimulate tumor progression in an orthotopic nude mouse model of pancreatic cancer. Future experiments will explore the mechanism of this very important phenomenon.

YM155, a Small-molecule Inhibitor of Survivin, as a Novel Therapy for Pancreatic Cancer

Q.P. Ly, J. Person, M. Mathiesen, G. Howell, M.G. Brattain — 2012-02-01

Introduction: Pancreatic cancer (PaCa) has the poorest survival rate due to the lack of effective therapies for advanced disease. Therefore, there is a compelling need to develop new therapies. Survivin is a protein that stabilized XIAP, an inhibitor of caspase activation leading to inhibition of apoptosis. Importantly, survivin is highly expressed in most human tumors and fetal tissue, but is completely absent in matured, differentiated cells. Thus, inhibiting survivin would have less effect on normal cells and potentially less adverse effects. Recently, YM155, a small molecule inhibitor of survivin transcription, has been shown to be effective in inducing apoptosis in a variety of tumor cell lines and xenografts but it has not been studied in pancreatic cancers. in this study, we showed that YM155 inhibits cell proliferation and induces apoptosis in both Smad4 wild-type and mutant pancreatic cell lines in vitro and in vivo. Methods: MiaPaCa2 and Capan1 cell lines were treated with YM155 at increasing concentrations (0, 1, 10 100 and 200 nM) while growing in DMEM with 10%FBS. Using MTT assays and DNA fragmentation analyses, proliferation and apoptosis were measured for each cell line to assess YM155 activity in these PaCa cell lines in vitro. With the approval of our IACUC, we also implanted MiaPaCa2 xenografts in athymic nude mice and treated the animals with YM155 using an Alzet pump to study the in vivo efficacy of YM155 in PaCa xenografts. the xenografts were harvested for immunohistochemical study with Ki-67 and TUNEL to study the biochemical markers of the tumor response to YM155. Results: Both MiaPaCa2 and Capan1 responded to YM155 treatment with decreased proliferation and increasing apoptosis (figure 1A and 1B). YM155 decreased the tumor growth rate of MiaPaCa xenografts at 5mg/kg/day (figure 1C). As with in vitro data, Ki-67 was decreased while TUNEL staining was increased in treated xenografts compared to lactic acid vehicle control confirming decreased cell proliferation and increased apoptosis, respectively. Conclusions: YM155, a first in its class drug that inhibit survivin, is efficacious in decreasing cell proliferation and increasing apoptosis in pancreatic cancer cell lines both in vitro and in vivo. YM155 has been shown to be safe in several phase I and phase II clinical trials in solid tumors such as prostate, lung and melanoma. Our data provide strong evidence and rationale for a phase II clinical trial in Smad4 wild-type patient with locally advanced unresectable or metastatic pancreatic cancer.

Multifunctional Gold Nanorods for Targeted Drug Delivery To Carcinoids

2012-02-01

Introduction: Carcinoids are gastrointestinal (GI) neuroendocrine (NE) tumors that secrete excessive hormones which cause carcinoid syndrome. Importantly, carcinoids are often metastatic at the time of diagnosis and no longer amenable to curative surgery. Therefore, novel therapeutic strategies are needed for both palliation of symptoms and prevention of tumor progression in patients with carcinoid neoplasms. Carcinoids are known to express high levels of somatostatin receptors (SSTRs) and somatostatin analogs such as octreotide (Oct) can bind to them effectively.Therefore, targeting SSTRs has the potential to significantly enhance therapeutic efficacy against carcinoids. In the present study, we have developed a multifunctional gold nanorod (Au NR)-based nanoplatform conjugated with octreotide capable of delivering an anticancer drug (for example, doxorubicin (DOX)) and assessed its specificity to target the carcinoid cancer cells. Methods: Bare gold nanorods were prepared by a two-step seeding growth approach followed by DOX or DOX-Oct conjugation. A human GI carcinoid cell line (BON) expressing SSTRs was treated with these nanoparticles (NPs), either non-targeted (Au-DOX) or targeted (Au-DOX-Oct), at a concentration of 1 mg/ml for 30 and 120 minutes separately. Using the fluorescent features of DOX (488nm and 575nm, excitation and emission, respectively), the cellular uptake of the NPs were analyzed using a FACSCalibur flow cytometer and confocal laser scanning microscope (CLSM). Results: A multifunctional, pH-responsive and tumor-targeting gold nanorod-based nanocarrier was developed for targeted anticancer drug delivery to carcinoids. DOX release profiles from NPs were strongly dependent on the environmental pH values, which can minimize systemic spread of toxic drugs during circulation while maximizing the efficiency of tumor-targeted anti-cancer drug delivery. BON cells incubated with Au-DOX-Oct exhibited higher cellular uptake than Au-DOX (14% and 44% increase after 30' and 120', respectively) measured by flow cytometry. Moreover, the observation under CLSM confirmed that the intracellular transport of Au-DOX-Oct is faster than Au-DOX. Conclusions: We have developed a multifunctional, pH-responsive and tumor-targeting gold nanorod-based nanoplatform for anticancer drug therapy. We demonstrated that the presence of the surface ligand Oct increased the cellular uptake of NPs (Au-DOX-Oct) substantially in a BON cell line compared to the non-targeted NPs (Au-DOX). Additionally, the targeting ligand sped up the intracellular transport making these NPs more efficient in reaching the nuclei of cancer cells. Thus, targeted delivery of anticancer drug (DOX) specifically to the tumor tissue could achieve therapeutic outcomes in treating carcinoid disease. Therefore, these NPs require further preclinical investigation so that the targeted nanoparticles may provide a successful delivery system of therapeutic agents specifically to tumor tissues in the future.

Changes in Leptin Hormone Serum Levels in Hyperparathyroid Patients Undergoing Minimally Invasive Parathyroidectomies

2012-02-01

Introduction: Hyperparathyroidism is a common endocrine problem affecting 1 in 500 individuals who are typically female and over the age of 60. With routine serum calcium screening now, many patients are treated with minimally invasive parathyroidectomy (MIP) before hypercalcemic sequelae develop. Discovered in 1994 as a member of the class 1 cytokine receptor family, leptin has generated considerable interest and implications in multiple calcium related metabolic processes which have led to numerous attempts to elucidate the relationships between leptin, vit D, parathyroid hormone (PTH), and obesity. We have biochemical evidence that leptin is produced in diseased parathyroid glands. In this study, therefore, we wished to clarify the clinical relationship between PTH, leptin, and BMI in a cohort of patients with hyperparathyroidsm. Methods: Last year, we enrolled 96 patients with hyperparathyroidism undergoing MIPs in a prospective clinical study to assess subsequent perioperative changes to serum leptin. Patients WITHOUT hyperparathyroidism undergoing hemithryoidectomies under identical surgical conditions were control subjects. Wilcoxon signed-rank test, non-parametric version of paired t-test, and pearson correlations were used to compare leptin level changes and with different clinical variables using SAS 9.2. Results: Our study subjects included 71 (76%) Adenoma, 13 (14%) Hyperplasia, and 12 (13%) controls. The median age was 59 years old and were mostly females (76%). This population was overweight (median BMI: 28) and pre-hypertensive (median SBP:131). Comparing measurements pre and post surgery, BOTH serum leptin and PTH levels decrease significantly in the whole cohort (p<0.001). Leptin decreases significantly in adenoma (p<0.001) and hyperplasia subgroups (p=0.002); it increases in controls (p=0.007). Decrease in Leptin was significantly associated with a decrease in PTH pre-post surgery (r=0.32, p=0.003) (r=0.55, p<.001 for adenoma subgroup). Bivariate analysis revealed several variables associated with changes in perioperative leptin and PTH levels. In multivariate analysis, parathyroid disease subtype, starting leptin levels, age, BMI, and calcium at diagnosis were significantly associated with changes in leptin. Conclusions: Our results provide interesting insight and implications into the relationship between leptin, obesity, and primary hyperparathyroidism (adenomas) as there is no previous research data elucidating the relationship between leptin, PTH, and the parathyroid gland.

Lymph Node Ratio Predicts Recurrence in Papillary Thyroid Cancer

D.F. Schneider, H. Mazeh, H. Chen, R.S. Sippel — 2012-02-01

Introduction: Lymph node metastases are common in papillary thyroid cancer (PTC) and are an independent risk factor for recurrence. The aim of this study was to determine how the lymph node ratio (LNR) may be used to determine the adequacy of a nodal dissection and predict the likelihood of disease recurrence. Methods: We conducted a review of patients undergoing total thyroidectomy for PTC at our institution from 2005 – 2010. A lymph node ratio (LNR – positive nodes to total nodes) was calculated. Binomial logistic regression was used to determine a total LNR and cLNR (central LNR) that best predicted recurrence. Multivariate logistic regression then determined the influence of LNR on recurrence. Kaplan-Meier analysis and the logrank test were used to compare differences in disease-free survival. Results: Of the 217 patients undergoing surgery for PTC, 69 patients had neck dissections. 16 patients developed pathologically proven recurrence, and 14 underwent subsequent operations; median time to recurrence was 11.8 months (range 4.3-59.5). Mean cLNR was 0.45±0.38, and mean total LNR was 0.35±0.30. A total LNR >0.7 and a cLNR >0.86 best predicted disease recurrence. When Kaplan-Meier disease-free survival curves were compared, patients with a total LNR >0.7 (p <0.01) or a cLNR >0.86 (p=0.04) had significantly worse disease free survival than patients with ratios below these values (Fig. 1). When combined with other known predictors of recurrence, we found that total LNR (OR 19.5, 95% C.I. 4.1-22.9, p<0.01) and cLNR (OR 18.1, 95% C.I. 1.5-22.2, p=0.02) were significantly associated with disease recurrence. Conclusions: An elevated total LNR and cLNR are strongly associated with recurrence of PTC after initial operation. LNR in PTC is a tool that can be used to assess the completeness of the initial surgery and to determine the likelihood of the patient developing recurrent disease.

Thiocoraline Regulates Neuroendocrine Phenotype and Inhibits Proliferation in Carcinoid Tumor Cells

2012-02-01

Introduction: Carcinoid tumors are rare, slow growing neuroendocrine neoplasms that typically arise in the gastrointestinal tract or bronchi. Hormone overproduction associated with these tumors adversely impacts patient quality of life. Also, due to the difficulty in obtaining an early diagnosis and relatively ineffective chemotherapy, metastatic disease frequently results. This precludes potentially curative surgical treatment. Therefore, novel treatment strategies targeted at reducing hormone secretion and tumor markers offer both palliative and therapeutic treatment options for patients. The present study was performed to assess the antiproliferative effects of thiocoraline, a compound derived from marine bacterium Verrucosispora sp., on human carcinoid cell lines in vitro. Methods: Human pancreatic carcinoid BON and pulmonary carcinoid H727 cells were treated with increasing thiocoraline concentrations (0 – 50 nM) for up to 6 days to determine the effect on cellular proliferation by 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Total cellular proteins isolated from thiocoraline treated cell lines were analyzed by Western blot for the neuroendocrine tumor markers human achaete-scute complex-like1 (ASCL1) and chromograninA (CgA), a clinically relevant test to monitor carcinoid serotonin hypersecretion. Western analysis was performed to determine the mechanism of growth regulation by analyzing the levels of cell cycle and apoptotic regulatory proteins. Results: Following 48 hour thiocoraline treatment, CgA and ASCL1 expression levels in both cell lines were reduced in a dose dependent manner. These changes correlated with the MTT cell viability assays, which showed both a dose and time dependent reduction in cell proliferation. Thiocoraline resulted an increase in poly(ADP)-ribose polymerase (PARP) cleavage, an apoptosis marker, and a decrease in X-linked inhibitor of apoptotic protein (XIAP) expression, an anti-apoptotic marker, suggesting that thiocoraline mediates growth inhibition via apoptosis. Conclusions: Our findings indicated the in vitro antiproliferative effect of thiocoraline on carcinoid tumor cell lines is via apoptosis. Tumor cell growth reduction with a concomitant fall in hormone secretion, reflected by decreased neuroendocrine tumor marker CgA expression, occurred with nanomolar dosing. These effects warrant further preclinical studies to identify the potential use of thiocoraline as a palliative or antineoplastic carcinoid tumor therapy.

MAPK Pathway Activation in Papillary Thyroid Cancer Contributes to Decreased Interferon-gamma Cellular Growth Inhibition

H. Cai, W. Gao, X. Zhang, M.T. Stang — 2012-02-01

Introduction: Papillary thyroid cancer (PTC) is often associated with one of several non-overlapping mutations within the RAS/RAF signaling pathway. Molecular alterations such as RET/PTC rearrangement or BRAF mutation lead to the activation of the MAPK signaling pathway. Type II interferon (IFN) signaling as seen with IFN gamma in the context of autoimmune thyroiditis has the profound effect of growth inhibition and/or induction of apoptosis in normal thyroid follicular cells. We hypothesize that MAPK pathway activation contributes to the decreased responsiveness of PTC to the effects IFN gamma. Methods: Papillary thyroid TPC-1 (RET/PTC1) and BCPAP (BRAFV600E) cell lines were treated with MEK inhibitors PD98059 (75 mM) and U0126 (10 mM) with and without INF gamma (100 Units/mL) in a time and dose dependent manner. Expression levels of IRF-1, IRF-2, total ERK 1/2 and p-ERK1/2 were analyzed by SDS-PAGE immunoblot. The effects of treatment on cell proliferation were analyzed by MTT assay in at least 3 separate experiments. Adenovirus vector transduction was utilized to achieve increased expression of recombinant IRF-2 under similar treatment conditions. Results: Both TPC-1 and BCPAP cell lines exhibit constitutive activation of MAPK signaling as evidenced by high baseline levels of p-ERK 1/2. The non-competitive, specific MEK1/2 inhibitors PD98059 and U0126 are both able to decrease the p-ERK1/2 irrespective of activating mutation. The expression of p-ERK1/2 is significantly diminished within 1 hour of treatment and maintains inhibition for up to 24 hours. MEK1/2 inhibition results in a higher expression of the key IFN gamma regulatory transcription factor IRF-1 following treatment with IFN gamma when compared to control conditions at 24 hours. MEK1/2 inhibition results in moderate growth inhibition in both cell lines by 24 hours when compared with that of control cells. BCPAP is inhibited 30.9% ±3.8% by PD and 24.4% ±5.9% by U0126. TPC-1 is similarly inhibited. IFN gamma inhibits BCAP by 9.9% ±6.8%. However, the combination of IFN-gamma stimulation and MEK inhibition results in a significant and synergistic effect on cell proliferation (BCPAP inhibited 64.8% ±0.6% by PD98059+IFN, 54.7% ±13.7% by U0126+IFN) when compared to MEK inhibitor or IFN-gamma treatment alone (P<0.01). Overexpression of IRF-2 abolishes this synergistic effect of combined MEK inhibition and IFN gamma treatment suggesting an IRF-1 dependent mechanism. Conclusions: This data suggests MAPK pathway activation contributes to the resistance of PTC to the growth inhibitory effect of IFN gamma irrespective of specific activating mutation. Targeted therapy of the RAS/RAF activating mutations may promote PTC tumor inhibition by innate type II interferon signaling.

Differentiation of Benign from Malignant Thyroid Nodules Using MicroRNAs Amplification in Leftover Cells Obtained by Fine Needle Aspiration Biopsy

H. Mazeh, Y. Levy, I. Mizrahi, N. Ilyayev, D. Halle, H.R. Freund, A. Nissan — 2012-02-01

Introduction: Fine needle aspiration biopsy (FNAB) is the most commonly used and reliable diagnostic tool to identify thyroid malignancy. Nevertheless, some FNAB results are not definite and malignancy is identified on final histo-pathology in 15-75% of these cases. The aim of this study was improve upon the accuracy of FNAB based cytology by amplification of microRNAs from the very same biopsy cells in differentiating benign form malignant thyroid nodules. Methods: Cells leftover in the needle cup following FNAB cytology were collected from 77 consecutive patients with thyroid nodules. RNA was extracted form all patients with cytology showing follicular lesion or suspicion for malignancy. cDNA was then constructed and the expression of microRNAs 21, 31, 146b, 187, 221, & 222 was determined using real time PCR. Results were compared to final surgical pathology. Results: RNA was successfully extracted from all FNAB specimens. Five patients had FNAB-cytology suspicious for malignancy. The microRNA panel was positive in all five specimens and all were found to have malignancy on final pathology. Six patients had follicular lesions on FNAB. The microRNA panel was positive in 3 out of 4 patients with confirmed malignancy and was negative in two patients with benign pathology results. This corresponded to a specificity of 100%, sensitivity of 88%, and accuracy of 90%. Conclusions: RNA extraction from FNAB leftover cells is feasible and a microRNA panel amplified from the extracted RNA, may be accurately used to predict malignancy in the same needle sample of undetermined FNAB results.

MicroRNA-9* Reduces Medullary Thyroid Cancer Cell Proliferation in Vitro and Under-expression is Associated with Lateral Lymph Node Metastases

J.S. Gundara, J. Zhao, B.G. Robinson, S.B. Sidhu — 2012-02-01

Introduction: Medullary thyroid cancer (MTC) contributes disproportionately to thyroid cancer related mortality and management options beyond surgery are presently still limited. MicroRNAs (miRNAs) influence post-transcriptional tumour cell regulation and are increasingly being shown to represent a novel class of potential biomarkers and therapeutic targets. miRNA profiling of many cancers (including MTC) has been undertaken and miR-9* has been shown to be suppressed in sporadic MTC. Methods: A MTC cell line (TT cells) was forward transfected with miR-9* and the effect upon cellular proliferation examined at 48h using MTS proliferation, and wound healing assays. The mechanism of the miR-9* effect was investigated using post transfection TT cell protein lysates and Western blotting for a marker of autophagy (LC3B). Patients with available lateral lymph node metastasis tissue (n=8) had nodal tissue RNA extracted and miR-9* relative expression levels (compared to primary tumour samples) quantified using qPCR methods. All experiments were performed in triplicate. Finally, primary tumour miR-9* expression was correlated with clinical outcomes in a cohort of MTC patients (n=45). Results: miR-9* transfection resulted in significantly reduced TT cell proliferation as observed in MTS (p<0.05) and wound healing assays (Figure 1). Western blotting densitometry identified significantly increased expression of LC3B when compared to negative control. miR-9* relative qPCR expression levels were significantly reduced in lymph node metastases as compared with primary tumours (p<0.05). Primary tumour miR-9* under-expression was associated with lateral lymph node metastases at diagnosis (p<0.1). Conclusions: miR-9* reduces MTC cell line proliferation in vitro. Suppression of cellular proliferation is associated with elevated expression of markers of autophagy, suggesting cell death through autophagy induction. This identifies miR-9* as a potential member of a new class of “autophagamiRs” that are of future therapeutic interest. miR-9* expression was found to be reduced in MTC lymph node metastases. Together with the finding that primary tumour under-expression correlated with a tendency to early development of lymph node metastases suggests that under-expression of miR-9* is also a negative prognostic indicator.

Leptin - A Novel Hormone of Parathyroid Neoplasms

2012-02-01

Introduction: Hyperparathyroidism (HPTH) is a common problem affecting 1 in 500 individuals who are usually female and over the age of 60. Combined effects of low calcium and vitamin D stimulating parathyroid activity and excess PTH secretion are well established, especially in secondary HPTH. Unclear is the mechanism behind the development of hypersecreting parathyroid adenomas, responsible for nearly 90% of HPTH. Discovered in 1994 as a class 1 cytokine receptor, leptin has mounted considerable interest as a mitogenic factor involved in the stimulation and growth of tumors. Also, leptin has been implicated in multiple calcium related metabolic processes which have led to numerous evaluations of the connections between vit-D, PTH, and obesity. Ultimately, these scientific findings, the prevalence of higher leptin levels in women with HPTH, and our hypothesis that leptin found with increased body weight contributes to the pathogenesis of HPTH, has led to our study on leptin production and its exogenous effect on parathyroids. Methods: Blood and tissue was collected from patients with HPTH undergoing minimally invasive parathyroidectomy (MIP) to colocalize leptin hormone and PTH by in-situ hybridization, immunohistochemistry, immunofluorescence, and electronmicroscopy. Cell culture and whole organ explant experiments in which surgically removed human parathyroid tissue were exposed to recombinant human leptin to functionally characterize the effect of leptin on PTH secretion. Parathyroid adenomas and hyperplasia were xenografted into nude rats and evaluated for the production of human leptin. Results: Leptin, leptin receptor, and PTH mRNA transcripts and protein were detected in an overlapping fashion in secretory (chief) cells in parathyroid hyperplasia and adenoma samples by ISH, IHC, IF (Figure), and EM studies. Immunofluorescent studies under spinning disc confocal microscopy and electron microscopy imaging confirm co-localization of endogenous leptin and PTH as well as active exogenous leptin uptake in cultured parathyroid cells. Tissue explant experiments show that PTH-secretion responds to recombinant leptin exposure in a dose dependent, statistically significant manner. Nude rats had positive human leptin at 2, 4, and 8 weeks in increasing trend for adenoma and decreasing trend for hyperplasia xenografts. Conclusions: Our results provide very strong evidence of a new functional and physiologically relevant parathyroid hormone. Until now, no previous research data identified the presence of leptin hormone in parathyroid glands; it remains unclear as to whether high exogenous leptin levels initiate hyperparathyroid disease, possibly acting mitogenically on parathyroid leptin receptors, or whether hyperparathyroid glands and elevated PTH serum levels precede elevated leptin production, either by endogenous parathyroid production or through downstream endocrine signaling of adipose tissue production. Based on our results and published reports, future studies will test our hypothesis that leptin play a central tumorogenic role in the etiology of parathyroid neoplasms.

The Cost-Effectiveness of Recombinant Human Thyroid Stimulating Hormone Administration Prior to Remnant Ablation for Treatment of Differentiated Thyroid Cancer

K. Zanocco, C. Sturgeon — 2012-02-01

Introduction: Radioiodine thyroid remnant ablation is routinely performed following near total thyroidectomy for differentiated thyroid cancer. Administration of recombinant human thyroid stimulating hormone (rhTSH) prior to remnant ablation eliminates the need for levothyroxine (LT4) withdrawal and symptomatic hypothyroidism. However, the use of rhTSH adds significant costs to the overall treatment strategy. The cost-effectiveness of rhTSH has not been examined from a third-party payer perspective in the United States. We hypothesized that rhTSH administration prior to remnant ablation would be cost-effective in patients with differentiated thyroid cancer who highly valued the avoidance of symptomatic hypothyroidism. Methods: Cost-effectiveness analysis was performed using a Markov transition-state model to compare LT4 withdrawal versus LT4 replacement with rhTSH administration prior to radioiodine remnant ablation. A decision model was developed based on a standardized reference case of a 40 year old patient with differentiated thyroid cancer who underwent near total thyroidectomy. Treatment outcomes were identified based on literature review. Costs were estimated using Medicare reimbursement data and wholesale drug prices and reported in 2011 US Dollars. Outcomes were weighted using quality of life utility factors, yielding quality-adjusted life years (QALYs) as a measure of effectiveness. A 3% annual discount rate was applied to the model. The threshold for cost-effectiveness was defined as an incremental cost-effectiveness ratio of $100,000/QALY. Univariate and multivariate sensitivity analysis and Monte Carlo simulation were used to examine the uncertainty of costs and utility estimates in the model. Results: The rhTSH strategy produced a gain of 0.0187 QALYs at an additional cost of $1,698.94 when compared to the LT4 withdrawal strategy. The incremental cost-effectiveness ratio for the rhTSH strategy was $90,090 per QALY gained. One way sensitivity analysis produced the following threshold requirements for cost-effectiveness of the rhTSH strategy: a total cost of rhTSH of less than $1,872.86, a quality adjustment factor for symptomatic hypothyroidism during LT4 withdrawal of less than 0.879, and duration of symptomatic hypothyroidism of greater than 50 days. Monte Carlo simulation showed the rhTSH strategy to be cost-effective in 535/1,000 hypothetical patients. The model was not sensitive to the cost of LT4, life expectancy, or the discount rate. Conclusions: This study demonstrates that avoidance of symptomatic hypothyroidism with the use of rhTSH administration prior to remnant ablation leads to a cost-effective increase in quality of life. However, the rhTSH strategy was cost-effective in this model only for those patients who would have a significant decrease in quality of life for greater than 50 days due to hypothyroidism.

Hesperetin Activates The Notch1 Signaling Cascade, Induces Cellular Differentiation, And Causes Apoptosis In Anaplastic Thyroid Cancer

P.N. Patel, Y. Xiao-Min, M. Kunnimalaiyaan, H. Chen — 2012-02-01

Introduction: Anaplastic thyroid cancer (ATC) is characterized by aggressive growth and extremely poor prognosis, for which there is no standardized therapy. In addition, the undifferentiated feature of ATC in which sodium iodide symporter (NIS) expression is reduced makes treatment such as radioactive iodine ineffective. Therefore, there is an urgent need for strategies that sensitize ATC cells to iodine treatment. It has been reported that the Notch1 signaling pathway, which affects cell proliferation and differentiation, is inactivated in ATC. However, it is unknown whether activation of Notch1 could yield therapeutic effects in ATC. Therefore, in this study we evaluated whether Hesperetin, a naturally occurring flavanone and potential Notch activator, effects ATC cell proliferation and possibly up-regulates thyroid-specific differentiation markers such as NIS. Methods: In-vitro studies were carried out to evaluate the drug effects using a human derived ATC cell line (HTH7). To determine whether Hesperetin activates Notch1 in ATC, expression of Notch1 mRNA and protein was evaluated upon treatment. In addition, mRNA levels of Notch1 response genes (Hes1 and Hey1) were measured, and the Notch1 activity was assessed using CBF-1-luc binding assay. Cellular proliferation was measured by viable cell counts every 24 hours post-treatment (0-200mM) for 72 hours, and the underlying mechanism was investigated by western blot. Expression of thyroid-specific differentiation markers including Thyroid Transcription Factor 1 (TTF-1), Thyroid Transcription Factor 2 (TTF-2), Paired Box Gene 8 (PAX-8), Thyroid Stimulating Hormone Receptor (TSHR), and NIS were also measured. Results: Hesperetin caused activation of the Notch1 signaling pathway. Treated ATC cells displayed elevated Notch1 protein and increased mRNA levels of Hes1 and Hey1, important Notch1 response genes. Treatment induced a dose-dependent increase in Notch1 activity as evidenced by increased luciferase activity. Hesperetin treatment also resulted in a time and dose-dependent decrease in cell proliferation: at 72 hours, 50mM and 100mM Hesperetin resulted in a 27% and 47% reduction respectively compared to control. Protein expression of apoptotic markers cleaved PARP, cleaved Caspase-3, and BAD increased with treatment. Importantly, Hesperetin up-regulated NIS mRNA levels, a thyroid-specific marker responsible for sensitivity to radioactive iodine. Other thyrocyte markers (TTF-1, TTF-2, PAX-8 and TSHR) were also increased, further evidence that Hesperetin induces differentiation in ATC. Conclusions: Hesperetin activates the Notch1 signaling cascade and has a significant antiproliferative effect on ATC that can be largely attributed to apoptosis. Hesperetin also induces differentiation in ATC, and by up-regulating markers such as NIS, could be useful in combination with radioactive iodine for treating ATC. Further investigation to elucidate the in-vivo effects of Hesperetin on ATC is warranted.

Prognostic Impact of Perineural, Blood and Lymph Vessel Invasion for Esophageal Cancer Patients

2012-02-01

Introduction: Esophageal cancer (EC) is one of the most aggressive tumor types and associated with a poor prognosis of the patients. This is mainly caused by its high and early metastatic potential. Only few studies with inconsistent results have investigated the prognostic impact of tumor-micro-invasion in blood (AI) and lymphatic vessels (LVI) and perineural invasion (PNI). Aim of the present study was therefore to investigate the value of the aforementioned factors as predictors for survival in a large cohort of patients with EC. Methods: Data from 644 patients with either adenocarcinoma (EAC) or squamous cell carcinoma (ESCC) of the esophagus, who underwent subtotal esophagectomy at University Medical Center Hamburg-Eppendorf between May 1992 and July 2009, were collected. Status of AI, LVI and PNI was determined by experienced pathologists and data were correlated with clinical and histopathological information and survival of the patients (Chi-square-test, Kaplan-Meier analysis, log-rank test). in addition, multivariate analysis was performed (Cox-regression). Results: Twelve percent of all specimen showed an AI (n=87), 32% a LVI (n=209) and 6% only a PNI (n=36). All three factors are significantly associated with tumor size (pT), nodal involvement (pN), metastatic disease (M), grading (G) and involvement of resection margin (R; see Table). Lymphatic vessel invasion and PNI were significantly more often in adenocarcinoma (EAC) compared to squamous cell carcinoma (ESCC). Angioinvasion and LVI are associated with positive bone marrow micrometastasis status. Kaplan-Meier survival analysis, stratified for cancer cell type, revealed a prognostic impact for AI and LVI in EAC and ESCC, while PNI revealed to be a significant factor in EAC, not in ESCC (Figure). Multivariate analysis showed that only LVI is an independent prognosticator for overall survival in EAC (HR 1.427, 95%CI 1.009/2.017; p=0.044). Neither LVI, nor AI and PNI revealed to be significant independent prognosticators in multivariate analysis of ESCC. Conclusions: This retrospective study demonstrates the prognostic significance of LVI for EAC patients and supports its inclusion into the TNM staging system of the UICC. Determination of LVI might help to identify those patients who profit from adjuvant treatment after surgical resection.

Role of Operative Therapy in Treatment of Metastatic and/or Recurrent Gastrointestinal Stromal Tumors

V. Zaydfudim, S.H. Okuno, F.G. Que, D.M. Nagorney, J.H. Donohue — 2012-02-01

Introduction: Operative resection of metastatic and recurrent gastrointestinal stromal tumor (GIST) is controversial. Current treatment strategies rely on response to systemic tyrosine kinase inhibitor therapies with individualized utilization of operative intervention. We investigated the role of operative therapy in patients with metastatic and/or recurrent GIST. Methods: This retrospective cohort study included all consecutive patients treated for metastatic and/or recurrent GIST between January 2002 and June 2011. Patients were stratified by use of operative therapy and by disease response to tyrosine kinase inhibitor (partial response; stable disease; progressive disease). Kaplan-Meier survival analyses with log-rank comparisons tested the effects of operative therapy and response to targeted systemic therapy on survival. Results: of 438 patients treated for GIST during the study period, 87 patients (mean age 60±14 years, 55% male) had metastatic and/or recurrent GIST (84% metastatic, 3% recurrent, 13% metastatic and recurrent). Half of these patients (n=43) had metastatic disease at the time of original diagnosis. Median time to development of metastases/recurrence in the other 44 patients was 29 months (IQR: 13-61 months). 54 of the 87 patients (62%) underwent operative exploration. Peri-operative mortality was zero. R2 subtotal resections for palliative debulking were performed in 19 patients (22%); 35 patients had gross total resections. Operative intervention was associated with improved disease-specific survival compared to systemic therapy alone (1-year: 98% vs. 80%; 5-year: 65% vs. 11%, p<0.001). One- and five year disease-specific survival after R2 resections (95% and 26%, respectively) did not differ from those patients treated non-operatively (p=0.190). A tyrosine kinase inhibitor was used before resection in 32 patients (59%) for a median of 7 months (IQR: 3-13 months): disease response was partial in 13 patients, stable in 10, and progressive in 9. One- and five year disease-specific survival was strongly associated with preoperative disease response to tyrosine kinase inhibitor (partial response: 100% and 100%; stable disease: 100% and 42%; progressive disease: 89% and 11%, respectively, p<0.001). Patients selected for metastasectomy (n=22) without preoperative tyrosine kinase therapy had similar disease-specific 1- and 5- year survival (95% and 87%, respectively) to patients with partial response (p=0.695) and better disease-specific survival than patients with stable disease (p=0.022). Conclusions: Patients with partial response or stable disease with tyrosine kinase inhibitor therapy demonstrate improved survival associated with operative treatment of recurrent or metastatic GIST. There is no survival benefit among patients who develop progressive disease on tyrosine kinase inhibitor or patients undergoing R2 resection.

Targeting Osteopontin Splice Variation to Increase Chemo-Sensitivity and Decrease Malignant Potential in Non-Small Cell Lung Cancer

J.S. Donington — 2012-02-01

Introduction: Cisplatin resistance by non-small cell lung cancer (NSCLC) is a vital medical problem resulting from deregulation of cell death and survival pathways. Osteopontin (OPN) is a ubiquitous protein associated with a wide range of normal and pathologic functions. It is a central regulator of the malignant phenotype in NSCLC and an instrument used to elude apoptosis when under stress, via αvB3integrin binding at OPN's central RGD domain. the three naturally occurring OPN splice variants are differentially expressed in NSCLC and have divergent impact on malignant potential. OPNa, the full length protein, is disproportionately expressed in tumors and cell lines, increasing invasion and decreasing apoptosis compared to the shorter splice variants. OPNa overexpression is also uniquely associated with down-stream gene-expression pattern consistent with EMT, a mechanism for cisplatin resistance in NSCLC.Conversely, OPNc, the shortest splice variant is not expressed in NSCLC tumors or cell lines, but inhibits invasion and platinum resistance when expressed. the critical RGD sequence is expressed in each of the OPN isoforms. the only structural difference between OPNa and OPNc is the transcription of exon 4 in the amino terminus. the hypothesis of our work is that the OPNa isoform alone contributes to the malignant potential in NSCLC and that the structural difference between OPNa and OPNc can be targeted to decrease invasion and cisplatin resistance. Methods: A 4-amino acid deletion (PDAV) was made in exon 4 of OPN by site-directed mutagenesis. the sequence was validated and functional impact evaluated by transfecting cDNA plasmids specific to OPNa, OPNc, PDAV deleted OPN or empty vector control into three NSCLC cell lines: H358 (low endogenous OPN) and A549 and H460 (high endogenous OPN). Pooled populations of cells were used in scratch closure, proliferation, invasion, and cisplatin treatment assays. Results: the PDAV deleted OPN activity mirrored that of OPNc expression, significantly decreasing proliferation, invasion, and migration and increasing cisplatin sensitivity compared to controls in cell lines with high endogenous OPN. (Fig. 1) Conclusions: the PDAV sequence in exon 4 of OPN has not been previously identified as a regulatory region, essential for OPN's effect on malignant phenotype. This work provides unique insight to the importance of splice variation as opposed to mutation, as a mechanism for controlling malignant potential in cancer cells. the fact that OPN acts extracellularly coupled with the fortuitous observation that retention of the PDAV sequences, rather than its deletion, leads to malignant behavior is unique and ideal for antibody or targeted small molecule therapies.

The Novel Role of the Tyrosine Kinase Inhibitor Sunitinib on Control of Myeloid-Derived Suppressor Cell Function and T-Regulatory Cell to Th17/Th1 Conversion

B.A. Coakley, D.Z. Kalir, G. Ma, J. Ozao, P. Pan, S. Chen, C. Divino — 2012-02-01

Introduction: Myeloid-derived suppressor cells (MDSCs) and T-regulatory cells (Tregs) have been shown to correlate with tumor prognosis. in a previous study, we have demonstrated the ability of the tyrosine-kinase inhibitor sunitinib, which is currently FDA-approved for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors, to inhibit MDSC development and to impair the ability of MDSCs to activate Tregs, favoring a Th1-type response in tumor-bearing mice. Here, we have further investigated the regulatory effects of sunitinib on these critical suppressor cells. Methods: Cells were purified via magnetic separation from tumor-bearing mice and cultured with sunitinib, IL-2, αCD3, αCD28 and TGFb and analyzed via ELISA, FACS, and suppression assays. in vivo assays were performed using sorted RFP cells derived from OT-II X FoxP3-RFP mice and transferred into OVA-B16 tumor-bearing mice, which were then administered sunitinib daily. Adoptively transferred cells were re-isolated from spleens, tumors and lymph nodes for analysis. Results: We found that sunitinib can inhibit STAT3 phosphorylation (p = 0.02) in MDSCs and induces apoptosis in M2-type macrophages (p = 0.002), resulting in selection for MDSCs, which had differentiated into classical, M1-type macrophages (p < 0.005). Treatment with sunitinib dramatically inhibited Treg proliferation (p < 0.001) and FoxP3 expression (p < 0.001) and significantly converting existing Tregs into ROR?t and T-bet expressing cells, as measured by IL-17 and IFN-gamma (both p<0.001). This effect is specific to Tregs and not normal effector T-cells, and is mediated through the inhibition of VEGFR3 activation and AKT signal pathways. This in turn abrogates FoxP3 expression and favors Treg conversion. These results indicate that multiple targets of TKIs like sunitinib can regulate both MDSCs and Tregs through different regulatory mechanisms. Conclusions: Although sunitinib has traditionally been used to target cancer directly, much of the mechanism behinds its phenotypic effects remains poorly described. We have identified that sunitinib controls MDSC differentiation to M1-type macrophages and additionally inhibits FoxP3 expression, while upregulating the inflammatory RORγt and T-bet transcription factors. This represents a novel role for sunitinib in the modulation of immune suppressor function in tumor-bearing hosts. Sunitinib's capacity to manipulate Tregs, Th17 and Th1 cells may be applicable not for the treatment of oncologic disease with novel tumor immunotherapies.

Inhibitor of Differentiation-1 (Id1) Regulates Tumor Growth and Metastases in An Orthotopic Nude Mouse Model

J.G. Trevino, S.A. George, S.J. Hughes, S.P. Chellappan — 2012-02-01

Introduction: Inhibitor of differentiation-1 (Id1) has demonstrated a role in tumor progression by promoting angiogenesis, invasion, and chemoresistance in a variety of tumors. Unfortunately, little is known as to the actual role of Id1 in pancreatic adenocarcinoma. We have previously demonstrated that nicotine, the addicting component of tobacco smoke which is a significant risk factor for pancreatic adenocarcinoma, induces Id1 expression through a Src-dependent signaling pathway promoting proliferation, invasion, and a chemoresistant phenotype in pancreatic cancer cells. Conversely, silencing of Id1 re-establishes gemcitabine sensitivity in acquired and innate gemcitabine-resistant cell lines. Therefore, our current schema is to further demonstrate role of Id1 in chemoresistance and tumor progression in vivo. Methods: Protein analyses, mRNA expression, and further cell lysate analyses were determined in PANC-1, MIA-PaCa-2, L3.6pl, L3.6plGemRes, and CD18 pancreatic cancer cells by western analysis and RT-PCR. L3.6pl metastatic pancreatic cancer cells were grown in culture and stably transfected with a luciferase expression vector (L3.6plLuc) and stable short hairpin RNA (shRNA) silencing of Id1 gene expression was established with appropriate controls. Animal studies were performed in nude mice with an orthotopic model with intraperitoneal injections of nicotine (1mg/kg, M/W/F) and gemcitabine (250 mg/kg, T/Th) with weekly monitoring of in vivo growth and metastases determined with the IVIS imaging system based on luminescent signal concentration. Confirmation of primary tumor and metastases was confirmed with H/E histological analyses and staining of tissues was performed with immunohistochemical techniques. Results: Our results demonstrate that nicotine promotes tumorigenic properties and chemoresistance through a Src-dependent Id1 signaling axis in innate and acquired gemcitabine-resistant pancreatic cancer cells. in vivo, nicotine significantly promoted tumor growth and metastases and while gemcitabine reduced the tumor burden, nicotine abrogates this inhibitory effect resulting in continued tumor growth and metastases. Additionally, stable knockdown of Id1 in vivo results in a significant decrease in primary tumor burden, regardless of nicotine exposure, when compared to controls, with no liver metastases demonstrating Id1 plays a significant role in primary tumor growth and metastases in an orthotopic model. Conclusions: in our current study, we demonstrate that Id1 contributes to tumor progression and nicotine, through a Src-dependent-Id1 signaling axis, promotes tumorigenic properties, metastases, and chemoresistance in an in vivo pancreatic cancer model.

The Role of MicroRNAs in Mediating the Oncogenic Effect of Notch Signaling in Melanoma

L. Yin, Q. Huang, H. Shao, O.C. Velazquez, Z. Liu — 2012-02-01

Introduction: We have previously demonstrated that aberrant Notch signaling is a crucial oncogenic promoter in the development and progression of melanoma. However, the molecular mechanism underlying the pathogenesis of Notch signaling with respect to melanoma progression remains largely unknown. We hypothesized that the oncogenic effect of Notch signaling is partially mediated by microRNAs (miRNAs), which in turn modulate expression of Notch targets. We aimed to identify these Notch-regulated miRNAs in human melanoma cells. Methods: Notch-regulated specific miRNAs were identified by examining miRNA profilesin response to Notch activation and inhibition in human melanoma cellsusingRT2-PCRArray. Enforced activation and suppression of the Notch pathway were achieved by transducing melanoma cells with viral vectors encoding activated Notch1 (NIC) and dominant-negative Mastermind-like 1 (DN-MAML1), respectively. the role of specific miRNA in mediating the oncogenic effect of Notch was studied bytesting whether alteration of miRNAexpression could reverse the Notch oncogenic effects (melanoma cell growth, survival and colony-formation in vitro, and lung metastasis in vivo). the potential target genes of the Notch-regulated specific miRNA were identified by searching available miRNA target databases and validated in melanoma cells by immunoblotting. Results: Six miRNAs specifically regulated by Notch signaling in melanoma cells were identified. of these, mi143 was selected for further studies because it was down-regulated 4.1-fold when the Notch1 pathway was constitutively activated; such down-regulation could be completely reversed with the suppression of Notch activation. Overexpression of mi143 antagonized Notch1-induced melanoma cell growth, survival and colony-formation in vitro as well as lung metastasis in vivo. the anti-oncogenic effect of mi143 is mediated by inhibiting Notch1-induced expression of Fibroblast Growth Factor-1 (FGF-1) in melanoma cells since expression of FGF-1 was upregulated by activation of the Notch1 pathway while downregulated by concurrentoverexpression of mi143. Consistently, a consensus mi143 targeting sequence has been mapped in the 3'-UTR of FGF-1 gene (based on published database). Supplementation of exogenous FGF-1 protein to Notch1-activated and mi143-overexpressing melanoma cells restored the oncogenic effects of Notch 1 on melanoma cells. Conclusions: Six miRNAs were identified as Notch-regulated specific miRNA in melanoma cells. Our study unveils a novel Notch1/mi143/FGF-1 cascade and demonstrates that Notch activation up-regulates FGF-1 expression through down-regulation of mi143 in melanoma cells. the mi143 is responsible for mediating, at least in part, the oncogenic effect of aberrant Notch signaling on promoting melanoma progression. This work uncovers a novel mechanism underlying the oncogenic effect of Notch signaling, and it provides potential novel targets for melanoma prognosis and therapy.

Assessment of Serum Platelet Factor 4 as a Predictive Marker for VTE and as a Prognostic Biomarker in Pancreatic, Colorectal, Breast, and Lung Cancer

2012-02-01

Introduction: Cancer is the second leading cause of mortality in the United States, with lung, colorectal, breast, and pancreas carcinoma comprising the four leading causes of cancer death. We previously demonstrated that platelet factor 4 (PF4) is an independent prognostic factor in pancreatic adenocarcinoma, with a significant association between elevated serum levels and increased venous thromboembolism (VTE) incidence and death. It is possible that high serum PF4 levels may be a useful biomarker for patient stratification for directing VTE prophylaxis in cancer patients. We sought to determine if PF4 is useful as a biomarker for prognosis and VTE incidence in other common cancers. Methods: We measured PF4 levels in sera from 39 patients with breast cancer, 48 with colorectal cancer, 50 with lung cancer and compared these results with data from 132 patients with pancreatic adenocarcinoma using a commercially available ELISA kit. Results: for all four cancers, age (P < 0.0001) and stage (P < 0.0001) were both significant predictors of survival. When examined in univariate Cox models, serum PF4 was a significant predictor of survival in breast (P = 0.018), colorectal (P = 0.01), and pancreatic (P = 0.003) cancer but not lung cancer (P = 0.65). After correcting for age and stage in multivariate analyses, PF4 remained a significant prognostic indicator in only colorectal (P = 0.008) and pancreatic (P = 0.001) cancers, with resulting hazard ratios for mortality of 1.53 and 1.105 respectively. Serum PF4 levels were significantly elevated in colorectal (P = 0.04) and pancreatic (P = 0.008) cancer subjects with VTE as compared to those without VTE, but this relationship was not present in breast (P = 0.81) or lung (P = 0.29) cancer subjects. When adjusted for age and stage, this association between serum PF4 level and VTE remained significant only in pancreatic cancer (P = 0.015). Conclusions: These initial results suggest PF4 may have utility as a clinical biomarker to predict survival in patients with pancreatic, and colorectal cancer and may be useful for patient stratification for VTE prophylaxis. We hypothesize that colon and pancreatic cancer patients with high serum PF4 levels may benefit from extended VTE prophylaxis. This question should be answered in a clinical trial.

Transformation of Mesenchymal Stem Cells into Cancer Associated Fibroblasts Within the Tumor Microenvironment

C.E. Weber, L.J. Talbot, P.Y. Wai, Z. Mi, M. Kundu, P.C. Kuo — 2012-02-01

Introduction: Cancer associated fibroblasts (CAF) in the tumor microenvironment (TMEN) potentiate tumor growth and metastasis. TGF-β mediates transition of mesenchymal stem cells (MSC) from local and distant sources into CAF . Osteopontin (OPN) is a secreted phosphoprotein predominantly expressed at the tumor-TMEN interface and is associated with tumor growth and metastasis. However, the underlying relationship between OPN and TMEN is unknown. We hypothesized that OPN interacts with MSC in the TMEN to mediate CAF formation. Methods: Human MSCs were stimulated with OPN to generate dose- (5- 320 ng/ml) and time- (0-48 h) response curves. Co-culture studies were performed using MSC and MDA-MB231 (OPN-high) or MCF7 (OPN-low). Active TGF-β (ELISA/qRT-PCR) and CAF markers (α-smooth muscle actin, tenascin-c, FSP-1, vimentin; qRT-PCR) were assayed. in selected instances, OPN signaling was ablated using an RNA aptamer (R3), integrin Ab and/or CD44 Ab. TGF-β was blocked using SB431542. in a murine NOD-scid xenograft model of Luc-MDA-MB231 co-implanted with GFP-hMSC, tumor size/bioluminescence was measured over 8 weeks; hMSC TGF-β and CAF markers from primary site and liver/lung metastases were determined at sacrifice. (Statistical analysis was performed using ANOVA or t-test, as appropriate. P values < 0.05 were considered significant.) Results: OPN significantly increased MSC expression of active TGF-β-1, 2 and 3, specifically TGF-β1, in a dose- and time-dependent fashion, peaking at 12 hours and was 4- (OPN 160 ng/ml) and 6-fold (OPN 320 ng/ml) higher vs. untreated (p<0.01). All CAF markers were significantly increased (> 2-6 fold) with OPN stimulation; OPN blockade with R3 or integrin Ab abolished TGF-β and CAF marker changes. in co-culture studies, CAF markers (>4-10 fold) and TGF-β (>15 fold) in MSC were significantly upregulated with MDA-MB231(OPN-high). TGF-β blockade ablated the increase in CAF markers; OPN blockade abolished the increase in both CAF markers and TGF-β. Minimal changes were noted with MCF7/MSC co-culture. in the xenograft model, MSC accelerated local growth and metastasis of MDA-MB231. MSC expression of CAF markers and TGF-β were significantly increased in primary and metastatic sites (lung/liver). OPN blockade with R3 significantly decreased local tumor growth and metastasis and abolished TGF-β and CAF markers in MSC. Conclusions: at the TMEN-tumor interface, tumor-derived OPN induces MSC expression of TGF-β to mediate autocrine MSC-to-CAF transformation. This then potentiates tumor growth and metastasis. OPN targeting may represent a unique therapeutic approach to address the TMEN component of oncogenesis.

IL-27 Mediated Inhibition of Epithelial to Mesenchymal Transition is Augmented by STAT1 Activator, 2-(1,8-naphthyridin-2-yl)phenol (2-NP) in Human Non-small Cell Lung Cancer

P. Kachroo, M. Lee, G. Lee, K. Krysan, S. Sharma, S.M. Dubinett, J.M. Lee — 2012-02-01

Introduction: Interleukin-27 (IL-27), a member of the IL-12 family, signals through the JAK-STAT pathway with resultant activation of transcriptional factors with opposing roles in carcinogenesis, STAT1 (tumor suppressor) and STAT3 (oncogene). IL-27 has been shown to have anti-tumor activity, but understanding of its mechanism is limited. Epithelial to mesenchymal transition (EMT) is a critical process in metastasis whereby cells change from an epithelial phenotype to a migratory, mesenchymal phenotype. While STAT proteins have been linked to epithelial differentiation, their role in EMT in lung carcinogenesis is poorly defined. We hypothesize that modulation of STAT1 and STAT3 is important in regulating EMT. to investigate this concept, IL-27 and 2-(1,8-naphthyridin-2-yl)phenol (2-NP), a known STAT1 activator, were used to modulate STAT1 and STAT3 activity and to evaluate their ability to inhibit EMT in human non-small cell lung cancer (NSCLC). Methods: A549, a human NSCLC cell line, was pre-treated with 2-NP (0.072-45μM) for one hour prior to IL-27 stimulation (50ng/mL). Activation of STAT1 and STAT3, demonstrated by tyrosine phosphorylation, was analyzed by Western Blot. to inhibit the STAT1 pathway, cells were transfected with STAT1 siRNA. Additionally, epithelial markers (E-cadherin, γ-catenin) and mesenchymal markers (N-cadherin, vimentin) were assessed by Western blotting. to study the effect of IL-27 and 2-NP on cell migration, an in vitro scratch assay was employed by creating a scratch in confluent A549 cells and monitoring for wound closure. Results: IL-27 treatment induced rapid STAT1 and STAT3 phosphorylation, whereas total STAT1 and STAT3 expression was not affected. We also detected increased levels of E-cadherin and γ-catenin and decreased levels of N-cadherin and vimentin in IL-27-treated cells as compared to untreated control. to evaluate the importance of the STAT1 pathway, STAT1 expression was suppressed by siRNA prior to IL-27 stimulation. This abolished upregulation of E-cadherin and γ-catenin, and downregulation of vimentin by IL-27. to further elucidate the dominant effect of STAT1 in EMT prevention, A549 cells were treated with combined IL-27 and 2-NP. This experiment demonstrated increased STAT1 and decreased STAT3 activation, which resulted in augmented expression of epithelial and reduced expression of mesenchymal markers compared to IL-27 alone control. An in vitro scratch assay confirmed that 2-NP augmented the inhibitory effect of IL-27 on cancer cell migration by maintaining the wound gap for a longer duration compared to IL-27 alone. Conclusions: We demonstrated that IL-27 activated both STAT1 and STAT3, but inhibited epithelial to mesenchymal transition in human lung cancer in a STAT1-dependent manner. IL-27-mediated inhibition of EMT is augmented by 2-NP through increased STAT1 and decreased STAT3 activation. We conclude that a STAT1 dominant pathway is important in modulation of EMT in carcinogenesis.

Withdrawn

2012-02-01

Reversal of Premature Delivery Via Cox-2 Inhibition and GPR109A Deletion in Cystathionine-b-Synthase (CBS) Deficient Mice

V.K. Bhalla, S. Sonne, V. Ganapathy — 2012-02-01

Introduction: Mice with a heterozygous deletion of the enzyme cystathionine-b-synthase (Cbs) have increased plasma levels of homocysteine and deliver two days earlier than wild-type mice. This process is associated with increased expression of cyclooxygenase-2 (Cox-2) and oxytocin in the uterus and placenta. However, both the administration of oral Cox-2 inhibitors to pregnant Cbs+/- mice prior to their anticipated delivery date and the deletion of GPR109A reverse this process, suggesting that this G-protein-coupled receptor regulates Cox-2 expression. Our aim was to investigate the effects of hyperhomocysteinemia on premature delivery through Cox-2 and oxytocin expression and its reversal through oral Cox-2 inhibitors and the deletion of GPR109A. Methods: Placental and uterine tissue was obtained from Cbs+/- female mice on the 16th day of gestation and compared to wild-type females of the same gestational day (Cbs+/+). Cox-2 and oxytocin levels were assessed by qPCR, western blot, and immunohistochemistry. in vitro studies were performed by measuring uterine muscle contraction. to reverse premature delivery, a Cox-2 inhibitor was administered orally for two days prior to the anticipated delivery date. to study the effects of GPR109A on premature delivery, Cbs+/-/Gpr109a-/- mice were obtained by crossing Cbs+/- mice and Gpr109a-/- mice, and the gestational period was compared between Cbs+/-/Gpr109a+/+ mice and Cbs+/-/Gpr109a-/- mice. Results: Cox-2 and oxytocin expression were significantly increased in the Cbs+/- pregnant females compared to pregnant wild-type females. These results were confirmed by qPCR, western blot, and immunohistochemistry. Oral administration of a known Cox-2 inhibitor to Cbs+/- females resulted in the reversal of premature delivery with a total gestation period similar to wild-type mice. GPR109A deletion also reversed premature delivery. Cbs+/-/Gpr109a+/+ mice delivered on gestational day (GD) 16.6 ±0.12 whereas Cbs+/-/Gpr109a-/- mice and wild-type mice delivered pups on GD 20 ±0.5 and 20 ±0.17, respectively. Additionally, overall expression of Cox-2 and oxytocin were lower in Cbs+/-/Gpr109a-/- mice, further supporting the idea that Cox-2 acts through GPR109A. Conclusions: Elevated levels of homocysteine cause premature delivery in mice through increased expression of Cox-2, which we believe acts through a G-protein coupled receptor called GPR109A. These Cbs+/- females have increased plasma levels of homocysteine and demonstrate increased expression of Cox-2 and oxytocin in their uterus and placenta, which supports the idea of increased contraction leading to premature delivery. Gpr109A is a niacin-dependent receptor that is obligatory for this process and has been proven to facilitate increased Cox-2 expression in human dendritic cells. We therefore conclude that homocysteine-induced premature delivery in Cbs+/- mice is mediated through Gpr109A and its deletion reverses the premature delivery process, as does oral treatment with Cox-2 inhibitors.

Essential Role of the Anti-Inflammatory Cytokine IL-10 in the Fetal Regenerative Phenotype is Mediated Via Stat3 and Hyaluronan Synthase: Implications for Scarless Wound Healing

2012-02-01

Introduction: Mid-gestation fetal skin heals without scar with high levels of hyaluronan (HA) in the extracellular matrix (ECM) and an attenuated inflammatory response. We have previously demonstrated an essential role of interleukin-10 (IL-10) in fetal wound healing and the ability of fetal fibroblasts (FFb) to produce a large HA-rich pericellular matrix (PCM). These data include 1)elevated IL-10 levels in fetal skin, 2)IL-10-/- fetal wounds heal with scar, and 3)IL-10 induces postnatal wounds to heal scarlessly. Combining these data with accepted IL-10 signaling pathways, we hypothesize that the regenerative effects of IL-10 in the fetus are partially mediated through HA-rich PCM formation dependent on IL-10 receptor 1 (IL-10R1), STAT3 and hyaluronan synthase (HAS1-3) function. We further hypothesize IL-10 can recapitulate this fetal regenerative PCM phenotype in adult fibroblasts (AFb). Methods: to determine the role of IL-10 in FFb PCM production, murine IL-10-/- (E14.5) fibroblasts were cultured for 24 hours and PCM assessed. to determine if FFb produced PCM is HA rich, FFB were treated with hyaluronidase. Next in a sequential series of loss-of-function experiments, murine FFb (E14.5) were treated with inhibitors of IL-10R1 (IL-10R1 neutralizing Ab), STAT3 (STA-21) and HAS 1-3 (4-methyumbelliferone, 4-MU) or PBS and analyzed for PCM production. AFb were cultured with IL-10 (200ng/ml) and PCM assessed. PCM was quantified using erythrocyte particle exclusion assay and computer assisted morphometric analysis which generated a PCM area/cell area ratio. Data expressed as mean±SEM. Results: FFb produce a robust PCM (2.82+1.02). Treatment with hyaluronidase results in a significantly attenuated PCM in FFb (FFb with treatment 1.60±0.11 vs FFb 2.79±0.35;p<0.01). FFB produce a significantly greater PCM than IL10-/- FFb (FFb 2.82±1.02 vs IL10-/- 1.58±0.15;p<0.01). Blockade of IL-10R1 significantly reduces PCM formation compared to FFb (FFb with IL-10R1Ab 1.41±0.05 vs FFb 2.82±1.02;p=0.01). Inhibition of STAT3 by STA-21 results in decreased PCM formation (FFb with STA21 1.24±0.08 vs FFb 2.82±1.02;p<0.001). Attenuation of HAS1-3 function by 4-MU results in decreased PCM formation (FFb with 4-MU 1.63±0.16 vs FFb 2.82±1.02;p<0.01). Treatment with IL-10 significantly increases PCM production in adult Fb (AFb 1.84±0.40 vs AFb+IL-10 2.77±0.72;p<0.01) to levels which recapitulate the fetal regenerative PCM phenotype (AFb+IL-10 2.77±0.72vs FFb 2.82±1.02;p=NS). Conclusions: IL-10 is essential to the fetal fibroblast's ability to produce an HA-rich PCM. FFb production of the PCM is dependent on IL-10R1, STAT3 and HA synthase function. IL-10 can recapitulate PCM component of the fetal regenerative phenotype in adult fibroblasts. These data suggest that IL-10 plays a unique role in the fetal regenerative response, not only as an immunomodulatory agent, but as a regulator of the ECM.

Early Fibrotic Injury in A Rat Model of Bile Duct Ligation: Further Evidence of Intrinsic Matrix Metalloproteinase 8

S.S. Ciullo, C.S. Muratore, Y. Zhou, T.F. Tracy — 2012-02-01

Introduction: Bile duct ligation (BDL) produces a reproducible pattern of periportal fibrosis comparable to the injury pattern seen in patients over the course of cholestatic diseases such as biliary atresia. Matrix Metalloproteinases (MMPs) are responsible for degradation of extracellular matrix, and Tissue Inhibitors of Metalloproteinases (TIMPs) inhibit MMPs by binding to them in a one-to-one stoichiometric ratio, blocking enzyme activity. MMPs have different matrix substrate specificities. MMP-8 is a collagenase we have demonstrated whose hepatic gene expression and protein level increases in bile duct ligated animals. Through unknown mechanisms, resorption of collagen begins only after biliary decompression at the onset of repair. the initial and late stages of cholestatic injury have not been studied previously, and we sought to determine the time course of MMP-8 production within the liver. We hypothesized that MMP-8 levels shortly after injury represent intrinsic hepatic collagenase and TIMP-1 and MMP-8 should be found in corresponding levels consistent with inhibition of collagenase activity. Methods: Male Sprague Dawley rats (wt 200-300g) were divided into two groups: sham and BDL. All animals underwent laparotomy and BDL animals had their common bile duct doubly tied and ligated as previously described. on postoperative day 1, 2, 3, 4, 5, 7, and 14 three animals from each group were sacrificed and their livers were harvested for analysis. Western blotting for MMP-8, in-situ collagen zymography, and real-time polymerase chain reaction (rt-PCR) were performed. Results: By rt-PCR, MMP-8 levels remained only mildly elevated from baseline until Day 4 at which point, levels increased until day 14 where they were 28 times baseline. TIMP-1 PCR levels increased and peaked at 21 times baseline by day 5, but then fell to 9 times baseline by day 14. Neither MMP-9 nor MMP-2 were elevated significantly above sham comparisons throughout the experiment. Western blotting showed an increase in MMP-8 protein expression over time. in situ zymography shows no collagenolytic activity at any time point in obstructed animals. Conclusions: We have demonstrated a rapid but not immediate rise in MMP-8 expression at both a genetic and proteomic level. Interestingly TIMP-1 gene expression does not parallel that of MMP-8, but it is likely that this is due to higher levels of TIMP-1 present at baseline. While an increase in neutrophils is found immediately after injury, MMP-8 levels increase on day 4 of injury, providing further evidence of an intrinsic source of MMP-8.

Outer Membrane Protein A (OmpA) Expression Prevents IgA Production in Cronobacter Sakazakii Induced Necrotizing Enterocolitis

C.N. Emami, R. Mittal, H.R. Ford, N.V. Prasadarao — 2012-02-01

Introduction: Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal emergency in neonates and is associated with a high mortality and morbidity. Cronobacter sakazakii(CS) is a gram-negative opportunistic pathogen, which has been implicated in outbreaks of NEC in neonatal ICUs. We have previously demonstrated that enteral feeding of CS to newborn mice results in intestinal inflammation reminiscent of NEC and that outer membrane protein A (OmpA) is a most important virulence factor that enables CS to induce NEC. We hypothesize that OmpA-expressing CS compromises the local immune response in the gut to induce NEC-like pathology. Methods: C57Bl6 wild-type (WT) and B cell deficient (m-/-) newborn mice were infected with 103 CFUs of OmpA+ CS or OmpA- CS on day 3. the lamina propria cells were separated from the digested intestinal specimens using Percol gradient. Recruitment of B cells to the lamina propria of WT mice was determined using flow cytometry. the mucosal IgM and IgA levels were measured by ELISA. Intestinal bacterial load was determined by plating homogenates on ampillicin-containing agar plates. in addition, enterocytes were isolated from the WT and m-/-infected mice using flow cytometry and apoptosis was examined using AnnexinV-7AAD staining. Nitric oxide levels were determined in the intestinal homogenates of WT and m-/- mice by Griess assay. Results: We observed that OmpA+ CS infected animals showed very low levels of intestinal IgA compared to OmpA- CS infected mice. There was no change in intestinal IgM levels in OmpA+ CS infected animals similar to uninfected animals. However, higher IgA levels were observed in OmpA- CS infected animals accompanied by decreased IgM levels, suggesting that Ig-class switching occurs. Further, m-/-mice succumbed to infection within 48 h post-infection and displayed very severe intestinal pathology. Higher bacterial count was observed in the intestines of these mice compared to wild-type infected mice. on par with these findings, greater percentage of enterocytes was found to be apoptotic accompanied with elevated levels of nitric oxide in m-/-mice. Conclusions: These results suggest that OmpA expressing CS prevents class switching from IgM to IgA, thereby avoiding potent host immune defenses in order to colonize host tissues and cause infection. Targeting OmpA epitopes, which interact with IgM and prevents class switching may open new avenues for developing effective preventive strategies against CS induced NEC.

TLR4 Signaling Negatively Regulates Intestinal Microvascular Perfusion in the Pathogenesis of NEC Which Can Be Reversed With Exogenous Nitrite

2012-02-01

Introduction: Necrotizing enterocolitis (NEC) is the leading cause of GI-related death in premies and its etiology remains unexplained. Previous research has focused on epithelial signaling in NEC; however, NEC is a disease of early ischemia and tissue necrosis, which sets it apart from other inflammatory bowel diseases. Our lab has shown that TLR4 signaling is fundamental to the pathogenesis of NEC, although the mechanism of action of TLR4 is incompletely understood. We now hypothesize that TLR4 signaling reduces intestinal perfusion leading to NEC, and enhancement of this perfusion may reduce the severity of the disease. Methods: TLR4 activation was induced in wild-type (WT) and TLR4-/- adult and newborn mice via LPS injection. to study intestinal blood flow in response to LPS, mice received intra-cardiac injections of fluorescent Tomato Lectin 5 minutes prior to death. Tissue was stained for PECAM and 3D-blood flow modeling was performed. PCR was used to asses for inflammatory markers (iNOS, TNFa) and vascular regulatory genes: eNOS, which produces the potent vasodilator NO, as well as Endothelin-1 (ET1), which is a potent vasoconstrictor via its interaction with Endothelin receptor-A (ETAR), but also induces eNOS expression via Endothelin receptor-B (ETBR). to assess reversibility, NEC was also induced in newborn WT mice through a combination of hypoxia and gavaged formula +/- the NO donor sodium nitrite (NaNO2). NEC severity was assessed via mucosal cytokines and ileal histology; and gut perfusion in NEC was also analyzed. Results: TLR4 activation in vivo significantly induced expression of inflammatory markers in WT adults but not TLR4-/-. WT mice demonstrated 44% impairment of intestinal perfusion in response to LPS compared to 5% in TLR4-/- (p<0.05). the WT mice exhibited a 3-fold increase in the vasoconstrictive ratio of ET1/eNOS expression. This suggests LPS-TLR4 signaling leads to impairment in gut perfusion. WT neonates also had a 40% increase in the ratio of ET1/eNOS expression in response to LPS. However, intestinal ETAR and ETBR expression was 3-4 fold higher in neonates vs. adults, potentially making the newborn gut more susceptible to vasoconstriction and ischemia in response to an increased ET1/eNOS ratio. in support of this possibility, feeding formula + NO-donor preserved normal levels of intestinal blood flow in the NEC model, which was significantly impaired in the formula-only fed animals. This corresponded to significant disease protection in the NaNO2 group as evident by PCR, WB, and histological data.

Regulation of Breast Milk Induced P-Glycoprotein Expression in Intestinal Epithelial Cells

E.M. Pontarelli, A.V. Grishin, H.R. Ford — 2012-02-01

Introduction: P-glycoprotein (Pgp), a transmembrane efflux pump, is found at the apical surface of intestinal epithelial cells. Because adult Pgp-deficient mice develop colitis in a bacteria-dependent manner, and newborn Pgp-deficient mice display increased susceptibility to necrotizing enterocolitis (NEC), Pgp is believed to play a key role in protecting the intestinal epithelium against bacteria and their by-products. Pgp expression is induced by a variety of stresses and xenobiotics. the mechanisms by which these stimuli induce Pgp expression are not fully understood. Previous studies have shown that ß-catenin, PI3-kinase, c-jun and other signaling intermediates may be involved in the induction of Pgp. Recently we have demonstrated that expression of Pgp in the intestinal epithelium is upregulated by breast milk. We hypothesize that factor(s) in breast milk induce Pgp via their cognate receptors and intracellular signaling pathways. Our goal is to elucidate the signaling pathways by which breast milk upregulates Pgp expression. Methods: Human breast milk was obtained from healthy volunteers. the crude fraction containing the Pgp-inducing activity was obtained by precipitation with ammonium sulfate at 40% saturation. Casein was precipitated using high temperature centrifugation and dialysis at pH 4.6. Cultured enterocytes were grown to confluence at 37ºC in medium supplemented with 20% FBS. Cells were serum starved for 12 hours prior to treatments. Specific inhibitors of MEK, p38, PKA, PKC, JAK, B-catenin, and PI 3-Kinase were added 1 hour prior to treatment with the enriched fraction of breast milk. 18 hours post treatment, cells were lysed with RIPA buffer and Pgp expression was analyzed using western blotting with the C219 monoclonal antibody. Appropriate controls were used with each inhibitor. Results: Inhibition of MEK, JAK, PKC, and PI 3-Kinase appeared to have no effect on breast milk-induced expression of Pgp. Inhibition of ß-catenin and PKA partially suppressed expression of Pgp. in contrast, inhibition of p38, which has previously been shown to decrease Pgp expression, resulted in a marked potentiation of Pgp in our model. Conclusions: Breast milk-induced expression of Pgp in the intestinal epithelium, appears to be mediated by multiple pathways. ß-catenin and PKA pathways may act as positive regulators, whereas p38 may act as a negative regulator. Understanding the pathway of breast milk-induced expression of Pgp will further elucidate the mechanisms by which breast feeding protects against NEC.

Developmental Changes of NADPH Oxidase Activity and Subsequent Effects on A Rat Model of Necrotizing Enterocolitis

S.R. Welak, R. Teng, K.A. Pritchard, R.M. Rentea, K.M. Fredrich, D.M. Gourlay — 2012-02-01

Introduction: Necrotizing enterocolitis (NEC) is a major complication of prematurity. Infection, vascular dysfunction, and inflammation contribute to the pathogenesis and severity of the disease. Previous studies have shown that superoxide (O2-) production is increased in a neonatal rat model of NEC. the sources of increased O2- remain unknown. NADPH oxidase (NOX) generates O2- by reducing molecular oxygen and has been implicated as a source of increased O2- in diseases such as atherosclerosis and hypertension. Previous research has shown that NOX is present in intestinal tissue and is increased during an inflammatory response. It is currently unknown if NOX contributes to the pathogenesis of NEC, nor how bowel injury alters NOX activity. the hypothesis of this study is that NOX activity increases on a daily basis, and that the increase in activity is exaggerated with intestinal injury. Methods: Sprague Dawley newborn rat pups were divided into two arms. Control pups stayed with the mother and were breastfed. Experimental pups were birthed one day prematurely by caesarean section and gavage fed a hypercaloric formula with LPS and subjected to hypoxia. Some rats were sacrificed from each group prior to feeding. Pups were sacrificed daily, and the small intestines harvested. Small intestinal homogenates were analyzed for O2- production using an NADPH-dependent lucigenin assay in the presence and absence a O2- scavenger (Tiron). the chemiluminescence was expressed in relative light units per milligram of tissue homogenate (RLU/mg). A paired t-test was used to determine statistical significance between control and experimental animals respective to each day. Results: in the absence of NADPH, chemiluminescence was undetectable. in controls pups there was no significant difference in chemiluminescence over the first five days of life. However, following birth by caesarean section, but before induction of NEC the preterm pups had increased chemiluminescence over controls (3675 vs 1057 RLU/mg, p < 0.05). the NEC pups had a significant decrease in chemiluminescence on day 2 of life, followed by a significant increase each day until reaching significantly increased chemiluminescence on the fifth day of life over controls (7578 vs 1300 RLU/mg, NEC vs control, respectively, p<0.05). Chemiluminescence inhibited by Tiron was significantly increased in NEC animals on day of life 1 (3441 vs 736 RLU/mg), 4 (2464 vs 75 RLU/mg), and 5 (5505 vs 2190 RLU/mg), but was significantly decreased on the day of life 2 (382 vs 14 RLU/mg). Conclusions: the development of NEC is associated with an NADPH-dependent increase in O2- production by the small intestine, and correlates with the severity of injury. the absence of chemiluminescence in the absence of NADPH indicates that the assay is completely dependent on NADPH as a substrate for O2- production, highly indicative of NOX as its main source. Further studies are needed to determine more specific details on the contribution of NOX to the pathogenesis of NEC.

Intestinal Alkaline Phosphatase Prevents the Systemic Inflammatory Response Associated With Necrotizing Enterocolitis

K.M. Riggle, R. Rentea, S. Welak, D.M. Gourlay — 2012-02-01

Introduction: Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates with an incidence of 0.5-2.4 cases per 1000 live births. the mortality rate is between 10-50% and has not improved significantly in the past two decades. Neonates affected by NEC develop a septic injury that is associated with an increased risk of chronic lung disease and neurological impairment due to intraventricular bleeding. Intestinal alkaline phosphatase (IAP) is an endogenous protein that has been shown to inactivate the endotoxin lipopolysaccharide (LPS), and has recently been used successfully as an adjunct to treat sepsis in adult patients. Our hypothesis is that systemic, exogenous IAP will mitigate the inflammatory response as measured by serum levels of pro-inflammatory cytokines in a rat model of NEC. Methods: Newborn Sprague Dawley rats were divided into groups. Control pups were breast-fed. in the remaining pups, NEC was induced by feeding formula containing LPS and exposure to intermittent hypoxia. NEC pups were also given intra-peritoneal injections of 4 or 40 glyceine units (U) of IAP or placebo twice daily. Rats were sacrificed on days 1-3. Intestine was harvested to grade intestinal injury and serum was collected for cytokine analysis using a bioplex assay as well as measurement of alkaline phosphatase (AP) activity. Results: Systemic IAP administration had no effect on increasing intestinal AP activity or preventing NEC as measured by histology. However, systemic IAP administration significantly increased serum AP activity in a dose and time dependent fashion. the serum AP activity for the 40 U IAP treated groups on days of life 1-3 were 0.285, 0.344, and 0.496 U/mL respectively, while the controls and NEC groups had serum activities ranging from 0.007-0.0489 U/mL on days 1-3 (p<0.05 for all). We also found that the pro-inflammatory cytokines (measured in pg/mL) TNF-a, IL-6, and IL-1b were significantly increased in NEC rats as compared to controls on days 2 and 3 (TNF-a: 1390 vs. 25 on DOL 2 and 2896 vs. 1332 on DOL 3; IL-1b: 3295 vs. 66 DOL 2 and 7010 vs. 2394 on DOL3; IL-6: 1718 vs. 21 on DOL2; (p<0.05 for all). Importantly, we found that treatment with 40 U of systemic IAP not only increased serum AP activity, but also decreased these pro-inflammatory cytokines back to near-control levels. (TNF-a: 26 on DOL 2 and 1018 on DOL3; IL-1b 183 on DOL 2 and 1339 on DOL 3; IL-6: 381 on DOL 3; (p<0.05 for all). Figure 1 shows serum TNF-a levels in control, NEC, and 40 U IAP treated pups. Conclusions: Systemic IAP administration appears effective in preventing the systemic inflammatory response associated with NEC, and may prove to be a valuable adjunctive treatment for NEC.

The Progressive Decline in Diabetic Skin Integrity Is Associated With Decreased Collagen Protein Content and Dysregulation of MicroRNA-29a

2012-02-01

Introduction: We have shown that the biomechanical properties of murine and human diabetic skin are significantly inferior to non-diabetic skin at baseline. the major structural component of the skin responsible for skin integrity is type I collagen. MicroRNAs (miRNAs) are regulatory molecules that inhibit the translation of mRNAs at the post-transcriptional level, and have been shown to play an important role in the regulation of collagen content. Specifically, MiRNA-29a has been shown to inhibit the translation of Col1a2 mRNA. We hypothesized that the impaired biomechanical properties of murine diabetic skin at baseline is associated with decreased collagen protein and is due, in part, to increased miRNA-29a production. Methods: to test this hypothesis, we obtained samples from 4 and 20 week old diabetic and non-diabetic murine skin. the skin was processed for isolation of total protein and total cellular RNA. Western blot analysis was performed for type I collagen and real-time PCR performed for Col1a2 and miRNA-29a. Results: at 4 weeks of age diabetic and non-diabetic skin had similar type I collagen protein content, however, by 20 weeks the diabetic skin had significantly decreased type I collagen compared to non-diabetic (p<0.05) or 4 week old skin (p<0.05). at 4 weeks diabetic skin had significantly increased col1a2 and miRNA-29a expression compared to non-diabetic skin, and a col1a2/miRNA-29a ratio of 7.6:1 in diabetics. at 20 weeks the diabetic skin also had increased col1a2 and miRNA-29a expression compared to non-diabetic skin, but the col1a2/miRNA-29a ratio was significantly decreased to 2.7:1 in diabetics. Conclusions: These findings provide the first evidence that the impaired biomechanical properties of diabetic skin may be due, in part, to increased miRNA-29a expression relative to collagen gene expression, resulting in decreased collagen protein production. These results represent a novel potential therapeutic target to improve collagen content and subsequent biomechanical properties of diabetic skin to prevent injury, as well as to improve healing following injury.

Transcriptional Regulation of BMP5 in Fracture Healing and Development

Z. Wang, C. Guenther, M.C. Tran, G.P. Yang, D.M. Kingsley — 2012-02-01

Introduction: BMP5 plays an important role during skeletal development and its loss leads to multiple defects in the size and shape of skeletal elements. in adults, BMP5 mutants also display defects in fracture repair with decreased bone formation. in development and fracture healing, bone forms both through endochondral ossification. We have previously identified multiple cis-regulatory elements that control bmp5 expression at specific anatomical locations during embryogenesis. in this study, we address the question of whether transcriptional control of bmp5 expression in fracture healing is the same as during development. Methods: A series of transgenic mice carrying bmp5 regulatory elements fused to a LACZ reporter gene were used to investigate bmp5 regulation during adult rib-fracture healing. Transgenic mice and non-transgenic littermate controls underwent open rib fracture, tibia fracture, and excisional skin wounding. Ribs, tibias and skin wounds were harvested at the indicated dates and stained for LacZ activity, and sectioned. Fracture and embryologic expression patterns were then compared. Results: Following rib fracture, bmp5 expression throughout the callus was driven by an enhancer element that had not been previously shown to drive bmp5 transcription in development. There was a small level of expression at day 2 with a peak at day 4 post-fracture, and diminishing by day 7. to determine if the fracture element was specific to rib, tibial fractures were performed and harvested at day 4 in concurrence with the time of peak expression following rib fracture. These data demonstrated that bmp5 transcription in fracture healing depended upon the same element regardless of skeletal location. Finally, we addressed whether this enhancer element in the BMP5 promoter was a generalized injury response element by performing excisional skin wounds. at 4 days post-surgery, these wounds demonstrated LacZ expression primarily in the leading edge of the keratinocytes, consistent with previous reports of bmp5 expression during wound healing. Conclusions: Long bone development and fracture healing progress through endochondral ossification and appear very similar histologically with formation of an undifferentiated mesenchyme followed by hypertrophic cartilage formation, vascular invasion and bony replacement. Our data have demonstrated that although the biological process appeared similar, the molecular control of genes important in this process was different. Furthermore, the enhancer element identified appeared to be a generalized injury response element that drove bmp5 expression during fracture in multiple skeletal sites and in the skin during wound healing.

Transplanting Whole Livers from Donors Less Than 6 Kilograms -- is it Prudent?

N.T. Nguyen, T.R. Harring, H. Liu, J.A. Goss, C.A. O'Mahony — 2012-02-01

Introduction: Our experience suggests transplanting whole liver allografts (WL) from donors weighing <6 kgs portends a worse prognosis for the recipient. to find objective measures of outcome, we compare the pt and allograft survivals of infants who receive livers from donors ≥6kg, <6 kg or split liver allografts (SL) from a retrospectively-maintained, US database. Methods: the United Network for Organ Transplantation (UNOS) database was queried for infant liver transplantations (recipients ≤ 2yrs) performed between Sept 1987 and Mar 2011. of 5,976 OLTs, 1,394 pts received SLT. 509 pts had WL procured from donors weighing <6 kgs,and 4,073 remaining pts with whole livers from donors weighing ≥6 kgs. Survival data were analyzed by the Kaplan-Meier method and compared by the log-rank test between pts with WL from donors weighing <6 kg and those recipients of donors ≥6 kg and those who received SL. All statistical analyses were performed by SPSS version 15.0 (IBM SPSS, Chicago, IL). Results: in pts who receive WL from donors weighing <6 kgs (n=509), pt survival mean was 5093 days +/-177 days. Overall pt survivals were 76.7%, 71.4%, 68.4% and 65.9% at 1-, 3-, 5- and 10-years, respectively. This is significantly worse compared to the group with donors weighing ≥6 kgs, both in overall pt and allograft survival (p < 0.00). Pts whose donors ≥6kg (n=5976), overall pt survivals were 82.1%, 78.7%, 77.3% and 75.4% at 1-, 3-, 5- and 10-years, respectively (see Table 1 for allograft survivals). When compared to infants who received SL, recipients from donors <6kgs have worse survival experience. Infants who received SL (n=1394) had an overall pt survival of 89.6%, 87.1%, 85.5% and 83.3% at 1-, 3-, 5- and 10-years, respectively. This significance of difference has a p-value <0.00. (Table1) Pts who receive WL from donors weighing <6kgs fare worse than pts who receive allografts from heavier donors and in fact, pts who receive SL had the best outcomes compared to both groups in regards to pt and allograft survivals (p <0.00, Graph 1). Conclusions: Upon evaluation of the US experience with liver transplantation, we find the small weight of the donor influences the infant pt outcome. Pts with allografts from donors weighing <6 kgs have a worse prognosis compared to those whose donors weigh ≥6kg, or pts who received SL, both in pt and graft survival. This data helps with decision-making during the organ allocation process by revealing the superior outcomes of SL and poorer outcomes for WL of donors weighing <6kgs.

Trends in Immunologic Risk Among Kidney Transplant Recipients in the United States

2012-02-01

Introduction: Despite advances in immunosuppressive therapy, immune factors remain important predictors of graft failure following renal transplantation. However, development of a composite measure of immunologic risk is necessary to better inform clinician decision making and organ allocation policy reform. Methods: We retrospectively examined 208,560 recipients of kidney transplants from the United Network for Organ Sharing (UNOS) database preformed between 1995 and 2010. Graft survival according to a composite (re-transplantation, panel reactive antibody >40%, human leukocyte antigen mismatch, recipient hepatitis C virus seropositive, and African American race) Immunologic Risk Index (IRI) was analyzed using Kaplan-Meier and log-rank methodology. Cox proportional hazards regression was utilized to examine both the performance of individual immune risk factors and the IRI as predictors of graft failure. Stratified analysis and calculation of the Rothman Synergy Index were used to characterize the interaction between immune risk factors. Results: A decline in unadjusted graft survival was observed with escalating Immunologic Risk Index score (Figure 1). After risk adjustment for donor and recipient covariates, the Immunologic Risk Index remains a significant predictor of graft failure (HR 1.21 per point, 95% CI 1.19-1.22, p<0.0001). Recipient Hepatitis C virus (HR 1.36, 95% CI 1.31-1.41, p<0.0001) was the strongest and panel reactive antibody >40% (HR 1.12, 95% CI 1.09-1.16, p<0.0001) the weakest predictor of graft failure. in contrast, the transplant era 2005-2010 (HR 0.90, 95% CI 0.86-0.95, p=0.0001) and high (>1000 cases) transplant center volume (HR 0.82, 95% CI 0.78-0.88, p<0.0001) protected against graft failure. Interestingly, the mean Immunologic Risk Index score increased from 1.47 in 1995 to a peak of 1.52 in 2003, but abruptly declined thereafter reaching a nadir of 1.40 in 2005. Conclusions: the Immunologic Risk Index is a superior risk predictor compared to individual immune risk factors. A decline in the Immunologic Risk Index after 2003 might explain the improvement in graft survival observed in the 2005-2010 transplant era. Immunologic risk aversion and/or the unintended consequences of changes made to kidney allocation policy in 2003 are implicated. Policy reform may be necessary to preserve equal access to renal transplantation among high immunologic risk patients.

Delayed Activation of Innate Immunity in Murine Model of Diabetic Wounds: Initiating An Aberrant Inflammatory Response

2012-02-01

Introduction: Diabetic wounds are a costly and morbid condition requiring continuous wound care, frequent debridement, and in some cases, amputation. Aberrant immune responses, characterized by delayed infiltration of inflammatory cells, paucity of growth factors and persistence of the inflammatory phase of wound healing, are well-documented manifestations in chronic diabetic wounds. the relationship of these findings has not been well described. Skin resident immune cells function as a first line of defense in the activation of the inflammatory wound-healing cascade. Skin resident γδ T cells, also known as Dendritic Epidermal T cells (DETC), recognize damage-associated molecular patterns. Upon activation, DETC recruit neutrophils and monocytes to the wound bed and stimulate fibroblast and keratinocyte proliferation. Abnormal activation and signaling from these cells would result in poor clearance of pathogens and debris, faulty ECM formation, and delayed keratinocyte migration. We hypothesize that wounds in leptin receptor deficient, diabetic mice will exhibit decreased activation of DETC resulting in atypical macrophage phenotypes. Methods: An excisional wound-healing model was implemented with leptin receptor deficient (db/db) mice and their wild type (WT) littermates. on post-operative days (POD) 3 and 7, wounds were photographed, and skin was harvested. Wound samples were dispersed in an enzyme cocktail to single cell suspension. Cells were stained with two antibody panels for 12-color Flow Cytometry (FC) to identify surface markers for leukocyte subtypes, skin homing, and activation states. Results: the leptin receptor deficient model of Type II Diabetes was validated with serial blood glucose and body mass readings (p<0.05) (n>5; 2-tailed Ttest). Delayed wound healing was observed in the db/db group with widened epithelial gap on photomicroscopy (p<0.05). DETC in wounds of db/db animals had lower levels of surface activation markers CD25 (p=0.03) and CD44 (p=0.02) on POD3. Activation of these cells in skin was comparable to WT on POD7. on POD3, Ly6G+ neutrophils were increased (p=0.006) and activated macrophages (F480+, MHCII+, CD11b high) (p=0.003) were decreased in db/db wounds. Activated macrophages were also decreased in the db/db wound on POD7 (p=0.02). Total wound macrophages were equivocal in both groups on POD3 and POD7. Conclusions: Diabetic mice show an impaired ability to activate DETC upon wounding. Blunted activation of DETC likely leads to delayed recruitment of neutrophils and inflammatory monocytes in the early phases of wound healing. the result is persistent neutrophils and less activated macrophages in the wound on POD3 and 7. Ineffective macrophage activation could lead to poor clearance of wound debris and pathogens as well as altered signaling within the microenvironment of the wound. These findings could explain the improper regulation of the inflammatory wound-healing cascade and the clinical manifestations of chronic diabetic wounds.

Depletion of Circulating Gamma-Delta(GD) T-Lymphocytes Increases Mortality in Hemorrhagic Shock

2012-02-01

Introduction: Hemorrhagic shock-resuscitation is an activator of the innate immune system with a resulting systemic inflammation response post-injury. the GD T-lymphocyte is a unique CD3+, CD4-/8- lymphocyte that functionally resides between the innate and adaptive arms of the immune system and plays a role in neutrophil activation and end-organ damage in reperfusion. the role of this cell-type in hemorrhagic shock and resuscitation has yet to be fully elucidated. We hypothesized that depletion of GD T-cells would decrease the inflammatory response following hemorrhagic shock-resuscitation. Methods: Control Sprague Dawley rats and those injected with TS2/9, a GD-depleting antibody, underwent controlled hemorrhage by cannulation of femoral vessels to a mean arterial pressure of 30mmHg. After 30 minutes the animals were resuscitated with normal saline and blood and survived to 6hr or until death. Following necropsy, leukocyte flow cytometric analysis, liver gene expression (84 targets), and serum cytokine profiling (23-plex) was performed. Statistical Methods: Significance defined as p<0.05. Results described as mean±SEM. Results: GD depletion was confirmed with flow cytometry (>95%). 100% of the control animals survived to 6hr, while only 37.5% of GD-depleted rats survived (p=0.016). Pre-shock GD-depleted rats had significantly higher serum levels of CXCL1 (GRO-KC: 1913pg/ml±172 vs. 1151pg/ml±136,p=0.006), IL-2 (1567±551 vs. 27±7, p=0.033), Leptin (24875±2206 vs. 14973±3057, p=0.019), and CCL3 (MIP-1a: 35±11 vs. undetectable, p=0.03). GD-depleted rats surviving to 6hr demonstrated significantly higher levels of IL-2 (1401±614 vs. 21± 1, p=0.011), and IL-4 (371±174 vs. 71±34, p=0.047) at 360min compared to the controls. Liver analysis revealed a 3.4-fold down-regulation of IL-11 (p=0.029) gene expression in all GD-depleted rats vs. controls at 6hrs/death. Conclusions: the GD-depleted group exhibited a significantly higher mortality compared to controls. This suggests that GD T-lymphocytes are more immunologically protective than detrimental in shock. Down-regulation of the IL-11 gene, an immunosuppressive cytokine, and the increases in serum pro-inflammatory cytokines, demonstrates a heightened inflammatory response and potentially increased risk for MODS and MOF. the pro-inflammatory cytokine elevations prior to initiation of hemorrhage suggest that GD-depletion may create a “primed” basal state. Thus, it appears that the presence of these cells confer protection in recovery from hemorrhagic shock. Further investigations to elucidate the full role of GD T-lymphocytes in the immunologic response and attributed morbidities/mortality in hemorrhagic shock-resuscitation are warranted.

Interleukin-23 Mediates Hepatic I/R Injury Through IL-17 and CXCL1/2

C.E. Bartels, A. Spadaro, S. Ueki, N. Murase, Q. Du, D.A. Geller — 2012-02-01

Introduction: Interleukin-23 (IL-23) is an inflammatory cytokine known to induce CD4+ cell differentiation into Th17 cells and promote secretion of interleukin-17 (IL-17). CXCL1 and CXCL2 are chemokines that recruit PMN leukocytes and hematopoietic stem cells. Their expression can be induced by IL-17. Previously, we have shown a crucial role for IL-23 that mediates liver ischemia/reperfusion injury (I/R) through the IFN-g/IRF-1 pathway. Since IL-23 is known to induce IL-17 secretion, we hypothesize that IL-23 also mediates liver I/R injury through IL-17 and CXCL1/2. Methods: Rat (Sprague-Dawley) hepatocytes (HC) and non-parenchymal cells (NPC), and mouse RAW cells were used for in vitro studies. Mouse liver transplant (OLTx) was performed in syngeneic C57BL/6 mice. in vitro hypoxia was induced in 1% oxygen incubator. Real-time PCR (qRT-PCR) was used to analyze mRNA expression. Western blotting and immunoflourescent staining were used to analyze protein expression. Results: (1) IL-23 expression is significantly induced early during hypoxia (1%) in rat HC and NPC co-culture system. IL-23 mRNA and protein expression were measured using qRT-PCR and Western Blotting following 1 hour hypoxia and subsequent normoxia. See table. (2) Hypoxia (1%) increased IL-23 receptor (IL-23R) expression on mouse RAW cells and rat NPC following 1 hour of hypoxia and subsequent normoxia in a time dependent manner coinciding with IL-23 cytokine expression. Receptor expression was detected by immunoflourescent staining. See image. (3) IL-23 treatment (20 ng/ml) of rat HC and NPC co-culture results in increased expression of IL-17. IL-17 expression was detected using qRT-PCR. See table. (4) IL-17 production is increased early (peak at 3 hours) in mouse OLTx I/R injury. IL-17 expression was measured using qRT-PCR. See table. (5) CXCL1 and CXCL2 production is increased early in mouse OLTx. Chemokine (C-X-C motif) ligand 1/2 (CXCL1 and CXCL2, respectively) production was detected using qRT-PCR. See table. Conclusions: These data identify that hepatic I/R injury in vivo and hypoxia in vitro induce IL-23 and IL-23R expression. IL-23 signaling induces IL-17 expression, which is known to induce chemokine expression of CXCL1/2. During in vivo experiments, IL-23, IL-17, and CXCL1/2 were unregulated earlyfollowing OLTx. Combined with our previous findings, these results indicate that IL-23 initiates hypoxic I/R injury in the liver through both the IFN-g/IRF-1 and IL-17/CXCL1/2 pathways.

REDD1 Mediates Liver Ischemia-Reperfusion Injury Through NF-kB in A TLR4-Dependant Manner

R.E. Eid, J. Evankovich, R. Zhang, G.W. Nace, H. Huang, A. Tsung — 2012-02-01

Introduction: Hepatic ischemia-reperfusion (I/R) injury is encountered during transplantation, trauma, shock, and elective liver surgeries. the novel protein REDD1 (Regulated in Development and DNA damage responses 1) has recently been found to be key regulator of various signaling pathways activated in hypoxia, cell stress and DNA damage. Depending on the cell context, REDD1 can either play a protecting or a detrimental role. However, little is known about the signaling pathways that regulate and implicate REDD1 function, especially in I/R. in this study we propose that a key link between the initial insult and the activation of inflammatory signaling pathways following liver I/R involves the induction of REDD1 with subsequent activation of NF- κB in a TLR4-dependant manner. Methods: Hepatocytes and non-parenchymal cells were isolated from C57BL/6 or REDD1 KO mice and cultured in normoxic vs. hypoxic (1%) conditions. Western blots and RT-PCR were used to quantify protein and mRNA levels respectively. in vivo studies were conducted using C57BL/6 and REDD1 KO mice. Mice were subjected to a nonlethal model of segmental (70%) hepatic warm ischemia for 1 hr, followed by reperfusion for various time points. Liver damage was assessed via measurement of serum ALT (sALT) levels in addition to histological analysis. Results: REDD1 KO mice exhibit significant protection following liver I/R when compared to WT mice. This is demonstrated by decreased sALT levels (figure1) in addition to decreased sinusoidal congestion and necrosis on liver histology. Tissue levels of the proinflammatory cytokines TNF-α and IL-6 were also significantly decreased in REDD1 KO mice compared to WT controls. in cultured hepatocytes and non-parenchymal cell (NPC), REDD1 levels were induced as early as 30 min by either hypoxia (1%) or treatment with reactive oxygen species (ROS) donors. Treatment of hypoxic hepatocytes or NPC with N-acetyl cysteine, an ROS scavenger, inhibited the expression of REDD1 both at the protein and mRNA level in a dose-dependant fashion. Oxidative stress-induced REDD1 expression was dependent on toll-like receptor 4 (TLR4) as treatment with a TLR4 inhibitor abrogated the induction of REDD1 in WT cells. to determine the mechanism of REDD1-mediated cellular injury, WT and REDD1 KO hepatocytes were stimulated with conditions of oxidative stress and phospho-p65 levels were analyzed. Phospho-p65, a marker of NF- κB activation, was significantly reduced in the REDD1 KO group compared to WT cells. Conclusions: These results demonstrate that REDD1 is a crucial mediator of inflammation and injury following liver I/R and implicate NF-κB as a downstream target through TLR4 signaling.

Calcium/Calmodulin-Dependent Protein Kinase IV Limits Organ Damage in Hepatic Ischemia/reperfusion Injury Through Induction of Autophagy

2012-02-01

Introduction: Sterile inflammatory insults such as ischemia/reperfusion (I/R) injury result from pathogenic factors including damage associated molecular pattern (DAMP) signaling, activation of innate immunity, and up-regulation of proinflammatory cytokines. at the same time, a number of pro-survival pathways are also activated during I/R. Autophagy is one such protective process thought to be involved in I/R injury. in this study we investigated the role of calcium/calmodulin-dependent protein kinase IV (CaMKIV) in hepatic I/R injury and provide evidence that this protein promotes a protective autophagic response in hepatocytes undergoing ischemic stress. Methods: A model of warm partial hepatic I/R was performed to study in vivo activation of CaMKIV by Western Blot. Organ damage, assessed by sALT, histopathology, and circulating cytokines, were measured in CaMKIV KO mice and their WT counterparts subjected to I/R. Autophagy signaling was assessed by transmission electron microscopy and LC3B Western Blot. Results: CaMKIV KO mice experience significantly worse organ damage after I/R, and this effect is independent of systemic cytokine and HMGB1 release. Hepatocytes in livers of CaMKIV KO mice after I/R showed marked deficiency in autophagic signaling as determined by decreased number and volume of autophagosomes by TEM, which correlated with increased accumulation of damaged mitochondria and necrosis. Rapamycin restored autophagic signaling in hepatocytes of CaMKIV KO mice after I/R and reduced organ damage to WT levels. in vitro, we show that CaMKIV activation by oxidative stress induces autophagy in mouse hepatocytes as evidenced by LC3B Western blot and increased LC3B punctae by immunofluorescent staining. CaMKIV KO hepatocytes or WT hepatocytes treated with CaMKIV siRNA or showed reduced autophagic signaling by LC3B Western Blot and were more sensitive to oxidative-stress induced cell death. Conversely, both CaMKIV activation and induction of autophagy protect hepatocytes from oxidative stress-induced cell death. Lastly, we show in vitro that the mechanism of CaMKIV-mediated autophagy in hepatocytes is dependent on CREB signaling. Conclusions: CaMKIV KO mice experience increased damage during I/R in a mechanism dependent on deficient autophagic signaling in hepatocytes, and restoration of hepatocyte autophagic signaling reduces damage to WT levels. CaMKIV mediated activation of autophagy in I/R is a protective mechanism that promotes hepatocyte cell survival during ischemic injury and thus represents a novel therapeutic strategy in I/R injury.

Differential Mediation of Cold Ischemia Reperfusion Injury By BH3 Pro-apoptotic Proteins in Steatotic Hepatocytes

2012-02-01

Introduction: the BH3 proteins Bim and Bid are well described mediators of apoptotic cell death which is active in lean hepatocytes during cold ischemia and reperfusion (CIR) injury. Their role in the setting of steatosis where necrosis is thought to predominate is unclear. We hypothesized that inhibition of Bim and Bid would improve steatotic hepatocellular viability during CIR. Methods: Steatotic hepatocytes were isolated from obese Zucker rats and cultured until confluent as our lab has previously reported. the steatotic phenotype was confirmed by measuring intracellular triglycerides. Lean Zucker hepatocytes were isolated as a control. SiRNAs against Bim and Bid were used to knock down the corresponding gene expressions. Cold ischemia was produced by replacing the media with HTK and maintaining the cells in an anaerobic chamber at 4°C for 6 hours. Reperfusion was recreated by replacing the hepatocyte culture medium and warming to 37°C for 6 hours. Bim, Bid and caspase 3 expressions were measured by qRT PCR. Flow cytometry and fluorescent microscopy assessed cell viability and apoptosis. Results: the cells isolated from obese rats maintained steatotic phenotype in culture. Bim and Bid siRNAs resulted in gene knockdown (KD). Apoptosis was more prominent feature following CIR in lean cells compared to steatotic (82.8% vs 58.2% apoptotic cells among dead cells, p= 0.04). Following CIR, the steatotic cells had a greater expression of Bim (8.85 vs 4.06 fold, p<0.05) and Bid (7.32 vs 3.8 fold, p<0.05) compared to lean. Lean cells had higher Caspase3 (3.55 vs 1.92 fold, p=0.04) expression. KD of Bid or Bim alone failed to protect the steatotic cell lines. However, KD of both Bim and Bid simultaneously significantly decreased apoptosis (42.7% vs 58.2%, p<0.05) and improved steatotic cell viability (53.3% vs 66.9%, p<0.05) following CIR. This is contrasted to the lean hepatocytes, which had protection with knockdown of Bim (30.1% vs 82.2%, p=0.03), Bid (32.8% vs 82.2%, p<0.05) and both (31.3% vs 82.2%, p<0.05). Conclusions: BH3 proteins are a major mediator of cell death following CIR in liver. the roles of BH3 proteins seem to differ between the lean and steatotic cells. in the lean hepatocytes, inhibiting the expression of either Bim or Bid can improve cell viability during CIR. Whereas in the steatotic cells, knockdown of both Bid and Bim was required to protect the cells from CIR injury. This data shows that the BH3 proteins are active mediators in CIR injury and they act differently in the setting of hepatic steatosis. Further investigation into this mechanism may yield potential targets for therapeutic intervention.

Adenosine Bolus Injection Prior to Cardioplegia Enhances Preservation of Heart Grafts in Rats

S. Lim, S. Lee, X. Peng, N. Shigemura, T. Billiar, Y. Toyoda, A. Nakao — 2012-02-01

Introduction: Adenosine opens potassium channels leading to rapid cardiac arrest; has antioxidant, anti-inflammatory and vasodilative properties; and has been used as protective cardioplegia during cardiac surgery. However, adenosine is not administered during organ procurement for cardiac transplants. in this study, we hypothesized that bolus injection of adenosine prior to cardioplegic induction could be an effective adjunct to cardioplegia infusion after cross-clamping the aorta, and may be a clinically accessible alternative to current heart procurement practices. Methods: We used 60-week old female Lewis rats as donors (7 per group). When harvesting the grafts, we injected saline (1 ml; control) or adenosine (0.1 or 0.2 mg/kg in 1 ml saline) before cardioplegia, perfused with 3 ml of cold Celsior,and stored in Celsior for 6 hours at 4°C. the grafts were heterotopically transplanted into syngenic, 12-16 week-old male recipients. Gross and microscopic appearance, serum troponin I and creatine phosphokinase (CPK), and graft proinflammatory mRNA levels were analyzed as markers of graft injury. Results: Bolus injection of adenosine resulted in significantly faster cardiac arrest after perfusion with cardioplegia and faster reanimation after reperfusion compared with controls. After 3 hours of reperfusion, tissue ATP content was higher in adenosine-treated group than in the saline-treated controls. Adenosine treatment significantly reduced myocardial injury, as indicated by serum troponin I, CPK, and TNF-α levels 3 hours after reperfusion. the grafts with adenosine treatment exhibited less upregulation of proinflammatory mRNAs and had fewer infiltrating macrophages. the 2 doses of adenosine tested elicited similar effects. Conclusions: Adenosine injection prior to perfusion of cardioplegia significantly reduced cold ischemia/reperfusion injury in cardiac grafts. This procurement protocol may be clinically feasible and should be considered in the clinical setting.

Donor Treatment With Inhaled Hydrogen Gas Induces Clara Cell Protein 16 and Mitigates Cold Ischemia/Reperfusion Injury in Rats

Y. Tanaka, T. Kawamura, K. Isse, N. Shigemura, T.R. Billiar, Y. Toyoda, A. Nakao — 2012-02-01

Introduction: Hydrogen is an important physiologic regulatory gas molecule with antioxidant, anti-inflammatory and antiapoptotic effects on cells and organs, which underlie its potential therapeutic benefits for various lung injuries. We have previously shown that donor treatment with inhaled hydrogen protects lung grafts from cold ischemia/reperfusion (I/R) injury and induces heme oxygenase (HO)-1. However, the mechanisms underlying hydrogen's protective effects against lung I/R injury remain largely unknown. We conducted a preliminary gene array analysis to begin to elucidate the mechanisms underlying the protective effects of preloaded hydrogen in lung grafts. One upregulated gene encoded the secretoglobin family member clara cell protein 16 (CC16; also called CC10 or uteroglobin), which is produced by the nonciliated cells of the tracheobronchial epithelial tree and has anti-inflammatory and antioxidant properties. the purpose of this study was to investigate the CC16 expression during lung cold I/R injury and hydrogen treatment to further understand how hydrogen mitigates lung cold I/R injury. Methods: Donor male LEW rats (6 per treatment) were exposed to mechanical ventilation with 98% oxygen plus 2% nitrogen or 2% hydrogen for 3 hours prior to harvest and the lung grafts were stored for 4 hours in cold Perfadex. the recipients received 100% oxygen during surgery and until sacrifice 2 hours after reperfusion for evaluation of the graft function and molecular analysis. the right lungs were analyzed by gene array analysis, western blot and immunohistochemistry. the left lungs were orthotopically transplanted into fully allogenic BN rat recipients. Results: Preloading hydrogen by mechanical ventilation resulted in a significant increase in CC16 mRNA and protein expression in the donor lungs, compared with the lungs ventilated with control nitrogen (Figure 1A and B). Immunohistochemical analysis revealed more prominent CC16-positive epithelial cells in the hydrogen-preloaded allografts compared with control allografts, although there were no obvious histopathological changes in routine histological analysis. CC16 expression correlated well with graft function after transplantation. Hydrogen improved blood oxygenation of the lung grafts (Figure 1C). I/R injury induced TNF-alpha mRNA in the control grafts; TNF-alpha upregulation was significantly attenuated by hydrogen (Figure 1D). Conclusions: Ventilation of lung donors with hydrogen significantly ameliorated lung cold I/R injury and was accompanied by increased expression of CC16 in the lung grafts. Our results showed that CC16 may be involved in the cytoprotection afforded by hydrogen and may define a novel mechanism underlying the protective effects of inhaled hydrogen.

Diabetes is Associated With Increased Mortality in Trauma Patients

2012-02-01

Introduction: Trauma patients with diabetes mellitus (DM) represent a unique patient population as they require aggressive insulin replacement, often independent of elevated blood glucose. Previous studies demonstrate DM is an independent predictor of mortality in patients with traumatic brain injury. We analyzed moderate to severely injured trauma patients without brain injury to determine if DM is associated with increased mortality after major trauma in the absence of brain injury. Methods: the National Trauma Data Bank (version 7.0) was queried for this retrospective analysis to compare mortality in patients with and without DM. Patients were included with injury severity scores >9 and excluded if missing data, age<10 years, major head injury (abbreviated Injury Scale (AIS) >3), or any AIS=6. Demographics were compared in patients with insulin dependent DM (IDDM), non-insulin dependent DM (NIDDM), and those without DM. Logistic regression modeled the association between mortality and DM. Results: Overall 166,103 patients with moderate to severe trauma were analyzed. Mortality was 7.6% and 4.4% in patients with and without DM, respectively (P < 0.0001). Mortality was 9.9% for patients with IDDM and 6.7% for NIDDM (P = 0.0002). Increased mortality associated with DM appeared to be independent of age related DM co-morbidities as mortality differences by decade between DM and no-DM was only significantly higher in patients in their 40's (5.6% v. 3.3%, p<0.001). Multivariable logistic regression analysis demonstrated DM was an independent predictor for mortality (AOR 1.14, 95% CI 1.01-1.28, P=0.036). Conclusions: DM was associated with significantly increased mortality in moderate to severely injured trauma patients. Our data determined that stepwise increases in insulin deficiency from no-DM, to NIDDM, to IDDM led to stepwise increases in mortality; this supports the importance of insulin after trauma.

The Severity of Disparity: Increasing ISS Accentuates Disparate Outcomes Following Injury

2012-02-01

Is the Kampala Trauma Score An Effective Predictor of Mortality in Low-Resource Settings? A Comparison of Multiple Trauma Severity Scores

2012-02-01

Introduction: Injury results in an estimated 5.8 million deaths worldwide annually, with the majority occurring in developing countries. in the developed world, multiple injury severity scores have been developed and validated for trauma patients. Despite widespread use in resource-rich settings, few studies have supported effectiveness of these scores in the developing world. As a result, the Kampala Trauma Score (KTS) was developed for resource-limited settings and has been shown to be a robust predictor of death. However, the literature supporting its utility over other tools is limited to retrospective analyses and small patient cohorts. This study evaluates trauma scores as predictors of mortality in a large prospective patient cohort from the emergency ward of a teaching and referral hospital in Cameroon. Methods: This prospective study was conducted in the emergency ward of the Central Hospital of Yaounde, the largest trauma volume hospital in the capital of Cameroon. Collected information included patient demographics, clinical presentation and outcome. for each patient, Kampala Trauma Score (KTS), Injury Severity Score (ISS), Revised Trauma Score (RTS), Glasgow Coma Scale (GCS) and Trauma Injury Severity Score (TRISS) were calculated. Scores were evaluated as predictors of mortality using logistic regression models. Comparisons between areas under ROC curves were made using DeLong's nonparametric jackknife method. Results: A total of 2855 patients were enrolled. 73% of patients were male; 60% presented after a road traffic injury. the median age was 28 years. 60% of patients had injuries classified as mild (<9) on the ISS scale, 29% moderate (9-15), 8% severe (16-24) and 3% profound (> 24). the mortality rate was 0.6%. 2472 patients with non-missing values for KTS, ISS, RTS, GCS, and TRISS were included in logistic regression analysis which showed each score to be a statistically significant predictor of mortality. ROC curves are shown. the area under the ROC for KTS as a predictor of mortality was 0.7748 (95% CI: 0.6285 - 0.9212). No pairwise difference between ROC areas of KTS and other scores was statistically significant. Similar results were found when analysis was limited to severe injuries. Conclusions: This comparison analysis of KTS to trauma scores from the developed world supports the adoption of KTS for injury surveillance and triage in resource-limited settings. As severity scores are a necessary tool for evaluating patient care, it is important to develop and validate scoring systems for use in the developing world. We show that the Kampala Trauma Score is as effective as other scoring systems in predicting patient mortality. As KTS is simple to administer and record, it is therefore a valuable tool available for resource-limited settings.

Population-Based Analysis of Blunt Splenic Injuries in Children: Using Operative Rate as a Quality of Care Indicator

M. Hsiao, B. Haas, D. Gomez, C. De Mestral, S. Sharma, J.C. Langer, A.B. Nathens — 2012-02-01

Introduction: in hemodynamically normal children with blunt splenic injury (BSI) and no other indications for abdominal surgery, the standard of care is non-operative management. Several studies have reported that non-pediatric hospitals and non-trauma centers have higher operative rates in children with BSI compared to pediatric hospitals and trauma centers. We propose using operative rate for BSI as a quality indicator for hospitals caring for pediatric trauma patients. Methods: We performed a population-based retrospective cohort study of children (≤ 18 years) with BSI admitted to all acute-care hospitals in Canada from 2002-2010. the main outcome measure was the rate of operative management for BSI (total splenectomy, partial splenectomy, splenorraphy). Regression models were constructed to evaluate the relationship between rate of operative management and different hospital types (pediatric vs. non-pediatric; trauma vs. non-trauma). These models also allowed for generation of hospital-level observed to expected (O/E) ratios for rate of operative management. Results: We identified 2,923 children with BSI. 80% were male, with a median age of 15 years (IQR 12-17) and a median injury severity score (ISS) of 9 (IQR 4-20). the most common mechanisms of injury were motor-vehicle collision (53%), fall (23%), and other blunt trauma (23%). the majority (73%) were isolated splenic injuries and the grade of splenic injury was specified in 44% of cases (39% grade I or II; 61% grade III, IV, or V). the overall operative rate was 11% (n=315), of which 9% were total splenectomy and 2% were spleen-preserving operations. the operative rate was higher in non-pediatric hospitals than in pediatric hospitals (13% vs. 2%, p<0.001). the operative rate was also higher in non-trauma centers than in trauma centers (16% vs. 8%, p<0.001). After adjusting for patient age, gender, mechanism of injury, splenic injury grade, and ISS, admission to a non-pediatric hospital was associated with a higher probability of receiving operative management (OR 8.7, 95% CI 4.3-17.6), as was admission to a non-trauma center (OR 3.0, 95% CI 2.2-3.9). Outlier status (CI of O/E ratios excluding 1) was determined to identify centers with higher or lower than expected operative rates (Figure 1). Conclusions: the operative rates for children with BSI are significantly higher in non-pediatric hospitals and in non-trauma centers. in these hospitals that do not routinely care for pediatric trauma patients and in hospitals with higher than expected operative rates, we have identified an opportunity for quality improvement initiatives including dissemination of operative rate benchmarks, hospital-level performance reports, and clinical practice guidelines.

National Trauma Volume and Mortality By Time of Arrival: Implications in the Era of Resident Duty Hour Restriction

A. Yaghoubian, C. De Virgilio, A. Kaji, B. Putnam, A. Neville — 2012-02-01

Introduction: Given the increasing restrictions on surgical resident duty hours, we sought to evaluate the National Trauma Data Bank (NTDB) to identify peak hours of trauma volume and severity to optimize personnel deployment. We also wanted to determine whether mortality was worse when patients were cared for by surgeons working late night shifts Methods: Retrospective review of NTDB from 2002 to 2006. Data was analyzed to determine hourly volume of: trauma arrivals, patients with injury severity score (ISS)> 15, operative trauma, penetrating trauma, and death. Peak hours for these variables were compared to nonpeak hours. Trauma volume, injury severity, need for surgery, and mortality, were also measured hourly and in 8 hour time shifts. Univariate and multivariable analysis were performed to determine predictors of mortality. Results: 1,043,320 patients were analyzed. 11% sustained penetrating injury, 19% had an ISS>15, and overall mortality was 4.3%. Trauma volume, number of patients with ISS > 15, those needing surgery, and trauma deaths all began rising at 1400 hours, and sequentially rose for 4 consecutive hours (Figure). the top 8 hours for these outcomes all occurred between 1400 and 2200 hours, and when compared to the 16 nonpeak hours, accounted for nearly 50% of overall trauma patients, the severely injured, and operative cases (all p<0.0001). (Table). Peak hours for penetrating trauma occurred between 1900-0300. on multivariable analysis adjusting for ISS > 15, penetrating trauma, and age, hour and time period of arrival were not predictive of mortality (OR 1.002 CI 0.99-1.01, p=0.7), whereas ISS (OR 15.8, 95%CI 15.4-16.3) and penetrating mechanism (OR = 3.4, 95%CI 3.4-3.6) were highly statistically significant predictors of mortality. Conclusions: Nearly 50% of all traumas, the severely injured, those needing surgery and deaths arrive between 1400 and 2200 hours. in the era of diminishing resident work hours, manpower should be aligned to match peak trauma volume and severity. Since mortality is overwhelmingly predicted by ISS and not by the time shift of arrival, further restricting work hours is unlikely to result in an improvement in trauma patient mortality.

Nonlinear Statistical Algorithms Improve Probability of Survival Prediction Using TRISS Variables

2012-02-01

Introduction: Assessment of probability of survival (Ps) based on injury severity is important for recognition of quality improvement opportunities and uniform comparisons between trauma centers. Since TRISS methodology was developed in 1987 using logistic regression modeling, other predictive modeling methods have been developed. We propose that newer nonlinear statistical methods may more accurately predict mortality using the same variables as TRISS and that the superiority of nonlinear statistics is greatest at the middle of the injury severity curve. Methods: This was a retrospective study including all trauma patients admitted to a level 1 trauma center from January 2008 to December 2009; thermal injuries were excluded. We collected demographic and outcome data; in particular the variables used to calculate TRISS. the Probability of Survival was calculated with TRISS variables using 3 different algorithms:Support Vector Machine (SVM), Naïve Bayes Classifiers (NB) and AdaBoost. the performance of these algorithms was compared to that of the TRISS methodology using area under the receiver operating characteristic curves (AUC), overall and by ISS ranges. Results: 6337 patients were included, 607 were excluded: 370 with thermal injuries and 237 with missing data. the median age was 29 (IQR: 16, 49) and 69% were male. Overall mortality was 4% (283/6337). the predominant mechanism of injury was blunt 87% (5524/6337). Regarding overall prediction of mortality, TRISS (AUC= 0.971) was equivalent to AdaBoost (AUC= 0.974; p = 0.16), and superior to both SVM (AUC= 0.94; p< 0.001) and Naïve Bayes (AUC=0.961; p = 0.05). in ISS ranges <15, TRISS (AUC=0.956) was equivalent to Naïve Bayes (AUC=0.934; p=0.24) and AdaBoost (AUC=0.949; p=0.67) but outperformed SVM (AUC=0.763; p<0.001). in the ISS ranges between 15 and 50, TRISS (AUC=0.918) was inferior to AdaBoost (AUC=0.942; p=0.03) superior to SVM (AUC=0.88; p = 0.02) and equivalent to Naïve Bayes (AUC=0.89; p=0.1). in the ISS ranges >50, TRISS (AUC=0.833) was equivalent to AdaBoost (AUC=0.8; p=0.65) and to Naïve Bayes (AUC=0.795; p=0.62) and superior to SVM (AUC=0.588; p=0.01). Conclusions: Using the same variables, other nonlinear statistical algorithms for survival prediction are not superior to TRISS. However in the middle ranges where mortality rates are neither very high nor very low, AdaBoost methodology outperforms TRISS.

Differences in Intra-Cranial Pressure Monitor Utilization and Subsequent Outcomes Between Insured and Uninsured Patients After Traumatic Brain Injury

2012-02-01

Introduction: Disparities in outcome after Traumatic Brain Injury (TBI) have been previously demonstrated for uninsured patients. However, the cause of this discrepancy is not well defined. We hypothesized that there are differences in Intra-Cranial Pressure (ICP) monitor utilization and subsequent outcomes between insured and un-insured patients after TBI. Methods: This was a retrospective of review of the most recent National Trauma Databank research datasets from 2007-2008. All patients that met ICP insertion criteria as set forth by the most recent Brain Trauma Foundation Guidelines were included in the analysis. Relevant demographic data, injury characteristics, invasive procedures, and outcomes were abstracted from the dataset. Patients were stratified by insurance into respective Private, Medicaid, and Uninsured groups. Multiple logistic regression was used to derive factors associated with ICP monitor insertion and stepwise logistic regression used to identify independent predictors of mortality. Results: A total of 25,585 patients met inclusion criteria with 14.5% of all patients having ICP monitors placed. Uninsured patients had the lowest rate of ICP monitor placement (13.2%, p< 0.05) despite being younger and presenting with a similar degree of head injury [Head AIS (mean all groups 4.1) and total GCS (mean all groups 3.9)] and overall injury burden [Total ISS (mean all groups 27.8) and SBP <90mm Hg (10.7%)]. After adjustment for confounding factors; uninsured patients were 20% less likely to receive ICP monitoring (AOR 0.8; CI 0.8-0.9) though there was no significant difference in craniotomy between groups. Stepwise logistic regression identified lack of insurance as an independent predictor for death after TBI (AOR 1.4; 1.3-1.5). Conclusions: Uninsured patients were noted to have a significantly lower association of ICP monitor placement after TBI and subsequently increased mortality. This discrepancy and its consequent affect on outcomes after TBI in the uninsured should be explored in future studies.

Elevated Admission Blood Pressure After Trauma: Tolerated in the Elderly

A. Gangi, M.B. Singer, M.A. Clond, M. Bukur, D.R. Margulies, A. Salim, E.J. Ley — 2012-02-01

Introduction: Hypotension predicts poor outcomes in elderly trauma patients. Recent studies also demonstrate that hypertension is an independent predictor of mortality in patients with traumatic brain injury (TBI). We undertook the current study to determine the impact of elevated admission systolic blood pressure (SBP) on elderly trauma patients without TBI. Methods: We conducted a retrospective review of the Los Angeles County Trauma System Database to identify all patients with moderate to severe traumatic injury (injury severity score >9) who required admission between 2003 and 2008. Patients with head abbreviated injury score ≥3 were excluded. Multivariate regression modeling was used to identify admission SBP thresholds that predict increased mortality in young (20-49 years), middle aged (50-69 years) and elderly patients (≥70 years). Results: 23,931 patients met inclusion criteria. Overall mortality was 8.6% and increased with age across the three groups. the admission SBP thresholds associated with significantly increased mortality in the young and middle aged were 190mmHg (AOR 1.54, CI 1.02-2.33, P=0.04) and 180mmHg (AOR 1.53, CI 1.09-2.14, P=0.01), respectively. in the elderly, we identified no admission SBP threshold associated with significantly increased mortality. Interestingly, several elevated admission SBP thresholds were associated with significantly reduced mortality in the elderly (≥150mmHg AOR 0.61, CI 0.48-0.78, P<0.0001; ≥160mmHg AOR 0.61, CI 0.47-0.78, P=0.0001; and ≥170mmHg AOR 0.72, CI 0.55-0.95, P=0.02). Conclusions: Elderly trauma patients tolerate higher admission SBP than their younger counterparts. in addition, multiple elevated SBP thresholds were associated with significantly reduced mortality in the elderly. These findings question the need for early hypertension treatment in elderly trauma patient without TBI.

Sex-Associated Differences in Access to Trauma Center Care: A Population-Based Analysis

2012-02-01

Introduction: Severely injured patients cared for in a trauma center have lower mortality and improved functional outcomes compared to patients receiving care at a trauma center. Access to trauma center care is dependent on the implementation of evidence-based field trauma triage criteria by EMS personnel. However, data from other healthcare settings suggest women are disadvantaged, with lesser access to high quality medical care than men. We postulated that based on the existence of evidence-based criteria for field trauma triage and inter-facility transfer, women would have equal access to trauma center care compared to men and thus set out to explore the existence of disparities across a regional trauma system. Methods: We performed a population-based retrospective cohort analysis of severely injured (ISS>15) adults surviving to reach hospital. Differential access to trauma center care was evaluated for females compared to males. EMS providers and emergency physicians both determine access to trauma center care – the former from the scene of injury and the latter from non-trauma centers through the process of inter-facility transfer. As such, these two decisions nodes were analyzed separately. the adjusted odd of trauma center care was determined using logistic regression models. Results: We identified 26,861 severely injured patients; 35% were women. 30-day mortality was 16%. Thirty-eight percent of patients were transported directly from the scene to a trauma center. of the remaining 16,524 patients who were initially transported to non-trauma centers, 31% were transferred to a trauma center within 24 hours of initial assessment. A significantly smaller proportion of women received trauma center care compared to men (57% vs. 62%, p<0.001). This association persisted after adjusting for age, number of comorbidities, mechanism of injury, and measures of injury severity: 13% lower odds of trauma center care among females compared to males (OR 0.87, 0.79-0.96). EMSpersonnel were 12% less likely to transport females from the field to a trauma center compared to males(33% vs. 41%, p<0.001; OR 0.88, 0.81-0.97). Similarly, emergency medicine physicians were 15% less likely to transfer females to trauma centers compared to males (24% vs. 36%, p<0.001; OR 0.85, 0.73-0.99). Conclusions: in this population based analysis of severely injured patients, women were less likely to receive care at a trauma center compared to men. Our results suggest that these sex-associated differences occur both at the level of the EMS provider and the physician making triage decisions in the ED. This inequity in access to care is particularly important given that the benefits of trauma center care for the severely injured are well established. Further exploration through qualitative approaches is required to understand the causes to better mitigate these inequities.

ORIF Vs. Arthroplasty in Femoral Neck Fractures: A Review of A National Database

2012-02-01

Introduction: Hip fractures represent the second leading cause of hospitalization for elderly patients. Total hip arthroplasty (THA), hemiarthroplasty (HA), and open reduction internal fixation (ORIF) are treatment options for hip fractures. Optimal surgical treatment for displaced femoral neck fractures remains unclear. the aim of this study is to compare 30-day post-operative outcomes of THA, HA, and ORIF among elderly patients with displaced femoral neck fractures within a national surgical database. Methods: A retrospective analysis of the American College of Surgeons National Quality Improvement Program for the periods January 2005 through December 2009 was conducted. Included were patients 65 years or older who underwent THA, HA, and ORIF, for displaced femoral neck femoral fractures. Patient demographics, co-morbidities, and complication rates were compared across the three treatment options. Logistic regression model was used to assess variation in overall morbidity and mortality following surgery, adjusting for age, gender, ethnicity, co-morbidities, functional status prior to surgery, behavioral risk factor, and time from admission to surgery. Results: A total of 3,172 patients met the inclusion criteria. of these patients, 534 underwent ORIF, 358 HA, and 2,271 THA, respectively. Most patients were White (87.5%, n=2,642), female (63.6%, n=2,017), and had an overall mean age of 76. Regarding pre-operative co-morbidities, patients who underwent HA had the highest rate of co-morbidities (91.4%, n=62), followed by ORIF (86.6%, n=96), and THA (23%, n=561). in bivariate analysis, patients who underwent HA had the lowest rate of post-operative complications. on adjusted multivariate analysis, patients who underwent THA had a lower likelihood of developing any complication, including infection, respiratory, sepsis, cardiovascular, and death when compared to ORIF (Table 1). Conclusions: Our study demonstrates that THA had less 30-day post-operative complications and a lower mortality rate than HA and ORIF in elderly patients with displaced femoral neck fractures.

Vagal Nerve Stimulation Blocks Burn Injury-Induced Priming of Peritoneal Macrophages

2012-02-01

Introduction: Large surface area burn injuries lead to activation of the innate immune system. Activated, hyper-responsive macrophages are thought to be responsible for the increased susceptibility to sepsis which is seen after severe burn. This supports a two-hit model in which the macrophage inflammatory response is dependent on pre-existing levels of immune cell activation. We, and others, have shown that activation of the parasympathetic nervous system via vagal nerve stimulation (VNS) can alter the inflammatory response to injury. We hypothesized that VNS would decrease peritoneal macrophage responsiveness to an inflammatory insult after severe burn injury. Methods: Male balb/c mice were subjected to a 30% total body surface area steam burn with and without electrical stimulation to the right cervical vagus nerve. Peritoneal macrophages were collected at various time-points after injury. to assess the degree of macrophage hyper-responsiveness, peritoneal macrophages were incubated in LPS or vehicle. Flow cytometry was then used to identify F4/80+ macrophages and to analyze the phosphorylation state of the NFkb pathway mediator, p65 Rel A in this population. Results: There was a significant increase in macrophage NFkb p65 Ser 536 phosphorylation in macrophages exposed burn injury, followed by LPS treatment. Neither burn alone, nor LPS alone was capable of increasing macrophage NFkb p65 Ser 536 phosphorylation. Increased NFkb p65 Ser 536 phosphorylation was seen within 4 hours after burn injury, with phosphorylation returning to baseline by 24 hours post-burn. Treatment with VNS significantly reduced NFkb p65 Ser 536 phosphorylation in burn-primed macrophages that were exposed to LPS. Finally, VNS mediated an unexpected reduction in baseline NFkb p65 Ser 536 phosphorylation levels in sham animals. Conclusions: VNS prevented priming of peritoneal macrophages after severe burn injury, characterized by decreased phosphorylation of NFkb p65 Ser 536. Moreover, VNS limited the baseline inflammatory state of macrophages in uninjured animals. These studies suggest that VNS mediates the inflammatory response in peritoneal macrophages by affecting the set point of LPS-responsiveness.

Beta Adrenergic Antagonists in Sepsis: Effects on Survival in A Murine Model

R.S. Friese, J. Weller, P. Rhee — 2012-02-01

Introduction: Distinct from their actions on the cardiovascular system, beta-blockers also have profound effects on modulation of the inflammatory response and metabolism. However, sepsis-induced myocardial depression has resulted in clinical avoidance of beta-blockers as treatment in severe sepsis. Potential benefits of beta-blockade in severe sepsis include blunting activation of the sympathetic nervous system resulting in decreased myocardial oxygen demand as well as modulation of the immune response. the purpose of this study was to examine the effect of beta-1 selective blockade on survival after endotoxemia. We hypothesized that beta-1 selective blockade initiated after septic insult would prolong survival in a murine model. Methods: 8-12 week old C57BL/6J male mice underwent intra-peritoneal injection of 12.5mg/kg of lipopolysaccharide (LPS). Four hours post LPS injection anesthetized animals underwent cannulation of the right external jugular vein with placement of an osmotic pump (Alzet; Cupertino, CA) in the subcutaneous space of the back. Pumps were primed to continuously deliver a beta-1 selective antagonist (esmolol hydrocholide; delivered at 6.7ug/kg/min) or an equal volume of normal saline (control). Primary outcome was survival at 120 hours (5 days) post LPS injection. Kaplan-Meier survival analysis with log-rank test was utilized to explore for mortality differences between groups. Significance was defined as p<0.05. Results: Mean pre-protocol weight was 25.6 + 2.2 mg. Continuous beta-1 selective antagonist infusion resulted in increased survival at five days post LPS injection. (Figure) Mean survival after LPS injection with beta blockade was 70.4 + 36.4 hours (n=24). While mean survival after LPS injection without beta blockade was 48.1 + 25.4 hours (n=23). Hazard ratio for mortality with beta-1 selective blockade is 0.40 (95% CI; 0.20-0.79). Conclusions: Continuous infusion of a beta-1 selective antagonist initiated after septic insult improves survival at five days in a murine model. Our findings support the use of beta-1 selective blockade in the treatment of sepsis. Further studies exploring mechanisms for this improved survival are warranted.

Single Nucleotide Polymorphisms and Type of Steroid Impact Functional Response of the Human Glucocorticoid Receptor

2012-02-01

Introduction: Clinical trials evaluating the use of steroids in septic shock have shown variable outcomes, yet little insight has developed as to why some patients improve with steroids and others do not. Studies have implicated polymorphisms of the human glucocorticoid receptor (hGR) coding sequence as a possible cause of altered steroid response. We previously identified a combination of 3 single nucleotide polymorphisms (SNP) of the hGR coding sequence that altered response. in order to further evaluate the role of hGR variations in humans, we further hypothesize that hGR polymorphisms along with type of steroid will influence the stress response and may lead to individualized tailoring of steroid therapy. Methods: Total RNA was isolated from 97 healthy human blood samples and surveyed for the hGR gene. the National Center for Biotechnology Information hGRα sequence was used as a reference for comparison. Two unique SNPs we previously studied, and shown to result in decreased hGRα activity in the absence of steroid (A214G and T962C), were selected for further evaluation with various steroids. Derivative hGRα isoform constructs which isolated the non-synonymous SNPs were created (A214G-hGRα and T962C-hGRα). Luciferase activity was measured to assess functional response of HEK293 cells transfected with these hGR isoforms using a reporter assay in the presence of graded concentrations of 0.9% saline, hydrocortisone(HYD), methylprednisolone(MPS) and dexamethasone(DEX). Significance was determined using ANOVA. Results: Each isoform had a unique dose-response curve to each steroid with a peak activity depending on concentration and type of steroid. the presence of either SNP A214G or T962C within hGRα, resulted in a decreased response when compared to hGRα when stimulated with HYD at a concentration range from 1x10−6 μM to 1 μM (P<.05). the same decreased response compared to hGRα occurred for the SNPs with DEX stimulation, but at a much lower concentration range than HYD (1x10−9 μM to 1x10−6 μM). in the presence of MPS, the presence of SNP A214G resulted in greater activity when compared to hGRα at a concentration range of 1x10−6 μM to 1x10−2 μM (P<.05), whereas the presence T962C resulted in activity equivalent to hGRα (Figure). Conclusions: SNPs alter the response of the hGR in both the presence and absence of steroids. in addition, different steroids appear to elicit unique response profiles depending on the specific SNP. A greater understanding of hGR polymorphisms and steroid response may further elucidate possible mechanisms explaining the variable response seen with patient treatment. Potentially, therapy could be individually tailored based on genetic polymorphisms and steroid type.

Neutrophil Extracellular Traps Are Generated By Reactive Oxygen Species Through Toll-Like Receptor 4

D.C. Miller, G.W. Nace, D. Chen, H. Huang, A. Tsung — 2012-02-01

Introduction: Neutrophil extracellular traps (NETs) are complexes of DNA, granular enzymes and histone proteins released by activated neutrophils during sepsis. NETs function to bind, disarm and destroy infectious microbes extracellulary following stimulation by lipopolysaccharide. However, the role of NET formation in non-infectious stimuli, such as oxidative stress, is unknown. Therefore, we sought to determine if neutrophils activated by reactive oxygen species (ROS) release NETs. Methods: Neutrophils were isolated from femurs and tibias of rats and HeJ (TLR4 Mutant) and HeOUJ (WT) mice using a percol gradient. Cells were treated at varying time points with xanthine oxidase and its substrate hypoxanthine (XO/HX), hydrogen peroxide, phorbol-myristate-acetate (PMA, positive control) and cell culture media (negative control). Slides were fixed and stained for DNA and histones. NETs release was verified by immunofluoresence and quantified by calculating the percentage of extranuclear histones with high field microscopy analysis. Results: NETs stained positively for DNA and histone proteins, and appeared as web-like structures extending extracellularly from neutrophils (Image 1). Neutrophils treated with hydrogen peroxide or XO/HX released NETs in a time dependant manner with peak release at 12 hrs post treatment, whereas neutrophils with media treatment alone released no NETs. Furthermore TLR4 WT neutrophils co-treated with XO/HX released NETs while TLR4 Mutant neutrophils released significantly less NETs. NETs stimulated with PMA, the positive control, appeared as extracellular histones and DNA in close proximity to the neutrophil of origin while oxidative-stressed NETs extended multiple diameters further and overlapped with other cells, suggesting that NETs released due to oxidative stress may be more intense than NETs release due to PMA in murine neutrophils. Conclusions: This study demonstrates that oxidative stress can induce the release of NETs from activated neutrophils. Further, NET generation involves TLR4, a receptor found to play a critical role in both infectious and non-infectious inflammatory conditions. These results provide evidence that NETs may play a previously unrecognized role in sterile inflammation.

Radiation Combined With Thermal Injury Results in Specific Alterations in Adaptive and Innate Immune Cell Populations

A.E. Mendoza, W.J. Brickey, C.J. Neely, J. Ting, R. Maile, B.A. Cairns — 2012-02-01

Introduction: Radiological catastrophes remain a legitimate threat to human health. the continued development of nuclear weapons and the potential of radiological accidents call for an improved understanding of the pathophysiology after radiation exposure and radiation combined with injury (RCI). RCI has been shown to increase morbidity and mortality at lower doses of ionizing radiation. We hypothesize that RCI will result in sustained reduction of lymphocyte populations and specific alterations of innate cell populations compared to irradiation or burn alone. Methods: C57BL/6 female mice age 10-12 weeks underwent either a 20% TBSA full thickness contact burn or sham procedure followed by a single whole body dose of 5 Gy radiation. the spleen and wound draining lymph nodes were harvested 14 days after sham, burn, radiation or RCI. Flow cytometry was performed on spleen and pooled wound draining lymph node (DLN) cell suspensions to identify adaptive and innate cells. Tukey's test was used to compare absolute cell number means. A P value of <0.05 was defined as significant (P <0.05*; P<0.01**; P<0.001***). Results: in our model of RCI, mice undergoing 5 Gy irradiation and 20% TBSA burn injury resulted in 30% mortality. Mice receiving 5 Gy irradiation alone all survived. Flow cytometry of DLN and spleen revealed significant changes in the adaptive and innate arms of the immune system. T cells were significantly decreased after irradiation (e.g. mean±SEM: 1.6x10^5±2.1x100 cells/DLN; n=5) and RCI (4.2x100±1.5x100cells/DLN; n=4) in lymph nodes when compared to sham (1.3x100±1.3x105cells/DLN; n=4, *) and burn (3.8x10^6±3.0x10^5 cells/DLN; n=4, ***). These differences remained significant in the spleen. Neutrophils were significantly decreased at this time point after radiation and RCI in the DLN and spleen (data not shown). A population of cells identified as GR.1+ CD11b+, which we and others have defined as immunosuppressive myeloid derived suppressor cells (MDSC), was significantly elevated in DLN of RCI mice (5.0x10^4±1.1x10^4cells/DLN) compared to both sham (1.3x10^4±1.2x103cells/DLN,*) and radiation alone (7.1x103±1.7x103cells/DLN,**). MDSC numbers did not differ between sham and radiation alone. These differences remained significant in the spleen. Conclusions: RCI results in significant T cell lymphopenia, neutropenia and expansion of immunosuppressive MDSC within the secondary lymphoid organs late after injury. Taken together, these specific changes likely explain the increased and sustained immunosuppression and mortality observed after this combination of injuries. This specific immune dysfunction signature only occurs in RCI, and not burn or radiation alone. These data define RCI as a chronic immunosuppressive outcome. Future studies need to address and develop specific immune countermeasures.

Activated Mesenchymal Stem Cells Increase Wound Tensile Strength in Old Mouse Model Via Macrophages

S. Lee, E. Szilagyi, L. Chen, L.A. DiPietro, A.M. Bartholomew — 2012-02-01

Introduction: Tissue damage with a subsequent delay in wound regeneration affects morbidity, mortality, and increases healthcare costs. This is especially true for older population groups where the natural ability to regenerate tissue has been diminished. We have previously observed that a treatment of 50,000 dose of autologous MSC activated with interferon gamma (aMSC) increased tensile strength in a younger mouse model. Drawing from these observations, we tested the extent to which aMSC could improve wound tensile strength in an older mouse model. in addition, we also explored the possible role of macrophages in aMSC's augmentation of wound healing. Methods: A B6 mouse model testing 5 therapies was used to determine the extent to which activated MSCs augmented wound tensile strength in older animals (>12 months). Therapies were injections of aMSC, aMSC after macrophage depletion with clodranate, only clodranate treatment, fibroblasts (3T3 cell line), and vehicle. Cell doses of 50,000 were injected into 4cm linear incisions on the dorsum on each animal and then sutured closed. Macrophage depleted animals were treated with intraperitoneal injections of clodranate for 5 days prior to wounding. After 7 days, histological analysis and tensile strength measurements of the wounds were conducted. Results: in older mice, it was found that the tensile strength of healing wounds was significantly lower as compared to younger animals (<2 months) when only vehicle was administered (100% +/- 11.4 vs. 381% +/- 24.3, p = 3.67E-4). Older mice treated with aMSC showed significant and substantial increases in tensile strength of healing wounds when compared to older control mice treated with only vehicle (100% +/- 16.4 vs. 270% +/- 43.3, p = 4.61E-5). Macrophage depleted older mice treated with aMSC had tensile strength not significantly different compared to controls (104% vs. 100% +/- 21.8, p = n/s). Lastly, there was less pronounced increase in tensile strength in older mice treated with fibroblasts when compared to older controls (100% +/- 9.81 vs. 164% +/- 47.9, p = 4.44E-2). Conclusions: When compared to vehicle only controls, aMSC treatment had a significant statistical increase in wound tensile strength in older mice. the application of aMSC brought the augmented tensile strength close to that of younger control mice, suggesting that aMSC may be an effective therapy option for enhanced healing in older animals with a naturally diminished ability to repair wounds. Macrophage depleted older mice did not differ significantly in wound tensile strength, suggesting that these cells may act as a mediator for the aMSC in enhancing wound healing. Further study of this mechanism is planned. aMSC also enhanced wound healing better than fibroblasts, a current clinical treatment option, suggesting a potential alternative therapy option. Taken together, this data provides encouraging support for the development of aMSC therapies for enhanced tissue regeneration, especially for older population groups.

XBP1 Inhibition Delays Wound Healing

2012-02-01

Introduction: Correct protein folding is essential to cells, and the unfolded protein response (UPR) is a protective measure against accumulation of misfolded proteins activated in the endoplasmic reticulum (ER). Ire1, a protein in the ER membrane, senses the presence of misfolded proteins in the ER lumen. Upon activation, IRE1 mediates mRNA splicing of Xbp1, leading to excision of 26bp fragment and creation of a new open reading frame. This causes the protein product to become an active transcription factor that promotes transcription of genes important in the adaptive response. Xbp1 activation has been implicated in a number of diseases, including cancer and diabetes, but has never been studied in wound healing. Due to increased protein translation that takes place during wound healing, we proposed that Xbp1 plays a role in skin wound healing. Methods: XBP1-LUC transgenic mice have a construct the luciferase (LUC) gene driven by Xbp1 enhancer sites and functions as a reporter. These mice underwent excisional wounding and were treated with irestatin, an Ire1 inhibitor, or DMSO as the control. the mice were imaged regularly to demonstrate activation of XBP1. the wounds were harvested at different time points, fixed and processed for histological analysis. Immunohistochemistry (IHC) for luciferase was used to identify cells expressing Luc as a surrogate for Xbp1 activation. Masson's trichrome staining and picro-sirius red staining was performed to evaluate collagen deposition and arrangement. in vitro scratch assay using mouse embryonic fibroblasts (MEFs) derived from XBP1 knockout mice and WT MEFs as control was performed, and imaged at various time points after scratch using phase contrast microscope. Results: Luciferase imaging indicated that Xbp1 was active in skin wound beginning at 8 days after wounding with a peak at 12-14 days. IHC demonstrated Xbp1 activation in basal keratinocytes and fibroblasts within the wound area. Treatment with irestatin was able to significantly decrease Xbp1 activation in the wound. Mice that had been treated with irestatin showed much delayed wound healing with full re-epithelialization not occurring until 22 days compared to 14 days for DMSO controls. Masson's trichrome staining revealed significantly decreased blue stain suggestive of decreased collagen deposition and this was confirmed using Picro-Sirius Red staining. in vitro scratch assay showed KO MEFs had a significant delay in coverage of the denuded area when compared WT MEFs with WT MEFs having closed the gap in 3 days while KO MEFs still had not at 6 days. Conclusions: Xbp1 plays a significant role during wound healing. Blocking activation with Irestatin leads to a significant delay in wound healing possibly through decreased deposition of collagen. Further mechanisms involving decreased fibroblast migration have also been implicated by our data. Our results suggest that modulation of the UPR can offer a new approach to wound healing therapies.

Use of Xenogenic Acellular Dermal Matrix As Resurfacing Graft to Promote Epithelialization of Avascular Wounds

H.R. Zahiri, J.A. Stromberg, R.P. Silverman, A.C. Greene, L. Holton, D.P. Singh — 2012-02-01

Introduction: the process of epithelialization restores the integrity of the epidermal layer after injury. This process requires angiogenesis to restore wound perfusion and promote granulation tissue formation. We investigated the promotion of angiogenesis by xenogenic acellular dermal matrix (XADM) as sole resurfacing graft for avascular full-thickness wounds of various sizes. Methods: Forty full-thickness 4x4 cm wounds were created paraspinally on the dorsum of 5 Yorkshire pigs, extending from dorsal thoracic to superior lumbar region. for experimental wounds, 1x1 cm or 2x2 cm silastic sheets were sutured to the muscle and fascia in the center of the wounds prior to resurfacing with XADM. Positive controls had no silastic sheets and negative controls had 4x4cm silastic sheets under XADM. Wounds either received negative pressure or bolster dressings. After 21 days, animals were euthanized and the central and lateral regions of each wound was biopsied. After immunohistochemistry with hematoxylin & eosin (H&E) and Factor VIII stains, the number of newly formed blood vessels per biopsy sample was randomly counted in a blinded manner using light microscopy. Groups were compared using one-way analysis of variance with Newman-Keuls multiple comparison posttest. Significance was P value ≤.05. Results: Grossly, after 21 days, granulation tissue and epithelialization was present peripherally and centrally for 1x1cm avascular wounds as well as for positive controls. for 2x2cm avascular wounds, granulation tissue and epithelialization was formed peripherally but had not yet reached central regions of viable XADM. Histological analysis revealed significantly more blood vessels per mm2 for 1x1cm avascular wounds compared to 2x2cm (27.8 vs. 13.3, p=0.005) and 4x4cm negative controls (27.8 vs. 8.7, p=0.005). No significant difference in angiogenesis was present between 1x1cm avascular wounds and positive controls (27.8 vs. 30.2, p>0.05). Conclusions: XADM as sole resurfacing graft has the potential to initiate and accelerate wound healing, including for avascular wounds caused by diabetes, vascular disease, trauma, or debilitation.

Notch1 Signaling Regulates G1-S and G2-M Phase in Keratinocyte Cell Cycle During Wound Healing

J. Stofflet, M. Roy, S.J. Schossler, B. Allen-Hoffmann, H. Chen, T.W. King — 2012-02-01

Introduction: Notch signaling, specifically Notch1, is known to play a key role in epidermal development; however, the role in tissue repair remains relatively unknown. to study the role of Notch signaling in wound healing, we used NIKS cells, a well-characterized, nontumorigenic human keratinocyte cell line. Western blot and QPCR analysis confirmed similar expression patterns for the Notch family of signaling molecules when comparing NIKS cells to independent primary keratinocytes. These data indicate that NIKS accurately represent the behavior of human keratinocytes and are therefore an ideal model to study Notch signaling during wound healing. Previous research from our lab suggests that DAPT, a widely used Notch inhibitor, causes a reduction in cell proliferation. However, since DAPT inhibits all four isoforms of mammalian Notch, we investigated the role of the specific Notch receptor that regulates keratinocyte proliferation. NIKS grown in 3D cultures form fully functional skin tissue and both Notch1 and 2 preferentially localize in the proliferating basal layer; with Notch1 expression being predominant. Therefore, we hypothesized that Notch1 signaling plays a key role in keratinocyte proliferation during wound healing. Methods: NIKS cells were transiently transfected with Notch1 siRNA to knockdown Notch1 expression or a non-specific siRNA as control; knockdown was confirmed by Western blot. Cell growth was studied using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell lysates of non-specific and Notch1 siRNA treated cells were analyzed for cell cycle arrest markers cyclin B1 and cyclin D1, and the apoptotic markers, activated caspase 3 and activated PARP, by Western blot to determine if reductions in cell growth were due to cell cycle arrest or due to apoptosis. Results: A 20% inhibition of growth was detected in the siRNA transfected cells for up to 72 hours following transient knockdown of Notch1 compared to non-specfic siRNA control (p<0.01). There was also a reduction in cyclin D1 (7-fold) and cylcin B1 (50%) protein levels at 24 hours in NIKS that were treated with Notch1 siRNA compared to control. Levels of total Caspase 3 and total PARP remained unchanged while activated caspase 3 and activated PARP were undetected in both control and Notch1 siRNA transfected NIKS. Conclusions: NIKS cell growth is inhibited when Notch1 signaling is attenuated. the reduction of cyclin D1 and cyclin B1 in Notch1 siRNA transfected cells suggests that Notch1 regulates G1-S and G2-M transition in keratinocyte cell cycle, respectively. in addition, the lack of apoptotic markers activated caspase 3 and activated PARP indicate that mechanism of keratinocyte growth inhibition resulting from diminished Notch1 signaling is due to cell cycle arrest. Understanding the molecular mechanisms of wound healing will help develop techniques that could modify levels of Notch receptor to heal wounds more efficiently.

Heparin-Binding EGF-Like Growth Factor (HB-EGF) Promotes Mesenchymal Stem Cell (MSC) Proliferation and Migration and Protects MSC from Injury

D.J. Watkins, C. Chen, G.E. Besner — 2012-02-01

Introduction: We have previously demonstrated that mesenchymal stem cell (MSC) administration protects the intestines from injury in a mouse model of intestinal ischemia/reperfusion (I/R) injury. We have also shown that heparin-binding EGF-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent in vivo, and our studies suggest that HB-EGF may protect the intestines via its effects on SC. the goal of the current study was to examine the effects of HB-EGF on MSCs in vitro. Methods: MSCs were isolated from the femurs of pan-EGFP mice, grown in MSC-specific culture medium, and purified by sequential passages based on the adherence properties of the cells. MSC pluripotency was confirmed by inducing differentiation into osteocyte and adipocyte lineages. MSCs were incubated with increasing concentrations of HB-EGF and proliferation quantified using the CyQuant cell proliferation assay kit. MSC chemotaxis was quantified using the CHEMICON QCM cell migration assay kit 24h after exposing 0.5x106 cells/ml to increasing concentrations of HB-EGF in serum-free media. to assess cell viability after injury, MSCs were incubated with increasing concentrations of HB-EGF and then subjected to 24h of anoxia (95% Nitrogen/5% CO2) followed by 24h of re-oxygenation. Apoptosis was then determined by caspase-3 immunohistochemistry (IHC), and quantified as the ratio of apoptotic cells to total cells. Results for cell proliferation and migration were normalized to the results for non-HB-EGF-treated cells which was set at 100%. Statistical significance was determined using the Student's t-test. Results: MSCs exposed to normoxia had significantly increased proliferation in the presence of HB-EGF compared to no HB-EGF (100 ± 5.8%) at HB-EGF concentrations of 10 ng/ml (155 ± 9.9%, p=0.001), 25 ng/ml (156 ± 17%, p=0.01) and 50 ng/ml (147 ± 23%, p=0.03). MSC migration was significantly increased compared to no HB-EGF (100 ± 14%) at HB-EGF concentrations of 5 ng/ml (118 ± 9.6%, p=0.01), 10 ng/ml (135 ± 11%, p=0.001), 25 ng/ml (140 ± 37%, p=0.05) and 50 ng/ml (132 ± 15%, p=0.008). Caspase-3 IHC demonstrated significantly decreased apoptosis compared to no HB-EGF (9.7 ± 3.2% apoptotic cells) at HB-EGF concentrations of 10 ng/ml (6.6 ± 0.8%, p=0.04), 25 ng/ml (4.3 ± 1.5%, p=0.02), 50 ng/ml (3.0 ± 1.7%, p=0.006) and 100 ng/ml (3.6 ± 2%, p=0.009). Conclusions: These data indicate that HB-EGF promotes MSC proliferation and chemotaxis, and decreases apoptosis in MSC exposed to anoxic stress. the effects of HB-EGF on MSC in vitro suggest that co-administration of HB-EGF and MSC in vivo may have synergistic therapeutic effects.

Mechanistic Importance of Oxidant Generation and Detection of Tyrosine Hydroxylation By Isotope Dilution Tandem Mass Spectrometry in Lung Contusion

D. Machado-Aranda, S.V. Madathilparambil, B. Yu, S. Pennathur, K. Raghavendran — 2012-02-01

Introduction: Lung contusion (LC) is an independent risk factor for ALI/ARDS and VAP. Previous work in our laboratory suggests that upregulation of superoxide dismutase (SOD) 1& 2 correlated with the resolution of hypoxia and inflammation in LC. Tyrosine is a major target of post-translational oxidative modification of proteins. Oxidation reactions of tyrosine include chlorination [to form chlorotyrosine; catalyzed by myeloperoxidase (MPO)], nitration (to from nitrotyrosine; mediated by reactive nitrogen species (RNS), and dimerization (to form dityrosine; mediated by tyrosine radicals/ROS).The following studies used liquid chromatography/isotope dilution tandem mass spectrometry (MS/MS) to accurately measure tyrosine oxidation markers as a surrogate for oxidant generation after LC in the lung samples. Additionally, in separate experiments we examined the role of cell permeable SOD mimetic (MnTBAP) in animals following LC. We hypothesized that MnTBAP will reduce lung injury/inflammation and markers of tyrosine oxidation will be significantly reduced. Methods: Rats with appropriate uninjured controls (n=7) received bilateral isolated LC from blunt trauma as previously described. the lung samples harvested at 5, 24 hr. was subjected to MS/MS. Levels of nitrotyrosine, chlorotyrosine and dityrosine averaged to tyrosine levels were measured. in separate experiments 10mg/ml MnTBAP (cell permeable and acts on SOD1, 2 and 3) was administered by i.p. injection concurrently with LC. the BAL fluid and lung samples at 5/24 hr. were assessed for qualitative/quantitative cytology and cytokine production. Additionally these lung samples were subjected to MS/MS. A statistical analysis using One-way ANOVA was performed and p<0.05 was considered significant (*). Results: Levels of nitrotyrosine and dityrosine were particularly sensitive markers of oxidant generation in LC. the number of neutrophils in the BAL (as determined by diff-Quik) was significantly lower in the rats with LC with MnTBAP (Figure). Additionally BAL albumin a sensitive indicator of permeability injury and IL-6 an indicator of increased inflammation were also significantly reduced in the same group. Additionally, levels of nitrotyrosine and dityrosine levels were significantly reduced at both 5 and 24 hr time points following the administration of MnTBAP. in summary the group receiving Mn TBAP had reduced lung injury and inflammation. Conclusions: These data clearly indicate that (1) Oxidants produced in LC are responsible in part for increased lung injury and inflammation (2) the feasibility to detect and quantify these novel protein oxidation biomarkers in lung tissue using highly sensitive and specific MS-based approaches.

Inhalation of Hydrogen Reduced Hyperoxic Lung Injury in Rat Through Heme Oxygenase-1 Induction

2012-02-01

Introduction: Highly concentrated oxygen is routinely administered to patients who can't breathe efficiently. Over a prolonged period, oxygen toxicity can occur, causing hyperoxic lung injury and respiratory failure. Successful abrogation of hyperoxic lung injury is critical and could strongly impact clinical practice. Recently, hydrogen has been recognized as a therapeutic medical gas and a gaseous signaling molecule. the purpose of this study was to determine if hydrogen could reduce hyperoxic lung injury and investigate underlying mechanisms. We highlighted the critical role of the redox-dependent transcription factor,NF-E2-related factor 2 (Nrf2), in regulating expression of heme oxygenase (HO)-1, a heme-degrading enzyme that is highly induced by oxidative stress and protects against oxidative damage including hyperoxia. Methods: We randomly assigned inbred male Lewis rats to four experimental groups and administered the following gas mixtures for 60 hours: air (normoxic conditions) with either 2% nitrogen or 2% hydrogen or 98% oxygen (hyperoxic conditions) with either 2% nitrogen or 2% hydrogen. We examined lung function by blood gas analysis of the arterial blood. We also examined pleural fluid volume, total protein present in bronchoalveolar lavage fluid, histology, mRNAs for inflammatory mediators, and expression of HO-1 and Nrf2-dependent genes. Results: Exposure to 98% oxygen/2% nitrogen for 60 hours markedly impaired lung function, as shown by poor gas exchange and lower pO2 in the arterial blood. These hyperoxic conditions induced lung edema and lead to the upregulation of the mRNAs for inflammatory mediators. Hydrogen treatment during exposure to hyperoxic conditions (98% oxygen/2% hydrogen) significantly improved pO2 levels and reduced inflammatory events (Table 1). Although hyperoxia induced HO-1, supplementation with 2% hydrogen further increased HO-1 mRNA and protein levels and HO-1 enzymatic activity (Figure 1A-C). Real-time RT-PCR revealed that the Nrf2-dependent gene, NAD(P)H dehydrogenase quinine (Nqo)-1 was upregulated (Figure 1D) in the lungs receiving hydrogen treatment during hyperoxic conditions. This suggests that hydrogen induced HO-1 via the Nrf2 signaling pathway. Conclusions: Inhalation of hydrogencan induce HO-1 and reduce hyperoxic lung injury. Our results suggest that hydrogen modulates Nrf2 and induces HO-1 and other antioxidant proteins. Administering hydrogen by providing gas for the patient to inhale is novel and may be feasible in clinical practice.

Stimulation of Carnitine Palmitoyltransferase 1 Improves Renal Function and Attenuates Tissue Damage After Ischemia/Reperfusion

J.P. Idrovo, W.L. Yang, J. Nicastro, G.F. Coppa, P. Wang — 2012-02-01

Introduction: Renal injury as a result of ischemia/reperfusion (I/R) is a severe clinical problem with a high mortality rate and its treatment still remains on supportive therapy. During I/R, cellular homeostasis is disrupted due to energy depletion. Stimulating energy production in insulted organs may facilitate their recovery from I/R-induced injury. Fatty acid oxidation or β-oxidation is the major metabolic pathway for generating ATP in the kidneys. Carnitine palmitoyltransferase 1 (CPT1) is a key enzyme to control a rate-limiting step of this pathway. C75 is a synthetic cell-permeable α-methylene-γ-butyrolactone compound that can stimulate CPT1 activity. Thus, we hypothesized that administration of C75 increases energy production and alleviates renal injury after I/R. Methods: Male adult rats were subjected to renal I/R by bilateral renal pedicle clamping with microvascular clips for 60 min, followed by i.v. administration of 8% DMSO (vehicle) or C75 (3 mg/kg BW). Blood and renal tissues were collected 24 h after reperfusion for measuring the levels of different markers of renal function, tissue damage and inflammation. Results: CPT1 activity and ATP levels decreased by 58% and 76%, respectively, in the vehicle group in comparison to the sham group, while those in the C75 treated group were restored to the levels comparable to the sham group (p < 0.05). C75 treatment significantly improved the microscopic structure of the kidneys in comparison to the vehicle group, judged by histological examination. Furthermore, C75 treatment also improved renal functions and reduced pro-inflammatory cytokine levels. the measurements among the sham, vehicle, and C75 treated groups are summarized in the table below. Conclusions: Stimulation of CPT1 activity by C75 administration enhanced ATP regeneration, prevented renal deterioration, lowered organ injury indexes, and inhibited the production of pro-inflammatory cytokines induced by renal I/R. Therefore, enhancing energy metabolism by C75 may provide a novel possible modality to treat patients with severe renal I/R injury.

Isoflurane Prevents Acute Lung Injury Through ADP-Mediated Platelet Inhibition

2012-02-01

Introduction: Growing evidence suggests platelets play an essential role in post-traumatic acute lung injury (ALI) and multiple organ failure. Halogenated ethers have recently been shown to interfere with the formation of platelet-granulocyte aggregates, further supporting the theory of the coupling of coagulation and innate immunity in ALI. Isoflurane decreases ALI in sepsis models, but the mechanism is not known. the potential benefit of isoflurane has not been investigated in trauma/hemorrhagic shock (T/HS) models. Therefore, we hypothesized that isoflurane anesthesia attenuates T/HS-mediated ALI through platelet inhibition. Methods: Sprauge-Dawley rats (n=32) were anesthetized by either 50 mg/kg pentobarbital or 0.5% inhaled isoflurane, and were subjected to control, trauma (laparotomy) and sham shock (T/SS), T/HS (laparotomy and hemorrhage-induced shock: MAP of 30 mmHg x 45 min), or were pre-treated with a P2Y12 (ADP) receptor antagonist and anesthetized with pentobarbital prior to T/HS (T/HS Pento ADP Inh). Animals were resuscitated with a combination of normal saline and returned shed blood. BALF protein was measured, and pulmonary immunofluorescence was performed to detect microthrombi. PlateletMapping™ was used to determine specific thrombin-independent inhibition of the ADP and AA pathways of platelet activation. Results: Animals undergoing T/HS, and anesthetized with pentobarbital, developed significant lung leak compared to control and sham animals (*,# p < 0.001) (Figure 1). T/SS animals had a modest increase in ALI (p < 0.001), but isoflurane use abrogated both T/SS and T/HS provoked lung leak (p < 0.001) (Figure 1). Isoflurane prevented pulmonary microthrombi formation following T/HS compared to the pentobarbital group (0.00 vs. 1227.23 microns2) (p < 0.001). PlateletMapping™ revealed specific platelet ADP-pathway inhibition with isoflurane (p < 0.001) without affecting the AA pathway (Table 1). Pentobarbital-anesthetized animals pre-treated with a P2Y12 (ADP) receptor antagonist decreased ALI to sham levels (Figure 1), and confirmed platelet ADP inhibition decreased lung injury without isoflurane. Conclusions: Isoflurane attenuates ALI through an anti-platelet mechanism, in part, through inhibition of the platelet ADP pathway. This platelet inhibition uncouples coagulation from innate immunity, which protects against ALI, and highlights the potential applications of this protective therapy in various ischemia/reperfusion clinical scenarios.

Calcium/Calmodulin-Dependent Protein Kinase II Is Activated By Reactive Oxygen Species During Liver Ischemia/Reperfusion Injury and Promotes Organ Damage Through Release of High Mobility Group Box Protein 1 from Hepatocytes

2012-02-01

Introduction: Hepatic ischemia/reperfusion (I/R) occurs in multiple clinical settings and involves activation of innate immunity, cytokine production, and release of endogenous danger signals such as HMGB1. the mechanisms that account for inflammation and local organ damage are only partially understood but include deranged calcium signaling and activation of Ca+2/Calmodulin Dependent Kinases (CaMK). the purpose of this study was to investigate the contribution of the CaMK II isoform to this process during liver I/R. Methods: WT, NADPH oxidase deficient (p47KO), or WT mice treated with the CaMKII inhibitor, AC3I, were subjected to partial warm ischemia of the left and median lobes of the liver. Liver damage was measured by serum ALT. Serum HMGB1 and tissue CaMKII activation determined by Western blot. HMGB1 localization was analyzed by immunofluorescent staining. in vitro, primary cell cultures were treated with AC3I or antioxidant and exposed to hypoxia (1% O2) or hydrogen peroxide (H2O2). Results: in vivo, hepatic I/R resulted in increased CaMKII activation. CaMKII inhibition with AC3I treatment resulted in protection from liver damage compared to vehicle treated control animals as measured by circulating ALT levels. Inhibition of CaMKII also resulted in decreased circulating levels of HMGB1 compared to controls. Using immunofluorescent staining, we found that hepatocytes of AC3I treated mice also demonstrated decreased HMGB1 nucleo-cytoplasmic translocation compared to control mice (Figure). to determine if the mechanism of CaMKII activation in liver I/R was mediated by oxidative stress, p47KO or WT mice treated with the antioxidant NAC were used. Both p47KO and WT mice treated with NAC were found to have decreased CaMKII activation compared to controls. in vitro, CaMKII activation was observed in hepatocyte and non-parenchymal cell (NPC) co-cultures exposed to hypoxia or H2O2. NPC cultures treated with AC3I and exposed to hypoxia had decreased inflammatory cytokine release compared to control cells. Transfer of cell culture media from oxidative-stressed NPCs to hepatocytes resulted in HMGB1 translocation and release, and this effect was abrogated by CaMKII inhibition in NPCs. Conclusions: CaMKII is activated during hepatic I/R and promotes organ damage through release of proinflammatory cytokines by NPCs and release of HMGB1 from hepatocytes. in addition, the activation of CaMKII occurs in response to NADPH oxidase-mediated oxidative stress. Inhibition of CaMKII leads to liver protection and therefore represents a potential therapeutic option against I/R injury.

The Lipid Mediator Lysophosphatidic Acid (LPA) Governs Microvascular Fluid Leak During Ischemia Reperfusion Injury

A. Garcia, A. Strumwasser, E.J. Miraflor, L.Y. Yeung, J. Sadjadi, G.P. Victorino — 2012-02-01

Introduction: Lysophosphatidic Acid (LPA) is a lipid mediator that disturbs endothelial barrier function and can precipitate endothelial cell loss. LPA is synthesized through the conversion of lysophosphatidylcholine to LPA catalyzed by the enzyme autotaxin (ATX). LPA initiates intracellular cascades by binding G-protein receptors on endothelial cells which leads to deterioration of endothelial barrier function. Our hypothesis was that LPA governs microvascular fluid leak and plays a critical role during ischemia-reperfusion injury (IRI). Our specific aims were: 1) to determine the effect of LPA on microvascular fluid leak, 2) to evaluate the impact of inhibiting LPA synthesis on endothelial cell monolayer permeability during anoxia/reoxygenation and on microvascular fluid leak during IRI, and 3) to evaluate the impact of LPA receptor blockade on microvascular fluid leak during IRI. Methods: in vivo mesenteric venular microvascular fluid leak (Lp) was examined using an intra-vital micro-occlusion technique in rats. First, Lp was analyzed in rats treated with increasing doses of LPA as well LPA + L-NAPSA, a selective LPA receptor antagonist. Next, in vitro monolayer permeability to biotinylated albumin in cultured bovine pulmonary artery endothelial cells was assessed using an absorbance assay during anoxia/reoxygenation with and without H2L9098, a selective inhibitor of ATX that prevents LPA synthesis. in vivo, Lp was analyzed in rats treated with and without H2L9098 during IRI. Finally, we treated rats with L-NAPSA, the selective LPA receptor antagonist, during the reperfusion phase of IRI and Lp was measured throughout IRI for each group as well as for controls. Results: In vivo, LPA increased Lp up to 4-fold in a dose dependent manner (p<0.001) and the LPA receptor antagonist completely blocked this response. Preventing LPA synthesis by ATX inhibition in vitro attenuated a 4.4-fold increase in monolayer permeability induced by anoxia/reoxygenation and returned monolayer permeability back to control levels (p<0.01). in vivo, IRI increased Lp approximately 8-fold. This increase was attenuated 37% by preventing LPA synthesis via ATX inhibition prior to and during IRI (p<0.05). Lp during IRI was decreased by 78% with the LPA receptor antagonist given during reperfusion (p<0.01). Conclusions: LPA governs microvascular fluid leak and plays a critical role during IRI. LPA induces microvascular fluid leak in a dose dependent manner. Preventing LPA synthesis through ATX inhibition attenuates microvascular permeability increases due to IRI. Additionally, LPA receptor antagonism decreases the microvascular fluid leak during IRI. the LPA cell surface receptor is an early common pathway for LPA induced endothelial barrier dysfunction and is a potential therapeutic target for treating microvascular fluid leak observed during IRI.

Nf-κB Promotes Pulmonary Endothelial Cell Survival During Ischemic Acute Kidney Injury

L.E. White, Y. Cui, C. M. Feltes Shelak, F.A. Moore, H.T. Hassoun — 2012-02-01

Introduction: Acute kidney injury (AKI) alters the host innate immune response and induces distant organ transcriptional and phenotypic changes including TNF-α and TNFR1-dependent pulmonary apoptosis. Our prior studies demonstrate activation of NF-κB during kidney ischemia-reperfusion injury (IRI), and we hypothesized that NF-κB plays a critical role in regulating lung apoptosis during ischemic AKI. Methods: Male 6-8 week old mice (C57BL/6J) were treated with Bay 11-7082, a NF-κB inhibitor, or vehicle and underwent 60 minutes of bilateral renal pedicle clamp (IRI) or sham laparotomy (sham). Animals were sacrificed at 24 hours, and 1) pulmonary endothelial cells (ECs) [CD45-CD31+] were isolated by novel tissue digestion techniques followed by antibody-coated magnetic bead sorting, 2) isolated ECs underwent RT-PCR “SuperArray” analysis of 84 apoptosis-related genes (mean relative fold change [FC] by 2-ΔΔCt method), 3) renal function was measured by serum creatinine (mg/dl), 4) lung tissue Complex I (NF-κB) and Complex II (caspase-8) activation was measured by western immuneblotting of nuclear and cytosolic fractions, respectively; and 5) lung cleaved (active) caspase-3 immunohistochemistry (IHC) was performed (relative FC of mean positive cells/hpf for 10 slides/sample). Results: Isolated lung ECs demonstrated altered transcription of genes related to NF-κB (Complex I) activation including DR5/killer (FC=3.05), Bcl10 (FC=2.22), Birc2 (FC=1.86), Traf1 (FC=-2.22), and Casp12 (FC=-2.31) during kidney IRI. Serum creatinine was increased during IRI in Bay 11-7082 mice (0.14±0.02 vs. 1.94±0.07*) and controls (0.18±0.02 vs. 1.94±0.17*) compared to sham. NF-κB (Complex I) activation in controls (1±0.2 vs. 1.93±0.1*) was effectively attenuated in Bay 11-7082 mice (1±0.09 vs. 0.28±0.04*), however neither control (1±0.03 vs. 1.04±0.06, p=0.21) nor Bay 11-7082 (1±0.47 vs. 0.98±0.28, p=0.97) mice demonstrated caspase-8 (Complex II) activation during ischemic AKI. Administration of Bay 11-7082 perpetuated the pro-apoptotic lung phenotype with increased cleaved caspase-3 IHC (FC=0.95±0.06 vs. 6.13±0.7*†) during kidney IRI compared to both sham and controls (1±0.11 vs. 2.96±0.64*). n≥5/group, *p<0.05 for IRI vs. sham, †p<0.05 for IRI in Bay-11 vs. control groups,n≥5/group. Conclusions: TNF-α/TNFR1 signaling mediates kidney-lung crosstalk during ischemic AKI, driving distant organ pulmonary apoptosis and microvascular dysfunction. NF-κB is an important regulator of the balance between cell survival and programmed cell death under these conditions.

Kidney-Lung Crosstalk During Ischemic AKI: Is the T Cell the Missing Link?

L.E. White, M.L. Lie, R.J. Santora, H. Rabb, H.T. Hassoun — 2012-02-01

Introduction: Kidney IRI induces TNF-mediated lung apoptosis which contributes to microvascular injury. T lymphocyte trafficking and activation play a critical role in mediating the local immune response during kidney ischemia-reperfusion injury (IRI), and we have previously shown CD3+ T lymphocyte activation and trafficking to the lungs during ischemic AKI. Utilizing T cell-deficitent (Tnu/nu) mice and adoptive transfer experiments, we investigated the specific role of T lymphocytes in mediating pulmonary apoptosis during kidney IRI. Methods: For adoptive transfer, T lymphocytes from 6-8 week old male C57BL6/J (WT) mice were isolated from spleen tissue utilizing the Pan T Cell Isolation Kit (Miltenyi Biotec). Tnu/nu mice received 5 x 106 isolated T cells IV and were allowed to reconstitute for 24 hours. WT, Tnu/nu, and adoptively-transferred Tnu/nu (Tnu/nu + T cells) mice underwent either 60 minutes of bilateral kidney ischemia or sham laparotomy. All animals were sacrificed at 24 hours, and assays included 1) flow cytometry of CD3+, CD3+CD4+, and CD3+CD8+ spleen lymphocytes to determine T cell reconstitution, 2) detection of lymphocyte activation markers CD25 and CD69, 3) measurement of serum creatinine (mg/dl), and 4) measurement of lung apoptosis by caspase-3 activity (IU/mg protein x 103). Results: Tnu/nu + T cells mice undergoing sham or IRI were effectively reconstituted with a total percentage of CD3+ (5.6±0.7 vs. 7.8±2.3), CD3+CD4+ (2.8±0.3 vs. 5.2±2.3), and CD3+CD8+ (2.5±0.6 vs. 2.2±0.2) T lymphocytes, however there was no increase in activation markers CD25 (8.4±0.6 vs. 6.2±0.9) or CD69 (52.3±7.9 vs. 37.2±5.2). Serum creatinine was increased in WT, Tnu/nu, and Tnu/nu mice + T cells during AKI compared to sham (Table 1). WT mice demonstrated an increase in lung caspase-3 activity during kidney IRI, and neither Tnu/nu nor Tnu/nu + T cells mice exhibited an increase in caspase-3 activity during ischemic AKI compared to sham. *p<0.05 compared to sham, n≥4/group. Conclusions: Pulmonary T cell trafficking and activation are sentinel events during ischemic AKI, likely contributing to the onset of pulmonary apoptosis. Tnu/nu mice demonstrated protection against pulmonary apoptosis, however adoptive transfer failed to restore the lung injury phenotype. Further studies regarding T lymphocyte-mediated pulmonary injury are needed to elucidate their specific role in kidney-lung crosstalk.

Platelet-activating Factor: A Critical Link Between the Inflammation and Coagulation Systems

H.B. Moore, M. Wohlauer, E. Gonzalez, A. Banerje, E.E. Moore — 2012-02-01

Introduction: Platelet-activating factor (PAF) is a tightly regulated inflammatory mediator in a variety of settings; including shock, trauma, and sepsis. A variety of cell types, many of which are central to the inflammatory and haemostatic systems, are capable of synthesizing PAF. the expression of PAF is tightly regulated involving crosstalk between endothelial, inflammatory and vascular cells. PAF, as its name implies, also induces platelet aggregation, however traditional clinical assays that monitor platelet activity commonly employ the agonists ADP and arachidonic acid and not PAF. We therefore hypothesized that PAF would have a potent, independent effect on platelet aggregation using TEG(c) Platelet Mapping, a modern platelet function assay. Methods: Whole blood was collected from healthy volunteers in heparin (68 U/4 ml). Platelet mapping was performed within 2 hrs of sample collection. Platelet function was determined using the TEG platelet mappingassay. A heparinized blood sample was collected, with 360 ml of the blood sample placed into the pre-warmed cup of the TEG analyzer, followed by 10 ml of the prepared activator solution, comprised of reptilase, factor XIIIa, and phospholipids. Reptilase cleaves fibrinogen to generate fibrin and is used to replace thrombin. Next, either ADP (2 μM final concentration, MAADP), or arachidonic acid (1 mM final concentration, MAAA) orPAF (12μM), was added as a platelet agonist. to determine the independent fibrin contribution to the clot, the activator solution was added to 360 ml of heparinized blood (MAFibrin). the maximum hemostatic activity (MAThrombin) was measured using a kaolin activated whole blood sample collected in citrate, rather than heparin. the percent platelet inhibition in response to either the ADP,AA, or TPA agonist was calculated using the following equation: (100 - [(MAADP(AA) - MAFibrin)/(MAThrombin- MAFibrin)× 100] Results: PAF (12μM) induced platelet aggregation was as effective as AA (1mM) and ADP (2μM) in promoting clot formation (29.6 ± 4.1 vs. 44 ± 5.0 MA (mm), p=0.3 PAF vs. AA; 29.6 ± 4.1 vs. 18.9 ± 2.6 MA (mm), p=0.1 PAF vs. ADP) [Table 1]. Conclusions: PAF signaling has a pivotal role linking inflammation and coagulation. Augmenting the current role of PAF in the laboratory as an inflammatory agonist, PAF-mediated platelet aggregation using TEG ® Platelet Mapping has potential to measure the bioactivity of blood and blood products.

Gene Networks Involved With Mechanoregulation of Cell Population in the Fibroblast-Populated 3D Collagen Matrix

M.A. Carlson, J.D. Eudy, L.M. Smith, M.A. Carlson — 2012-02-01

Introduction: The 3D collagen matrix has been used to study mechanical states in wound healing and tissue engineering. Human foreskin fibroblasts (HFFs) in a mechanically-stressed matrix (MS; attached to culture plate) increase their population, while HFFs in a stress-released matrix (SR; detached, floating in medium) decrease their population. Our intention was to use gene array data to uncover relevant gene networks which might mediate the decrease in matrix cell number associated with the MS-SR transition. Methods: HFFs (106/mL) were cultured in bovine collagen (1.5mg/mL; 0.2mL matrix vol; medium = 5% FBS in DMEM). Differentially-expressed (DE) genes in MS vs. SR matrices were identified at 6 and 24hr after SR with gene arrays (3 HFF strains; nonpooled mRNA; 6 chips). DE was corroborated with immunoblotting, ELISA, and qPCR. DE genes were analyzed with third-party software for putative gene networks associated with the MS-SR transition. Relevance of putative networks was tested by augmenting/inhibiting network participants and then measuring matrix cell number 48 and 72hr after SR. Results: There were 187 and 425 DE genes identified at 6 and 24 hr, respectively, including a 10-fold induction of IL6 and IL8 24hr after SR; NF-kB was not DE, but many of its targets were. the software analysis of the DE genes identified >20 putative networks as highly significant in the MS-SR transition; central participants included IL6, IL8, NF-kB, TGF-B1, MAP kinases, Akt, p53, and cyclins. With this data, we initially hypothesized that gene networks with IL6, IL8, or NF-kB helped mediate the decrease in matrix cell number after SR, and then began testing. Matrix treatment with IL6 or IL8 did not affect cell number, but treatment with neutralizing antibodies to either cytokine decreased matrix cell number (Table). As expected, matrix cell number decreased in SR with respect to MS matrices using medium only. Control IgG had minimal effect. Treatment of matrices with TNF-α (stimulant of canonical NF-kB signaling) produced a large decrease in cell number in MS matrices, with minimal effect in SR matrices. Conclusions: It can be speculated that IL6 and/or IL8 release was a counter-regulatory mechanism that mitigated the decrease in matrix cell population occurring after SR, as suggested by (1) the late induction, (2) networks generated from the DE genes, and (3) cell number downregulation after antibody neutralization. NF-kB signaling may have helped drive the population decrease after SR, as suggested by (1) modulation of multiple NF-kB targets, (2) generated networks, and (3) cell number downregulation after TNF-α treatment in the MS matrix. Future studies will focus on dissection of these and other gene networks active in the MS-SR transition.

Modulation of Phenotypic Differentiation Accounts for the Durable Inhibition of Neointimal Hyperpasia By Nitric Oxide

E.S. Moreira, A.K. Vavra, J. Martinez, V.R. Lee, M.R. Kibbe — 2012-02-01

Introduction: Neointimal hyperplasia is a major cause of restenosis following vascular interventions, often leading to procedure failure. Periadventitial delivery of the nitric oxide (NO) donor PROLI/NO provides durable inhibition of neointimal hyperplasia for up to 6 months despite its very short half-life. the aim of this study is to characterize the effect of NO on the phenotypic differentiation of vascular cells following injury. We hypothesize that modulation of cell phenotype within the arterial wall accounts for the durable efficacy of NO at inhibiting neointimal hyperplasia. Methods: The rat carotid injury model was performed in 10-week-old male Sprague Dawley rats. Groups included control, injury, or injury +PROLI/NO (10 mg). Arteries were harvested at 1, 2, 7, 14, and 56 days for immunohistochemistry. Proliferation was assessed by BrdU incorporation. Desmin was used to detect contractile vascular smooth muscle cells (VSMC), non-muscle myosin heavy chain (NM-MHC) was used to detect myofibroblasts (MF), and α -smooth muscle actin (α -SMA) was used to detect MF and VSMC (both synthetic and contractile phenotypes). Results: Overall, injury induced a significant increase in cell proliferation at 3 days in all layers. in the intima and in the media, NO inhibited proliferation for up to 8 weeks. Whereas in the adventitia, NO induced a surprising 60% increase in proliferation at 2 weeks (p<0.05). Assessment of the phenotypic markers revealed interesting patterns. Injury caused a significant increase in all three phenotypic markers (p<0.05). However, while contractile VSMC continued to increase by 8 weeks, MF returned to basal levels. Assessment of the individual layers of the arterial wall revealed distinct effects of NO on cell phenotype. in the intima, NO inhibited VSMC and MF to a similar extent up to 1 week (desmin 80%, α-SMA 80%, and NM-MHC 80% inhibition, p<0.05). However, by 8 weeks, NO inhibited MF to a greater extent than VSMC (NM-MHC 70% vs. desmin 50% inhibition, p<0.05). in the media, NO predominately inhibited the contractile VSMC phenotype (80% at 8 weeks; p<0.05), with little effect on MF (p=0.556). in the adventitia, NO appeared to have no effect on either VSMC or AF. Conclusions: The short-acting NO donor PROLI/NO inhibits neointimal hyperplasia by modulating proliferation and phenotypic differentiation in the arterial wall. While NO inhibited both VSMC and MF in the intima, NO predominately inhibited contractile VSMC in the media, and had no effect on VSMC or MF in the adventitia. Thus, the increase in proliferation observed in the adventitia is due to regulation of cell types other than VSMC and MF, such as endothelial cells of the vasa vasorum or resident or circulating progenitor cells. Further studies are needed to elucidate the effect of NO on the adventitia and its role in inhibiting neointimal hyperplasia.

EphB4 Activates Endothelial Nitric Oxide Synthase in An Akt-dependent Mechanism to Regulate Vein Graft Adaptation

2012-02-01

Introduction: EphB4, a receptor tyrosine kinase, is a marker of venous identity in adult veins that also controls wall thickness during vein graft adaptation to the arterial environment. Since the role of endothelial nitric oxide synthase (eNOS) in vein graft adaptation is not well understood, we evaluated whether eNOS is a downstream effector of EphB4 mediated signaling that controls vein graft thickness. Methods: Intrathoracic inferior vena cava was harvested from wild-type (WT) or eNOS knockout (KO) mice and placed as an interposition vein graft into the infrarenal aorta of WT mice. Vein graft thickness was assessed weekly by ultrasound. After 3 weeks, vein grafts were harvested and analyzed by histology. EphB4 was activated in WT or Akt-KO mouse lung endothelial cells (MLEC) in vitro using the bivalent ligand Ephrin-B2/Fc in a time course fashion for up to 60 minutes. Cell lysates were examined via Western blotting for differences in eNOS-phosphorylation. in some experiments WT MLECs were pre-treated with Wortmannin for 60 minutes prior to Ephrin-B2/Fc stimulation. EphB4 colocalization with phospho-eNOS in WT MLECs was assessed by immunofluorescence microscopy following 30 and 60 minutes of Ephrin-B2/Fc stimulation. Nitric oxide (NO) production was assessed using a NO-specific chemiluminescence analyzer. Results: Vein grafts derived from eNOS-KO mice showed significantly less wall thickening compared with WT vein grafts (n=7, p=0.04). EphB4 activation resulted in increased NO production from WT MLECs (176.0pmol vs 111.7pmol; p=<0.001; n=3) and co-localization of EphB4 with phosphorylated eNOS. Interestingly, EphB4 activation stimulated eNOS phosphorylation in a novel bimodal distribution, with maximal increases in eNOS phosphorylation at both 1 and 60 minutes (n=5; p<0.005). the initial, but not the late peak of eNOS phosphorylation was inhibited by wortmannin (n=2). Similarly, Akt-KO MLECs showed an early but not a late peak of eNOS phosphorylation (n=2). Conclusions: Eph-B4 stimulates eNOS phosphorylation in adult venous cells via a novel intracellular signaling pathway dependent on Akt. Both EphB4 and eNOS are critical for normal vein graft adaptation to the arterial circulation, and eNOS may be the mechanism by which EphB4 mediates vein graft adaptation. Selective promoters of eNOS phosphorylation may also promote normal vein graft adaptation and reduce vein graft failure.

Cytoplasmic Tyrosines 653 and 774 Are Critical for Eph-B4 Signaling to ERK1/2 and Akt

C.D. Protack, A. Muto, M.J. Collins, C. Jadlowiec, A. Dardik — 2012-02-01

Introduction: Eph-B4, a receptor tyrosine kinase that determines vascular venous fate during vasculogenesis, is downregulated during vein graft adaptation to the arterial circulation. We have previously shown that stimulation of Eph-B4 with Ephrin-B2/Fc prevents vein graft thickening. However, the mechanisms by which Eph-B4 activation prevents vein graft thickening are unknown. Similarly, we have also shown that Eph-B4 phosphorylation is critical to Eph-B4 action; therefore we examined whether several of the 15 phosphorylation sites in the cytoplasmic portion of Eph-B4 are critical to Eph-B4 stimulation of intracellular signal transduction pathways. Methods: A wild-type Eph-B4 plasmid vector was created, followed by three individual point mutations at tyrosine 581, 653, and 774, with substitution of phenylalanine for tyrosine. Mutations were confirmed by sequencing. Cos-7 cells were transfected with wild-type or mutant Eph-B4 (Y581F-, Y653F-, and Y774F-Eph-B4). Transfected cells were starved for 24 hours and then stimulated with Ephrin-B2/Fc (1 min). Cell lysates were examined via Western blotting for differences in tyrosine-phosphorylation, and phospho-Akt, phospho-ERK1/2, and phospho-FAK activity between wild-type and mutant Eph-B4. Double-labeled immunofluorescence of wild-type and mutant Eph-B4 transfected Cos-7 cells was utilized to examine Eph-B4 tyrosine phosphorylation and colocolization of Eph-B4 with caveolin-1. Results: Stimulation of wild-type Eph-B4 in transfected Cos-7 cells results in phosphorylation of Eph-B4, Akt, and ERK1/2, but not FAK. Tyrosine phosphorylation of Eph-B4 is decreased with the loss of Y774, but not with the loss of Y653 and Y581. Loss of Y653 and Y774 resulted in decreased phospho-AKT activity compared to wild-type Eph-B4 and a loss of Y774 resulted in decreased phospho-ERK1/2. phospho-FAK activity was equivalent between wild-type and all mutant Eph-B4 transfected cells. Loss of Y581 did not influence phosphorylation of Akt, ERK1/2, or FAK. Double-labeled immunofluorescence confirmed the above findings. Wild-type Eph-B4, but not mutant Eph-B4, colocalized with caveolin-1 after Ephrin-B2/Fc stimulation. Conclusions: Eph-B4 downstream cell signaling requires tyrosine residues 653 and 774. Their loss results in decreased phospho-ERK1/2 and phospho-Akt, but not phospho-FAK, activity, and also prevents colocalization of activated Eph-B4 with caveolin-1, showing that tyrosine phosphorylation of these residues is critical to Eph-B4 function. These results suggest novel molecular targets to modulate Eph-B4 activity.

A Novel All-Trans Retinoic Acid Perivascular Wrap Reduces Neointimal Hyperplasia Following Arterial Balloon Injury

E.K. Gregory, J. Martinez, M.E. Flynn, Q. Jiang, A. Webb, G.A. Ameer, M.R. Kibbe — 2012-02-01

Introduction: All-trans retinoic acid (ATRA) is a vitamin A derivative that has many beneficial effects on the vasculature, including inhibition of vascular smooth muscle cell (VSMC) proliferation and stimulation of nitric oxide (NO) release. Thus, through a multidisciplinary collaboration, we developed a novel ATRA perivascular wrap utilizing a biodegradable citric-acid based polymer, poly (diol-citrate) (POC). The goal of our current study was to characterize the properties of ATRA wraps in vitro and evaluate the efficacy of our ATRA wrap to inhibit neointimal hyperplasia in vivo. We hypothesized that treatment with ATRA perivascular wraps would decrease the formation of neointimal hyperplasia with a concomitant decrease in proliferation. Methods: ATRA wraps were fabricated using POC wraps loaded with ATRA dissolved in DMSO to a final concentration of 20 mg/ml. ATRA release was measured over time and was confirmed using HPLC. VSMC proliferation was assessed by measuring DNA content. The rat carotid injury model was performed in 10 week old male Sprague Dawley rats. Treatment groups included: control, injury, and injury +ATRA wrap (1cm x 1cm). n=6-7/group. Arteries were harvested at 14 days. Morphometric analysis was performed on H&E stained cross-sections utilizing Image J software. Immunohistochemistry was performed to assess for BrdU incorporation and macrophage infiltration (ED1 staining). Results: In vitro, ATRA release from the wraps occurred for 14 days, with the greatest release in the first 7 days. ATRA wraps inhibited VSMC proliferation by approximately 75% at 7 days (p<0.05) compared to controls. In vivo, ATRA wraps resulted in significant inhibition of neointimal hyperplasia, with a 56.3% reduction in intimal area (p<0.001) and a 57% reduction in the intima/media area ratio (p<0.001) as compared to injury alone. We observed no significant changes in the lumen or medial area. With respect to proliferation, ATRA wraps resulted in a decreasing trend in proliferation for both the intimal and medial layers, with a slight increase in the adventitia. However, none of these changes reached statistical significance. With respect to inflammation, treatment with ATRA wraps resulted in increased macrophage infiltration compared to injury alone (1.8-fold, p<0.001). Conclusions: Neointimal hyperplasia was significantly inhibited by the citric acid-based biodegradable ATRA-eluting perivascular wrap without significant effect on vessel wall remodeling. This therapy represents a novel method to deliver ATRA to the vasculature to inhibit neointimal hyperplasia. the increased inflammatory response, while not anticipated, is mostly likely due to mass effect of the wrap in the small neck of the rat. Further study of this therapy is warranted in a large animal model.

Effective Inhibition of Neointimal Hyperplasia Using A Nitric Oxide-diffusible Balloon Catheter

2012-02-01

Introduction: The long-term durability of vascular interventions is limited by the development of neointimal hyperplasia. the goal of our lab is to develop nitric oxide (NO)-based therapies that will prolong the efficacy of vascular interventions. Toward that end, our lab has demonstrated that periadventitial delivery of NO results in durable inhibition of neointimal hyperplasia. However, this method is not conducive to endoluminal delivery. Since NO is a highly diffusible gaseous molecule, the aim of the current study is to evaluate the efficacy of delivering NO to the site of injury to inhibit neointimal hyperplasia using a NO-diffusible balloon catheter. Using this method, NO will be delivered to the luminal surface of the arterial wall during inflation of the balloon catheter for only 5 minutes. Our hypothesis is that short-duration endoluminal delivery of NO at the time of injury will effectively inhibit the development of neointimal hyperplasia. Methods: 10-week-old male Sprague-Dawley rats underwent the carotid artery balloon injury model with inflation of a 2F Fogarty catheter to 5 atmospheres of pressure for 5 minutes. Treatment groups included: 1) control, 2) injury, 3) injury + periadventitial delivery of NO (PROLI/NO 10 mg), and 4) injury + NO-diffusible balloon catheter. n=6 rats/group. for the balloon diffusion group, PROLI/NO (300 mM) was prepared in a buffered solution and used to inflate the balloon catheter. for all other treatment groups, saline was used to inflate the balloon catheter. Arteries were harvested 2 weeks following injury for morphometric assessment and immunhistochemistry for inflammation. Results: As expected, balloon injury caused a significant increase in neointimal hyperplasia compared to control arteries, as assessed by the intima/media area ratio (I/M 1.07 vs. 0.11, resp., p<0.001). Periadventitial application of NO caused an expected reduction in the I/M area ratio compared to injury alone (55% decrease, p<0.001). Interestingly, only 5 minutes of endoluminal delivery of NO through the balloon catheter resulted in a more profound decrease in the I/M area ratio compared to injury alone (65% decrease; p<0.001). Similarly, endoluminal delivery of NO resulted in a greater reduction of intimal area than periadventitial delivery of NO (64% vs. 46%, respectively; p<0.001). Neither NO-based therapy effected medial area. Only periadventitial delivery of NO increased luminal area (64% increase; p<0.001) whereas only endoluminal delivery of NO increased arterial circumference (6% increase; p<0.001). Both NO-based therapies inhibited macrophage infiltration compared to injury alone (p<0.05). Conclusions: These data demonstrate that an extremely short-duration endoluminal delivery of NO via a balloon catheter can significantly inhibit the development of neointimal hyperplasia after an arterial intervention. This minimally invasive route of NO delivery could serve as an intriguing focus for clinical interventions in the future.

Protein Kinase C Delta Promotes Adventitial Cell Migration to Neointima by Upregulation of Monocyte Chemoattractant Protein-1 in Smooth Muscle Cells

Y. Si, J. Ren, X. Shi, K. Kent, B. Liu — 2012-02-01

Introduction: Accumulating evidence suggests that adventitial cells directly contribute to neointima formation by migrating into the intima. We have previously reported that gene transfer of Protein kinase C delta(PKC δ) attenuates intimal hyperplasia by inducing apoptosis of smooth muscle cells (SMCs). More recently, we showed that PKCδ mediates expression of monocyte chemoattractant protein-1(MCP-1). in the current study, we tested the hypothesis that PKCδ promotes adventitial cell migration by stimulating SMCs to produce chemokines such as MCP-1. Methods: In vitro migration of isolated adventitial cells was evaluated by chemotaxis assay. Gene transfer to SMCs was achieved by intraluminal perfusion with adenoviruses expressing PKCδ(AdPKCδ) or empty vector (AdNull) following rat carotid angioplasty. Adventitia cells were labeled by administration of AdLacZ via pluronic gel on the adventitial surface prior to wound closure. Results: Media conditioned by PKCδ-overexpressing SMCs stimulated migration of adventitial cells, which was blocked by quenching MCP-1 with a neutralizing antibody or by knocking down the MCP-1 receptor, CCR2. Similarly, gene transfer of PKCδ to injury arteries stimulated adventitial cell migration in vivo. Fourteen days following injury, accumulation of adventitial cells in the neointima was close to 10 times higher in AdPKCδ-infected arteries than in AdNull-infected arteries(LacZ positive area in pixel:769±67*103 vs 84±11*103, p<0.01). In addition, PKCδ gene transfer increased MCP-1 expression in the media by ∼4 folds (MCP-1 positive areas in pixel:397±20*103 vs 99±18*103, p<0.05). to further prove that PKCδ mediates migration of adventitia cells through MCP-1, we randomly divided AdPKCδ-treated rats into two groups, one injected with a neutralization antibody to MCP-1 and the other with nonimmunized IgG. Neutralizing MCP-1 significantly reduced the number of adventitial cells accumulated in the neointima (LacZ positive area in pixel:357±37*103 vs 894±42*103, p<0.01). Furthermore, combining PKCδ gene transfer and anti-MCP-1 treatment inhibited I/M ratio by 62% as compared to a 36.3% and 30.8% reduction produced by a single treatment of PKCδ and anti-MCP-1, respectively. None of the treatments altered macrophage infiltration, at least at the time points (7 and 14 days) we assessed. Conclusions: PKCδ appears to play dual functions during arterial injury response. in addition to inducing apoptosis, PKCδ promotes migration of adventitial cells by stimulating MCP-1 expression in SMCs. A combined strategy targeting both SMC apoptosis and chemokine production may be an efficient approach to inhibit intimal hyperplasia.

The Effects of Polyunsaturated Fatty Acids on Monocyte-Endothelial Cell Interactions

S.M. Grenon, J. Aguado-Zuniga, M.S. Conte, M. Hughes-Fulford — 2012-02-01

Introduction: Diet is known to have an important impact on cardiovascular health. Polyunsaturated fatty acids (FAs) are postulated to alter the interactions between monocytes and endothelial cells, a critical step in the initiation of atherosclerosis. In this experiment, we examined the effects of arachidonic acid (AA), an ω-6 FA and eicosapentanoic acid (EPA), an ω-3 FA on monocytes and endothelial cells interactions. Methods: A cellular model using endothelial cells (EA.hy.926) and monocytes (human leukemic myelomonocytic U937) was used in this study. Confluent endothelial cells were treated with AA or EPA, in the presence of TNF-α or vehicle-alone for 4 hr and 24 hours. Adhesion of monocytes to the endothelial monolayer was performed with the Molecular Probes Vybrant cell adhesion assay kit and read with Fluoroscan II plate reader. Adhesion assays were repeated after pre-treatment with indomethacin. Fluorescence microscopy was done with FAs and cytokine treatment. For gene expression, reverse transcription followed by real-time quantitative polymerase chain reaction was performed. Results: There was a significant increase in adhesion of monocytes to the endothelial monolayer with ω-6 FAs, both in the presence or absence of TNF-α (Figure 1a & b). The adhesion of monocytes to the endothelial monolayer was decreased with ω-3 FAs in the presence or absence of TNF-α at 24 hours (Figure 1b). There was a 3-fold increase in IL-6 (p=0.0016) and 1.3-fold increase in ICAM-1 (p=0.0003) in activated endothelial cells treated with ω-6 FAs compared to ω-3 FAs. Pre-treatment of endothelial cells with indomethacin led to a significant decrease in monocyte to endothelial adhesion in the presence of ω-6 FAs (Figure 1c). Conclusions: We conclude that AA (ω-6) leads to increased adhesion of monocytes to endothelial cells in a model using EA.hy.926 and U937 monocytes, while EPA (ω-3) leads to a decreased adhesion. the increased adhesion in the presence of AA is reversed by pre-treatment with a COX-2 inhibitor. the mechanisms of increased adhesion with ω-6 FAs appears to be related to an increased expression of ICAM-1 and IL-6. Taken together these data indicate that the AA can promote monocyte adhesion to endothelial cells, suggesting that the dietary intake of ω-3 and ω-6 FAs could have important consequences for early events in atherosclerosis.

Inhibition of CXCL4-CCL5 Chemokine Interaction Ameliorates the Development of Abdominal Aortic Aneurysm in Mice

Y. Iida, B. Xu, J.R. Schultz, C.R. Turner, H. Ogino, R.L. Dalman — 2012-02-01

Abdominal aortic aneurysm (AAA) is a macrophage-driven arterial degenerative disease. Given the importance of CXCL4-CCL5 chemokine interaction in macrophage/monocytes migration into inflamed arteries, we evaluated the effect of a novel synthetic peptide inhibitor to CXCL4-CCL5 interaction on AAA progression in the porcine pancreatic elastase (PPE)-induced AAA mouse model.

Regional Versus General Anesthesia for Carotid Endarterectomy: The NSQIP Perspective

M.A. Schechter, C.K. Shortell, J.E. Scarborough — 2012-02-01

Introduction: The ideal anesthetic technique for carotid endarterectomy (CEA) is a source of continued controversy. Most studies comparing anesthesia used during CEA are retrospective in nature and draw from relatively small numbers of patients from single centers. With its inclusion of prospectively collected information about surgical patients from over 250 participating hospitals, the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database offers the opportunity to re-evaluate the potential impact of anesthesia modality on outcomes after CEA. Methods: All ACS-NSQIP patients undergoing CEA for carotid stenosis from 2005-2009 were included for analysis unless they had undergone a prior operation within 30 days of CEA or received another major procedure concurrent with CEA. Preoperative patient characteristics and postoperative outcomes were compared for patients undergoing CEA with general (GEN) versus regional (REG) anesthesia. Multivariate logistic regression was then used to determine each patient's propensity for receiving regional anesthesia, using a number of variables to reflect patient demographics, comorbidities, physical status, and carotid disease characteristics. These propensity scores were used to create a cohort of GEN and REG patients matched on their propensity for undergoing CEA with REG anesthesia. The outcomes of the matched cohort were compared using Wilcoxon signed rank tests for continuous variables and McNemar's chi square tests for categorical variables. Results: 24,716 patients were included for analysis (20,670 in GEN group versus 4,046 in REG group). Univariate analysis revealed no difference in 30-day postoperative composite Stroke/MI/Death rates based on anesthesia modality (2.2% REG vs. 2.6% GEN, p = 0.13). Propensity matching resulted in the creation of a cohort of 4,025 pairs that were well-matched for all preoperative variables. Analysis of this cohort revealed no difference in 30-day composite Stroke/MI/Death rates (2.2% REG vs. 2.4% GEN, p = 0.66), but a trend towards a lower overall complication rate in patients receiving regional anesthesia (2.8% vs. 3.6%, p = 0.07). Procedures performed under regional anesthesia were associated with significantly shorter operative (99 ± 36 minutes vs. 119 ± 53 minutes, p <0.0001) and anesthesia times (52 ± 29 minutes vs. 64 ± 37 minutes, p<0.0001). Patients receiving regional anesthesia were also significantly less likely to require postoperative hospitalization for more than 1 day (23.0% vs. 35.6%, p<0.0001). Conclusions: Our analysis demonstrates no significant association between anesthesia modality during CEA and 30-day composite Stroke/MI/Death rate, confirming the findings of a previous randomized controlled trial. Our findings suggest that regional anesthesia may be associated with a slightly lower overall complication rate, and significantly shorter operative/anesthesia times and length of postoperative hospitalization.

An Integrated Biochemical Prediction Model of All-Cause Mortality in Patients Undergoing Lower Extremity Bypass Surgery for Advanced Peripheral Artery Disease

C.D. Owens, J. Kim, W. Gasper, M.A. Creager, M.S. Conte — 2012-02-01

Introduction: Patients with advanced peripheral artery disease (PAD) have a high prevalence of cardiovascular (CV) risk factors and shortened life expectancy. However, CV risk factors poorly predict midterm (<5 years) mortality in this population. This study was designed to test the hypothesis that baseline biochemical parameters would add clinically meaningful predictive information in patients undergoing lower extremity bypass. Methods: This was a prospective cohort study of subjects with clinically advanced PAD undergoing lower extremity bypass surgery. the Cox proportional hazard was used to assess the main outcome of all-cause mortality. A clinical model was constructed with known cardiovascular risk factors and the incremental value of the addition of clinical chemistry, lipid, and a panel of 11 inflammatory parameters were investigated using c-statistic, the integrated discrimination improvement (IDI) index and Akaike information criterion (AIC). Results: 225 subjects were followed for a median 893; IQR 539-1315 days). in this study 50 (22.22%) subjects died during the follow-up period. By life table analysis (expressed as percent surviving ± standard error), survival at 1, 2, 3, 4, and 5 years respectively was 90.5 ± 1.9%, 83.4 ± 2.5%, 77.5 ± 3.1%, 71.0 ± 3.8%, and 65.3 ± 6.5%. Compared with survivors, decedents were older, diabetic, had extant CAD, and were more likely to present with CLI as their indication for bypass surgery, P<.05. After adjustment for the above, clinical chemistry and inflammatory parameters significant for all cause mortality were albumin, HR .43; P=.001, estimated glomerular filtration rate (eGFR), HR .98, .023, high sensitivity C-reactive protein (hsCRP), HR 3.21, P=.019, and soluble vascular cell adhesion molecule (sVCAM), HR 1.74, P=.034. of all inflammatory molecules investigated, hsCRP proved most robust and representative of the integrated inflammatory response. Albumin, eGFR, and hsCRP improved the c-statistic and IDI beyond that of the clinical model and produced a final c-statistic of .82. Conclusions: A risk prediction model including traditional risk factors and parameters of inflammation, renal function and nutrition had excellent discriminatory ability in predicting all cause midterm mortality in patients with clinically advanced PAD undergoing bypass surgery.

Rosyln Award Winner: Augmenting Immunotherapy Strategies Against Glioblastoma Multiforme by Targeting the Immune Microenvironment

M. Lim — 2012-02-01

AAS Foundation Award Winner: MT1-MMP Cooperates With Kras G12D to Promote Pancreatic Fibrosis Through TGF-? Activation of Pancreatic Stellate Cells

S. Krantz — 2012-02-01

Kidney-Lung Crosstalk: Identifying Pulmonary Endothelial Cell-Specific Changes During Ischemic Acute Kidney Injury

Y. Cui, L.E. White, C.M. Feltes-Shelak, F.A. Moore, H.T. Hassoun — 2012-02-01

Introduction: Despite advancements in renal replacement therapy, acute kidney injury (AKI) harbors a grave prognosis, likely due to extra-renal organ dysfunction. Kidney ischemia-reperfusion injury (IRI) activates cellular and soluble mediators that incite a distinct distant pulmonary transcriptional response in vivo, leading to TNFR1-dependent pulmonary apoptosis, increased microvascular permeability and lung injury. Hypothesizing that pulmonary endothelial cell (EC) structural changes could result in lung microvascular barrier dysfunction in vivo, we performed phenotypic assessment of cytoskeletal derangements, apoptosis, and pathway-focused genomic analyses of candidate genes to characterize in vitro pulmonary EC-specific changes during kidney IRI. Methods: Male 6-8 week-old Sprague-Dawley rats (200-250 g) underwent 60 min of bilateral kidney ischemia or sham laparotomy and serum was collected upon sacrifice at 24 hrs. Rat lung microvascular endothelial cells (RLMVECs, Vec Technologies) were grown to confluence on fibronectin-coated flasks. RLMVECs were treated with 5%, 10%, and 25% sham or IRI serum for 2, 4, and 24 hrs, and in a separate experiment, RLMVECs were pre-treated with Etanercept (Enbrel, Immunex Corporation) or vehicle to determine the role of TNF-α/TNFR1 signaling in EC changes during ischemic AKI. Assays included 1) phalloidin staining for assessment of the actin cytoskeleton, 2) measurement of apoptosis by poly (ADP-ribose) polymerase (PARP) assay (relative absorbance at 450 nm), and 3) RT-PCR of candidate TNF-superfamily, pro-apoptotic, and inflammatory genes to assess the EC-specific transcriptional response during ischemic AKI. Results: RLMVECs treated with IRI serum demonstrated dose-dependent actin stress fiber formation and increased apoptosis at 2, 4, and 24 hours when compared to sham serum (0.65±0.22 vs. 2.12±0.56*, 0.24±0.06 vs. 1.71±0.44*, 0.24±0.07 vs. 1.33±0.36* respectively). RLMVECs displayed early and sustained dose-dependent increases in transcription of TNF-superfamily (TNFα, TNFR1, TNFR2, and FasL), pro-apoptosis (Dapk1) and pro-inflammatory (E-selectin, ICAM-1, IL-6 and Rho-B) candidate genes when treated with IRI vs. sham serum, and the pro-inflammatory transcriptional response was attenuated by TNF-α/TNFR1 signaling inhibition with Etanercept (Figure 1). *p<0.05 vs. sham, n≥4/group. Conclusions: Ischemic AKI incites a distinct pulmonary EC-specific transcriptional response, resulting in cytoskeletal derangements, EC apoptosis, and inflammation. Future endeavors to characterize the EC-specific response and effector mechanisms induced by the postischemic kidney may reveal potential therapeutic targets for deleterious kidney-lung crosstalk.

Hydrophilic Polymers Enhance Early Functional Outcomes After Interposition Grafting

K.W. Sexton, G.A. Del Corral, L.B. Nanney, R.B. Shack, W.P. Thayer — 2012-02-01

Introduction: Traumatic neuropathies (TN) are a common source of morbidity in the United States reported to affect approximately 5% of all admissions to level one trauma centers. at our institution, over 400 operations are performed yearly for TN of the upper extremity. Approximately 12% of these operations are for patients with segmental nerve loss and these are commonly treated with interposition nerve grafting. It has been reported that less than 50% of these injuries will obtain a functional recovery and less than 5% will obtain a full recovery. We sought to improve the rate of functional recovery following interposition nerve grafting using hydrophilic polymers. Methods: Female Sprague Dawley rats were anesthetized with isoflourane. The right sciatic nerve was exposed in a muscle sparing fashion. Baseline compound action potentials (CAPs) were stimulated and recorded using a Powerlab data acquisition system (ADInstruments). The nerve was then transected in 2 areas creating an injury to mimic segmental nerve loss. the segments removed were interposed in an end-to-end fashion using microsurgery. A series of solutions (Calcium free Kreb's solution [0.5 mM EGTA in 99 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 10 mM Na ascorbate, 10 mM dextrose, pH 7.35, 295 mM], 1000 μm Methylene Blue in Calcium free Kreb's solution, and 190 mM Polyethylene glycol 3.35 kD in distilled water) were applied for one minute each. the wound was then irrigated with Kreb's solution containing 2 mM CaCl2. After 3 minutes, solution CAPS were obtained in the same manner above. the rats were then given ketoprofen (10 mg/kg) and allowed to recover. Six rats received solution therapy (experimental), and six rats received microsurgical repair without solution therapy (control). At 24 hours and 72 hours postoperatively, the rat's hind paws were differentially inked and the rat traversed a wooden beam covered with white paper. Sciatic Functional Index (SFI) was calculated based on measurements averaged from 6 sequential paw prints for each rat. Results: All 6 of the experimental group had return of CAPs after solution therapy and there was no statistically significant difference from baseline amplitude, while none of the control group had measureable CAPs after microsurgical repair. (Figure 1a) Rats in the experimental group demonstrated a significantly improved SFI at 24 and 72 hours postoperatively. (Figure 1b) Conclusions: Hydrophilic polymer solution therapy improves early functional outcomes after interposition grafting. Further work needs to be done to see if this improvement is permanent and if hydrophilic polymers potentially improve outcomes in humans.

Surgical Research Society of Australasia - Diannexin Reduces Tissue Factor Positive Microparticles, Microvascular Obstruction and Endothelial Cell and Myocyte Necrosis, in Cardiac Ischemia Reperfusion Injury

Connor O'Meara — 2012-02-01

Warm Humidified Carbon Dioxide Gas Insufflation for Laparoscopic Appendicectomy in Children: A Double-Blinded Randomised Controlled Trial

T. Yu, J. Hamill, A. Liley, A.G. Hill — 2012-02-01

Introduction: Pneumoperitoneum during laparoscopy is typically created with dry, room temperature carbon dioxide (CO2) gas. As a consequence, desiccation-related structural and inflammatory alterations occur to the peritoneal mesothelial lining and this has been clinically linked to additional postoperative pain and delayed recovery. Warm humidified insufflation gas is hypothesised to improve postoperative outcomes by preventing peritoneal desiccation. This study investigated the benefits of warm humidified CO2 gas insufflation during laparoscopic appendicectomy on postoperative pain and recovery in children (8-14 years of age). Methods: A single-institution, double-blinded, randomised controlled trial was implemented. Participants in the intervention group received warmed (37°C) humidified (98% relative humidity) insufflation gas while control participants received standard room temperature (20°C) gas with 0% relative humidity. Perioperative analgesia and anaesthesia was standardised and intraoperative core body temperature was monitored at ten-minute intervals. At the conclusion of surgery, severity of laparoscopic camera lens fogging was rated by the primary surgeon. Postoperative opiate usage was expressed as Morphine Equivalent Daily Dosages (MEDD) and pain intensity at rest and on moving was rated using visual analogue scales. Postoperative recovery and return to normal activities on day 10 were assessed using a questionnaire. Results: Between February 2010 and March 2011, 190 participants were randomised. Intervention (N = 95) and Control (N = 95) groups were matched at baseline and intraoperative core body temperature variation was statistically similar. There were no statistically significant differences in postoperative MEDD and pain intensity scores. Postoperative recovery parameters and the severity of camera lens fogging were also found to be statistically similar. Conclusions: Warm humidified CO2 gas insufflation for laparoscopic appendicectomy has no short-term clinical benefits in paediatric patients.

Deconstructing Intra-operative Communication Failures

Y. Hu, A.F. Arriaga, S.E. Peyre, K.A. Corso, E.M. Roth, C.C. Greenberg — 2012-02-01

Introduction: Communication failure is a common contributor to adverse events. in the operating room (OR), where a high level of coordination is required, communication is of particular importance. We sought to characterize communication failures during complex operations. Methods: We video-recorded and transcribed 6 complex, high-acuity operations, representing 22 hours of patient care. Communication events, each defined as a verbal exchange between two or more team members with a unique communicative intent or theme that occurred at a specific point in time, were characterized by the participants involved and the content discussed (Halverson, 2011, Surgery). Failures were classified using a previously validated instrument (Lingard, 2006, QSHC) into 4 non-mutually exclusive types: audience (the exclusion of key individuals from a conversation), purpose (the non-resolution of a discussed issue), content (insufficient or inaccurate information), and/or occasion (a discussion rendered futile by timing). We added a systems category to reflect communication occurring at the organizational level. The impact of each identified failure was then described. Results: We observed communication failures in every case (mean 29, median 28, range 13-48) at a rate of every 7 minutes. Cross-disciplinary exchanges resulted in failure nearly twice as often as intra-disciplinary ones. Discussions about or mandated by hospital policy (20%), personnel (18%), or other patient care (17%) were most error-prone. Audience and purpose each accounted for >40% of failures. A substantial proportion (26%) also reflected flawed systems for communication, particularly those used for disseminating policy (29% of system failures), coordinating personnel (27%), and conveying the planned procedure (27%) or the equipment needed (24%). In 81% of failures, inefficiency (extraneous discussion and/or work) resulted, although resource waste (19%) and workarounds (13%) were also frequently seen. Conclusions: During complex operations, communication failures occur frequently and lead to inefficiency. Improving synchronous, cross-disciplinary communication intra-operatively may prevent many of these events. Given the rate of failure among discussions about or mandated by policy, standardized interventions for accomplishing this must be carefully designed. System-level support for asynchronous peri-operative communication (e.g. to continually address staff scheduling or OR set-up issues) may provide additional benefit by streamlining OR coordination and preparation efforts.

Reliability of Evaluating Hospital Quality Using Surgical Site Infections After Colorectal Procedures

E.H. Lawson, J.L. Adams, C.Y. Ko, B. Lee Hall — 2012-02-01

Introduction: Policymakers are increasingly focusing on surgical site infections (SSI) for public reporting and pay-for-performance because they are a common and costly cause of patient morbidity. Our objective was to determine if superficial and deep/organ-space SSI occurring after colorectal procedures should be considered independently or aggregated when measuring hospital quality. Methods: Colorectal cases in the National Surgical Quality Improvement Program (NSQIP) were identified by CPT code, producing 25,455 cases from 236 hospitals in 2009. Hierarchical multivariable logistic models were developed for (1) superficial SSI, (2) deep/organ-space SSI and (3) all SSI. Models were adjusted for procedure and case-mix. for each model, hospitals' observed/expected (O/E) ratios were calculated then ranked and split into equal decile groups. Hospital decile was compared between the models. The reliability of each O/E was calculated on a scale from 0 to 1, with 0 indicating that variation is due to measurement error (“noise”) and 1 indicating that variation is due to real differences in performance (“truth”). The relationship between reliability and sample size was estimated using the Spearman-Brown prophecy. The 2009 Nationwide Inpatient Sample was used to estimate the number of colorectal procedures performed in US hospitals. Results: There was poor agreement between hospitals' decile rank using the superficial vs. deep/organ-space SSI models (weighted kappa 0.080). The mean O/E reliability was 0.731 for superficial, 0.620 for deep/organ-space, and 0.704 for all SSI. Reliability increased with increasing sample size (see figure). the estimated number of cases a hospital is projected to require for different levels of reliability and number of hospitals nationwide that performed the specified caseload in 2009 are reported in the table. Conclusions: for high-stakes quality comparisons such as pay-for-performance and public reporting it is essential that measures be valid and reliable - meaning high and low quality is accurately and consistently identified. We found a decrease in value when combining superficial and deep/organ-space SSI into one quality measure. Hospital risk-adjusted performance for these SSI types is not correlated, and grouping does not confer an advantage for the reliability of the quality measurement. Policies and initiatives aimed at reducing SSI should consider superficial and deep/organ-space SSI as two distinct entities when measuring quality.

Importance of Perioperative Glycemic Control in General Surgery: A Report from the Surgical Care and Outcomes Assessment Program

S. Kwon, R.E. Thompson, P. Dellinger, T. Rogers, D. Flum — 2012-02-01

Introduction: There is limited evidence on the impact of perioperative hyperglycemia and insulin administration in patients undergoing general surgical procedureas on adverse outcomes, in patients with or without known diabetes. Methods: The Surgical Care and Outcomes Assessment Program is a Washington State quality improvement benchmarking-based initiative using clinical data from over 55 hospitals. We evaluated the relationship of perioperative hyperglycemia (> 180 mg/dL) and insulin administration on mortality, operative interventions, and infections for patients undergoing elective colorectal and bariatric surgery at 38 participating hospitals between 4th quarter of 2005 and 4th quarter of 2010. Results: Of the 11,633 patients (55.4±15.3 yrs; 65.7% women) with a serum glucose either on the day of surgery, postoperative (POD) 1, or POD 2, 29.1% of patients were hyperglycemic. After controlling patient and clinical factors, those with hyperglycemia had a significantly increased risk of infection (OR 2.0; 95% CI 1.63-2.44), reoperative interventions (OR 1.8; 95% CI 1.41-2.3), anastomotic failure (OR 2.43; 95% CI 1.38-4.28), and mortality (OR 2.71; 95% CI 1.72-4.28). Those with hyperglycemia on both PODs postoperatively had the highest risk of infection (OR 3.1; 95% CI 1.72-5.59), and we observed increasing risk of reoperative interventions and infections with increasing glucose levels. Those with hyperglycemia on the day of surgery who received insulin had no significant increase in infections (OR 1.01; 95% CI 0.72-1.42), reoperative interventions (OR 1.29; 95% CI 0.89-1.89), or deaths (OR 1.21; 95% CI 0.61-2.42). However, 26% of patients with hyperglycemia on the day of surgery did not receive insulin. Conclusions: Perioperative hyperglycemia was associated with and adverse outcomes in patients with and without diabetes, but patients receiving insulin were not at greater risk than those with normal blood sugars. Perioperative glucose evaluation and insulin administration in patients with hyperglycemia are worthwhile QI targets.

Effect of Metabolic Syndrome on Peri-Operative Outcomes Following Liver Surgery: A National Surgical Quality Improvement Project (NSQIP) Analysis

2012-02-01

Introduction: The problem of obesity has risen to epidemic levels in the United States. A subset of patients (pts) with obesity will have metabolic syndrome (MetS), defined as a constellation of clinical features including hypertension, elevated fasting glucose and high body mass index (BMI). the impact of MetS on outcomes following liver resection remains ill defined. We sought to examine the impact of MetS on the risk of morbidity and mortality among a large cohort of pts who underwent hepatic resection. Methods: All pts included in the National Surgical Quality Improvement Program (NSQIP) dataset who underwent hepatic resection between January 2005 and December 2008 were identified. Data on clinical characteristics, co-morbidities, operative details, as well as post-operative complications and mortality were collected and analyzed. Patients with BMI > 30 kg/m2 who also had hypertension and diabetes were defined as having MetS; the impact of MetS on morbidity and mortality was then assessed. Results: 3,973 pts who underwent a liver resection were identified. Half of pts were male (n=1962, 49%) with a mean age of 58 yrs (range, 17-89). Overall mean BMI was 28 kg/m2; 2.5% (n=98) pts were underweight (BMI < 18.5 kg/m2), 32% (n = 1289) had a normal BMI (18.5-24.9 kg/m2), 33% (n = 1327) were overweight (25.0-29.9 kg/m2), and 31.7% (n=1259) were obese (>30 kg/m2). of the 1259 pts who were obese, 256 (20.3%) also had both diabetes and hypertension and therefore were defined as having MetS. the mean BMI in the non-MetS group was 27.5 kg/m2 vs 35.3 kg/m2 in the MetS group (p<0.0001). Pts with MetS were less likely to have had a major hepatectomy (≥ hemi-hepatectomy, 36% vs. 43%; p=0.01) but had a higher mean number of red blood cell transfusions (1.6 vs. 1; p=0.02). Pts with MetS were more likely to have HCC (n=75, 29%) vs non-MetS pts (n=596, 16%) (p<0.001). the incidence of post-operative complications following hepatectomy was 23.1% (n = 919). Pts with MetS had a higher risk for re-intubation (OR = 1.9; p=0.02), ventilator dependence for >48 hrs (OR=2.0; p=0.003), myocardial infarction (OR=5.5; p=0.01) and superficial surgical site infections (OR=1.7; p=0.01) vs. non-MetS pts. Overall, the post-operative mortality was 3%. MetS was associated with an increased risk of post-operative death (OR=2.7; p=0.001). Conclusions: The incidence of MetS in a large, nationally-representative cohort of pts undergoing hepatic resection was 6.4%. The presence of MetS was associated with a higher risk of peri-operative complications. In addition, pts with MetS had nearly a three-fold increase risk of death following hepatic resection.

Do Surgeons Expect Patients to Buy-in to Postoperative Life Support Preoperatively? A National Survey of Surgeons Who Routinely Perform High Risk Operations

M.L. Schwarze, A.J. Redmann, G.C. Alexander, K.J. Brasel — 2012-02-01

Introduction: “Surgical Buy-In” describes the process of surgeons negotiating or contracting with patients preoperatively for their participation in life supporting therapy postoperatively. the objective of this study is to assess whether the practice of buy-in is generalizable to a large sample of surgeons who routinely perform high risk operations. Methods: We designed a survey to determine the prevalence of surgical buy-in, the consequences of buy-in regarding withdrawal of postoperative life supporting therapy and surgeon characteristics associated with this practice. We surveyed 2100 randomly selected vascular, cardiothoracic and neurosurgeons via US mail. We used bivariate and multivariate analysis to identify surgeon characteristics associated with a) negotiating for time or creating an informal contract with the patient describing agreed upon limitations of life support and b) declining to operate on patients with a specific request to limit life support postoperatively. Results: The adjusted response rate was 56%. Of surgeons faced with a patient at moderate risk for prolonged mechanical ventilation or dialysis who had a preoperative request to limit postoperative life supporting therapy 62% would create an informal contract with the patient describing agreed upon limitations of aggressive therapy and 72% of surgeons would negotiate for a time period after which life supporting therapy could be discontinued. 60% might decline to operate on such a patient. Surgeons who favored negotiating for time were more likely to be female (84 vs. 71%, p = 0.04) and reported fewer years of experience (77 vs. 66%, p = 0.02). Surgeons who were willing to withdraw life support on postoperative day 14 (86% of respondents) were twice as likely to negotiate or contract preoperatively about life-supporting therapy (OR: 2.0, 95%CI: 1.3-3.0 and OR: 2.1, 95%CI: 1.4-3.2 respectively) and almost half as likely to decline to operate (OR: 0.6, 95%CI: 0.4-0.9) than surgeons who would rarely withdraw life support on postoperative day 14. Conclusions: A majority of surgeons will negotiate or contract with patients preoperatively about postoperative life supporting therapies if the patient has a specific request to limit these treatments. Surgeons who believe that it is rarely acceptable to withdraw life supporting therapy on or before postoperative day 14 are more likely to decline to operate than negotiate with such a patient.

Assessment of the Implementation of a Surgical Preoperative Checklist

2012-02-01

Introduction: Peri-operative checklists are mandated by many hospitals based on the reduction in morbidity and mortality seen with utilization of the World Health Organization's (WHO) “Surgical Safety Checklist.” Although an adapted peri-operative checklist was implemented within our hospital system without formal system-wide training, compliance with the checklist is reported to be 100%. We hypothesize that compliance does not measure fidelity of implementation, in that all items on the checklist are not performed as intended. Methods: Over a 10 week period, a prospective study was performed evaluating the completion of the 12 pre-incision components of the surgical checklist. Pediatric surgical operations, occurring in the operating room or in neonatal and pediatric intensive care units, were randomly selected for direct observation. Emergent cases were excluded. The evaluated checkpoints include essential parties present, team members identified, patient name/procedure verified, incision site confirmed, team member concerns addressed, administration of appropriate antibiotics (if applicable), essential imaging displayed (if applicable), anticipated case length stated, anticipated risk of blood loss stated, and sterility indicator confirmed. Essential parties included anesthesiology attending or fellow, pediatric surgical attending or fellow, circulating nurse, and scrub technologist. Results: 142 pediatric surgical cases were observed. Hospital data demonstrated 100% compliance with the pre-incision phase of the checklist for these cases. Our observation revealed that in 3.5% of cases the checklist was not performed at all. None of the cases completely executed all items on the checklist, and the average number of checklist items performed in the observed cases was five. the most commonly performed checkpoints were the confirmation of patient name and procedure (99.3%) and administration of antibiotics (88.1%). the rest of the checkpoints were performed in less than 60% of cases (figure). Conclusions: These data show that despite the 100% documented completion of the pre-incision phase of the checklist, most of the individual checkpoints are not routinely performed. These findings demonstrate lack of fidelity in implementing the checklist, which may be a reflection of the poor strategy in disseminating and implementing this patient safety practice. Failure of the system to measure the appropriate implementation metrics and to fully adopt the evidence-based intervention could lead to failure to achieve the intended outcomes. The impact and etiology of non-compliance with all components of the surgical checklist requires further investigation.

Predictors of Nursing Home Admission, Severe Functional Impairment or Death One Year After Surgery for Non-Small Cell Lung Cancer

S.E. Billmeier, J.Z. Ayanian, Y. He, M.T. Jaklitsch, S.O. Rogers — 2012-02-01

Introduction: Patients perceive long-term disability as one of the most undesirable complications of lung cancer treatment. Therefore, we assessed factors associated with nursing home admission, severe functional impairment, or death one year after surgery for stage I-IIIa non-small cell lung cancer (NSCLC). Methods: A population- and health system-based sample of patients newly diagnosed with NSCLC between 2003-2005 in 5 geographically defined regions, 5 integrated health care delivery systems and 15 Veterans Affairs hospitals was surveyed 4 and 12 months after surgery. Separate logistic regression models were fitted to determine adjusted predictors of severe functional impairment, nursing home admission or death at 1 year after surgery. Recursive partitioning was used to create a risk index based on preoperative factors. Results: Of 1007 patients who underwent surgery for stage I-IIIa NSCLC, 34 (3%) patients were admitted to a nursing home, 124 (12%) died, and 146 (15%) experienced the composite outcome of death or admission to a nursing home in the first year after surgery. in unadjusted analyses, the rate of death or nursing home admission for patients age 75 or older was nearly double the rate of younger patients (20-25% vs. 11-12%, p<0.001). Additional unadjusted resultsare shown in Table 1. After adjustment, patients age 75 or older were significantly more likely to experience nursing home admission or death (OR 2.01, 95% confidence interval [1.07, 3.77], and OR 2.85 [1.50, 5.43] for ages 75-79 and 80 and over, respectively, p<0.001). Patients with severe comorbidities (OR 2.22 [1.07, 4.62], p=0.01) and patients with stage II or stage IIIa disease were also more likely to experience the composite outcome (OR 2.76 [1.76, 4.33] and OR 4.24 [2.44, 7.36], respectively, p<0.001). Among 759 patients who were alive at one year and completed a follow-up survey, 38 (5%) reported inability to get out of bed, dress or wash themselves, or perform usual activities. Fifty-one patients (7%) were admitted to a nursing home or reported severe functional impairment, with higher risk among those with moderate comorbidities (OR 3.86 [1.97, 7.56], p<0.001) or lack of a high school diploma (OR 2.95 [1.12, 7.78], p=0.02). The risk of nursing home admission, severe functional impairment or death was low (16%) for patients under age 75 and for those age 75 or older with stage I disease, intermediate (33%) for patients age 75 years or older with stage II-IIIa disease and no or mild comorbidities, and high (60%) for those age 75 years or older with stage II-IIIa disease and moderate or severe comorbidities. Conclusions: Patient's risk of long-term disability should be incorporated in preoperative counseling and decision-making.

Accuracy of Histological Classification of Needle Biopsy/Aspirate Specimens in Patients With Non-Small Cell Lung Cancer (NSCLC)

R. Sharma, S. Chandan, T. Demmy, S. Dhillon, S. Yendamuri — 2012-02-01

Introduction: NSCLC histology has emerged as an important predictor of disease response and treatment related toxicity over the last few years. Increasing use of limited biopsy material obtained by image guidance or bronchoscopy for diagnosis has been co-incident with this trend. There is paucity of data in the published literature regarding the correlation of histology of the preoperative limited biopsy with that of the final resected specimen. We looked at this correlation at our institution, a tertiary care cancer center, and studied the factors that influence this correlation. Methods: All patients with initial diagnosis of NSCLC on limited biopsy undergoing definitive surgical lung resection between Jan 1, 2008 to Dec 31, 2010 were reviewed. Those with insufficient limited biopsy specimens for diagnosis of malignancy, final diagnosis other than NSCLC and pathological complete response on final pathology were excluded. Demographics, smoking status, neoadjuvant chemo and radiation status and initial and final histological classification were noted. All patients had the initial biopsy and final resection specimen pathology reviewed at our institution. Results: 201 patients meeting above criteria were included in the present study. Demographic, treatment and histological (final pathology) characteristics are summarized in Table 1. Overall 36.4% had squamous cell carcinoma and 51.9% had adenocarcinoma. IHC was performed in 48.5% of biopsy specimens and 37.9% of final specimens. in 25.9% (52/201) of patients, initial diagnostic biopsy was either insufficient (15.4%) or inaccurate (10.5%) for histological classification. This discordance existed independent of place of biopsy (our institution vs. outside), use of IHC or use of small vs. large limited biopsies. in 155 patients that had adenocarcinoma or squamous cell carcinoma diagnosed by initial biopsy, 12.3% of patients had a different final histology (Table 2). Also, among these patients, neoadjuvant therapy was associated with greater discordance (20.5% vs. 9%; p=0.05). Conclusions: Histological subclassification is important to guide therapy in NSCLC. Even in a tertiary referral cancer center, the rate of inaccuracy of histological subclassification based on limited biopsy material is significant, especially when neoadjuvant therapy is used. Clinicians managing patients with NSCLC need to recognize this discordance. Further research is needed to develop new biomarkers capable of using limited biopsy material to subclassify NSCLC accurately.

Segmentectomy and Brachytherapy Mesh Implantation for Clinical Stage I Non-Small Cell Lung Cancer (NSCLC).

2012-02-01

Introduction: Sublobar wedge resection is associated with an increased risk of locoregional recurrence (15-20%) when compared with lobectomy for early NSCLC. We have previously shown that the addition of brachytherapy mesh at the time of sublobar resection might decrease the risk of local recurrence in this setting, equivalent to that of lobectomy [Santos et al. Surgery 2003; 134: 691-7]. In the current study, we evaluate the impact of brachytherapy mesh implantation following formal anatomic segmentectomy on local recurrence rates in the management of clinical stage I NSCLC. Methods: Retrospective review of 391 patients undergoing anatomic segmentectomy for clinical stage I NSCLC from 2002-2010 with (n=152) or without (n=239) the use of I131 brachytherapy mesh applied over the staple line. the primary endpoint was local recurrence. Secondary endpoints include morbidity, mortality and recurrence-free survival. Results: Patients undergoing brachytherapy mesh implantation were older (71.1 vs. 68.7 years, p=0.006) and had larger tumors (2.3 vs. 2.0 cm, p=0.003) compared to those treated without mesh. There were no differences noted in gender, histology or tumor stage. Overall mortality was 1.0% (Mesh-0.7%, No Mesh 1.3%). Perioperative morbidity was higher in patients receiving mesh (43.4% vs. 32.2%, p=0.03). At a mean follow-up of 32 months, the overall local recurrence rate was 5.9%. There was no difference noted in local recurrence rates between groups [Table]. Five year actuarial freedom from local recurrence was 92% in the mesh group, and 90% in patients undergoing segmentectomy without mesh (p=0.24) [Figure]. Conclusions: It appears that the local recurrence noted with non-anatomic wedge resection is not an equivalent concern when anatomic segmentectomy with adequate margins are obtained. This implies that adjuvant brachytherapy following anatomic segmentectomy is not required for local control, thus avoiding the costs of radiation therapy and its associated potential toxicity. These data suggest that proper anatomic segmentectomy alone may be associated with local recurrence rates similar to those of anatomic lobectomy in the setting of clinical stage I NSCLC.

Surgical Outcomes for Type-A Aortic Dissection in Septuagenarians and Octogenarians

2012-02-01

Introduction: Surgery for acute type-A aortic dissection (TAAD) in the elderly is associated with high morbidity and mortality. These older patients are often denied surgery. the natural history of TAAD is extremely poor without surgical treatment. We reviewed our surgical experience among patients 70 years of age and older. Methods: A retrospective review was conducted from 1996 to 2010 of patients 70+ years of age that underwent surgical repair of TAAD. Data were obtained through query of the STS database and chart review. Sixty-seven patients met inclusion criteria (male=51%, female=49%). Average age was 77(±4.6). Twenty-two (33%) patients were 80 years of age or older. Average ejection fraction was 53(±10). Comorbid conditions included: diabetes=10%(7/67), hypertension=84%(56/67), CHF=18%(13/67), smoking=39%(26/67), COPD=24%(16/67), history of myocardial infarction=27%(18/67), cardiogenic shock=18%(12/67), and PAD=27%(18/67). Multiple logistic regression was used to test for predictors of operative mortality. Interaction effect with p-values lower than 0.10 underwent follow-up with simple effect analysis. Long-term survival was tracked and evaluated using Kaplan-Meier survival analysis. Results: Operative mortality was 21%(14/67; for 70-80 group=18%[8/45], for 80+ group=27%[6/22]). One, three, five, and ten-year survival rates were 51%, 45%, 36%, and 18% for the study as a whole and 41%, 33%, 25%, and 8% for the 80+ group. Perioperative complications included myocardial infarction=3%(2/67), reoperation for bleeding=6%(4/67), neurologic impairment=19%(13/67), prolonged ventilator requirement=52%(35/67), and renal failure=19%(13/67). Despite a relatively small sample size, there was evidence of an interaction between ejection fraction (EF) and patient age that approached statistical significance. Because this interaction was hypothesized, we proceeded to estimate the odds associated with increases in EF at various points in our patient age range (figure). Higher EFs were statistically significantly protective (OR: 0.859, 95%CI 0.745-0.991) at the youngest ages, but the protective effect was attenuated and not statistically significant as age increased. Conclusions: Data suggest that despite high morbidity and mortality, satisfactory long-term survival can be achieved for 70- and 80-year-old patients requiring surgical treatment for TAAD. However, interpretation of this study, and other similar ones, must account for a likely high degree of bias in selection of only the good-risk (elderly) patients for surgery. in elderly patients treated surgically for TAAD, EF predicts mortality in the younger portion of this (elderly) group. This trend diminishes as age increases.

Right Ventricular Outflow Tract Reconstruction Using Valved Homografts in Non-Ross Patients: Small Versus Large-Size Conduit Comparison

M. Ruzmetov, D.M. Geiss, R.S. Fortuna — 2012-02-01

Introduction: To evaluate the late outcomes and effectiveness of right ventricular outflow tract (RVOT) reconstruction with homografts in non-Ross patients with complex RVOT obstruction or insufficiency. Methods: A retrospective study (1986-2011) included 156 consecutive non-Ross patients who received homografts (standard cryopreserved, 30 aortic and 105 pulmonary; decellularized, 21 pulmonary) for RVOT reconstruction. Median age at implantation was 2 years (0-67 years). Prosthetic choice included small-size homografts (<16mm, n=71; SSH) and large-size homograft (>17mm, n=85; LSH). Follow-up was greater in number in large-size homografts (LSH, 10.4±5.8 years vs SSH, 7.3±6.1 years; P=0.01). Results: Overall patient survival was 81% at 15 years. There was no homograft-related death. Early and late mortality were significant better in LSH group (LSH, 7% vs SSH, 32%; P<0.001). Freedom from dysfunction was improved at 5 and 15 years with LSH (LSH, 90% and 61% vs SSH, 59% and 31%; P<0.001). Conduit failure trended higher in the SSH cohort at 5 and 10 years (SSH, 68% and 43% vs LSH, 84% and 65%; P=0.006). Freedom from explantation at 15 years was significantly better for LSH patients (LSH, 70% vs SSH, 43% P<0.001). Freedom from distal conduit stenosis was similar (LSH, 87% vs SSH, 76% P=0.10). the time interval between first and second homograft reoperation was not significant different (P=0.92). Conclusions: the homograft as a tool for RVOT reconstruction in non-Ross patients displays high dysfunction and failure rates. Conduits larger than 17 mm have improved performance. Longer follow-up will define structural integrity and efficacy of this prosthesis.

Changes in Coronary Microvascular Reactivity After Cardioplegic Arrest in Patients With Uncontrolled Versus Controlled Diabetes

J. Feng, Y. Liu, N. Dobrilovic, A.K. Singh, L.M. Chu, F.W. Sellke — 2012-02-01

Introduction: We investigated the effects of cardioplegic arrest and reperfusion (CP-Rep) on coronary arteriolar responses to endothelium-dependent and -independent vasodilators in controlled, uncontrolled, and non-diabetic patients. Methods: Human right atrial tissue was harvested pre- and post-CPB/CP-Rep from non-diabetic (ND) patients (n = 12, HbA1C = 5.3 ± 0.2), controlled diabetic patients (n = 12, HbA1C = 6.1 ± 0.15) and uncontrolled diabetic patients (n = 8, HbA1C = 8.3± 0.3) undergoing coronary artery bypass grafting. Coronary arterioles (90-160 μm in diameter) were dissected from the harvested tissues for in-vitro microvesel study. In-vitro relaxation responses of pre-contracted coronary arterioles were examined in the presence of the endothelium-dependent vasodilators adenosine 5'-diphosphate (ADP) and substance P and the endothelium-independent vasodilator sodium nitroprusside (SNP). The gene expression of endothelium markers and the expression of oxidative stress protein of atrial tissues were analyzed by microarray analysis and western blotting. Results: The pre-CP-Rep vasodilatory responses of uncontrolled, but not controlled diabetic arterioles, to ADP (10−5 M), substance P (10−8 M) and SNP (10−5 M) were significantly diminished compared with those of non-diabetics (P<0.05), respectively. The post-CP-Rep arteriolar responses to ADP, substance P and SNP were significantly impaired in all three groups compared to pre-CPB responses (P<0.05). However, these impairments were more pronounced in the uncontrolled diabetic group (P<0.05 vs. non-diabetics). There were no significant changes in the gene expression of endothelium markers between diabetics and non-diabetics. in contrast, oxidative stress protein was significantly up-regulated in the atrial tissues of uncontrolled diabetics compared to non-diabetics (P<0.05).

Regeneration of in Utero Fetal Myocardial Infarction is Associated With Repopulation of the Infarct With Nkx2.5+ Cells

2012-02-01

Introduction: The adult response to myocardial infarction (MI) results in the loss of normal cardiac architecture, fibrosis, and decreased function. In contrast, the fetal response to MI is regenerative, with restoration of tissue architecture, lack of fibrosis, and restoration of left ventricular (LV) function. Nkx2.5 is a nuclear transcription factor involved in cardiomyocyte differentiation and a marker of cardiac progenitor cells (CPC). We hypothesized that the regenerative response to MI in fetal sheep is associated cellular repopulation of infarct with nkx2.5+ CPCs. Methods: To test our hypothesis, a 25% apical infarct was created in fetal sheep(65-76 days gestation) by ligation of the distal LAD. Control animals underwent sham operations without coronary artery ligation. the animals were sacrificed 3 days or 4 weeks after MI. Echocardiography was performed pre-and post-infarction, and prior to sacrifice to assess LV function and infarct size. Immunohistochemistry was performed for nkx2.5 and dapi. Total number of nuclei and nkx2.5+ nuclei were averaged from five representative images of apical infarcts per specimen and averaged. Significant difference determined by student T-test (p<0.05). Results: At 3 days, echocardiography demonstrated akinetic myocardium in the region of the infarct and decreased myocardial function. At 4 weeks fetal cardiac function had returned to baseline and there was no akinetic myocardium. Cell counting studies at 3 days, demonstrated significantly decreased cell numbers within the infarct compared to sham myocardium (29±11 vs 187±1.1 cells/HPF, p=0.0065) of which 9% and 63% expressed nkx2.5 respectively. One month after infarction, apical cell counts showed no significant difference in cell number between infarct and sham (184 ± 15 cells/HPF vs 193 ±30cells/HPF, p=0.85) with 64.5% and 57.2% staining positive for nkx2.5 respectively. Conclusions: This study demonstrates that the fetal regenerative response to MI is associated with increased CPC recruitment which repopulates cells lost following infarction. Strategies to increase cardiac progenitor cell numbers by increasing their proliferation and recruitment have tremendous potential in preventing or reversing the negative sequelae of ventricular remodeling following adult MI. This novel mammalian model of cardiac regeneration provides a powerful tool for the development of these strategies.

Is Peroxynitrite a Therapeutic Target in the Management of Pulmonary Hypertension?

Y. Ravi, K. Selvendiran, P. Kuppusamy, C.B. Sai-Sudhakar — 2012-02-01

Introduction: Peroxynitrite (PN) stimulates proliferation of pulmonary artery smooth muscle cells (PSMC) and human smooth muscle cells (HSMC) by activation of Akt and mediators of PI3K pathway. Our novel therapeutic compound HO-3867, a curcumin analog, may help in scavenging of PN by stabilizing Akt/PTEN and may prevent the progression of pulmonary hypertension. Methods: Two cell lines, human vascular smooth muscle cells and pulmonary artery smooth muscle cells were used in this study. HSMC and PSMC were maintained in culture and exposed to PN solution at various concentrations. (0.1, 0.25, 0.5, 1, 2.5, 5 μM) for different time points (at 24, 48, and 72 hours). Cell viability was determined using MTT assay and cell proliferation by BrdU assay. PN activity was determined spectrophotometrically at 37oC in particle-free supernatants by measuring the decrease of absorption at 340 nm. AKT and PTEN gene associated proteins and mRNA levels were measured by Western blot, immunoprecipitation and RT-PCR. Furthermore, we verified AKT induction and modulation of PTEN expression by PN in PSMC cells by using their siRNA and cDNA transfection studies. Results: Low concentration of PN (0.1 to 1 μM) stimulated the proliferation of HSMC and PSMC when compared to untreated cells. Cell proliferation was accompanied by activation of upstream (Fak) and downstream mediators (Akt, PTEN, p21, p38) of PI3K pathway on Western Blot analysis. Our novel synthetic anti-cancer and anti-oxidant compound, HO-3867 significantly inhibits both HSMC and PSMC proliferation by stabilizing the AKT/PTEN pathways. Conclusions: PN stimulates proliferation of PSMC and HSMC by activation of Akt and mediators of PI3K pathway. Our novel therapeutic compound HO-3867, a curcumin analog, may help in scavenging of PN by stabilizing Akt/PTEN and may prevent the progression of pulmonary hypertension.

NLRP3 Inflammasome is Upregulated in Thoracic Aortic Aneurysms and Dissections

2012-02-01

Introduction: Inflammation plays a critical role in the progression of thoracic aortic aneurysms and dissections (TAAD). Inflammasomes are intracellular proteins that help regulate the inflammatory response. Activated by cellular stress signals, inflammasome molecules form multiprotein complexes and function as molecular platforms to mediate the activation of inflammatory caspases, the secretion of proinflammatory cytokines, and cellular injury. Within the subfamily of NLRP (nucleotide-binding oligomerization domain-like receptor proteins) inflammasomes, NLRP3 is the most fully characterized inflammasome. In this study, we tested the hypothesis that NLRP3 inflammasome expression is altered in human TAAD. Methods: Descending thoracic aortic tissues were obtained from 10 patients with thoracic aortic aneurysms caused by chronic dissection (TAD), 10 patients with thoracic aortic aneurysms without dissection (TAA), and 8 organ donors without aortic diseases. The protein expression of the NLRP3 inflammasome was detected by Western blot, immunohistochemistry, and immunofluorescence. Human aortic smooth muscle cells (SMCs) were treated with palmitic acid and NLRP3 siRNA, and the expressions of NLRP3 and MMP9 were detected by Western blot. Results: Western blot analysis revealed that the expression of NLRP3 was significantly greater in TAA and TAD aortas than in control aortas (Figure A). Similarly, immunohistochemistry staining revealed higher NLRP3 levels in TAAD tissue, especially in dedifferentiated vascular SMCs in the media (Figure B) and inflammatory cells in the adventitia. Double staining confirmed NLRP3 expression in macrophages and SMCs. in cultured aortic SMCs, palmitic acid induced the cleavage of MMP9 from its pro-form to the active form (Figure C); knocking down NLRP3 decreased MMP9 cleavage, suggesting a critical role of NLRP3 in MMP9 activation and tissue destruction. Conclusions: NLRP3 inflammasome levels are increased in TAAD. NLRP3 may play an important role in vascular inflammation and protease-mediated tissue destruction in TAAD.

Cardiothoracic Training: Current Perceptions and Expectations of Residents and Faculty

2012-02-01

Introduction: The cardiothoracic surgery specialty is going through a phase of re-evaluation and adaptation. Our aim was to evaluate and compare perceptions and expectations relating to cardiothoracic training among residents and faculty. Methods: A content-validated, 13-item survey was distributed electronically between August 14 and August 24, 2010, to 728 cardiothoracic surgery residents, recent program graduates (on or after June 2006), cardiothoracic surgery chairpersons, program directors, and section heads. Results: The response rate was 34% (244/728). on a scale of 1 (not valuable) to 5 (very valuable), faculty willing to teach in the operating room was ranked as the most valuable aspect of a training program (mean, 4.9), whereas strict adherence to the 80-hour work week ranked as least valuable (mean, 2.3). from a list of 11 routine patient-care and housekeeping tasks, on a scale of 1 (definitely not scut work) to 5 (definitely scut work), dictating discharge summaries was most widely perceived as scut work (mean, 4.2). Responding to questions about 16 major cardiothoracic procedures of varying complexity, 46% of respondents (109/236) stated that a resident should be satisfied if allowed to perform <50% of a high-complexity case, whereas only 1% (2/238) indicated that a resident should be satisfied if allowed to perform <50% of a low-complexity case. There were many differences between the responses of residents and faculty, most notably with regards to expectations about how much of a procedure, across the complexity spectrum, residents should be allowed to perform and what constitutes scut work. Finally, 63% (150/238) of respondents (residents (72%), faculty (43%)) felt that the increasing scrutiny of outcomes has adversely affected training. Conclusions: Reconciling residents' expectations, duty-hour restrictions, and high-stakes procedures will require the development of novel educational approaches to improve resident learning.

Intravascular Retained Surgical Items: A Multi-Center Study of Risk Factors

2012-02-01

Introduction: Retained surgical items (RSI) have been previously studied in patients undergoing major surgical procedures. However, little information is available regarding RSI associated with vascular procedures. This is the first case-control study that specifically examines potential risk factors for intravascular RSI. Methods: Multi-center retrospective review of 83 RSI events was performed. Among these, 13 cases involved intravascular RSI (ivRSI). Cases were compared to procedure-matched controls to determine potential risk factors for ivRSI including procedural factors (urgency, complicating factors); patient factors (BMI); equipment failure and safety variances. Fisher's exact testing were performed. Results: Thirteen ivRSI cases and 14 controls were examined. There were no differences between the two groups with regards to age, gender, or BMI. ivRSI items included guidewires (8/13), catheter/catheter fragments (4/13) and a coil (1/13). The incidence of unexpected procedural factors was significantly higher among ivRSI cases (10/13) than among controls (3/14, p<0.007). Equipment failure occurred in 5 ivRSI cases, with no such incidences among controls (p><0.016). There were no differences between the two groups with regards to proportion of urgent procedures, bleeding >500 mL, procedure between 5pm-7am, or trainee involvement. Both groups had a very high proportion of safety variances (8 in ivRSI and 11 in control group, p=ns). In addition, 7/13 ivRSI were missed on confirmatory post-procedural imaging. Conclusions: Unexpected procedural factors and equipment failure are significantly associated with ivRSI. of concern, over half of all ivRSI were missed on confirmatory post-procedural imaging. Strict adherence to established safety protocols and incorporation of more stringent radiographic review for intravascular procedures.

Progression of Acute Appendicitis to Perforation: Examination of a Two-Variant Disease Model in 683,590 Patients

D.J. Papandria, D. Rhee, A. Gorgy, G. Ortega, Y. Zhang, F. Abdullah — 2012-02-01

Introduction: Recent studies have suggested that acute appendicitis may represent two discrete disease entities, one associated with frequent early perforation and the other associated with a low overall likelihood of perforation. One implication of this two-variant disease model has been greater emphasis by some authors on entirely nonsurgical management of non-perforated appendicitis. The present study examines the clinical evolution of acute appendicitis - from the time of hospital admission until appendectomy - to assess rates of perforation as they relate to the delay preceding appendectomy in a nationwide sample. Methods: A cross-sectional descriptive analysis was performed using the National Inpatient Sample (NIS) and Kids' Inpatient Database (KID) data from 1988-2008. Patients were selected if they received a diagnosis of acute appendicitis (perforated or non-perforated) at discharge and received surgical treatment within seven days of admission. Patients were sub-grouped by age and by delay of surgical intervention and rates of perforation were compared. Patients admitted electively and those receiving a drainage procedure prior to appendectomy were excluded. Logistic regression analysis was then used to estimate the risk of perforation associated with increasing delay of surgery. Results: Of the 683,590 patients with acute appendicitis that were included, 30.3% received a discharge diagnosis of perforated appendicitis. Overall, 27.9% of patients underwent laparoscopic appendectomy with the remainder receiving an open procedure. A total of 81.1% patients proceeded to operation on the day of admission, with 15.8%, 1.7% and 0.7% of operations performed on the second, third and fourth hospital days, respectively; operations on days five through eight accounted for less than one percent of cases. for patients undergoing appendectomy on the day of admission, the perforation rate was 28.8%, and for appendectomy later in the hospital stay, the perforation steadily increased from 33.3% on hospital day two to 78.8% by hospital day eight (p<0.001). After adjustment for patient demographics, type of surgery, hospital characteristics and admission year, the hazard ratio for perforation associated increased from 1.20 (95% CI 1.18 - 1.23) for adults and 1.08 (95% CI 1.05 - 1.11) for children on hospital day two to 4.76 (95% CI 3.62 - 6.27) for adults and 15.42 (95% CI 8.69 - 27.35) for children by hospital day eight. Conclusions: Greater time intervals separating hospital admission and appendectomy are associated with increased rates of perforation in both children and adults within this large population-based study. These findings are more consistent with the conventional model of progression of uncomplicated acute appendicitis to perforation in the absence of surgery.

Predictors of Long-Term Pain After Laparoscopic Ventral Hernia Repair: Results from a Prospective, International Study

V.B. Tsirline, P.D. Colavita, I. Belyansky, A.E. Lincourt, B. Heniford — 2012-02-01

Introduction: Incisional hernias occur in up to 20% of patients after a laparotomy. Technique refinement and the use of mesh have resulted in low recurrence rates. Issues surrounding quality of life remain suboptimally defined and in the past have been difficult to predict. The International Hernia Mesh Registry – a prospective multicenter database of patient-reported quality of life outcomes - was analyzed to determine the factors that may limit the success of laparoscopic ventral hernia repair (LVHR) in the short- and long-term. Methods: Patients undergoing LVHR from October 2007 to March 2011 were compared based on the presence of post-operative symptomatic pain, movement limitation, and mesh sensation as measured by the Carolinas Comfort Scale (CCS) in order to determine preoperative and operative factors predictive of outcome. T-test and Chi-square were used to compare continuous and discrete values respectively. Results: Of the over 1000 VHRs performed, 352 were repaired laparoscopically. Follow-up surveys were available from 257 (73%) at 1 month, 191 (54%) at 6 months, 187 (53%) at 1 year, and 81 (23%) at 2-year follow-ups. at 1-month, heavy weight mesh, fixation with glue or transfascial sutures, and larger defects were associated with significantly more pain. at 6 months, there were no such associations. At 1-year follow-up, patients had more pain if they underwent recurrent hernia repairs or if the defects were multiple. However, the most consistent predictor of long-term pain was the presence of preoperative pain. from 6 months onward, patients with preoperative discomfort, activity limitation, or overall symptoms had significantly more pain (38-40% vs 9-13%, p<0.0001 for all factors). These patients had significantly more pain at 1-year (35-38% vs 12-16%, p<0.01) and 2-year follow-ups (47-52% vs 9-12%, p<0.01). Patients who did not have preoperative symptoms showed a significant decrease in postoperative pain from 1 month to 6 months, while those with preoperative symptoms remained at their early postoperative level long-term. Conclusions: There are several factors that contribute to early postoperative pain after LVHR, including type of mesh fixation, mesh choice, and larger defects; however, preoperative symptoms emerge as the most consistent predictor of long-term pain. While most patients' discomfort typically resolves by 6 months, two-thirds of patients with preoperative pain continue to experience pain for at least 2 years after LVHR.

Predicting Complicated Choledocholithiasis: Improved Management Through Risk Stratification

2012-02-01

Introduction: Gallstone disease remains prevalent in the United States affecting 10% of the population. Choledocholithiasis is less common but can result in much greater morbidity and mortality secondary to acute pancreatitis or cholangitis. Management of choledocholithiasis exhibits wide variation in care and often requires transfer to a specialty facility which may delay diagnosis and treatment. the purpose of this study was to link patient-specific characteristics with the outcome measure of complicated choledocholithiasis to identify high risk patients who may require expedited treatment or transfer to a higher level of care. Methods: Patients with a discharge diagnosis of choledocholithiaisis (CDL) were identified from the 2009 Nationwide Inpatient Sample (NIS). Specific patient demographic and comorbid conditions were analyzed for the primary outcome measure of developing complicated choledocholithiasis (cCDL), defined as acute pancreatitis or cholangitis during the admission for CDL management. the association between cCDL and mortality was also evaluated. Patient demographics and comorbidities were compared using univariate and adjusted analyses. Results: We identified 123,990 discharges with a diagnosis of CDL. the overall incidence of CDL was 314 per 100,000 NIS discharges. Forty-one percent of CDL discharges were for cCDL (acute pancreatitis 31%, cholangitis 12%). Risk factors for cCDL included age (risk increases 0.8% per year after age 18), male gender (Odds Ratio (OR) 1.2, 95%CI 1.1-1.2), alcohol abuse (OR 1.5, CI 1.3-1.8), diabetes (OR 1.1, CI 1.0-1.2), hypertension (OR 1.1, CI 1.0-1.2), obesity (OR 1.2, CI 1.1-1.3), non-elective admission (OR 2.3, CI 2.0-2.6), and Asian/Pacific Islander race/ethnicity (OR 1.2, CI 1.0-1.5). Patients with cCDL had increased odds of mortality (OR 1.5, CI 1.2-2.0). Conclusions: Increased age, non-elective admission, and several comorbid conditions predispose to development of complicated choledocholithiasis, which in turn is associated with increased mortality. These factors can be used to stratify patients at higher risk of pancreatitis or cholangitis and to streamline CDL management by expediting treatment or transfer of high risk patients to a specialty center.

Modeling Anesthetic Times. Predictors and Implications for Short Term Outcomes

P. Kougias, G. Pisimisis, N. Barshes, P.H. Lin, C.F. Bechara — 2012-02-01

Introduction: Little is known about predictors of anesthetic times, and the impact of anesthetic and operative times on patient outcomes. Methods: We documented operative case length (OCL) - defined as the time from incision to time of dressing placement -, and anesthetic time length (ATL) - defined as the sum of anesthetic induction time length (time from the patient's entrance in the operating room to the time of skin incision) and anesthetic recovery time length (time from dressing placement to time of patient's exit from the operating room), in 1771consecutive patients who underwent elective vascular surgical interventions. We recorded patient and procedure-related characteristics that might influence the anesthetic time length, including a variable for possible July-effect. Forward stepwise linear regression was employed to model the length of anesthetic times. Forward stepwise logistic regression was used to model the impact of ATL and OCL on a composite outcome of perioperative (30-day postoperative) death, myocardial infarction, cardiac arrhythmias, stroke, and congestive heart failure. A single level random effects model was used to account for patient clustering into individual procedure types. Results: The OCL (mean: 145 minutes, SD: 100min) demonstrated higher dispersion that the ATL (mean: 64 minutes, SD: 25 minutes). Predictors of decreased ATL included regional or local anesthesia (p<0.001), decreasing surgeon's experience (p<0.001), and endovascular intervention type (p<0.001). Predictors of prolonged ATL included operative case length (p<0.001), and the presence of COPD (p<0.006). There was a trend for diminished ATL in the months of July and August (p<0.08). the random effects model improved the overall fit, and diminished the level of statistical significance for surgeon's experience as predictor of ATL. Surgeon's experience, endovascular procedure type, and operative case length accounted for 39% of the variability of the anesthetic time length. Predictors significantly increasing the odds of the composite outcome included the operative case length (OR: 1.005, 95%CI: 1.002 to 1.007, p<0.001), revised cardiac risk index (RCRI) of 3 (OR: 2.2, 95%CI: 1.18 to 4.07, p<0.01), and RCRI of 4 ((OR: 2.64, 95%CI: 1.15 to 6.02, p<0.02); whereas local anesthesia decreased the odds of the composite outcome (OR: 0.25, 95%CI: 0.09 to 0.66, p<0.006). Anesthetic induction and recovery time did not impact the composite outcome (OR: 1.004, 95%CI: 0.991 to 1.017, p<0.47). All these associations were minimally changed after accounting for patient clustering into individual procedure groups. Conclusions: Surgeon and patient related variables account for a large portion of the variability in anesthetic induction and anesthetic recovery time. In this patient population prolonged operative case length and increased RCRI were associated with increased likelihood of death and perioperative adverse cardiovascular events.

Blood Transfusion and Cancer Surgery Outcomes: A Continued Reason for Concern

W.B. Al-Refaie, H.M. Parsons, A. Markin, J. Abrams, E.B. Habermann — 2012-02-01

Introduction: The known adverse effects of blood transfusion after cancer surgery have recently been challenged in older persons with anemia or substantial intra-operative blood loss. We hypothesized that intra-operative blood transfusions adversely impact short-term cancer surgery outcomes in all age groups regardless of pre-operative hematocrit (Hct) levels. Methods: Using the 2005-2008 ACS NSQIP Participant Use File, we identified 38,926 patients who underwent thoracic, abdominal, or pelvic cancer surgery. Pre-, intra-, and post-operative factors were compared by number of blood transfusion units. Stratified multivariable analyses, by age and Hct level, were performed to assess the impact of blood transfusion on short-term operative outcomes, adjusting for covariates. Results: Up to 14% of patients received intra-operative blood transfusion. Of those, over 60% received only 1-2 units of blood. Overall, less than 5% experienced intra-operative events. Receipt of intra-operative blood transfusion predicted significantly higher rates of 30-day operative mortality, major complications, total number of complications, and prolonged length of stay (p< 0.0001). Stratified and sensitivity analyses continued to show these adverse operative outcomes in all age groups and in persons with low to normal Hct levels. (Table) Conclusions: The present study demonstrates that intra-operative blood transfusion adversely impacts short-term cancer surgery outcomes across all age groups and in those with low to normal Hct levels. While supporting previous investigations, these findings also provide insightful implications on the patterns of blood transfusion during cancer surgery that are in need of further investigation.

Why SCIP-Based Antibiotic Prophylaxis is Inadequate to Prevent Surgical Site Infections?

2012-02-01

Introduction: The Surgical Care Improvement Project (SCIP) recommends appropriate spectrum and timing of antibiotic prophylaxis to prevent surgical site infections (SSIs). However, emerging data has demonstrated that despite increased compliance, SSIs are not decreasing. Furthermore, there is an all-or-none phenomenon associated with SCIP infection guidelines. We hypothesized that despite the routine administration of antibiotic prophylaxis in pediatric surgery patients, compliance with appropriate evidence-based practice does not occur in a majority of cases. Methods: Data was prospectively collected, over a 10 week period, in randomly selected pediatric surgical cases occurring in operating rooms, neonatal and pediatric intensive care units. Emergent cases were excluded. Information was obtained from direct observation of cases and review of electronic medical records. Five aspects of antibiotic usage were evaluated for each case to determine compliance with evidence-based pediatric surgery service guidelines at a university-affiliated hospital including appropriate administration when indicated, type, weight-based dose, time between infusion and surgical incision, and redosing (if applicable). Results: During this study, 143 cases were observed. In 141 cases (98.6%), antibiotics were given or withheld appropriately, in one case unnecessary antibiotics were given, and in one case, indicated antibiotics were withheld. of the 100 cases (69.9%) requiring antibiotic prophylaxis, only 48% of cases were compliant with all four guidelines (figure). The most common failures in compliance occurred because of dosing or timing errors. Conclusions: There is significant room for improvement in the appropriate administration of prophylactic antibiotics to reduce SSIs. The perceived failure of increased compliance with SCIP infection guidelines may be linked to lack of attention to other important aspects of antibiotic prophylaxis. The importance of these components that are not addressed by SCIP on SSI rates remains to be determined.

Follow-Up of Pancreatic Cysts: is Yearly Imaging Really Necessary?

Xourafas D., W. Jiang, R.S. Lipsitz, A. Tavakkolizadeh, W.S. Ashley — 2012-02-01

Introduction: The prevalence of invasive cancer in non-resected asymptomatic pancreatic cysts is low and the ideal interval for follow-up imaging remains to be determined. International consensus guidelines suggest a yearly follow-up for lesions <1cm in size, 6-12 monthly for lesions between 1-2cm and 3-6 monthly if lesions >2cm. Symptomatic cysts that undergo resection have a potential risk of malignancy and local recurrence and should be followed but the frequency of recurrence and imaging surveillance following resection is also unclear. Methods: We identified 375 patients diagnosed with pancreatic cysts between 1992 and 2010. Patients with cystadenomas and pseudocysts were excluded. 163 patients with potentially malignant cysts were classified based on surgical or conservative management. Demographics and co-morbidities, cyst characteristics, time to recurrence and follow-up were registered. Imaging surveillance performed every 6 months was used to detect progression for the surveilled and recurrence for the resected cysts. Kaplan Meier curves were used to estimate the distribution of time to recurrence and log rank test to investigate factors associated with recurrence. Results: 92 patients incidentally diagnosed with pancreatic cysts were followed conservatively while 71 underwent surgery confirming premalignant pathology. Patient and cyst characteristics are illustrated in Table-1. at the end of the study, none (0%) of the surveilled patients experienced any significant increase in lesion size compared to baseline evaluation; (median follow-up of 35 months). for patients undergoing resection, local pancreatic recurrence was detected after the 1st year of follow-up. the Kaplan-Meier curve for recurrence demonstrates that the estimated probability of recurrence by year 1 is 0%; by year 2 is 4% and by year 3 is 12%, (figure-1A). Using the long-rank test we found that type of cyst (IPMN vs. MCN) was the only statistically significant factor associated with recurrence (p<0.05). at 2 years of follow-up, the estimated probability of recurrence for MCNs is 0% while for branch-duct IPMN is 8% CI(2.5%-33.8%) (Figure-1B). Conclusions: Our data suggest that the imaging follow-up of non resected asymptomatic pancreatic cysts with diameter of <3cm could be safely lengthened to 3 years from diagnosis resulting in a more efficient use of medical resources. Predominantly symptomatic patients with resected non invasive lesions should have an imaging surveillance at 2 years from surgery to detect early local recurrence which may benefit from further resection.

Age and Mortality After Injury: Is the Association Linear?

2012-02-01

Introduction: In 2008, 39.6 million persons were aged 65 years or older. As demographic transition progresses this group of elderly Americans is expected to number 72.1 million by 2030. Injury is the ninth leading cause of death for Americans aged 65 or older. Additionally, multiple studies have demonstrated a linear association between advancing age and mortality after injury. An inflection point, or age at which outcomes begin to differ, has not been previously described. We hypothesized that the relationship between age and mortality after injury is non-linear and an inflection point exists. Methods: We performed a retrospective cohort analysis of the trauma registry at our urban level one center from 2007 through 2009. All patients aged 65 and older with the admission diagnosis of injury were included in the analysis. Nonparametric logistic regression was used to identify the functional form between mortality and age. Multivariate logistic regression controlling for injury mechanism, Injury Severity Score (ISS), initial blood pressure, Glasgow Coma Score, hospital length of stay, head abbreviated injury score (AIS), need for any operative procedure, and need for intensive care unit (ICU) admission was utilized to explore the association between age and mortality. Age 65 was used as reference. Significance was defined as p<0.05. Results: 1,107 injured patients, 65 years and older were included in the analysis. 54% were male. A majority of patients (97%) sustained blunt mechanism. One third required ICU admission and 48% had traumatic brain injury. 229 patients (20.6%) were 84 or older. Overall mortality was 7.2% and median ISS was 9 [4, 16]. Our model indicates that mortality is a quadratic function of age. (Figure) Specifically, after controlling for confounders, age is associated with mortality with a regression coefficient of 1.08 for the linear term (p=0.02) and a regression coefficient of -0.006 for the quadratic term (p=0.03). The model identified 84.4 years as the inflection point at which mortality rate begins to decrease. Odds ratio for death at 65 years was 1.0 (reference); 70 years, 2.99 (1.34-6.34); 84.4 years, 11.78 (2.25-54.57); and 90 years, 9.24 (1.96-43.58). Conclusions: The risk of death after injury varies linearly with age until 84 years. Over 84 years of age mortality rates decline. These findings may reflect varying severity of co-morbidities in elderly trauma patients. Specifically, our injured patient population less than 84 years old may have more complex co-morbidities contributing to an increased mortality after trauma. Whereas those injured patients over 84 years old, by virtue of reaching this advanced age, may in fact have less complex or severe co-morbidities at the time of injury contributing to less risk of death.

Advanced Age is an Independent Predictor for Increased Morbidity and Mortality After Emergent Diverticulitis Surgery.

M.E. Lidsky, J. K. Marosky Thacker, S.A. Lagoo-Deenadayalan, J.E. Scarborough — 2012-02-01

Introduction: The goal of our analysis was to identify predictors of 30-day postoperative mortality after emergency surgery for perforated diverticulitis in the elderly. Methods: All patients from the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) Participant User File from 2005-2009 who underwent emergent surgery for diverticulitis were included for analysis. First, multivariate logistic regression was used to determine the association between advanced age and postoperative mortality and morbidity. Next, patients ≥ 65 years old were included in a separate multivariate regression model in order to identify predictors of postoperative mortality in the Elderly. Super-Elderly classification (age ≥ 80 years) was included as a predictor in this second regression model, as were preoperative demographic and comorbid characteristics, intraoperative variables, and the presence of specific postoperative complications. Results: 2,264 patients undergoing an emergent operation for diverticulitis were identified, of which 1,267 (56%) were Non-Elderly, 648 (28.6%) were Elderly, and 349 (15.4%) were Super-Elderly. Advanced age was associated with significantly higher postoperative mortality after adjustment for other patient- and procedure-related characteristics [1.7% in Non-Elderly (Reference), 9.7% in Elderly (AOR 4.37, 95% CI 2.12-9.03, p<0.0001) and 17.8% in Super-Elderly (AOR 9.85, 95% CI 4.41-22.0, p<0.0001)], with the affect of age on mortality appearing to be most pronounced among those patients who suffered postoperative complications. For the subgroup of patients aged ≥ 65 years, several predictors of 30-day postoperative mortality were identified, including several specific postoperative complications [Table]. Even after adjustment for preoperative characteristics and postoperative complications, age ≥ 80 years remained a significant independent predictor of death. Conclusions: Our analysis confirms advanced age as an independent predictor of increased mortality after emergent surgical management of diverticulitis, and identifies those postoperative complications that are most strongly associated with subsequent death. We also find that age ≥ 80 years remains an independent predictor of mortality even after adjustment for preoperative variables and the presence or absence of postoperative complications, providing support for the theory that Super-Elderly surgical patients may have an inherently lower physiologic reserve when undergoing emergency surgery.

The Use of ECMO for Persistent Pulmonary Hypertension of the Newborn: A Decade of Experience

2012-02-01

Introduction: Despite improvements in the management of persistent pulmonary hypertension of the newborn (PPHN), a number of infants with inadequate gas exchange may benefit from treatment with extracorporeal membrane oxygenation (ECMO). The objectives of this study were to utilize the Extracorporeal Life Support Organization (ELSO) Registry to review the outcomes of neonates with PPHN receiving ECMO and to identify pre-ECMO variables that may be associated with mortality. Methods: The study is a retrospective analysis of all PPHN ECMO patients reported to the ELSO registry from 2000 to 2010. Data is presented as a mean with SEM. Prematurity was defined as < 37 weeks gestation. Univariate analysis was performed using Student's t-test or Fisher's exact test. Variables found to be statistically significant underwent multivariate analysis by logistic regression. Kaplan-Meier survival curves were generated to assess duration of therapy and patient survival. Results: A total of 1,569 neonates with PPHN received ECMO support during the study period at an average age of 3.1 days of life (DOL) and for a duration of 6.9 days. Overall survival among neonates with PPHN receiving ECMO support was 81%, and those receiving support for 7, 10, 14, and 21 days survived at rates of 88%, 78%, 55%, and 25%, respectively. By logistic regression, prematurity (p<0.01), pre-ECMO pH ≤7.2 (p=0.02), pre-ECMO SaO2 ≤65% (p=0.01), and duration of ECMO greater than or equal to 7 days (p<0.001) were independent predictors of death in this group. An average of 2.2 complications occurred per patient, with cardiovascular, mechanical, and renal complications being the most common. Conclusions: This study affirms, that over the past decade, PPHN patients have excellent survival after ECMO therapy. Prematurity, acidosis, and profound hypoxia are associated with increased mortality. Furthermore, prolonged ECMO therapy portends a worst prognosis.

Surgery for Benign Lesions of the Liver: Are we Improving the Quality of Life for Our Patients?

2012-02-01

Introduction: Indications for surgical management of benign hepatic lesions are ill defined. While symptom relief is commonly the main therapeutic goal, there is no published data on patient quality of life (QOL) after surgery. We sought to define patient (pt) reported outcomes following surgery for benign hepatic tumors. Methods: Pts who underwent surgery for benign liver lesions were invited to complete a QOL survey. the survey was designed using elements of validated assessment tools (SF-36, McGill Pain Score, EORTC QLQ-30 & QLQ-LMC-21). Clinical and pathological data were collected from liver databases of participating centers. Data were analyzed using univariate and multivariate analyses. Results: Of 255 surveys sent, 179 (70%) responses were received. Median age was 49 years and the vast majority of pts were female (n=158, 88%). Preoperative imaging revealed benign cystic (n=75, 42%) or solid (n=85, 48%) tumors in most pts; 20 (11%) pts had indeterminate lesions. Most pts had a solitary lesion (n=128, 72%) with a median size of 7.5 cm (range, 1-22 cm). Presenting symptoms included abdominal pain (n=131, 73%), fatigue (n=70, 39%), early satiety (n=50, 28%), nausea/vomiting (n=38, 21%), or other (n=13, 7%). Preoperatively, 98 (55%) pts described “moderate - severe” pain and 73 (41%) pts had symptoms for > 6 months. Reported reasons for surgery included impaired lifestyle (n=87; 49%), fear of malignancy (n=105, 59%), and anxiety about future complications (n=156; 90%). Most pts underwent partial hepatectomy (n=129, 72%); laparoscopic approach was utilized in 73 (41%) pts. Perioperative morbidity was 16%, mortality zero. Pathology revealed: adenoma (n=30, 17%), focal nodular hyperplasia (n=35, 20%), hemangioma (n=35, 20%), simple cyst (n=64, 36%), or other (n=16%). Following surgery, pts reported less activity limitations (pre 40% vs. post 27%; p=0.001) and depressed moods (pre 36% vs. post 22%, p=0.003), improved energy levels (pre 43% vs. post 57%; p=0.001) and work productivity (pre 64% vs. post 84%; p=0.001). Overall, 168 (94%) pts reported satisfaction with their treatment; 81 (45%) pts reported a “better” or “much better” QOL. Presence of preoperative “moderate - severe” pain was the strongest predictor of overall improved QOL (OR 18.2, p=0.001). Reported pain decreased dramatically as a function of time elapsed from the operation (Figure). At 1-year, only 11 (6%) pts reported persistent “moderate - severe” pain. Conclusions: Surgery for benign liver lesions is associated with high patient satisfaction and improved quality of life. Patients with preoperative “moderate - severe” pain derive the most benefit from surgery. Most patients have resolution of symptoms by 6-12 months following surgery.

Trans-cutaneous Closure of Central Defects (TCCD) in Laparoscopic Ventral Hernia Repairs (LVHR)

M.L. Clapp, S.S. Awad, A. Subramanian, M.K. Liang — 2012-02-01

Introduction: LVHR has been reported to have lower recurrence rates, fewer surgical site infections, and shorter hospital stays compared to open repair. Despite improved surgical outcomes with standard LVHR (sLVHR), seroma formation, eventration (or bulging), and hernia recurrence remain common complications. in a 2010 pilot study, we have previously reported that TCCD may lower seroma, eventration, and hernia recurrence rates following LVHR. Our objective was to evaluate outcomes with LVHR-TCCD, including patient satisfaction and functional status. Methods: A retrospective review of one hundred fifty-nine patients that underwent elective LVHR between January 2008 and December 2010 was performed. of the 159 patients, 37 (23%) had the LVHR-TCCD procedure and 122 (77%) had sLVHR. Patient demographics, co-morbidities, hernia characteristics, operative details, imaging data, and complications were collected. Patient satisfaction (using a 10-point, Likert-type scale), late post-operative pain (using the visual analogue scale), and patient functional status (using the Activities Assessment Scale; AAS) were analyzed. Continuous data was analyzed using unpaired Student t-test and Fischer's exact test was used to compare categorical data. Results: Patient demographics, comorbidities, hernia size, and surgical histories were similar between the two groups with the following exceptions: the LVHR-TCCD group had more obese patients (BMI 34±1.4 vs 32±0.5, p <0.05), more patients with COPD (38% vs 9%, p=0.006), and more incisional hernias (78% vs 56%, p=0.09). Compared with sLVHR, patients who underwent LVHR-TCCD had significantly lower rates of seroma formation (8% vs 34%, p=0.02), hernia recurrence (0% vs 11%, p=0.02), mesh eventration (4% vs 41%, p=0.0003), tissue eventration (4% vs 37%, p=0.001), and total eventration (8% vs 52%, p=0.0001). Post-operative infectious complications and early complications were similar between both groups. Median follow-up in the LVHR-TCCD group was 25 months (range 7-30 months) and 24 months in the sLVHR group (range 7-38 months). Patient overall satisfaction (8.8±0.4 vs 7.8±0.3, p<0.05) and cosmetic satisfaction (8.7±0.4 vs 7.6±0.3, p<0.05) were higher in the LVHR-TCCD group. Late post-operative pain scores (2.4±0.5 vs 2.8±0.4, p=0.59) and chronic pain (9% vs 10%, p=1.00) were similar in both groups. Patients who underwent LVHR-TCCD had improved total AAS scores (20.9±1.9 vs 25.4±1.3, p<0.05). Conclusions: The incidence of seroma, mesh and tissue eventration, and hernia recurrence was significantly lower following LVHR-TCCD when compared to sLVHR. Subjective improvement in overall patient satisfaction, cosmetic satisfaction, and functional status was reported with closing the central defect. LVHR-TCCD may be a superior technique for treating ventral hernias due to lower complication rates and higher patient satisfaction and functional status.

Scheduled, Intermittent Oximetry Fails to Detect Postoperative Hypoxemia After Bariatric Surgery

K.L. Haines, J.B. Downs, M.B. Mullen, J.A. Klonsky, S.F. Gallagher — 2012-02-01

Introduction: Postoperative respiratory depression is a common, life-threatening sequela of patient-controlled analgesia (PCA), especially in bariatric patients with obstructive sleep apnea (OSA). Guidelines for postoperative monitoring of such patients usually include intermittent oximetry, despite the lack of evidence to support the efficacy of scheduled, intermittent, postop pulse oximetry. the aim of this study was to evaluate scheduled, intermittent, postop pulse oximetry. Methods: During two prospective, blinded, IRB-approved, pilot studies we monitored bariatric patients for 24 hours after gastric bypass using continuous pulse oximetry with arterial blood analyses every 4 hours in the first study and randomly in the second with continuous transcutaneous PtCO2. On the floor, patients received scheduled, intermittent vital sign recording including oximetry, every 2-4 hours per Hospital policy, in addition to blinded study monitoring. Data are Mean±SD. Results: Thirty-two patients (n=32, BMI 49±6 kg/m2) were monitored for 24 hours, postoperatively. All patients were screened for OSA, 80% confirmed, and all but one of those required CPAP. While all 32 patients had at least one episode of hypoxemia lasting more than 30 seconds, 25% (n=8) experienced continuous hypoxemia (SpO2<90%) for more than 10 minutes, two of whom were not in the group diagnosed with sleep apnea. Supplemental oxygen did not prevent episodes of hypoxemia. No arterial blood analyses revealed hypercarbia or hypoxemia. No patient had significant alteration in documented vital signs. Finally, not one scheduled, intermittent, spot pulse oximetry check recorded by nursing staff indicated hypoxemia. Conclusions: Despite repeated and prolonged hypoxemic episodes-identified in every patient with continuous oximetry-scheduled, intermittent oximetry did not identify a single episode of hypoxemia in any bariatric patient on the floor in the first 24 hours after gastric bypass. Furthermore, despite aggressive preoperative diagnosis of OSA and treatment with CPAP, severe, prolonged, postoperative, arterial hypoxemia was a consistent finding. in both studies, the degree and frequency of desaturation was unexpected, and no patient ever experienced significant alteration in vital signs that was recorded by intermittent monitoring. Our data showed no value in scheduled, intermittent, pulse oximetry. Similarly, scheduled arterial blood sampling was ineffective, compared to continuous monitoring with pulse oximetry. Scheduled, intermittent pulse oximetry was essentially useless in detecting hypoxemia. Our data supports continuous monitoring of oxygen saturation for at least 24 hours after gastric bypass. CPAP is indicated at all times when patients with obstructive sleep apnea might conceivably sleep. In conclusion, unbuffered, continuous pulse oximetry, with an alarm for saturation less than 90% for ten seconds, would have detected 100% of significant hypoxemic episodes, thus permitting appropriate diagnosis and therapy.

Remodeling Characteristics and Collagen Distribution in Biologic Scaffold Materials Explanted From Human Subjects After Abdominal Wall Reconstruction: An Analysis of Scaffold Remodeling by Host Characteristics and Surgical Site Classification

2012-02-01

Introduction: the purpose of this study was to perform a systematic histologic analysis of an allogeneic biologic scaffold explanted from abdominal wall repair sites of human subjects at the time of a clinically-indicated abdominal re-exploration. We hypothesized that higher collagen type I:III ratios and more favorable remodeling scores of explanted scaffolds would inversely correlate with subject Charlson Comorbidity Index (CCI), ACS Wound Class, and CDC National Nosocomial Infections Surveillance (NNIS) Risk Index assessed at time of both scaffold implantation (T1) and explantation (T2). Methods: Biopsies of the allogeneic scaffolds were obtained from the abdominal wall repair sites of n=15 patients during a subsequent abdominal re-exploration for comparision. De novo allogeneic scaffolds and biopsies were stained with H&E, and evaluated according to a semi-quantitative scoring system for characteristics of remodeling [cell infiltration, cell types, host extracellular matrix (ECM) deposition, scaffold degradation, fibrous encapsulation, and neovascularization] and a mean composite score. De novo allogeneic scaffolds and biopsies were also stained with sirius red (SR) for collagen I & fast green (FG) for collagen III, photographed under polarized light, and analyzed to determine the SR:FG ratio. Results: Mean subject age at T2, M:F ratio, mean duration of scaffold indwelling, and ratio of onlay:inlay:sublay location were 55.9 years, 7:8, 681.3 days, 3:1:11, respectively. SR/FG significantly correlated with ACS wound class at T1 (p=0.01), CCI at T1 (p=0.02), and CCI at T2 (p=0.02). Cell infiltration significantly correlated with subject age at T2 (p=0.00), CCI at T1 (p=0.00), and CCI at T2 (p=0.00). Fibrous encapsulation significantly correlated with scaffold location (p=0.03). Neovascularization significantly correlated with subject age at T2 (p=0.00). When duration of scaffold indwelling was categorized as 0, 1-6, 7-12, 13-18, 19-24, and >24 months, greater indwelling durations significantly correlated with greater composite remodeling score (p=0.00), cell infiltration (p=0.00), cell type (p=0.01), extracellular matrix deposition (p=0.03), and neovascularization (p=0.02). Conclusions: As hypothesized, greater ratios of collagen I:III significantly correlated with greater ACS wound class at T1, and greater CCI at both T1 and T2. Mean composite scores for remodeling did not significantly correlate with subject characteristics or wound class. However, significant correlations were found between greater cell infiltration and greater CCI at both T1 and T2; and greater fibrous encapsulation and inlay location. With greater indwelling duration, scaffolds demonstrated significantly greater overall remodeling, cell infiltration, favorable cell types, ECM deposition, and neovascularization, but not scaffold degradation or fibrous encapusulation. Host characteristics and surgical site assessments may predict degree of remodeling following abdominal wall repair for costly biologic scaffolds.

Early Graft Failure After Lower Extremity Arterial Bypass: Results from More Than 200 Hospitals

G. Soma, D.Y. Greenblatt, M.T. Nelson, J. Havlena, C.C. Greenberg, K.C. Kent — 2012-02-01

Introduction: Early graft failure (EGF) is a serious complication after lower extremity arterial bypass. EGF has not been examined using national data since the widespread adoption of percutaneous treatments for LE arterial occlusive disease. To address this gap, we utilized data from the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP), which includes more than 200 academic and community hospitals. Methods: Patients who underwent lower extremity arterial bypass from 2005 to 2009 were selected from the ACS NSQIP database. The frequency of 30-day EGF NSQIP variable “OTHGRAFL” was determined. Univariate and multivariate methods were utilized to identify risk factors for EGF. Results: Of 13,751 patients who underwent open lower extremity arterial bypass, 733 (5.6%) had EGF. Patients who suffered EGF had a longer mean length of hospital stay (11.4 vs. 6.4 d, p<0.001), and had higher rates of reoperation (83.3% vs. 14.8%, p<0.001) and 30-day mortality (5.6% vs. 2.2%, p<0.001). The rate of other complications in patients who suffered EGF was 37.2%, compared to 19.1% in those who did not have EGF (p<0.001). In patients who had both EGF and another complication, the majority (77.3%) experienced the other complication subsequent to EGF. In a multivariable model, factors associated with EGF included younger age, female gender, smoking, prior operation, femoral to tibial bypass, prosthetic graft, and emergent operation (Table). Conclusions: In an era of increased utilization of percutaneous techniques, EGF after open lower extremity arterial bypass remains a common event, with serious consequences. EGF is strongly associated with developing other complications, reoperation, and mortality. We have identified a number of risk factors for EGF, which may be of use for pre-operative counseling and decision making.

Diagnosis of Venous Thromboembolism as an Outpatient in Patients Undergoing Surgical Treatment for Malignancy: An Analysis of ACS NSQIP Data 2005-2008

C.E. Reinke, G.C. Karakousis, R.A. Hadler, J.A. Drebin, D.L. Fraker, R.R. Kelz — 2012-02-01

Introduction: the relationship between malignancy, surgery and venous thromboembolism (VTE) is well established. Previous studies have reported that the incidence of VTE varies by tumor type. Current guidelines provide recommendations on use of inpatient pharmacoprophylaxis for surgical oncology patients. the practice of continuing pharmacoprophylaxis after discharge is reserved for the “high-risk” patient and the use of this practice varies widely. Little is known regarding rates of outpatient VTE diagnosis by tumor site for surgical patients. the current study was designed to determine the likelihood of VTE diagnosis after hospital discharge by site of neoplasm to help inform surgeons when deciding upon the duration of pharmacoprophylaxis for their cancer patients. Methods: We performed a retrospective cohort study of patients entered into the ACS-NSQIP database from 2005-2008 who underwent a surgical procedure and had a post-operative neoplasm diagnosis. The incidence of VTE was calculated by tumor site for malignant neoplasms and the median post-operative day of diagnosis of VTE and median post-operative day of discharge were calculated and compared across cancer types. We report 30-day VTE outcomes and censored length of stay at 30 days. Patients with neoplams in the field of general surgery, gynecologic and genitourinary surgery were analyzed. Patients with VTE diagnosis on the day of discharge were excluded from the analysis of outpatient status (n=13) as well as patients who were missing day of diagnosis of VTE (n=6). For VTE patients, diagnosis status (inpatient versus outpatient) was determined and the percent of VTE patients diagnosed as an outpatient was compared across groups. An incidence of post-discharge VTE diagnosis was determined by multiplying the incidence and the percent of patients with outpatient discharge for each malignancy site. Results: Out of 130,284 patients who underwent surgery for neoplasm, there were 92,072 with malignant diagnosis by pathology, of which 76,479 were included in the analysis. The incidence of VTE varied substantially by site of malignancy (0-51 events per 1000 cases). Percent of VTE events diagnosed as an outpatient varied as well, ranging from 0 to 100% (see Table). The absolute incidence of VTE diagnosed as an outpatient ranged from 0 to 10.1 per 1000 cases, with malignancies of the pancreas, stomach and prostate having the highest rates. Conclusions: Extended VTE pharmacoprophylaxis should be considered for patients with malignancies with a high incidence of VTE diagnosis after discharge, such as malignancies of the pancreas, stomach and prostate. Future studies to determine the impact of extending VTE prophylaxis on rates of VTE and re-admission or complications of bleeding are needed.

Cost-effective of Laparoscopic Versus Open Groin Hernia Surgery in Colombia: An Institutional Perspective

F.E. Pinzon, L.C. Dominguez, F.M. Rincon, E.L. Espitia — 2012-02-01

Introduction: Laparoscopic repair (LR) reveal clinical benefits above open repair (OR) of the groin hernia (QALY: LR: 0.84; OR: 0.83); however their cost-effectiveness is controversial. In both techniques, lightweight meshes (LWM) provide less postoperative pain, earlier labor return and QoL that heavyweight meshes (HWM). In Colombia, the OR+HWM has been currently performed, but LR (TAPP and TEP) is increasing. As well, there is not available information in Colombia of the cost-effectiveness of LR versus OR and available LWM. the aim of this study is to perform a comparison of four scenarios for treatment, based on technique and meshes, to identify the economic impact of the intervention through a cost-effective analysis. Methods: Retrospective analysis of economically active patients with primary unilateral hernia, submitted to OR+HWM from 2005-2010. Three economical scenarios were simulated (OR+LWM, TAPP+LWM and TAPP+HWM) and compared with OR+HWM, by the use of institutional costs. Direct and indirect costs, cost per QALY gained and incremental cost-effectiveness radio (ICER) were determinate for interventions. Work return in LR was assumed five days less than OR according to literature. the intervention was considered cost-effective if QALY value was less than three annual GNP per capita (29.000 USD). Results: 503 patients were submitted to OR+HWM. A mean of 12.3 days of labor return was identified. Total cost of treatment per patient was 564 USD (direct: 344; indirect: 220). Cost of treatment per patient on simulated scenarios was: OR+LWM: 607 USD (direct: 387; indirect: 220); TAPP+HWM: 1242 USD (direct: 1099; indirect: 143); and TAPP+LWM: 1285 USD (direct: 1142; indirect: 143). Main direct cost of treatment in LR was associated to surgical devices (fixation and harmonic scalpel). ICER for LR groups per QALY gained was highest that reference value (TAPP+HWM: 75.500 USD; TAPP+LWM:79.743 USD). The optimal ICER for LR (TAPP) is 638 USD. Conclusions: LR (TAPP) in Colombia is not a cost-effective treatment for groin hernia. Based in clinical advantages for the patient and face to the advances of LR, significant adjustments in direct cost by industry and negotiation policies among health system stakeholders are indispensable. Continuous training among surgeons is critical for develop of the LR. Further studies are necessary to evaluate the cost-effectiveness of other techniques as TEP in Colombia.

Technetium-99m-sestamibi Imaging: Are the Results Dependent on the Reviewer?

M. Richards, E.R. Slavin, S.W. Tamarkin, R.N. Machekano, C.R. McHenry — 2012-02-01

Introduction: Minimally–invasive parathyroidectomy (MIP) is dependent on accurate preoperative parathyroid localization. We hypothesized that surgeon recognition of subtle differences in radiotracer accumulation would increase the sensitivity of technetium-99m-sestamibi imaging and result in more frequent use of MIP. Methods: Technetium-99m-sestamibi scans completed at our institution for patients who underwent resection of a solitary parathyroid adenoma were reviewed by a surgeon and a radiologist who were blinded to the identifying information of the patient, the prior interpretation of the scan and the results of operation. For each scan, the reviewer determined whether there was abnormal radiotracer accumulation and documented its location. Results were correlated with the outcome of operation and the final pathology. The blinded interpretations of the surgeon and radiologist were compared to each other and to the original radiologic interpretation. Results: From 1994-2009, 274 patients with primary hyperparathyroidism (HPT) had technetium-99m-sestamibi imaging prior to parathyroidectomy.149 patients with a single adenoma underwent curative parathyroidectomy and had scans available for review. The original interpretation of the sestamibi images were completed by 17 radiologists who reviewed an average of 11 ± 14 scans (range = 1-61). The sensitivity of technetium-99m-sestamibi imaging was 81% (74-87%, 95% CI) for the blinded surgeon compared to 70% (62-77%, 95% CI) for the blinded radiologist and 65% (57-72%, 95% CI) for the original radiologists (p<0.05). There was no difference in the false positive rates (blinded surgeon=5% and radiologist=6%, original radiologists=5%, p<0.05). Conclusions: Radiologists were less likely to call a scan positive. Surgeon recognition of subtle anatomic asymmetry increases the sensitivity of technetium-99m-sestamibi imaging and the successful completion of MIP.

Electronic Discharge Summaries for Surgical Patients: Evaluating Risks and Benefits

C.E. Reinke, C. Baillie, A. Norris, S.L. Schmidt, J. Myers — 2012-02-01

Introduction: As electronic discharge summaries (EDS) become more prevalent, analysis of summary quality after implementation remains important. Current literature evaluating EDS quality has mixed results, and no prior studies have studied surgical discharges or evaluated the use of copy and paste. We sought to assess the quality of surgical discharge summaries after implementation of an EDS and the impact of copy and paste on quality. Methods: We conducted a retrospective study of a sample of discharge summaries from surgical admissions in an urban university teaching hospital before and after the implementation of an EDS program. All patients discharged from the hospital with a discharge summary authored by a surgical intern were potential study subjects, and a random sample of 200 summaries (100 before and 100 after EDS implementation) was chosen for grading. Summaries were evaluated on several items including time to summary completion, summary length, and quality which was measured on a 13-item scoring tool. Electronic summaries were also evaluated for use of obvious copy and paste and its relationship to the quality and readability of summaries. Interrater reliability was analyzed with Crohnbach's alpha. Results were analyzed using the chi-squre test, students t-test, Fisher's exact test, and ranksum test as appropriate. Results: After exclusion of 5 patients who died, 195 discharge summaries were evaluated. Discharge summaries before and after EDS implementation were similar in admission type, length of stay, and discharge destination of the patients. Compared with dictated summaries, there was a significant decrease in time to completion and length of electronic discharge summaries. Electronic discharge summaries were equal in overall quality, with improvements in provision of follow-up phone number, follow-up instructions, pending results, and a complete discharge medication list. All other specific quality aspects, such as discharge diagnosis, history of present illness, and significant findings had equivalent scores, with the exception of readability. Obvious use of copy and paste was identified in 8% of discharge summaries, and resulted in decreased readability (score 2.0 vs 1.4, p=0.02). Conclusions: The implementation of electronic discharge summaries for patients discharged from surgical services improved the timeliness of summary completion without sacrificing quality. Excessive copy and paste can reduce the readability of discharge summaries and strategies to discourage this practice should be employed.

Sometimes You Can't Make it on Your Own: The Impact of a Professionalism Curriculum on the Behaviors, Attitudes, and Values of an Academic Plastic Surgery Practice

C.S. Hultman, M.O. Meyers, P. Rowland, E.G. Halvorson, A.A. Meyer — 2012-02-01

Introduction: Professionalism is now recognized as a core competency for graduate medical education and maintenance of certification. However, few models exist in plastic surgery that define, teach, and assess professionalism as a competency. the purpose of this project was to evaluate the effectiveness of a professionalism program in an academic plastic surgery practice. Methods: We created and conducted a 6-week, 12-hour course for health care professionals in plastic surgery (faculty, residents, nurses, medical students). Teaching methods included didactic lectures, journal club, small group discussions, and book review (Forgive and Remember, by Charles Bosk). Topics included 1) Professionalism in Our Culture, 2) Leadership Styles, 3) Modeling Professional Behavior, 4) Leading Your Team, 5) Managing Oneself, and 6) Leading While You Work. Using Kirkpatrick methodology to assess perception of the course (Level 1 data), learning of the material (Level 2 data), effect on behavior (Level 3 data), and impact on the organization (Level 4 data), we compiled participant questionnaires, scores from pre- and post-tests, and such metrics as incidence of sentinel events (defined as infractions requiring involvement by senior administrators), number of patient complaints reported to Patient Relations, and patient satisfaction (Press-Gainey surveys), for the 6 months before and after the course. Results: 30 health care professionals participated in a 6-week course, designed to improve professionalism in plastic surgery. Level 1 data: Although only 54.4% of respondents felt that the course was a “good use of my time,” 72.7% agreed that the course “will help me become a better professional” and 81.8% “would recommend the course to others.” Level 2 data: Post-test scores increased from 48% to 70% (p<0.05), and the ability to recall all 6 competencies increased from 22% to 73% (p<0.01). Level 3 data: the number of sentinel events in our division decreased from 12 to 3. After the course, one resident was placed on probation and resigned, and two other employees left the division after being counseled on issues of professionalism. Interestingly, these participants did very well on the post-test but were not considered to be “team players.” Level 4 data: Patient complaints decreased from 14 to 8, and patient satisfaction increased from 85.5% to 90.5%. Conclusions: A focused curriculum in professionalism may improve the knowledge of participants and overall behavior of the group, but may not affect individual attitudes. Nevertheless, efforts toward assessing, teaching, and influencing professionalism in plastic surgery are very valuable and should be pursued by educators, to help satisfy GME/MOC requirements and to improve the performance of the organization.

Perceived Barriers to Professional Advancement in Academic Surgery: A Structured Analysis of Gender-Based Differences

T. Hauschild, W.B. Elder, L.A. Neumayer, K.J. Brasel, M. Crandall, A. Cochran — 2012-02-01

Introduction: Women represent roughly 50% of U.S. medical students and one-third of U.S. surgery residents. Within academic surgery departments, however, women are disproportionately underrepresented, particularly at the more senior levels; 2010 data showed that 16% of Associate Professors and 8% of Professors of Surgery are women. We hypothesized that female academic surgeons perceive different barriers to professional advancement relative to their male colleagues. Methods: A modified version of the Career Barriers Inventory was administered to senior surgical residents (R) and early career surgical faculty (F) at a diverse cohort of 8 academic medical centers via an online survey tool. Likert scales were used to measure respondents' agreement with each survey item. Fisher's exact test was used to identify significant differences based upon gender. IRB exemption was obtained. Results: Respondents included 85 R (44 female, 41 male; 74% response rate) and 69 F (26 female, 43 male; 37% response rate). Women responding to this survey anticipated or perceived active discrimination in the form of being treated differently (41% R, 77% of F) and experiencing negative comments about their sex (23% R, 58% F) in academic surgical practice, findings that were notably different from their male counterparts (different treatment: 17.5% R, p=0.001, 14% F, p<0.001; negative comments: 7.5% R, p=0.001, 7% F, p<0.001). Non-conscious bias or implicit resistance to women holding academic surgical jobs manifested in the form of negative attitudes; 23% of female residents and no male residents strongly agreed or agreed that negative attitudes based upon sex as a barrier to career aspirations (p=0.009) as did 50% of female faculty and 2% of male faculty (p<0.001). the presence of overt and implicit bias results in a sense that sex is a barrier to women surgeon's career development in academic surgery (Table 1). No differences were observed between male and female respondents with regard to career preparation or structural barriers to advancement. Conclusions: Women in academic surgery experience a number of difficulties that are perceived by them to be different from their male counterparts. Women who participated in this study report feeling excluded from the male culture in Departments of Surgery, which are more male-dominated than any other medical school department. This study may help to guide plans that will ultimately transform the culture of Departments of Surgery, such as the creation of professional development programs, education of those in leadership regarding the specific barriers women face in their careers in academic surgery, and deliberate recruiting of women into academic positions.

The Financial Burden of Training Surgical Residents: Who Should Bear the Cost?

S. Patil, R. Chamberlain — 2012-02-01

It is widely believed that involvement of surgical residents prolongs operative time and increases costs. to date, there is little published in regard to the veracity of this belief and the actual additional cost of care in cases involving surgical residents is poorly documented. This report analyses costs associated with five commonly performed surgical procedures with and without surgical resident assistance. 7146 surgical procedures performed between May 2004 and February 2011 were analyzed. Data pertaining to operative time, type surgery (open or laparoscopic), and training level of residents, if any, were abstracted. Two major groups and 11 procedure related subcategories were formed. Attending only (AO) cases did not involve residents, whereas cases designated as AR, involved attendings and residents. the difference in the operative times between these groups coupled with the hospital OR charges per minute of time ($15/min after the initial 30 minutes) was used to calculate the additional cost of care. the impact of resident training level on OR time was assessed separately for each procedure related subcategory. Statistical analysis was conducted using IBM SPSS® 18.0.1 Windows version. 28 surgeons and 121 residents were involved in 7146 surgical procedures performed over 81 months. 6666 (93.3%) cases involved residents and 480 (6.7%) cases did not involve residents. Procedure related subcategories included 339 U/L laparoscopic inguinal hernia repair (INH), 131 B/L laparoscopic INH, 1138 U/L open inguinal INH, 38 B/L open INH repair, 3127 laparoscopic cholecystectomy (LC), 1032 laparoscopic appendectomy, 125 open appendectomy, 280 laparoscopic colectomy, 226 open colectomy, 324 total thyroidectomy, and 386 partial thyrioidectomy. Although an increased OR time was seen in nearly all cases in which residents were involved, it was significant for only LC cases involving PGY2 residents, p< 0.001. Involvement of PGY5 residents in open INH (both U/L and B/L) decreased the OR time, p>0.5. the additional time required to perform 6666 surgical procedures with the involvement of residents was 483951 minutes and the corresponding increase in the OR cost was $7255941 over the period of 81 months. Involvement of surgical residents significantly increases OR time and cost, especially in laparoscopic cholecystectomy cases. the increased operative time also represents lost opportunity costs for attending surgeons. the training of surgical residents is an important public health endeavor and as such, further research into the finances, lost opportunity and time required for resident training should be performed in order to permit graduate medical education bodies to better determine appropriate reimbursement for teaching institutions.

Learning Preferences of General Surgery Residents: Implications for Education Curricula Development in the Era of Work Hour Restriction

2012-02-01

Introduction: Learning preferences define an individual's preferred sensory modality for the intake of new information. Teaching is most effective when there is concordance between teaching styles and learning preferences. This is even more important in the era of work hour restrictions where time is limited, and vast amounts of information must be communicated in short periods of time. Surgical education is therefore being supplemented by complementary educational techniques including the development of online lectures, educational web sites, and skills labs. Such strategies would likely be most effective if they were congruent with the learning preferences of the general surgery residents. However, the learning preferences of general surgery residents remain poorly defined. It is therefore the purpose of this study to determine the learning preferences of general surgery residents with the overarching aim of curricula development, evaluation, and modification. Methods: With exemption from the Institutional Review Board, the Visual-Aural-Read/Write-Kinesthethic (VARK) questionnaire was anonymously administered to 85 general surgery residents of our academic, tertiary, urban training program. Statistical analysis was performed using the Z-test of proportions. A p value of less than 0.05 was considered significant. Results: General surgery residents are significantly more likely to have a multimodal learning preference compared to a unimodal preference (58 vs. 27, p <0.0001, 95% CI 0.22-0.42). of these, 20 had a preference for two (34.8%) modalities, 12 (20.7%) for three, and 26 (44.8%)for all four modalities. A significantly higher proportion of residents preferred the kinesthethic mode (61 of 85, p=0.0038) compared to the visual mode (49 of 85, p<0.001), the aural mode (42 of 85, p=0.008) or the read/write mode (50 of 85, p=0.008). Resident preferences did not vary with the postgraduate year, gender of the resident, location of medical school (US vs. International) or type of residency position (categorical vs. preliminary). Conclusions: General surgery residents have a predominantly multimodal learning preference, which is consistent across the entire training spectrum. Further, a significant proportion had a preference of a kinesthethic mode of learning. These findings suggest the need for the development of multimedia education curricula with a strong skills lab component

Show Me the Money: Cost Comparison of Virtual Reality Versus Immersive Part Task Laparoscopic Cholecystectomy Simulation-Based Training

J. Paige, A. Marr, L. Stuke, J. Hunt, C. Hilton — 2012-02-01

Introduction: Cholecystectomy (CCY) is one of the most common procedures performed by general surgeons, 90% of which are done via a laparoscopic approach. Simulation-based training (SBT) of laparoscopic CCY provides an opportunity for junior residents to gain “experience” in a safe learning environment outside the operating room (OR). Such training can be accomplished using either a virtual reality (VR) machine or an inanimate procedural trainer (PT) containing anatomically correct models and disposable components. The upfront cost of each device, however, is very different. Whereas VR machines are quite expensive, PT devices are more reasonably priced. We conducted a cost comparison of conducting a laparoscopic CCY SBT program using a VR machine versus an immersive PT model. Methods: From January to June, 2011, 21 postgraduate (PGY) 1, 2, and 3 level general residents underwent training in a laparoscopic CCY SBT curriculum consisting of structured mental rehearsal followed by SBT using either a virtual reality (VR) machine or PT model housed within a realistic laparoscopic virtual OR (VOR) suite (i.e., immersive PT SBT). This PT model required replacement of the gallbladder model and overlying skin after use. Training was distributed over two sessions for each resident. Upon completion of the program, the cost of training using each format was determined and compared based on the number of sessions conducted and the number of residents completing the full curriculum. Additionally, residents completed surveys containing questions using a 5-point Likert-type scale (1=Definitely no to 5=Definitely yes) related to the effectiveness of the training device they used. Mean scale scores were determined for each group and compared using t-test. Results: Nineteen of the 21 residents completed the full laparoscopic CCY curriculum. Nine residents (PGY 1 = 4, PGY 2 = 3, PGY 3 = 2) underwent VR SBT over 12 half day sessions, and 10 residents (PGY 1 = 4, PGY 2 = 4, PGY 3 = 2) underwent immersive PT SBT over 11 half day sessions. Without including institutional overhead, total cost for conducting the program was higher for the immersive PT SBT group ($750 per resident) compared to the VR SBT group ($560 per resident). PT SBT was still higher even when room costs were equalized. Residents in both groups equally felt that training on the device was relevant to their professional development (4.44 for VR SBT versus 4.50 for PT SBT, p =NS). Conclusions: Although the initial upfront cost for a VR machine is high, SBT using it can cost less compared to less expensive PT models due to disposable costs. the costs for both forms of SBT, however, are minimal compared to those related to biliary injury during laparoscopic CCY. Residents found both SBT formats equally relevant to their development as surgeons.

Disorganized Care: The Findings of an Iterative, In-Depth Analysis of Surgical Morbidity and Mortality

C. Graham, K. Gohil, C. Anderson, B. Mosher, K. John — 2012-02-01

Introduction: Performance improvement driven by the review of surgical morbidity and mortality is often limited to critiques of individual cases with a focus on individual errors. Little attention has been given to an analysis of why a decision seemed right at the time or to lower level root causes. the application of scientific performance improvement has the potential to bring to light deeper levels of understanding of surgical decision-making, care processes, and physician psychology. Methods: A comprehensive retrospective chart review of previously discussed morbidity and mortality cases was performed with an attempt to identify areas where we could better understand or influence behavior or systems. We avoided focusing on traditional sources of human error such as lapses of vigilance or memory. An iterative process was used to refine the practical areas for possible intervention. Definitions were then created for the major categories and subcategories. Results: On comprehensive review, two areas of possible intervention emerged: Technical failures and disorganized care. Of a sample of 152 presented cases, 53 (35%) were related to technical failures and 71 (47%) were related to disorganized care. 52 (34%) cases had multiple causes. In addition, 53 (35%) were strictly related to patient disease with no other contributing causes. Of the 71 cases identified with disorganized care, 40 (56%) were related to undisciplined treatment strategies, 15 (21%) were related to failures in critical thinking, 7 (10%) were related to failures in situational awareness, and 9 (13%) were related to failures in resource allocation. Twenty-two (55%) of the undisciplined treatment strategies were related to sepsis management. Four (10%) were related to failures of venous thromboembolic disease prophylaxis. Conclusions: On a comprehensive review of cases presented at the morbidity and mortality conference, the failure to deliver appropriate care as based on standardized guidelines was frequently identified to be the result of disorganized care as well as technical failures. The disorganization resulted from failures of resource allocation, critical thinking, situational awareness, and undisciplined treatment strategies. Future research may determine if focused training in these areas improves patient outcomes.

Tackling Technical Skills Competency: A Surgical Skills Rating Tool

T.J. Wade, T.P. Webb — 2012-02-01

Introduction: The 2011 ACGME common program requirements have mandated a competency-based assignment of duties and progression to independent practice. In order to satisfy these directives, valid and reliable assessment tools must be developed to determine resident competence in a variety of duties, including technical skills. This study evaluated a novel rating tool to evaluate competence in basic surgical suturing skills. Methods: A technical skills exercise consisting of the closure of three incisions, each three centimeters long was devised in 2006. the incisions were closed with simple stitches with two-handed knots, vertical mattress stitches with instrument knots, and a running stitch with one-handed knots respectively. Fifteen minutes were allotted to complete the exercise. A rating instrument with 17 competency markers worth one point and a global 5-point Likert scale competency score (1=not competent, 5=expert) was used to evaluate the performance. Twelve first week PGY1 surgical residents completed the exercise in 2006, and 16 final month PGY1 surgical residents completed the exercise in 2011. All tasks were video recorded and scored on video review. Statistical analysis included descriptive statistics, t-score analysis of total competency scores, rank sum analysis of global competency scores, Cohen's kappa coefficient for inter-rater reliability, and Cronbach's alpha for internal consistency. Results: The mean total score (11.8 vs 13.9, p=0.002) and median global competency rating (1 vs 3, p<0.001) were lower for the first week cohort than final month resident cohort. Inter-rater reliability was high with a Cohen's kappa coefficient of 0.77. Cronbach's alpha measure of internal consistency was 0.87. Conclusions: This rating form is a valuable tool to evaluate resident technical skill competency. the form displayed construct validity with improvement in both total score and competency global rating from early to late PGY1 residents. the form also demonstrated excellent internal consistency and inter-rater reliability. This form may be used to evaluate technical skill competency in an efficient technical skills exercise.

Elderly Surgical Patients: Are There Gaps in Residency Education?

T.J. Wade, J. Petronovich, K. Denson, D. Simpson, D. Brown, T. Webb — 2012-02-01

Introduction: the geriatric population is the fastest growing demographic in healthcare and is projected to exceed 20% of the U.S. population by 2030. Geriatric specific education is essential to ensure the competency of surgical graduates to care for this population. However, competing educational demands and constricting duty hours may leave an educational “gap” in geriatric training. This study assesses resident and faculty perceptions of resident geriatric related competencies and correlates these perceptions to the actual management of injured geriatric patients. Methods: A resident/faculty geriatric competency survey and geriatric chart review template were developed based on a review of the APDS and Surgical Council on Resident Education (SCORE) curricula. the 24 question competency survey was framed within the ACGME Competencies and sent electronically to 40 general surgery residents and 57 Department of Surgery faculty members (Likert scale where 5=excellent). Five general markers of clinical care were identified for the chart review: goals of care documentation, identification of delirium, pain control, anticoagulation management, and medication management based on institutional geriatric medicine management guidelines. A retrospective chart review was then performed of 22 injured, hospitalized patients > 65 years old with at least 3 rib fractures. Results: 30/40 (75%) residents and 22/57 (39%) faculty completed the survey. Residents were more likely to rate themselves as overall “competent in geriatric care” than faculty (90% vs. 68%, p=0.048). Residents rated their competency higher than faculty on all 16 ACGME Competency related questions (p=0.0002). Four questions had a mean faculty rating <3 (below acceptable): screening guidelines, delirium management, contraindicated medications, and medication adjustments. on chart review: code status was documented in 7/22 (32%) patients, goals of care in 1/22 (5%) patients. Delirium was documented in only 1 patient. Pain control included rib block or epidural in 14/22 (64%) patients with pneumonia developing in 3/11 (27%) patients who received neither therapy. Anticoagulation was present in 3/22 (16%) patients on admission with 1/22 (5%) patients requiring correction. Contraindicated medications were prescribed in 13/22 (59%) patients. Conclusions: A competency based needs assessment of geriatric training in a general surgery residency has identified educational “gaps” for further training. Faculty and resident surveys identified target areas including medication and delirium identification and management. the chart review corroborated these clinical care deficiencies and identified two additional areas: discussion of goals of care and pain management. This needs assessment supports the implementation of geriatric education initiatives in our general surgery program.

The Adequacy of Dictated Operative Reports

N.P. Zwintscher, M.J. Martin, J.A. Maykel, S.R. Steele — 2012-02-01

Introduction: the surgeon's clinical note has been previously shown to poorly reflect both physician-centered and patient-centered outcomes. We hypothesized that dictated operative reports do not adequately demonstrate surgeons' work-load, preoperative involvement, clinical decision-making or core competencies. Methods: We retrospectively reviewed operative reports in the month of January for the years 2007-2011. Operative reports were dictated by general surgery residents (R1-R5), rotating interns and surgical staff (general, vascular and cardiothoracic). All intern and resident reports were approved by staff surgeons. We qualitatively assessed each for fifteen items that encompassed physician-centered outcomes (preoperative physical exam, preoperative objective data, surgical indications, and postoperative plan), patient-centered outcomes (presenting complaint, intraoperative complications, and procedure tolerance), and Joint Commission/Medicare required fields (provisional diagnosis, postoperative diagnosis, practitioner name, procedure performed, operative findings, procedure description, estimated blood loss (EBL), and specimens removed). Groups were compared to each other with one way ANOVA with Bonferroni correction. Results: We reviewed 999 dictated operative reports. of these only 21.2% included a preoperative physical exam and 16.3% documented a postoperative plan. 80.7% listed the patient's presenting complaint; however, only 57.3% listed whether or not there were any complications. All charts included a procedure description and the surgeon's name. Only half recorded operative findings. 67.4% documented EBL. the mean number of fields missed based on level of surgical training was R1: 4.83, R2: 4.46, R3: 3.68, R4: 3.35, R5: 3.29 and staff: 3.09. There was no difference between interns and second-year residents (p=0.210), but they missed significantly more data fields than upper-level residents and staff (p<0.0001). Staff surgeons also missed fewer data fields than third year residents (p=0.004). There was no statistical difference between R4, R5 and surgical staff (p=1.000). Conclusions: The dictated operative report does not accurately document preoperative surgeon involvement, clinical decision-making, maintenance of core competencies or full compliance with Joint Commission regulations. Focused education and enhanced staff oversight of junior-level dictated operative reports may be required to improve quality.

Who's Guarding Whom? the Changing of the Guard: A Study of Pediatric Abdominal Examinations

R.J. Doiron, C.W. Hartin, D.E. Ozgediz, P.L. Glick — 2012-02-01

Introduction: Pediatric abdominal examination requires a delicate balance of distraction and elicitation of findings while preventing undue discomfort to the patient. True involuntary guarding in children may be difficult to discern from voluntary guarding, but the distinction is important. Other findings are also essential to establish a differential and guide treatment. We hypothesize that there is a significant difference in findings on abdominal examination in a pediatric emergency department between two examiners, a general surgery resident (GS) and an emergency department resident (ED). Methods: The records of 100 consecutive children evaluated for possible appendicitis by both an ED and GS resident at a children's hospital were reviewed. Critical components of an abdominal examination between the two examiner groups were recorded and tested for agreement. Fisher's exact was used to compare the inclusion of each component of the physical examination between the groups. Results: Table 1 demonstrates the significant differences found between the groups of examiners for the same children. the ED examiners tested for and documented rebound tenderness (p = 0.0072) and bowel sounds (p < 0.0001) significantly more than the GS examiner. the GS examiners tested for and documented the presence of a soft abdomen, guarding, peritoneal signs, psoas sign, Rovsing's sign, and obturator sign significantly more than the ED examiner (all with p value of ≤ 0.001). There was no difference in the recording of masses, distention, or tenderness to palpation. When the ED examiner recorded that the child was guarding, only two of 19 specified whether guarding was voluntary or involuntary compared to 21 of 36 GS examiners (p = 0.0006). Conclusions: An unacceptable degree of variability and disagreement exists in pediatric abdominal examination, which may contribute to excessive diagnostic testing and radiation exposure, as well as diagnostic delay and potential patient morbidity. Guarding should be documented as voluntary or involuntary, and rebound tenderness should probably not be elicited in a child. These results suggest that closer scrutiny of fundamental principles in pediatric abdominal physical examination may be warranted in medical school and residency curricula, and additional multidisciplinary training may be needed in this specific area.

Novel Technique for Gallbladder Retraction During Single, Incision Cholecystectomy: Initial Experience

A. Echeverria, U. Garza, A. Kaul, C. Galvani — 2012-02-01

Introduction: Single-incision laparoscopic cholecystectomy (SIL-C) is evolving as an improved technique in minimally invasive surgery. Nevertheless, some problems have arisen due to the space minimization and as a consequence, the reduction of the degree of freedom of surgical movements and the impossibility to use assistants during the surgery. Consequently, one of the main setbacks is the lack of retraction to allow adequate exposure, which is so vital in laparoscopic procedures. A novel, self-retaining, internal retractor was developed to improve gallbladder retraction. Herein, we present our initial experience using a total intracorporeal device for retraction of the gallbladder. Methods: Exclusion criteria included; morbidly obese patients, patients with acute cholecystitis, patients with known common bile duct stone or biliary disease, and patients with significant medical conditions. Procedure; A 2.5cm umbilical incision was used in every patient. A single port platform was used for access. the initial version of the retraction device was developed by attaching a Lone Star stay hook (Lone Star Medical products, Stafford, TX) to the back of a laparoscopic bulldog clamp. (Aesculap, Tuttlingen, Germany). Later in the series, a modified bulldog clamp, the Cinch Organ Retractor System, was used (Aesculap, Tuttlingen, Germany). to retract the gallbladder, the internal retractor was introduced through a 12mm trocar and deployed at the gallbladder fundus using a bulldog applicator. Using a laparoscopic needle driver, the hook needle was grasped and attached to the parietal peritoneum of the right hemidiaphragm, thereby enabling cephalad retraction of the gallbladder. the internal retractor was repositioned as needed. to carry out dissection, one straight bariatric-length instrument and a flexible-tip “L” hook cautery were used. Results: Between July 2010 and August 2011, a total of 26 patients underwent SIL-C using the internal retractor at our Department of Surgery. There were 22 female (85%) and 4 male (15%). Average age was 45 years (range 24-85 years), and the average BMI was 27.68 ± 4.3 kg/m2(range 20.1-36.3 kg/m2). Postoperative diagnosis included 18 (69%) cholelithiasis, 6 (23%) chronic cholecystitis, and 2 (8%) gallbladder polyp. Operative time was 54 ± 22 minutes (range 20-118). Estimated blood loss was 8 ± 7 cc (range 2-35cc). Time average to discharge patients was 2 ± 2 hours. Postoperative pain score was 2 ± 2 (Scale 1-10). No intraoperative complications were observed due to lack of exposure or from the retractor. No conversions were documented. Conclusions: In this initial experience, the use of this novel retraction device demonstrated to be reliable, safe and easy to use. the retraction device allows the surgeon to perform the cholecystectomy within acceptable operative time, and no added complications. the advantages of this new device are that it is versatile, reusable, reduces the number of incisions, and is re-adjustable. in addition, more retractors can be added if necessary. the adaptability of this novel device will potentially broaden the range of procedures that can be performed through a single-incision.

Non-Cosmetic Benefits of Single-Incision Laparoscopic Sigmoid Colectomy for Diverticular Disease: A Case-Matched Comparison to Standard Multi-Port Laparoscopic Technique

V. Vasilakis, C.E. Clark, H.T. Papaconstantinou — 2012-02-01

Introduction: Single-incision laparoscopy (SIL) is an advance in laparoscopic colectomy technique and has gained significant momentum. Its popularity is due in part to improved cosmesis. Studies have shown feasibility and safety of SIL colectomy; however, the benefits of SIL colectomy are not well defined. the purpose of this study is to directly compare outcomes of SIL sigmoid colectomy for diverticular disease with standard multi-port laparoscopic (LAP) technique. Methods: We retrospectively reviewed data from our first 20 consecutive elective SIL sigmoid colectomy for diverticular disease. Cases were matched for patient age, gender, body mass index (BMI), ASA score, and presence of previous laparotomy to an equivalent number of most recent LAP sigmoid colectomy cases for diverticular disease. All operations were performed by the same surgeon. Operative outcomes measured included operative time, estimated blood loss (EBL), procedure conversion, and incision length. Short-term outcomes include length of hospital stay (LOS) and 30-day hospital readmission. Maximum post operative pain score using visual analog scale (VAS) was obtained from nursing records for POD1 and 2, and time of discharge. Results: Data in Table 1 is expressed as mean ± stdev. Twenty SIL and LAP sigmoid colectomy cases for diverticular disease were analyzed. the mean age, BMI, and ASA score were similar between groups. Each group had 8 (40%) female patients, and 7 (35%) patients with a previous open laparotomy. Operative time and EBL were similar between the two groups. There were 2 (10%) conversions in both the SIL (2 to open) and LAP (1 to hand-assisted laparoscopy and 1 to open) groups. the mean incision length for SIL and LAP (4.9 cm vs. 5.1 cm) groups were not significantly different. LOS was 3.7 days for the SIL group, which was over 1 day shorter than the LAP group (5.0 days; p<0.05). Hospital readmissions were similar between groups with 1 (5%) in the SIL group and 3 (15%) in the LAP group. Maximum VAS pain score was significantly lower in the SIL group on POD 1 and 2 (table 1). Pain at time of discharge was similar between groups. Conclusions: These data indicate that the SIL technique is a safe and effective approach for sigmoid colectomy in patients with diverticular disease. There was no increase in operative time, EBL, conversion rates and 30-day readmission when compared to standard LAP technique. Furthermore, these data show that SIL approach is associated with a shorter LOS and decreased early postoperative pain. Collectively, these data suggest that SIL sigmoid colectomy has the benefit of improved patient recovery and may become the preferred technique for laparoscopic colectomy. Further prospective studies are required to fully realize the benefits of SIL colectomy.

Improving Efficiency and Productivity in the Operation Room: Implementing a Process Flow Control System

D.A. Anaya, V. Tiwari, D.H. Berger — 2012-02-01

Introduction: The AHRQ and National Science Foundation have highlighted the critical role of Systems Engineering for HealthCare delivery. the operating room (OR) is a complex system requiring coordination across multiple units; efficient execution of its processes is hampered by fragmented communication. Process flow control systems providing real-time information to users have improved quality and efficiency within numerous industries. Our goals were to implement a process flow control system in the OR and to determine its effects on OR efficiency and productivity. Methods: The OR Control System (ORC) was implemented on April 2010. Specialized hardware equipment was installed in all perioperative areas and flow control software was tailored to hospital practices. OR efficiency was measured by the proportion of first case delays (FCD), mean turn-around time (TT) and percent time of room utilization (RU); productivity was measured by surgical volume. Pre-ORC and current OR data were compared using Spearman rank correlation test. Results: The ORC system collected information for over 6,000 procedures derived from a 14-room OR unit (12-month period). the FCD and TT decreased by 22% and 18% (p<0.05), respectively. RU increased by 4% (p=0.06). These improvements were associated with a significant increase in the surgical volume representing 180 additional surgical cases during the last 6-month period. Conclusions: The ORC system improves communication among OR users facilitating daily OR operations. This resulted in improved efficiency and higher productivity. Systems engineering such as this have a high impact on the quality, efficiency and productivity of essential healthcare delivery processes within the OR.

Near-Infrared Imaging for Intra-operative Assessment of Perfusion in Vascularized Bone Flaps

2012-02-01

Introduction: Free vascularized bone transfers are a versatile and valued technique for reconstructing large bone defects resulting from postoncologic resections, severe trauma, infections, and congenital defects. It is clear, however, that vascularized bone transfers come with significant risk of complications. Recent literature reveals that patients with total flap loss after vascularized bone flap reconstruction is reported to be as high as 14% with complication rates found to range from 21 to 43%. Therefore, it is imperative that we establish techniques to effectively monitor the viability of vascularized bone grafts to decrease the failure rate of these flaps. Near Infrared (NIR) fluorescence imaging system is capable of providing a composite intraoperative evaluation of perfusion in real time and has been shown to assess tissue perfusion. This study was designed to investigate the ability ofNear Infrared (NIR) fluorescence imagingto effectively monitor and assess perfusion and viability on harvested vascularized bone flaps. Methods: Vascularized bone grafts were created (N=10) using well established models for pig fibular bone and forelimb osteomyocutaneous flap allotransplantation. Bone quality and perfusion was studied at the time of flap harvest by clinical observation and confirmed intra operatively with doppler ultrasound. Intraoperative perfusion was also assessed with NIR fluorescence imaging. Animals were injected with indocyanine green and NIR fluorescence excitation (745-779 nm) light are generated by light emitting diodes (LEDs) over a 15 cm area. Intermittent clamping trials were performed on the vascular pedicle to assess perfusion. NIR fluorescence images were obtained simultaneously with color video and merged images were formed. Results: Our composite fibular and forelimb flaps appear to be viable and well perfused upon examination. (Figure 1A) NIR fluorescence reveals that while clamped, our cross section of vascularized bone does not emit fluorescence when injected with ICG. (Figure 1B) Once clamp is removed, our vascularized bone segment emits fluorescence indicating perfusion along the periosteum on cross section. (Figure 1C and composite image Figure 1D) Conclusions: Currently there are no established techniques to effectively monitor the viability of vascularized bone grafts. in our initial pilot study, results suggest that NIR fluorescence has the capability to assess perfusion and viability of bone. This emergent technology shows promise in the ability to assess tissue and bone perfusion during the elevation and harvest of vascularized bone in addition to monitor perfusion after microsurgical anastamosis. NIR fluorescence imaging proves to be highly valuable in reconstruction of large bone defects.

Assessment of Perfusion in a Partial Face Transplanation Model With a Near-Infrared Imaging System

2012-02-01

Introduction: Composite tissue allografts (CTAs) have many applications in microsurgery including partial face composite tissue allotransplantation. Partial face CTA has recently been achieved in human subjects with success, however, significant risk of complicationsstill exists with great concern for tissue ischemia and rejection. As a result, it is imperative that we establish techniques to effectively monitor the viability of transplanted microsurgical grafts to decrease the failure rate of composite tissue flaps. Near Infrared (NIR) fluorescence imaging system is capable of providing a composite intra-operative evaluation of perfusion in real time and has been shown to assess tissue perfusion. This study was designed to investigate the ability ofNear Infrared (NIR) fluorescence imagingto effectively monitor and assess perfusion and viability on harvested partial face CTAs. Methods: Hemi-facial CTAs were created (N=4) using a well established model for hemifacial transplantation. the Hemi-facial flap consists of ear cartilage, nerve, parotid gland, lymphoid tissue, muscle and skin. the composite tissue flap is supplied by the carotid artery and external jugular vein. the elevated hemi-facial flap was assessed intra-operatively by clinical examination and confirmed with doppler ultrasound. Intraoperative perfusion was also assessed with NIR fluorescence imaging. Animals were injected with indocyanine green (ICG) and NIR fluorescence excitation (745-779 nm) light are generated by light emitting diodes (LEDs) over a 15 cm area. NIR fluorescence images were obtained simultaneously with color video and merged images were formed. Results: Hemi-facial flaps are elevated from our pigs excluding the nose and the eyelids(Figure 1A). Flap perfusion is then assessed with injection of ICG. With NIR imaging, arterial perforators emit fluorescence indicating perfusion along the surface of the skin paddle (Figure 1B). Perfusion in the vascular pedicle can be discerned through an arterial and venous phase (Figure 1C and 1D respectively). Quantitative metrics for arterial and venous perfusion were assessed. Conclusions: The ability to evaluate and assess perfusion and viability of composite tissue is highly valuable in facial transplantation. in our initial pilot study, our results suggest that NIR fluorescence has the capability to assess perfusion and viability of partial facial CTAs. This emergent technology shows promise in the ability to assess tissue perfusion during the elevation and harvest of a composite flap in addition to monitor perfusion after microsurgical anastamosis. NIR fluorescence imaging proves to be highly valuable in monitoring the viability of transplanted microsurgical grafts to decrease the failure rate of composite tissue flaps.

Optical Imaging of Rat Sentinel Lymph Nodes With a Clinical Photoacoustic and Ultrasound System

J.A. Margenthaler, T.N. Erpelding, L. Jankovic, M. Pashley, L.V. Wang — 2012-02-01

Introduction: Sentinel lymph node biopsy has become the standard method of axillary staging for patients with early breast cancer and clinically negative axillae. Although sentinel lymph node identification rates are high with the use of methylene blue dye and/or radioactive tracer, it requires an invasive surgical procedure for pathologic analysis with associated morbidity. Axillary ultrasound has emerged as a diagnostic tool to evaluate the axilla, but it can only assess morphology and cannot specifically identify sentinel lymph nodes. in this pilot study, we have investigated the utility of a non-invasive photoacoustic and ultrasound sentinel lymph node identification system. Methods: Animal protocols were approved by the institutional animal studies committee. Photoacoustic and ultrasound images of the axillary lymph nodes were collected before and after 0.1 mL intradermal injection of 1% methylene blue dye into the left forepaw pad of 7 healthy Sprague-Dawley rats using a photoacoustic imaging system adapted from a Philips iU22 clinical ultrasound. to investigate clinical translation, the imaging depth was extended up to 5 cm by adding chicken breast on top of the rat skin surface. Three-dimensional photoacoustic images were acquired by mechanically scanning the ultrasound transducer and light delivery fiber bundle along the elevational direction (Y axis). Co-registered images displayed photoacoustic signals using a pseudo-colormap ranging from blue to red over grayscale B-mode ultrasound images. Results: Pre-injection photoacoustic B-mode images featured photoacoustic signals from superficial blood vessels and skin surfaces. Five minutes post-injection, methylene blue accumulated in sentinel lymph nodes, as detected photoacoustically. When imaging depth was increased by adding biological tissue on top of the rat skin surface, lymph nodes were able to be identified at 5 cm depth. Three-dimensional photoacoustic images illustrated the propagation of methylene blue from lymph vessels to sentinel lymph nodes. Optical spectra based on photoacoustic signals from sentinel lymph nodes closely matched the optical absorption spectrum of methylene blue, confirming the presence of methylene blue in detected lymph nodes (R = 0.995). Conclusions: Co-registered photoacoustic and ultrasonic images demonstrate the ability to combine functional (photoacoustic) and structural (ultrasonic) features for sentinel lymph node mapping. These results support the clinical investigation of photoacoustic and ultrasound imaging in the identification of sentinel lymph nodes with the potential for accurate, non-invasive staging of the axilla in breast cancer patients.

Mechanism for Expansile Nanoparticle Uptake in Mesothelioma

D.M. Gilmore, K.V. Zubris, M.W. Grinstaff, Y.L. Colson — 2012-02-01

Introduction: Mesothelioma is an aggressive form of cancer affecting the pleura, pericardium and peritoneum. Despite aggressive surgical resection, 5-year survival rates are less than 40% and recurrence rates reach 80%. Previously, we have demonstrated anti-tumor efficacy of paclitaxel-loaded pH-responsive expansile nanoparticles (Pax-eNP) both in vitro and in in vivo murine models of malignant mesothelioma with significantly prolonged survival in Pax-eNP treated animals. We have hypothesized that these nanoparticles enter mesothelioma tumor cells via endocytosis however, until now the mechanism of cell uptake has not been determined. Methods: Human malignant pleural mesothelioma cell lines MSTO-211H (ATCC, Manasses, VA) were cultured for24 hours before being incubated for 1 hour in media containing inhibitors of macropinocytosis, general endocytosis, cholesterol dependent, caveolae-mediated, or clathrin-mediated endocytosis. Expansile nanoparticles were labeled with rhodamine to allow for intracellular visualization. Rhodamine-labeled expansile nanoparticles (Rho-eNP) were added to the media of each treatment group and co-incubated for an additional 4h. Cells were collected and washed in FACS buffer and the intracellular presence of Rho-eNP within tumor cells was studied using flow cytometry (LSRFortessa, BD Biosciences Bedford, MA). Results: MSTO cells rapidly demonstrate uptake of Rho-eNP within 4h. in the presence of inhibitors of macropinocytosis (wortmannin and hexamethylene amiloride) the uptake of Rho-eNP is reduced by greater than 40%. in contrast, MSTO cell uptake of Rho-eNP were not affected by inhibitors of general endocytosis, cholesterol dependence, caveolae-mediated or clathrin mediated pathways of endocytosis, as Rho-eNP uptake remained greater than 95% of untreated controls. Conclusions: Uptake of expansile nanoparticles by tumor cells utilizes pathways of macropinocytosis. This pathway is non-receptor dependent thus allowing rapid nanoparticle uptake to occur in tumors of various cell origins; thus explaining similar anti-tumor efficacy against breast and ovarian carcinoma in vivo.

Vascularized Adipose Tissue Flaps Engineered With Minicircle DNA Provide a Platform for Sustainable Transgene Expression

2012-02-01

Introduction: Several single gene disorders, including factor IX deficiency, require gene delivery strategies that would provide a stable, safe and sustainable systemic protein release. Previous techniques relying on viral or plasmid gene delivery are clinically limited by risk of insertional mutagenesis or poor efficiency due to gene silencing. Minicircle (MC) DNA represents an ideal gene delivery system that is episomal and resistant to in vivo gene silencing. MCs are engineered through removal of the bacterial backbone of plasmid DNA, preventing immunogenicity and subsequent transgene elimination. Adipose tissue flaps are ideal candidates for MC transfection due to their excellent vascularization, peripheral location and expendability. We propose MC transfection of an adipose tissue flap as a safe, reversible and clinically translatable gene delivery system. Methods: In vitro, murine adipocytes and adipose derived stem cells (ASCs) were isolated from inguinal fat pads and transfected with MC DNA encoding for luciferase or GFP via nucleofection. In vivo, naked MC DNA was injected into murine inguinal fat pads at two doses (10ug or 30ug, n=6), and compared to injection of PBS alone (n=4). for all experiments, successful transgene expression was determined using fluorescence microscopy for GFP and bioluminescent in vivo luciferase imaging (IVIS) for luciferase MCs. Results: In vitro nucleofection resulted in efficient transgene expression in both murine ASCs and adipocytes. In vivo luciferase MC DNA injection showed a localized, initial maximum luminescence of 1.81x106 and 9.44x105 photons/s/sr/cm2 at 30 and 10ug, respectively, compared to 1.02x104 photons/s/sr/cm2 in the negative control (p=0.02). Although luciferace expression declined initially, it remained localized to the fat pad and was traceable over a mid-term period of time. Conclusions: Vascularized adipose tissue flaps are particularly suitable for sustainable gene delivery approaches since they are easily accessible and expendable. We successfully show that transfection with MC DNA is a viable and safe in vivo gene delivery system that provides efficient mid-term transgene expression with minimal adverse effects. This has significant clinical implications, specifically with respect to the treatment of single gene disorders where a constant systemic release of a factor is required.

Manipulation of the Intrinsic Apoptotic Pathway to Enhance Stem Cell Survival Towards an Osteogenic Lineage

D. Lo, J. Hyun, D. Montoro, M. Grova, D.C. Wan, M.T. Longaker — 2012-02-01

Introduction: There are significant hurdles to overcome for the ultimate widespread clinical application of stem cells. the exact fate of these cells when implanted in vivo remains a challenging and important question. the clinical application of these cells often involves placement into a wound environment that is hypoxic with an upregulation of inflammatory mediators. Within this hostile environment, a large percentage of the implanted progenitor cells undergo apoptosis. the Bcl-2 family is a group of proteins that function as one of the main mediators of cellular apoptosis and survival. These proteins are divided into pro-survival and pro-apoptosis groups and the balance of expression between the two groups is a critical component in determining cellular fate. in this study, we overexpressed Bcl-2, a pro-survival protein, in order to evaluate whether it would increase cellular survival during osteogenic differentiation. Methods: Human adipose-derived stromal cells (hASC) were obtained from lipoaspirate according to Stanford IRB protocols. Human embryonic stem cells (hESC) were obtained through existing federally mandated cell lines. the intrinsic apoptosis pathway was manipulated through the over-expression of the pro-survival protein, Bcl-2 in hASC and hESC. the ability of hASC and hESC with Bcl-2 over-expression to undergo in vitro osteogenic differentiation was tested using qPCR, western blotting, immunocytochemistry and staining for alkaline phosphatase and Alizarin Red after placement in osteogenic medium. Enhanced cellular survival with Bcl-2 overexpression in hASC and hESC was evaluated by placement of these cells under hypoxic conditions and through the induction of apoptosis using a pro-apoptotic chemotherapeutic agent. Western blotting and ELISA were employed to evaluate levels of pro-apoptotic mediators cytochrome c and Caspase 3, 7, and 9. Results: Bcl-2 over-expression in hASC and hESC did not compromise the ability for these stem cells to differentiate down an osteogenic lineage in vitro as shown by comparable expression of early osteogenic genes, alkaline phosphatase and Runx2. Bcl-2 overexpression also showed comparable mineral deposition as shown by Alizarin Red staining. However, Bcl-2 over-expression significantly decreased mediators of apoptosis in vitro under hypoxic conditions and during the introduction of a pro-apoptotic chemotherapeutic agent. Specifically, Bcl-2 over-expression inhibited the apoptotic mediators cytochrome c and Caspase 3, 7, and 9 (*p<0.05). Conclusions: These data show that overexpression of Bcl-2 can modulate effectors of apoptosis leading to increased survival of stem cells directed towards an osteogenic lineage. the ability to increase survival of implanted progenitor cells will be a critical step for the widespread use of these cells in regenerative medicine.

Alginate Gels Treated With Nonthermal Plasma: A Novel Wound Dressing

2012-02-01

Introduction: Treatment and prevention of wound infection remains a challenge in medicine. Currently, many dressings are used with varying efficacy as antimicrobials and barriers to contamination. Alginate hydrogels are often used because they are soft, inert, have hemostatic and bacteristatic properties, and can absorb large amounts of exudate relative to their size. Alginates are often modified with bactericidal materials (chlorhexidine or silver ion) to improve their efficacy. Generally, the more effective antimicrobial agents are more irritating to tissue, thus the most effective antimicrobial dressings cannot be placed on open wounds. Methods: We used non-thermal plasma discharge to confer antimicrobial properties to an alginate hydrogel. Non-thermal plasma energizes electrons, which then produce excited species, free radicals, and ions that have biocidal effect. We used colony count assays to determine bacterial killing and ViaCount assays to determine cellular toxicity. Results: Alginate treated with non-thermal plasma for 1 minute sterilized 10^9CFU/mL E. coli. By placing a time interval between exposure to plasma and bacteria, we found that the antimicrobial properties after a 3-minute plasma treatment remain undiminished for 21 days. We also found a wide spectrum of activity: treated hydrogels inactivated every antibiotic resistant organism tested. Finally, we exposed epithelial cells and fibroblasts to treated hydrogels, chlorhexidine-containing dressings, and silver-containing dressings. the hydrogels were less toxic than chlorhexidine-containing dressings and comparable to silver-containing dressings. Conclusions: These results suggest that this novel product has potential for clinical use. Future research will assess the toxicity of plasma-treated gels in vivo as well as the efficacy in treating clinically infected wounds.

Vodka and Wine Consumption in a Swine Model of Metabolic Syndrome Alters Insulin Signaling Pathways in the Liver and Skeletal Muscle

N.Y. Elmadhun, A.D. Lassaletta, L.M. Chu, C. Bianchi, F.W. Sellke — 2012-02-01

Introduction: The purpose of this study was to examine the effects of vodka and wine consumption in the context of metabolic syndrome on insulin signaling pathways in the liver and skeletal muscle. Methods: Male Yorkshire swine developed metabolic syndrome by consuming a high calorie, high-fat/cholesterol diet for 4 weeks. Pigs were then split into three different groups according to diet supplementation for an additional 7 weeks. the control group was continued on a hypercholesterolemic diet alone (HCC) (n=9). the hypercholesterolemic vodka (HCVOD) and hypercholesterolemic wine (HCW) groups were supplemented with 112 mL of vodka (40% EtOH/V) (n=9) and 375 mL of red wine daily (12.5% EtOH/V) (n=8) respectively. Animals underwent intravenous dextrose challenge prior to euthanasia and tissue collection. Protein expression in the liver and skeletal muscle was analyzed by western blot. Results: At baseline, HCC, HCVOD and HCW groups had similar blood glucose levels (67.00, 66.13, 62.78 mg/dl respectively). the mean BMI of the HCC, HCVOD and HCW groups were also not statistically different (35.02kg/m2, 32.44kg/m2and 33.17kg/m2respectively). the dextrose challenge showed that HCVOD and HCW groups had significantly higher blood glucose levels at 30 minutes (210 and 206.89mg/dl) and 60 minutes (147.13 and 125.89mg/dl) compared to HCC (166.29mg/dl and 94.57mg/dl). Western blot analysis in skeletal muscle demonstrated a marked increase in the markers involved in the insulin signaling pathway including phosphorylated insulin receptor substrate 1 (p-IRS1) (Ser612), IRS2, AKT, AMP-activated protein kinase (AMPKα), peroxisome proliferating activated receptor α (PPARα), Fox01, and glucose transporter type 4 (GLUT4). There was a decrease in the p-AKT (Thr 308) level in skeletal muscle. in the liver, there was only a moderate increase in the markers of insulin signaling in HCW including AKT, AMPKα, and GLUT4 (see table). Conclusions: Vodka and wine consumption in a swine model of metabolic syndrome increases glucose intolerance compared to the control. the western blot data demonstrates an altered activation of the insulin signaling pathway in the liver and skeletal muscle. Whereas an inhibitor of the insulin signaling pathway p-IRS-1 is elevated and an activator of the pathway p-AKT is decreased, there are also elevated levels of other markers involved in propagating insulin signaling. the mixed signals may result in the observed glucose intolerance. Alcohol seems to have a complex and negative effect on the insulin signal transduction pathway.

Salivary VEGF Plays an Essential Role in Oral Mucosal Wound Healing

2012-02-01

Introduction: Palatal mucosal wound healing occurs more rapidly than in cutaneous wounds. Vascular endothelial growth factor (VEGF) induced angiogenesis is critical for cutaneous wound healing. We have previously demonstrated 1) salivary glands produce high levels of VEGF and 2) an essential role for salivary VEGF (salVEGF) in the intestinal response to injury. This has led us to hypothesize that salVEGF is critical in oral mucosal wound healing and neovascularization. to test this hypothesis, we performed a loss-of-function experiment by selectively inhibiting salVEGF using a novel adenoviral construct (AdVegfTrap) and a gain-of-function experiment by supplementing oral VEGF following submandibular gland (SMG) resection. Methods: In the loss-of-function experiment, C57/BL6 mice underwent SMG duct cannulation and were treated with 1x108 PFU AdVegfTrap, AdLacZ or PBS (n=5/group). 1.5 mm palatal mucosal wounds were created. in the gain-of-function experiment, C57/BL6 mice underwent SMG sialoadenectomy (n=4) or sham operation (n=5). 1.5 mm palatal wounds were created and oral VEGF protein was supplemented. AdVegfTrap was not used due to anticipated consumption of the orally replaced VEGF. in both experiments, salVEGF protein levels were quantified by ELISA. Wounds were harvested at 3 days and analyzed for epithelial gap and vessel density. Data were expressed as mean+SEM. Results: Retrograde SMG duct injection of AdLacZ results in 100% transduction efficiency confirming effective transgene delivery. SMG duct cannulation, SMG administration of AdLacZ and sham operation controls do not result in significantly decreased of salVEGF (PBS513pg/ml±141 vs AdLacZ353pg/ml±56 vs sham472pg/ml±40 vs control514pg/ml±89;p=NS). Administration of AdVegfTrap results in depletion of salVEGF comparable to SMG sialoadenectomy (AdVegfTrap122pg/ml±42 vs SMG resection136pg/ml±15.9;p=NS). Selective inhibition of salVEGF by AdVegfTrap impairs wound closure (AdVegfTrap947μm±22 vs AdLacZ411μm±21 vs PBS355μm±31;p<0.001) and vessel density (AdVegfTrap15.95caps/hpf±0.83 vs AdLacZ30.87caps/hpf±1.1 vs PBS30.5caps/hpf±0.95;p<0.001). Oral replacement of VEGF in SMG resected mice rescues the impaired reepithelialization and neovascularization phenotype (SMG with oral VEGF194μm±43 vs sham350μm±46,p=NS; SMG with oral VEGF24.0caps/hpf±1.4 vs sham18.6caps/hpf±2.1,p=NS). Conclusions: Selective inhibition of salVEGF significantly impairs palatal reepithelialization and neovascularization. Oral supplementation of VEGF rescues the impaired wound healing phenotype in SMG sialoadenectomized mice. Taken together, these findings suggest that salVEGF is an essential growth factor in oral mucosal wound healing which may be a surrogate for intestinal wound healing. the palatal wound model may be a valuable tool in understanding the critical role of salVEGF in the alimentary tract response to injury.

Intestinal Alkaline Phosphatase Administration in Newborns Decreases Inflammatory Cytokine Expression in a Neonatal Necrotizing Enterocolitis Rat Model

R.M. Rentea, S.R. Welak, K.M. Friedrich, K.A. Pritchard, K.T. Oldham, D. Gourlay — 2012-02-01

Introduction: Our previous research has shown supplementation of Intestinal Alkaline Phosphatase (IAP), an endogenous protein expressed in the intestines, decreased the severity of Necrotizing Enterocolitis (NEC) associated intestinal injury. We hypothesized that IAP administration prevents the development of NEC related intestinal inflammation. Methods: Full-term newborn Sprague-Dawley rat pups were sacrificed on day of life four after vaginal or cesarean birth. Control pups were vaginally delivered and breast-fed, while the cesarean delivered rat pups were exposed to intermittent hypoxia in addition to feeds containing LPS (NEC). Select NEC pups received either 40, 4 or 0.4 units/kg of bovine IAP (NEC+ IAP40u, IAP4u or IAP0.4u) once daily in formula. Serum cytokine analysis for IL-1β, IL-6, IL-10 and TNF-α were performed. Data were analyzed by paired two-tail t-test and expressed as mean +/- SEM and p≤0.01 (0.05/5) considered significant after correcting for multiple comparisons using the Bonferroni method. Results: Supplemental, enteral IAP was associated with a decrease in NEC related intestinal injury in a dose dependent fashion IAP treated pups had intestinal injury grades of <1 on a 4 point scale (IAP40u 0.6, IAP4u 0.8 vs NEC 2, p≤0.002 and 0.0001). This resulted in a significant increase in serum TNF and IL-1 (6 and 4 fold change p≤0.00006 and p≤0.0008) in NEC vs Control which returned to control level with increasing doses of supplemental enteral IAP. Conclusions: We conclude that NEC is associated with an increase intestinal and systemic inflammation and that IAP administered enterally can prevent NEC related injury through mitigation of intestinal inflammation. Prophylactic enteral IAP may prove to be a useful strategy to prevent NEC.

Neuregulin-4 is Protective Against Necrotizing Enterocolitis in a Rat Model

S.L. Castle, A.V. Grishin, H.R. Ford, M.R. Frey — 2012-02-01

Introduction: ErbB4 is a member of the receptor tyrosine kinase family that includes epidermal growth factor receptor (EGFR). in human inflammatory bowel disease and adult murine colitis models, ErbB4 levels are elevated. Furthermore, ErbB4 expression protects cultured colon epithelial cells from cytokine-induced apoptosis. However, while activation of the related receptor EGFR is protective in a rat model of NEC, the role of ErbB4 in this disease is unknown. We hypothesized that ErbB4 would be upregulated in experimental NEC, and that administration of neuregulin-4 (NRG4), an ErbB4-specific ligand, would protect against NEC in our established rat model. Methods: Timed-pregnant Sprague-Dawley rats were allowed to deliver spontaneously. Pups were immediately separated from their mothers and randomized to formula feeding (FF), formula feeding with added NRG4, or breastfeeding alone (BF). Breastfed animals were returned to their mothers and allowed to nurse ad lib. FF and NRG4 groups were gavage-fed with formula and subjected to hypoxia (5 min at 5% oxygen) and hypothermia (10 minutes at 4 °C) per our established protocol. the NRG4 group was given 100ng/pup NRG4 with each feed. Animals were examined frequently for signs and symptoms of illness and euthanized upon the development of severe disease. Terminal ileum was collected for immunofluorescent analysis of ErbB4 expression. to examine the effect of ErbB4 activation in vitro, IEC-6 rat ileal epithelial cells were treated with 108 cfu/mL Cronobacter sakazakii, a bacterium that has been implicated in both human and experimental NEC, and 100 ng/ml of the ErbB3/4 shared ligand, heregulin, or the ErbB4-specific NRG4. After 6h, apoptosis was assessed using a luminescent caspase activity assay. Results: Immunofluorescence analysis showed increased ErbB4 in FF animals compared to BF animals. NRG4-treated animals showed levels comparable to the BF group, presumably reflecting ligand-induced receptor downregulation. Development of NEC in rat pups treated with FF plus NRG4 was delayed compared to pups fed with formula alone, with 86% of treated animals surviving beyond 36 hours, compared to only 29% of the FF group (p=0.02). in vitro, the ErbB4 ligands heregulin and NRG4 reduced bacteria-induced IEC-6 cell apoptosis by 31+/-1.0% and 45+/-5.6%, respectively. Conclusions: NRG4, a ligand specific for ErbB4, is protective against bacteria-induced apoptosis in vitro, and against early development of NEC in a neonatal rat model. As ErbB4 has a uniquely focused set of downstream binding partners compared to other ErbB/EGFR family members, it may represent a highly selective therapeutic target to prevent or attenuate NEC in humans.

Resolvin E1 Reduces Intraabdominal Adhesions by Inhibiting Macrophage Chemoattraction into the Peritoneum

H. Kosaka, A.F. Stucchi, A. Kantarci, H. Hasturk, T.E. Van Dyke, J.M. Becker — 2012-02-01

Introduction: Postoperative adhesions occur in more than 94% of patients after abdominopelvic surgery and can lead to serious complications including small bowel obstruction and infertility. We have previously demonstrated that a robust inflammatory response ensues within hours of abdominal surgery and that elevated levels of the proinflammatory mediators such as the neutrophil chemoattractant CXCL1 and CXCL2 and their receptor CXCR2 underlie this response. Macrophage chemoattractant CCL1 and its receptor CCR8 are also implicated in adhesion formation. the early recruitment of these inflammatory cells is thought to contribute to the suppression of peritoneal fibrinolytic activity and initiate adhesiogenesis. Resolvin E1 (RvE1) is a potent proresolving lipid mediator of inflammation derived from the omega-3 fatty acid eicosapentaenoic acid that acts locally to inhibit leukocyte recruitment and promote resolution. the goal of this study was to examine the effects of RvE1 on postsurgical adhesion formation and the pathways described above. Methods: BALB/c mice underwent laparotomy and cecal cautery applied with a bipolar forceps (5 Watts for 1 second) to induce peritoneal adhesions using our previously described model. Mice received a single intraoperative intravenous dose of either RvE1 (200ng/mouse) or 0.9% saline. A sham operated group received similar laparotomy and treatment with RvE1 without cecal cautery. Animals (n=7-8/group) were sacrificed at 0, 3, 6, 12, 18 and 24 hours and cecum was collected and RNA extracted to measure the mRNA levels for CCL1, CCR8, CXCL1, CXCR2, interferon-gamma (IFN-γ) and plasminogen activator inhibitor-1 (PAI-1), a key mediator of peritoneal fibrinolytic activity, relative to actin mRNA in cecal tissue by real-time quantitative PCR. in separate experiments, adhesions were induced and animals were administered with RvE1 as above, and sacrificed on day 7. Adhesions to the site of cecal cautery were scored 0–5, based on a validated scoring system accounting for adhesion number and tenacity. Results: RvE1 administration significantly decreased adhesion formation by 34% compared to saline controls (5.0±0. vs 3.3±0.6; p<0.05). RvE1 significantly reduced CCL1 (8.7±6.2 vs 0.6±0.2; P<0.05) and CCR8 (5.8±2.6 vs 1.1±0.5 P<0.05) mRNA levels 3 hours after surgery compared to saline controls. CCL1 and CCR8 mRNA levels were unaffected by RvE1 administration in the sham laparotomy group. CXCL1 (24.9±5.6 vs 18.9±4.0), CXCR2 (1.6±0.6 vs 2.1±1.1), IFN-γ (39.9±22.2 vs 24.4±10.3) and PAI-1 (5.5±1.3 vs 4.8±0.3) mRNA levels were unaffected by RvE1 treatment compared with the saline-treated control mice. Conclusions: These data indicate that the inflammation-resolving lipid RvE1 mediates adhesiogenesis in part by regulating macrophage chemokines and suggest that RvE1 is a potential therapeutic adjunct for adhesion prevention.

The Histone Deacetylase Inhibitor Valproic Acid Decreases Postoperative Adhesions With One Intraoperative Dose

2012-02-01

Introduction: Postoperative adhesions are a formidable source of morbidity. Previous studies from our laboratory have shown that peritoneal inflammation and reduced peritoneal fibrinolysis are implicated in adhesiogenesis. the histone deacetylase (HDAC) inhibitor valproic acid (VPA), in addition to its efficacy in seizure and mood disorders, has been shown to have novel anti-inflammatory and anti-proliferative properties. We hypothesized that VPA could reduce postoperative adhesions. Methods: 72 male Wistar rats underwent laparotomy with creation of six peritoneal ischemic buttons to induce adhesions. Rats received intraperitoneal (IP) administration of either 1ml of 0.9% saline (control, n=24) or 1ml of saline containing VPA at 200mg/kg (n=6), 100mg/kg (n=12), 50mg/kg (n=12), or 25mg/kg (n=6) at 24 hours preoperatively, intraoperatively, and 24 hours postoperatively. An additional group received a single intraoperative dose of VPA at 50mg/kg (n=12). on postoperative day (POD) 7, animals were sacrificed and adhesions were quantified as percent of ischemic buttons with adhesions. to investigate mechanisms, ischemic buttons were created in 12 rats and either 50mg/kg VPA or 0.9% saline was administered in one intraoperative dose. 24 hours later, animals were sacrificed, peritoneal fluid was collected, and fibrinolytic activity was measured using a kinetic bioassay for tissue plasminogen activator (tPA). in addition, 4 nonoperated (nonop) rats were given one IP injection of 50mg/kg VPA and four untreated rats served as nonop controls. to assess intestinal wound healing, 12 rats underwent laparotomy with complete colonic transection and single layer anastomosis. Rats received either one intraoperative dose of VPA at 50mg/kg or 0.9% saline. on POD7, the colonic segment containing the anastomosis was resected and burst pressure was measured. Results: Given in three doses, VPA significantly reduced postoperative adhesions by 40% (62.5% vs. 37.5%) and by 38% (71.3% vs. 44.4%) at doses of 100mg/kg and 50mg/kg respectively (p<0.001). There was no significant reduction in adhesions in animals administered doses of 200mg/kg or 25mg/kg. A single intraoperative dose of 50mg/kg VPA significantly reduced postoperative adhesions by 45% (71.3% vs. 39.4%; p<0.001). A single IP injection of VPA in nonop animals increased peritoneal fibrinolytic activity by 213% (0.83U/ml vs. 1.77U/ml) compared to nonop controls (p=0.03). in operated animals, peritoneal fibrinolytic activity was not significantly different between animals administered saline and VPA (2.61U/ml vs 2.08U/ml, respectively). There was no significant difference in colonic burst pressure between animals administered a single dose of 50mg/kg VPA and those given saline (221 mmHg vs. 264 mmHg). Conclusions: the significant reduction in postoperative adhesions by a single intraoperative dose of VPA indicates a novel mechanism of action for this HDAC inhibitor and suggests a new role for HDAC inhibitors in adhesion prevention.