Abstracts Presented for the Thirty-Fourth Annual Meeting of the Association for Academic Surgery

Journal of Surgical Research 93, 306 –377 (2000) doi:10.1006/jsre.2000.5989, available online at http://www.idealibrary.com on

ABSTRACTS Abstracts Presented for the Thirty-Fourth Annual Meeting of the Association for Academic Surgery Hyatt Regency Hotel, Tampa, Florida, November 2– 4, 2000

PLENARY SESSION 1. Identification of SURF2, a Ubiquitin E3 Ligase for TGF-␤ Signal Transducer SMAD2/3. X. Lin, Ph.D, and X. H. Feng, Ph.D. Departments of Surgery and Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas. Loss of the antiproliferative responsiveness to TGF-␤ is often considered as a major step in tumor progression. Tumor suppressors SMADs are often inactivated in colorectal and pancreatic cancers. SMADs are also negatively regulated by ubiquitin/proteasomedependent degradation. The objective of this study is to identify a ubiquitin E3 ligase that controls the degradation of TGF-␤responsive SMADs and to determine whether it physically interacts with SMAD2, SMAD3, or SMAD4 to promote their degradation. Methods. Partial cDNA sequences for an E3 ligase were identified in GenBank databases using BLAST program. We designated this E3 ligase SURF2 (for SMAD ubiquitin regulatory factor 2). Comparison with other E3 ligases using GAP program allowed us to design primers to amplify the full-length cDNA sequence for SURF2 in a PCR using total placenta cDNA as templates. SURF2 cDNA was then subcloned into an expression vector for subsequent sequencing, in vitro translation, and expression in mammalian cells. Interactions of SURF2 with SMADs were determined using yeast two-hybrid analysis and GST pull-down in vitro binding assay. Results. We cloned and sequenced the full-length cDNA of SURF2. Sequence analysis indicated that it has highest identity (83%) with Smurf1, a Xenopus SMAD1-specific E3 ligase. In yeast two-hybrid assays, we found that SURF2 interacted with SMAD2 and weakly with SMAD3. GST pull-down analysis also demonstrated that SURF2 directly interacted with SMAD2 and SMAD3, but not SMAD4, implying that SURF2 is an E3 ligase specific for SMAD2/3 degradation. Conclusion. We identified SURF2, an E3 ligase that specifically interacts with SMAD2 and SMAD3. Further characterization of their roles in SMAD2/3 degradation will help understand how positive and negative regulation of SMADs contributes to TGF-␤-induced tumor suppression. 2. Evidence for Receptor-Mediated Signaling by Sphingosine 1-Phosphate in Enteric Glia. B. J. Segura, M.D., R. A. Cowles, M.D., L. Xiao, M.D., and M. W. Mulholland, M.D., Ph.D. Departments of Physiology and Surgery, University of Michigan Medical Center, Ann Arbor, Michigan. Purpose. Sphingosine-1-phosphate (S1P) is a unique product of sphingomyelin hydrolysis that reportedly serves as an extracellular signaling molecule by activation of a new family of G protein-coupled

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receptors (GPCRs): endothelial differentiation gene (EDG) receptors. Our previous studies indicate S1P selectively causes dose-dependent calcium signaling in glia (not neurons) of the enteric nervous system (ENS) by mobilizing intracellular calcium (Ca i2⫹) stores. We hypothesized that S1P induces Ca i2⫹ signaling in enteric glia via inositoltrisphosphate receptors (IP 3Rs) on Ca i2⫹ stores through putative S1P GPCRs (EDG-1, EDG-3, EDG-5). Methods. Mixed primary cultures of myenteric plexus glia and neurons were isolated from guinea pig Taenia coli. Ca i2⫹ changes, ⌬[Ca 2⫹] i (nM), were quantified with Fura2AM (2 ␮M). Results represent data derived from over 50 cells and are expressed as mean ⫾ SEM (*P ⬍ 0.05 vs control by ANOVA). RT-PCR and sequencing analysis of plexus cultures was performed using primers generated from known human, rat, and mouse EDG sequences. Results. The cell-permeable IP 3R antagonist, 2-aminoethoxydiphenyl borate (2APB), dose-dependently inhibited S1P (1 ␮M)-generated Ca i2⫹ transients (see table). Additionally, EDG-1, EDG-3, and EDG-5 were found by RT-PCR in the guinea pig

TABLE—ABSTRACT 2 S1P ⫹ 2APB (␮M)

% Pos cells ⌬[Ca 2⫹] i

Control (S1P alone)

25

75

125

200

86 ⫾ 10 170 ⫾ 13

83 ⫾ 9 124 ⫾ 5*

41 ⫾ 6* 77 ⫾ 4*

53 ⫾ 8* 60 ⫾ 5*

6 ⫾ 2* 33 ⫾ 3*

ENS (82–92% nucleotide and amino acid sequence homology to human, rat, and mouse sequences published in GenBank). Conclusions. S1P causes Ca i2⫹ signaling in enteric glia by mobilizing IP 3Rsensitive Ca i2⫹ stores. This response may occur as a consequence of putative S1P receptor(s) activation. 3. Tumor Cell Adhesion to Endothelial Cells Is Increased by Endotoxin via an Upregulation of ␤-1 Integrin Expression. E. J. Andrews, M.B., B.Ch., D. C. Winter, M.D., J. H. Wang, Ph.D., W. E. Laug, M.D.,* and H. P. Redmond, M.Ch. Department of Surgery, Cork University Hospital, Cork, Ireland; and *Department of Pediatric Oncology, Childrens’ Hospital of Los Angeles, Los Angeles, California. Introduction. Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS postoperatively. We postulated that LPS increases tumor cell expression of ␤-1 integrins and that this leads to increased adhesion. Methods. The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were then treated with LPS for 1, 2, and 4 h (n ⫽ 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVEC), to allow adherence. Adherent tumor cells were counted in three fields on each plate using fluorescent microscopy. Expression of ␤-1 and ␤-2 integrins was assessed using flow cytometric analysis with specific monoclonal antibodies. These experiments were carried out in the presence or absence of a functional blocking ␤-1 integrin monoclonal antibody (4B4). Results. Tumor cell ␤-1 integrin expression was significantly (P ⬍ 0.05) enhanced after incubation with LPS for 2 and 4 h. Tumor cell adhesion to HUVEC was significantly increased. There was no increase in ␤-2 integrin expression after exposure to LPS. Addition of the ␤-1 integrin blocking antibody reduced tumor cell adhesion to control levels (see table). Conclu-

TABLE—ABSTRACT 3 Number of Adherent Tumor Cells to HUVEC after Adhesion of 120 min

Control LPS LPS and 4B4mAb

1-h incubation

2-h incubation

4-h incubation

10.8 (SE ⫾ 1.1) 28.4 (SE ⫾ 1.4)*

16.3 (SE ⫾ 1.6) 33.2 (SE ⫾ 2.1)*

24.3 (SE ⫾ 1.9) 53.2 (SE ⫾ 2.3)*

14.4 (SE ⫾ 1.4)*

17.3 (SE ⫾ 1.2)*

26.2 (SE ⫾ 1.2)*

* P ⬍ 0.05. sions. Exposure to LPS increases tumor cell adhesion to the endothelium through a ␤-1-integrin-mediated pathway. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth. 4. Modified AdCTLA4 Vector Blocks Alloimmune Response in Vitro. G. Zamir, M.D., A. E. Gelman, B.S., X. Que, M.D., X. Aldeguer, M.D., F. Debonera, B.A., A. Shaked, M.D., and K. M. Olthoff, M.D. Department of Surgery, University of Pennsylvania, Philadelphia, Pennsylvania. Gene transfer of the costimulatory blockade molecule CTLA-4Ig into cold-preserved rat liver allografts results in indefinite allograft survival. Despite local delivery, this mode of immunomodulation is associated with systemic immunosuppression. In an effort to restrict immunosuppression to the graft, we have constructed a novel adenoviral vector, AdCTLA4ex-TAG, in which the Ig sequence of CTLA-4Ig DNA has been deleted aiming to destabilize the gene product to promote rapid extrahepatic degradation while maintaining its immunosuppressive activity within the graft milieu. Methods. (1) Vector construction: Mouse CTLA-4 extracellular binding domain (CTLA-4ex) was prepared by PCR-based cloning methods and then fused in frame to a genetic element encoding an epitope TAG allowing for the in vivo identification of the transgene product CTLA4exTAG. CTLA-4exTAG was then subcloned into a shuttle vector (pAC-CMV) enabling the isolation of AdCTLA4exTAG. (2) AdCTLA4exTAG was transfected into Hep-G2 cell line. Supernatant was recovered for Western blot and MLR analysis over a period of 5 days. (3) In vitro alloimmune response was characterized by MLR using Balb/c responders and C57BL/6 stimulators. Results. Expression and secretion of the recombinant protein were documented in HepG2 cells infected with AdCTLA-4exTAG. Cell culture supernatant containing CTLA-4exTAG resulted in marked blunting of alloreactive

T-cell response in MLR assay. Conclusions. These results show efficient in vitro expression of CTLA-4exTAG after transfection with AdCTLA-4exTAG. The modified protein retains its ability to abrogate in vitro alloimmune response. These findings set the grounds for utilization of this novel adenoviral construct in an in vivo transplant setting, hoping to achieve restricted site immunomodulation. 5. Factors Affecting Choice of Surgical Residency Training Program between Men and Women. K. L. Mayer, M.D., R. V. Perez, M.D., and H. S. Ho, M.D. Department of Surgery, UC Davis School of Medicine, Davis, California. A significant problem facing American surgery today is the lack of participation from women and minorities. In 1995 and 1996, 15.1 and 15.8% of U.S. general surgical residency graduates were women. Of our 71 graduates in the past 12 years, 38% were women. The aim of this study is to identify the factors influencing their choice of training program and the reasons why our program has a high percentage of female graduates. Between 1989 and 2000, 27 women and 44 men completed general surgical training at our university, and 44/71 (59%) responded to our survey. The age at residency completion was 34 ⫾ 2.2 years for men and 33.9 ⫾ 2.8 years for women. Fifty-five percent of men and 30% of women went on to fellowship training, and 36% of men and 20% of women are in academia. Factors influencing our graduates’ selection of training program are shown in the table. Only 23% of men had a female faculty as their mentor, while 90% of women had a male faculty as their mentor during training. Only 59% of men but 80% of women

TABLE—ABSTRACT 5 Factors

Men (n ⫽ 22)

Chair Clinical experience Resident morale Program reputation Institution reputation Faculty Geographic location Gender mix

20 (91%) 20 (91%) 20 (91%) 21 (95%) 16 (73%) 15 (68%) 17 (77%) 4 (18%)

Women (n ⫽ 20) P value 19 (95%) 20 (100%) 19 (95%) 18 (90%) 16 (80%) 15 (75%) 18 (90%) 15 (75%)

NS NS NS NS NS NS ⬍0.05 ⬍0.05

(P ⬍ 0.05) agree that female medical students need role models of successful female faculty members. Fifty-five percent of men and 45% of women would encourage a female medical student to choose surgery as a career, while 82% of men and 50% of women would encourage a male medical student to do so. Ninety-one percent of men and 85% of women would choose surgery as a career again. A surgical residency training program with strong leadership, good clinical experience, and high resident morale will equally attract both genders. Women may pay more attention to the program’s gender mix and geographic location. 6. Identification of Regulatory Elements in the Distal Promoter Region of the Clara Cell Secretory Protein Gene. A. S. Y. Chang, M.D.,* P. L. Ramsay, M.D.,†‡ M. J. Reardon, M.D.,* F. and J. DeMayo, Ph.D.† ,‡ Departments of *Surgery, †Pediatrics, and ‡Cell Biology, Baylor College of Medicine, Houston, Texas. Objective. The Clara cell secretory protein (CCSP) is the major secretory product of Clara cells, nonciliated bronchiolar epithelial cells, and plays a major role in modulating lung inflammation. A complex promoter, including an extensively studied proximal region (⫺-166 to ⫹1 bp) and a less analyzed distal region (⫺800 to ⫺166 bp),

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

regulates about 90% of endogenous CCSP expression in the transgenic mouse model. This study was designed to define the key regulatory elements in the distal promoter. Methods. Mouse transformed Clara cells previously generated by clonal expansion of tumor cells from transgenic mice expressing SV40 large T antigen controlled by the CCSP promoter were utilized for distal promoter analysis. We transiently transfected the combined promoter region (800 bp) and three deletion constructs (729, 588, and 166 bp) and then assayed for luciferase reporter gene activity. Results. Transfection analysis shows 800 bp has a fivefold increase in luciferase activity over 166 bp. We observed a significant reduction in luciferase activity between 800 and 729 bp (P ⬍ 0.05) and a secondary reduction between 588 and 166 bp (P ⬍ 0.05). A gel shift assay demonstrated a single specific DNA–protein complex in the ⫺800 to ⫺729 region that was also found to be upregulated by hyperoxia. Preliminary sequence analysis of this area revealed a putative orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF4). Conclusions. We identified two distal promoter regions that significantly regulate the CCSP gene and may contain a known regulatory element. Further analysis of the potential role of this novel site will enhance our understanding of CCSP regulation and expression during infection, cardiopulmonary bypass, or hyperoxia and could provide an opportunity for intervention to modulate the deleterious effects of the pulmonary inflammatory cascade. 7. Voltage-Gated Potassium (K v) Channel Blockade Blunts Oxygen-Induced Vasodilation in Pulmonary Arterioles from Term Fetal Rats. J. R. Gosche, M.D. Department of Surgery, Yale University School of Medicine, New Haven, Connecticut. Oxygen is an important stimulus for pulmonary vasodilation in the perinatal period. Several K ⫹ channels have been implicated as sensors or effectors of the oxygen-induced pulmonary vasodilator response. We have examined the effect of blocking K ⫹ channels on oxygen-induced vasodilation in pulmonary arterioles taken from fetal rats at term. Third generation pulmonary arterioles were isolated from fetal rats at term (day 22 of gestation). Arterioles were cannulated and pressurized in a tissue chamber and perfused with a modified Krebs solution. Internal diameters were measured at baseline (BL), following “hypoxic” (pO 2 ⬍ 40 mm Hg) preconstriction for 30 min and at 10-min intervals during a 30-min period of “normoxic” (pO 2 ⬎ 100 mm Hg) resuffusion. Responses were recorded in the presence of no blockers (controls) or in the presence of the voltage gated K ⫹ channel blocker (K v) 4-aminopyridine (4-AP, 10 mM), the ATP sensitive K ⫹ channel (K ATP) blocker glibenclamide (Glib, 10 ␮M), or the calcium-sensitive K ⫹ channel (K Ca) blocker tetraethylammonium (TEA, 10 mM). Responses were expressed as percentage reversal of hypoxic preconstriction. Differences between the control group and each treatment group were detected by one-tailed t tests. Values shown are group means ⫾ SEM. After 30 min of hypoxic exposure, pulmonary arterioles were preconstricted by 57 to 65% of the BL diameter in all four groups. Thirty minutes of reexposure to normoxic conditions resulted in an 83 ⫾ 19% reversal of hypoxic preconstriction in control arterioles (n ⫽ 9). After 30 min of reexposure to normoxic suffusion in the presence of 4-AP (n ⫽ 8), Glib (n ⫽ 7), or TEA (n ⫽ 8), hypoxic preconstriction was reversed by 44 ⫾ 9, 80 ⫾ 16, and 56 ⫾ 8%, respectively. Only the responses of the 4-AP treatment group were significantly different (P ⬍ 0.05) from controls. These data are consistent with a role for K v channels, but not K ATP or K Ca channels, as sensors or effectors for the oxygen-induced vasodilator response in rat pulmonary arterioles during the perinatal period. 8. Transforming Growth Factor ␤ 2 Lowers the Incidence of Incisional Hernias. M. G. Franz, M.D., M. A. Kuhn, M.D., K. Nguyen, B.S., X. Wang, M.D., Ph.D., F. Ko, B.S., T. E. Wright, M.D., FACS, and M. C. Robson, M.D., FACS. University of South

Florida, Tampa, Florida; and The Institute for Tissue Regeneration, Repair and Rehabilitation, Bay Pines VAMC, Bay Pines, Florida. A total of 200,000 incisional hernia repairs are performed in the United States each year. A biological intervention at the host:wound level designed to optimize fascial healing may prevent incisional hernias. Methods. A rodent incisional hernia model was used. Seventy rats underwent 5-cm midline celiotomies and were closed with fine, fast-absorbing sutures. Group 1 received no other treatment. The fascia in groups 2 and 3 was injected immediately prior to incision with 100 ␮l of vehicle alone or vehicle containing 1 ␮g of TGF-␤ 2. Results. After 28 days, incisional hernias developed in 88% of untreated incisions compared to 79% in the vehicle alone group and none in the TGF-␤ 2-treated incisions (P ⬍ 0.05) (see table). Standard histology and immunohistochemistry demonstrated enhanced macrophage and fibroblast chemotaxis and increased colla-

TABLE—ABSTRACT 8 Treatment modality

N

Incisional hernias (%)

Untreated Vehicle prophylaxis TGF-␤ 2 prophylaxis

40 14 16

35 (88) 11 (79) 0* (0)

gen I and III production in the TGF-␤ 2-treated incisions. Conclusions. Treatment of abdominal fascial incisions with TGF-␤ 2 prevented the development of incisional hernias in this rat model. TGF-␤ 2 stimulates fascial macrophage and fibroblast chemotaxis as well as collagen production. A biological approach such as this may reduce the incidence of incisional hernia formation in humans. 9. Pharmacological Induction of HSP 27 Attenuates Intimal Hyperplasia in Vivo. E. M. Connolly, FRCSI, C. J. Kelly, FRCSI, C. Gang, M.B., T. O’Grady, Bsc., E. Kay, FRCPath, A. Leahy, FRCSI, Mch., and D. J. Bouchier-Hayes, FRCSI, Mch. Departments of Surgery and Pathology, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland. Intimal hyperplasia (IH) is the major cause of restenosis postvascular intervention. Induction of cytoprotective heat shock proteins (HSPs) by thermal preconditioning reduces IH. Thermal preconditioning has limited clinical applications. Herbimycin A is a known HSP inducer. We hypothesized that induction of arterial HSPs by herbimycin A would attenuate IH in the rat carotid balloon injury model. Twenty-seven Sprague–Dawley rats were randomized into three groups. Stress proteins were induced 18 h preoperatively by hyperthermia (heat stress group) or pharmacologically with herbimycin A (herbimycin group). Arterial HSP 70 and HSP 27 expression was assessed using Western blot and immunohistochemistry. All groups underwent balloon injury (BI) to the left carotid artery with an embolectomy catheter. Two weeks later animals were sacrificed and the carotid intima/media area ratio (I/M ratio) calculated using

TABLE—ABSTRACT 9 Groups

BI

BI ⫹ heat stress

BI ⫹ herbimycin

I/M ratio (mean ⫾ SEM)

1.84 ⫾ 0.2

1.14 ⫾ 0.165*

0.87 ⫾ 0.19*

* P ⬍ 0.06 vs BI, ANOVA.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS computerized planimetry. Arterial HSP 72 and HSP 27 were increased post-heat stress. HSP 27 was increased post-herbimycin A. Heat stress and herbimycin A significantly reduced the I/M ratio (see table). harmacological preconditioning of the vasculature prior to surgical intervention may be an effective mechanism to reduce restenosis. 10. Mitochondrial Reactive Oxygen Species: A Link between Endothelial Hypoxia and IL-6 Production. D. P. Pearlstein, M.D., M. H. Ali, B.A., and P. T. Schumacker, Ph.D. Department of Surgery, University of Illinois at Chicago Metropolitan Group Hospitals, and Department of Medicine, Section of Pulmonary and Critical Care, The University of Chicago, Chicago, Illinois. Prolonged hypoxia results in increased endothelial IL-6 production. This increase in IL-6 is attenuated with antioxidants, suggesting that reactive oxygen species (ROS) play a signaling role in the hypoxic endothelium. We tested the hypothesis that the source of these ROS is the mitochondria and that these ROS lead to increased IL-6 production by mediating a transcriptional event. Human umbilical vein endothelial cells (HUVEC) were exposed to prolonged hypoxia (2% O 2 for 6 h) in the presence of inhibitors of the electron transport chain (ETC) (rotenone and DPI), and DCF fluorescence was measured. Additionally, DCF fluorescence was measured in the presence of inhibitors of other potential sources of ROS, xanthine oxidase (allopurinol), and NADPH oxidase (apocynin). Similar experiments were performed in which the HUVEC RNA was isolated and subjected to Northern blot analysis with a probe for IL-6 mRNA. Finally, an ELISA assay was performed after the cells were exposed to these conditions to quantify their IL-6 secretion. Hypoxia-induced increases in DCF fluorescence returned to baseline in the presence of ETC inhibitors. However, inhibitors of xanthine oxidase or NADPH oxidase did not significantly affect DCF fluorescence. The ETC inhibitors also prevented increases in IL-6 mRNA transcription. (see figure) Moreover, the ETC inhibitors prevented hypoxia-induced increases in IL-6 secretion (hypoxia 143 ⫾ 7 pg/ml; hypoxia ⫹ rotenone 21 ⫾ 1 pg/ml; hypoxia ⫹ DPI 19 ⫾ 3 pg/ml), a result not seen with

309

Years after Surgery. C. Y. Ko, M.D., L. C. Rusin, M.D., D. J. Schoetz Jr., M.D, L. Moreau, M.D, J. C. Coller, M.D., J. J. Murray, M.D., and P. L. Roberts, M.D. UCLA Medical Center, Los Angeles, California; and Lahey Clinic, Burlington, Massachusetts. Introduction. Health-related quality of life (HRQL) is clearly affected by the immediate postoperative changes in bowel function (BF). However, while studies have reported that mean HRQL levels generally return to preoperative levels within 12 months, investigations have not (a) assessed how varying degrees of BF might affect HRQL, (b) described which specific aspects of BF affect HRQL, and (c) performed long-term follow-up. The current study reports how eight HRQL domains are affected by varying levels of BF—more than 5 years after surgery. Methods. All patients more than 5 years status post-ileal pouch anal anastomosis (IPAA) procedure for familial polyposis (FP) at a single institution were studied. FP was chosen because patients are routinely asymptomatic preoperatively. BF (e.g., stool frequency, anal leakage) and HRQL (using the eight health domains of the SF-36) were assessed by patient interview. Student’s t tests and multivariate regressions were used to analyze associations between BF and HRQL. Results. The sample included 25 patients (14 male). Mean age was 39 years; mean follow-up time was 11 years. Although mean scores for the eight individual HRQL domains were not statistically different from the general U.S population, regression analyses of the different domains did demonstrate significant associations with varying levels of BF. While controlling for age and gender, the analyses show that the physical function domain is improved with the ability to pass flatus independent of stool, and physical role and mental health domains are improved with decreased stool frequency. The social function domain is improved with increased stool retention time, while the perception of general health is improved with less diaper usage and less sexual dysfunction. Conclusions. This study shows that (1) a current stateof-the-art surgical procedure (e.g., IPAA) does affect HRQL levels in the long term. This finding is clearly important when choosing between different surgical options for FP as well as ulcerative colitis patients. (2) Merely using mean levels to describe HRQL may not only misrepresent the overall clinical picture, but also fail to elucidate meaningful relationships between important clinical outcomes, such as function and HRQL. 12. Modified “Israeli” Repair Using Polypropylene Mesh Onlay for Treatment of Complicated Incisional Hernias. K. G. Chisholm, M.D., and S. R. Schell, M.D., Ph.D. Department of Surgery, University of Florida; and Malcolm Randall Veterans Affairs Hospital, Gainesville, Florida.

allopurinol (123 ⫾ 8 pg/ml) or apocynin (133 ⫾ 10). These results indicate that, in response to prolonged hypoxia, endothelial mitochondria increase production of ROS. These ROS then appear to participate in an intracellular signaling process that ultimately leads to IL-6 transcription and release.

PARALLEL SESSION I Clinical Trials and Outcomes 11. Long-Term Outcomes of the Ileal Pouch Anal Anastomosis: The Effect of Bowel Function on Quality of Life 5

Purpose. Incisional hernia following laparotomy represents a significant risk for morbidity to patients who develop this complication. This study examines treatment success and outcomes using our modification of the “Israeli” incisional hernia repair with an extrafascial onlay using polypropylene mesh in male VA patients. Methods. Seventy-eight male (mean age 58.1 ⫾ 1.9 years; median 62.2 years) patients underwent a total of 90 procedures for repair of incisional hernia (82.1%), recurrent incisional hernia (15.4%), and umbilical hernia (2.6%) following operation for benign, malignant, or vascular disease. Hernia sizes ranged from 9 to over 270 cm 2 (“giant”). Preoperative morbidity included COPD (20.5%), wound infection (6.4%), and immunosuppression (2.6%). Forty-nine (62.8%) patients underwent modified “Israeli” repair with mesh onlay (MOR), 7 patients (9.0%) underwent standard mesh repair (SMR), and 22 (28.2%) underwent repair without mesh (NMR). Results. Median follow-up was 50.7 (mean 47.7 ⫾ 1.6) months. There were no deaths related to hernia repair. Seven patients (9.0%) died from unrelated causes during the follow-up period. There were no recurrences following MOR repair. There were two recurrences following NMR and one following SMR (recurrence rates of 9.1 and 14.3% respectively).

310

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

These recurrences were successfully rerepaired using MOR, without further recurrence. Three patients developed new ventral hernias geographically separate from their initial defect and repair and were treated with SMR (2 patients) and MOR (1 patient) without further recurrence. One patient treated with MOR developed a wound infection successfully treated with local dressing changes and did not require mesh removal. Conclusions. Our modified “Israeli” repair using polypropylene mesh onlay provides excellent repair of incisional hernia with low risk of complications. This procedure may prove valuable for treatment of higher risk patients who develop incisional hernia after laparotomy. 13. Predictors of Myocardial Recovery in Patients with Ventricular Assist Devices. B. A. Bruckner, M.D., S. J. Stetson, B.S., P. Razeghi, M.D., G. P. Noon, M.D., M. L. Entman, M.D., G. Torre-Amione, M.D., O. H. Frazier, M.D., and K. A. Youker, Ph.D. M. E. DeBakey Heart Center, Baylor College of Medicine and the Texas Heart Institute, Houston, Texas. Recent clinical observations in patients supported with left ventricular assist devices have demonstrated the possibility that failing myocardium may recover cardiac function even to the point that patients may be weaned off the device. In this study we examined paired myocardial biopsy specimens (pre- and post-LVAD implantation) from 23 patients (ages 24 – 64 years) with end-stage cardiomyopathy. The study population included 9 patients who subsequently recovered clinically and had the device explanted without transplantation and 14 patients who were bridged to cardiac transplantation. Echocardiographic data were also collected and included ejection fractions immediately prior to device implantation and at time of removal. Despite nearly identical preimplant ejection fractions ⬍20% in all patients, the recovered group had significantly greater improvements in EF (31 ⫾ 16%) after LVAD support than the nonrecovered group (18 ⫾ 9%). Next, we determined myocyte size changes by computerized edge detection and total collagen content in the pre- and post-LVAD biopsies by a semiquantitative analysis of positive picrosirius red-stained areas. We found that 23/23 patients had significant reductions in collagen content with a mean reduction of 59%. The recovered patients demonstrated a statistically significant lower average collagen content at the time of LVAD implantation when compared to the nonrecovered patients. Myocyte size was decreased in all patients studied with an average reduction of 59% of the initial pre-LVAD myocyte size. Myocyte size at time of device implantation was also found to be significantly smaller in the recovered patients than in the nonrecovered patients (P ⬍ 0.001). Thus, despite the fact that initial clinical presentations were identical, significantly less fibrosis and hypertrophy existed in those who subsequently recovered. This may allow stratification and outcome prediction in stage IV heart failure and may be useful in selection of patients suitable for LVAD weaning. 14. Women Have Higher Operative Mortality after Coronary Artery Bypass Surgery: Is It Sex or Something Else? Raymond H. Chen, M.D., Ph.D., Alexander Kadner, M.D., John G. Byrne, M.D., Lishan Aklog, M.D, and David H. Adams, M.D. Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Objective. To clarify whether gender alone or associated preoperative risk factors contribute to higher mortality in women after coronary artery bypass graft (CABG) surgery. Methods. From January 1993 to December 1998 4215 patients (median age 66 years, 28% female) underwent primary CABG. Multivariable logistic regression was used to evaluate the risk of operative death to gender after controlling for potential confounders. Results. The overall operative mortality was 2% (3.5% in females; 1.4% in males) (P ⱕ 0.0005). Women were more likely to be older and smaller, with diabetes mellitus, renal failure, hypertensive, stroke, COPD, recent

MI, high cholesterol, cardiomegaly, worse functional class, and urgent status, while men were more likely to have lower EF, smoking history, three-vessel disease, and unstable angina. Univariable odds ratio for females for operative death was 2.7 (95% CI 1.7, 4.1; P ⱕ 0.0005). When potential confounders and gender were entered into the multivariable model, female gender was not a significant predictor of operative mortality following CABG (OR 1.3, 95% CI 0.76 –2.4; P ⫽ 0.31). In analyses stratified by gender, stroke (OR 2.6, 95% CI 1.2–5.6; P ⫽ 0.01), urgency (OR 2.3, 95% CI 1.2– 4.6; P ⫽ 0.01), and cardiomegaly (OR 2.3, 95% CI 1.1– 4.9; P ⫽ 0.04) were predictive of operative mortality in women, while EF ⬍ 30% (OR 3.6, 95% CI 1.8 –7.5; P ⫽ 0.001), hypertension (OR 2.8, 95% CI 1.2– 6.4; P ⫽ 0.02), urgency (OR 2.6, 95% CI 1.3–5.0; P ⫽ 0.006), mitral insufficiency (OR 2.2, 95% CI 1.1– 4.3; p ⫽ 0.03), age ⱖ 75 years (OR 2.1, 95% CI: 1.1-4.3; P ⫽ 0.03) predicted mortality in men. Conclusions. Gender does not predict operative mortality following CABG. Instead, gender-based mortality differences reflect preoperative clinical status. 15. Impact of Gender, Age, and Functional Capacity on Functional Outcome Following CABG. R. D. Stewart, M.D., N. C. Namour, B.A., E. Dziadik, B.A., S. Levitsky, M.D., and C. T. Campos, M.D. Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. The purpose of this study is to determine if gender, older age, and preoperative functional capacity are associated with functional outcome following coronary artery bypass surgery (CABG). The functional status of 202 consecutive patients undergoing CABG in 1999 was determined at the time of operation and at six months using the Duke Activity Status Index (DASI). Complete data were available for 164 patients (81%). Functional outcomes were categorized as either “good” (DASI equal or higher than baseline at 6 months) or “poor” (lower DASI). Outcomes were compared between genders, between younger and older patients, and between patients from each quartile of baseline functional capacity. Fisher’s exact was used for all tests of association. Overall, 114 (70%) patients had a good functional outcome and 50 (30%) had a poor outcome. There was no difference in functional outcome between men (78 of 116 (67%) good) and women (36 of 48 (75%) good, P ⫽ 0.4). There was also no significant difference between patients younger than 70 years (70 of 94 (74%) good) and those 70 years and older (44 of 70 (63%), P ⫽ 0.13). There was, however, a significant difference in the outcome between patients in the highest quartile of baseline functional status (21 of 42 (50%) good) and patients from the lower three quartiles (34 of 42 (81%), 30 of 40 (75%), and 29 of 40 (73%), first, second, and third quartiles, respectively, P ⫽ 0.016). This relationship persists for both genders and for both younger and older patients. Most notably, among patients 70 years or older in the highest baseline functional quartile, only 3 of 11 (27%) had a good outcome 6 months following CABG. Most CABG patients have an improvement in their functional capacity, regardless of age or gender. However, 30% of all patients do not improve, and the higher functioning patients are at greatest risk of failing to achieve at least their baseline capacity following CABG. 16. Learning Curves and Breast Cancer Lymphatic Mapping: Institutional Volume Index. E. Dupont, M.D., C. E. Cox, M.D., S. Shivers, Ph.D., C. J. Salud, B.A., K. Nguyen, B.S., A. Cantor, Ph.D., and D. S. Reintgen, M.D. Department of Surgery, University of South Florida, Tampa, Florida. Studies to date have analyzed breast lymphatic mapping (LM) success with respect to individual surgeons. However, LM and sentinel lymph node biopsy (SLNBx) are procedures that require multidisciplinary cooperation between the departments of radiology, pathology, and surgery. Thus, it is important to evaluate these procedures with respect to institution. This study examines 30 insti-

311

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS tutions to clarify the value of the institutional volume index (IVI) (cases/month) to the outcome of LM and SLNBx in breast cancer. From July 1997 to July 1999, 30 institutions participated in a national breast LM trial. All participants underwent a 2-day training course for surgeons, nuclear medicine physicians, and pathologists. The records for each institution were prospectively accrued and submitted to a database. IVI was calculated for each institution. A logistic regression model plots the relationship between IVI and failure rate. Using a multivariate analysis, mapping failure was analyzed as a function of case number with respect to the individual surgeon and the institution as a whole. Mapping failures were found in all but 7 of the 30 institutions whose data were complete. There were 71 mapping failures among 68 surgeons over 555 cases which yielded an overall failure rate of 12.79%. The logistic regression model revealed an inverse relationship between IVI and institutional failure rate. However, the multivariate analysis revealed that the individual surgeon was a more significant factor in determining institutional mapping success. Failure to map can be a function of multiple factors including surgical skill, surgical volume index, injection method, and pathologic evaluation of the SLN, all under the quality control of an institution. While differences in mapping success exist across institutions, we feel that this disparity is not due to factors associated with an institution as a whole, but lie with the individual surgeon. 17. Racial Differences in Breast Cancer Survival: The Effect of Residual Disease. A. T. Mancino, M.D., R. Landes, M.S., R. H. Tillman, M.D., L. F. Smith, M.D., H. J. Spencer, M.S., L. Erkman, C.T.R., I. T. Rubio, M.D., and V. S. Klimberg, M.D. Departments of Surgery and Biostatistics, University of Arkansas, Little Rock, Arkansas. The purpose of this study was to elucidate the differences between African-American (AA) and caucasian (C) women with breast cancer in regards to patient characteristics and outcomes. We performed a retrospective analysis of 1345 women with newly diagnosed breast cancer who were entered into our tumor registry from October 1980 to December 1998. The association between stage at presentation and race was significant, as was the difference in the median survival between C and AA women. The data revealed no significant difference in survival between C and AA women presenting with Stage I or II disease. However, the differences between the median survival times for AA and C women presenting with Stage III and IV were both highly significant (see table). A significantly lower percentage of AA women became “disease free” after initial therapy as compared to C women (P ⬍ 0.001); interestingly, only differences in Stage III and IV disease were significant. In conclusion, AA women

tend to present at a later stage and have poorer survival from later stage disease than C women do. This poorer survival appears to be related to the decreased ability to achieve disease free status for AA women. The causes of differences in treatment outcome between AA and C are under evaluation.

18. Early Results of Breast Cancer Lymphatic Mapping: No Axillary Recurrence in Breast Cancer Patients after a Negative Sentinel Lymph Node Biopsy. S. Dessureault, M.D., E. Dupont, M.D., A. Shons, M.D., C. Berman, M.D., N. Ku, M.D., C. Cox, M.D., and D. S. Reintgen, M.D. H. Lee Moffitt Cancer Center and Research Institute at the University of South Florida, Tampa, Florida. While complete axillary lymph node dissection (CALND) remains the standard of care for the management of invasive breast cancer, lymphatic mapping and sentinel lymph node (SLN) biopsy promises to be a successful and accurate alternative with significant cost savings and reduction in operative morbidity. We present here an update on the lymphatic mapping experience at the H. Lee Moffitt Cancer Center and Research Institute. A total of 1356 consecutive patients with suspected node-negative breast cancer were mapped between April 1994 and April 1999. Lymphatic mapping was performed using Tc 99m-labeled sulfur colloid and isosulfan blue dye. A SLN was defined as any blue node and/or any hot node with ex vivo radioactivity counts [symbol:Math Ext/99]10 times an excised nonSLN or in situ radioactivity count [symbol:Math Ext/99]3 times the background counts. Lymphatic mapping was successful in identifying the SLN in 1302 patients (96.0%). A total of 373 patients (28.6%) were found to have metastatic disease either by hematoxylin and eosin stains or by cytokeratin immunohistochemistry. A total of 929 patients (71.4%) had negative sentinel lymph node biopsies. The first 120 patients in this group all underwent CALND: One patient was found to have metastatic disease in the nodal basin (false negative rate ⫽ 1/120 ⫽ 0.83%). The subsequent 809 patients have been observed and followed without further nodal dissection. All patients treated with breast conservation had radiation therapy to the breast. Most patients received adjuvant chemotherapy. We have not seen any recurrences to date (mean follow-up 20 months). Previous studies have reported 10-year nodal failure rates of 17–28% in patients treated without axillary dissection or radiation. One study reported a median time to axillary failure of 17.2 months, with 70% of axillary recurrences occurring by 3 years. We conclude, therefore, that SLN biopsy is a sensitive alternative to CALND for the detection of nodal metastases in patients with invasive breast cancer and can be used to select patients who do not need CALND for local control.

TABLE—ABSTRACT 17 Stage

Race (%) C AA Med surv (months) C AA P Dz free % C AA P a

n a ⫽ 1020 n a ⫽ 264 Overall 128.2 78.0 ⬍0.001 Overall 93.0 81.6 ⬍0.001

0

I

II

III

IV

7.7 7.6

32.5 17.8

41.6 41.3

11.0 18.2

7.2 15.1

P ⬍ 0.001, ␹ 2

177.7 n/a 0.80

110.5 121.0 0.70

91.3 36.0 ⬍0.01

24.0 8.9 ⬍0.001

Log rank

99.1 97.9 0.41

97.6 98.2 1.0

95.5 77.1 ⬍0.001

29.7 12.5 0.04

Effective sample size excluding patients with missing stage data.

Fisher’s exact

312

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

PARALLEL SESSION I Peripheral Vascular I 19. Contraction-Coupled Nitric Oxide Release: A New Paradigm for Local Vascular Control? H. W. Kim, Ph.D, and A. G. Greenburg, M.D., Ph.D. Department of Surgery, Brown University/The Miriam Hospital, Providence, Rhode Island. Hemoglobin (Hb), a nitric oxide (NO) scavenger, elicits an additional contraction in norepinephrine (NE)-contracted isolated rat thoracic aorta. Does vascular contraction induce endothelial NO release? We tested this hypothesis using rat thoracic aortic rings prepared with or without the endothelium (E). Vessel rings (N ⫽ 6/group) bathed in an oxygenated Krebs buffer at 37°C were contracted with 25 nM NE, 40 mM KCl, 1.5 ␮M vasopressin (VP), 3.4 ␮M prostaglandin F 2␣ (PGF), or 0.2 mM serotonin (5HT). NO release was probed using a Hb contraction assay. Additional groups of vessel rings pretreated with 0.4 mM nitro-L-arginine methyl ester (NAME), a NO synthase inhibitor, were similarly tested. Changes in vessel ring isometric tension were compared (Student’s t test at P ⫽ 0.05). In all groups treated with various pressor agents, 2 ␮M Hb elicited significant additional contractions (see table). In contrast, Hb failed to elicit significant contractions in vessel rings without functional

but paradoxically attenuate aneurysmal enlargement following elastase infusion in the Anidjar/Dobrin AAA model. Distal AVF may affect both aortic wall shear stress (WSS) and relative wall strain. Both shear and strain trigger relevant regulatory gene expression in cell culture models. The impact of femoral AVF creation on aortic wall strain is unknown. We proposed that distal AVF may alter aortic wall strain. Male Sprague–Dawley rats underwent either left common femoral AVF creation or sham groin procedure. Rats were

TABLE—ABSTRACT 20 Flow (ml/min) 3 days AVF(n ⫽ 10) Sham (n ⫽ 9) 7 days AVF (n ⫽ 10) Sham (n ⫽ 7) 21 days AVF (n ⫽ 12) Sham (n ⫽ 5)

WSS (dynes/cm2)

DIA (mm)

Wall strain (%)

34 ⫾ 13* 3.96 ⫾ 1.27* 1.71 ⫾ 0.14* 16 ⫾ 6 2.89 ⫾ 0.73 1.49 ⫾ 0.11

8.95 ⫾ 2.77* 5.35 ⫾ 1.05

40 ⫾ 10* 3.23 ⫾ 1.74 15 ⫾ 6 2.72 ⫾ 1.63

2.02 ⫾ .31* 1.52 ⫾ .19

8.66 ⫾ 4.02 6.71 ⫾ 2.34

49 ⫾ 21* 2.70 ⫾ 1.16 10 ⫾ 5 1.21 ⫾ 0.50

2.24 ⫾ .40* 11.78 ⫾ 5.88 1.56 ⫾ .37 8.73 ⫾ 3.55

* Differ from SHAM, P ⬍ 0.05.

TABLE—ABSTRACT 19 Hb Induced Additional Contractions (g) in Vessel Rings Treated with Various Pressor Agents (Mean ⴞ SD) Drugs

⫹E

⫺E

⫹E ⫹ NAME

NE KCl VP PGF 5HT

0.7 ⫾ 0.34** 0.44 ⫾ 0.16** 0.34 ⫾ 0.20** 0.43 ⫾ 0.24** 0.33 ⫾ 0.22*

0.01 ⫾ 0.02 0.03 ⫾ 0.03 0.03 ⫾ 0.07 0.08 ⫾ 0.10 0.06 ⫾ 0.09

0.0 ⫾ 0.0 0.13 ⫾ 0.14 0.09 ⫾ 0.10 0.10 ⫾ 0.14 0.06 ⫾ 0.10

** P ⬍ 0.01, *P ⬍ 0.05 compared with 0 g tension increase (no change). endothelium (⫺E). Similarly, pretreatment with NAME prevented Hb-induced additional contractions. We thus conclude that, in the isolated rat thoracic aorta and perhaps other vessels, endothelial NO release may be coupled to contractile stimulus. This vascular property appears to render a unique local control mechanism independent of the baroreflex and other central mechanisms. 20. Shear and Strain Mediate Aortic Enlargement Following AVF Creation. J. K. Karwowski, M.D., C. C. Yeh, B.S., and R. L. Dalman, M.D. Stanford University School of Medicine, Stanford, California. Femoral arteriovenous fistulae (AVF) mediate aortic enlargement

sacrificed at 3, 7, and 21 days with simultaneous measurement of intraaortic mean arterial pressure (MAP), infrarenal aortic blood flow, and real-time continuous aortic diameter (DIA) measurement via sonomicrometry. Relative wall strain was defined as (MAX DIA ⫺ MIN DIA)/MIN DIA averaged over 14 s at stable heart rate and MAP. MAP did not differ between groups at all time points. Aortic blood flow was significantly elevated in AVF rats compared to SHAM at all time points. Consequent aortic enlargement in AVF rats was present by POD 3. Aortic WSS and relative wall strain were elevated on POD 3, but by POD 7 and 21 the differences between AVF and SHAM groups were no longer significant (see table). As expected, progressive aortic enlargement following AVF creation normalizes elevated wall shear stress. Moreover, this diameter enlargement also normalizes the early elevation in wall strain seen on POD 3. This suggests a potential regulatory role for wall strain in aortic remodeling. Paradoxical attenuation of aneurysmal enlargement following distal AVF creation in the elastase infusion model may result from increased wall strain, wall shear stress, or some combination of the two forces. 21. Oscillatory Flow Induces DNA Synthesis in Endothelial Cells and Requires Distinct Signaling to a Translational Control Pathway. L. W. Kraiss, M.D., T. M. Ennis, M.S., T. M. McIntyre, Ph.D., and G. A. Zimmerman, M.D. Departments of Surgery, Experimental Pathology and Medicine, University of Utah, Salt Lake City Utah. Fluid flow regulates transcription of numerous endothelial genes but a change in phenotype requires translation of mRNA into protein. P70/P85 S6 kinase (pp70 S6k) mediates signaling in a transla-

TABLE—ABSTRACT 21

Static Flow P value

0.1% serum (n ⫽ 22)

RAP (n ⫽ 6)

PD98059 (n ⫽ 5)

20% serum (n ⫽ 5)

7,440 ⫾ 2,344 11,560 ⫾ 3,367 0.01

1,028 ⫾ 540 1,080 ⫾ 324 0.94

901 ⫾ 512 4,645 ⫾ 2,028 0.15

47,658 ⫾ 14,028 — —

313

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS tional control pathway essential for mitogenesis and is activated by fluid flow. We hypothesized that oscillatory fluid flow (without exogenous mitogens) would induce endothelial cells to synthesize DNA via activation of pp70 S6k. For comparison, we also studied the ERK1/2 transcriptional signaling pathway. Confluent HUVEC were exposed to oscillatory flow (12 dynes/cm 2 peak shear stress; 3.3 Hz) or kept static in medium containing only 0.1% serum. Rapamycin (RAP, 10 nM) or PD98059 (10 ␮M) was used to inhibit pp70 S6k or ERK1/2 activation, respectively. Oscillatory flow activated pp70 S6k and ERK1/2 (appearance of phosphoisoforms on Western blots). RAP blocked activation of pp70 S6k but not ERK1/2 while PD98059 blocked ERK1/2 but not pp70 S6k. CDK1 mRNA (ribonuclease protection assay) was upregulated by fluid flow but inhibited by RAP. DNA synthesis was measured by [ 3H]thymidine uptake (cpm/well ⫾ SEM) (see table). Oscillatory flow induces DNA synthesis by HUVEC. Inhibition of either pp70 S6k (RAP) or ERK1/2 (PD98059) reduced basal [ 3H]thymidine uptake but RAP more effectively blocked flowinduced DNA synthesis. Separate and distinct signaling to a translational control pathway is necessary to mediate flow-induced DNA synthesis by endothelial cells. 22. Upregulation of Intrarenal and Adrenal Angiotensin II (AII) Production in Chronic Renovascular Hypertension (RVH). J. G. Modrall, M.D., K. Puttaparthi, Ph.D., G. P. Clagett, M.D., R. H. Turnage, M.D., and M. Levi, M.D. Departments of Surgery and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas. The mechanism by which hypertension is maintained in RVH remains poorly defined. Since plasma AII does not correlate with blood pressure (BP) in RVH, we postulated that activation of tissuespecific renin–angiotensin systems may upregulate local production of AII and maintain hypertension in chronic RVH. RVH was induced using a two-kidney, one-clip (2K1C) rat model in which a partially occlusive clip was placed on the left renal artery. BP was measured using a tail cuff. Hypertension was defined as systolic BP ⱖ 150 mm Hg. At sacrifice 1, 6, or 12 weeks postoperatively, plasma and tissue samples were collected for AII purification and quantification by RIA. BP was significantly elevated in 2K1C animals at each time point, compared to sham animals (P ⬍ 0.05). The results of AII assays are summarized in the table. These data show that despite normal plasma AII levels, RVH induced an immediate and sustained increase in AII in both clipped and unclipped kidneys of 2K1C animals and an increase in adrenal AII in chronic 2K1C animals. In conclusion, tissue AII production is upregulated in multiple tissues involved in BP regulation, providing a potential mechanism for maintaining hypertension in RVH. 23. Liposome-Mediated Gene Delivery to Endothelial Cells to Prevent and Treat Thrombosis. R. R. Dickason, M.D., Ph.D., A. B. Weinfeld, M.D., S. Almohammed, M.D., A. M. Amer, Ph.D., E. Yuksel, M.D., M. Kattash, M.D., and S. M. Shenaq,

M.D. Division of Plastic Surgery, DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas. Thrombosis is a significant and challenging problem in many surgical diseases and procedures. Current treatment protocols for thrombosis-related diseases rely on the delivery of recombinant thrombolytic proteins. Therapies based on systemic delivery of such proteins have a significant risk of cerebral and pulmonary hemorrhage. Thus, local gene therapy is an attractive alternative as it eliminates the risk of altering systemic coagulation parameters. Additionally, enhancing the thromboresistant potential of vascular tissue with the introduction of thrombolytic genes may offer a prolonged therapeutic affect and reduce rebound thrombosis. In our past experiments we have effectively prevented thrombosis in an animal model using local delivery of the tissue plasminogen activator (TPA) gene via an adenoviral vector. Currently we are investigating DOTMA:Chol liposome-mediated gene delivery because of the decreased immunologic complications associated with nonviral vectors. In our experiments cell culture was used to evaluate transfection efficiency with a ␤-galactosidase marker gene and functional TPA expression with an American Diagnostica (Greenwich, CT) chromogenic assay. Transfection in whole vessel segments in organ culture was evaluated in rabbit and human tissue using luciferase and IL-12 marker genes, respectively. Our results demonstrate: (1) 22.6% ⫾ 2.5% peak transfection efficiency in cell culture, (2) expression of functional TPA protein in cell culture, (3) transfection of whole rabbit arterial (n ⫽ 6) and venous (n ⫽ 6) tissue in organ culture, and (4) transfection of human venous tissue (n ⫽ 7) in organ culture. Our results suggest that liposome-mediated delivery of the TPA has significant potential as a local gene therapeutic approach to delivering genes to endothelial cells and deserves further evaluation in our gene therapy research program directed at the prevention and treatment of thrombosis. 24. Gene Delivery to Veins Using Adeno-Associated (AAV) Viral Vectors: Long-Term Expression with Minimal Host Response. K. K. Rhynhart, M.D.,* ,† S. P. Gangadharan, M.D.,* ,† R. O. Snyder, Ph.D.,† X. X. Sui, M.D.,* ,† and M. S. Conte, M.D.* ,† *Department of Surgery, Brigham and Women’s Hospital; and the †Harvard Institute for Human Genetics, Boston, Massachusetts. The potential application of gene transfer to modify vein graft biology will require a vector that confers stable gene expression with minimal host response. Recent studies employing AAV vectors have shown promising results. The purpose of these studies was to further characterize transgene expression and host responses following AAV exposure to vein segments in situ. NZW rabbits (N ⫽ 11) were anesthetized with cannulation of bilateral jugular veins. Each paired control was distended in situ with either AAV vector (7 ⫻ 10 9 infectious particles/ml) or normal saline for 30 min followed by restoration of venous flow. The AAV vector encoded the reporter gene ␤-galactosidase. Vessels were explanted 14 –140 days postinfection

TABLE—ABSTRACT 22 1 week

6 weeks

12 weeks

Tissue, N ⱖ 4/group

Sham

2K1C

Sham

2K1C

Sham

2K1C

Plasma Clipped kidney Unclipped kidney Adrenal gland

23 ⫾ 7 31 ⫾ 3 40 ⫾ 3 523 ⫾ 81

38 ⫾ 7 63 ⫾ 8* 59 ⫾ 6* 494 ⫾ 52

33 ⫾ 4 92 ⫾ 14 57 ⫾ 4 1179 ⫾ 131

64 ⫾ 9 163 ⫾ 18* 205 ⫾ 32* 1542 ⫾ 94

23 ⫾ 7 59 ⫾ 9 62 ⫾ 11 401 ⫾ 59

40 ⫾ 17 147 ⫾ 38* 130 ⫾ 32* 1129 ⫾ 149*

Note. Data are expressed as means ⫾ SEM of AII concentration (fmol AII/ml plasma or fmol AII/mg tissue); *P ⬍ 0.05 versus sham; Student’s t test.

314

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

for analysis of gene expression (direct en face examination and grading) and immunohistochemistry (CD31, CD5, RAM-11, ICAM1, VCAM1). DNA PCR was performed on solid organs for evidence of remote viral dissemination (10 2 copies/100 ng genomic DNA). Serum was analyzed for neutralizing antibodies. All animals survived post infection and all veins were patent at explant. Gene expression was visualized in all vector treated veins from 14 to 140 days with maximum seen at the latest time point examined (140 days). Transgene expression was localized primarily to the media. A confluent CD31⫹ endothelial monolayer was present in all veins. No discernible intimal hyperplasia or significant local inflammation was seen in 10/11 animals. PCR analysis revealed no evidence of viral dissemination to lung, liver, or gonads at 3 months. Neutralizing antibodies were seen at all time points post exposure. AAV-mediated gene transfer to in situ veins yields significant long-term expression with minimal apparent local inflammation or neointimal formation. This study supports further investigation of adeno-associated viral vectors for potential application to vein bypass grafting. 25. Adenoviral-Mediated Uteroglobin Gene Transfer to the Adventitia Reduces Arterial Intimal Hyperplasia. J. V. Lombardi, M.D., M. Naji, R. A. Larson, M.D., A. Naji, M.D., Ph.D., B. Koeberlein, and M. A. Golden, M.D. Harrison Department of Surgical Research, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania. Purpose. Investigate the efficacy of uteroglobin, a potent antiinflammatory and immunomodulatory agent, in reducing neointimal hyperplasia via adenoviral-mediated gene transfer to the adventitia in the mouse carotid ligation injury model. Methods. Forty-five C57bl/6NHSD mice were anesthetized and left common carotid artery ligation was performed. Adenoviral vector encoding the uteroglobin gene (Ad.UG; 15 ␮l of 5 ⫻ 10 12 p/ml) was applied to the adventitia of the injured artery in 16 mice. In our control groups, 16 mice received adenoviral vector encoding the ␤-galactosidase reporter gene (Ad.lacZ; 15 ␮l of 5.4 ⫻ 10 12 p/ml) and 13 mice received PBS only. Six mice from each group were sacrificed at 4 days for carotid protein extraction and Western blot analysis. The remainder were harvested at 30 days for histologic and morphometric analysis. The intima/media area ratios were calculated for each artery. The results were analyzed and compared using ANOVA and Bonferroni post hoc testing. Results. Two mice from the lacZ group and 1 from the PBS group died before the 30-day endpoint. Uteroglobin expression was demonstrated in the Ad.UG-treated arteries by Western blot analysis. Morphometric analysis demonstrated a statistically significant reduction in the intima/media area ratio of Ad.UG-treated carotids compared to controls. There was a reduction of intima/ media ratio with Ad.UG treatment of 68% compared to Ad.lacZ treatment (P ⬍ 0.0001) and 62% compared to PBS treatment (P ⫽

0.0006). There was no statistical difference between the control groups (see figure). Conclusion. Adenoviral-mediated gene transfer via the adventitia is an effective mode of gene delivery. Adventitial uteroglobin gene transfer using an adenoviral vector induces uteroglobin protein production and significantly reduces neointimal hyperplasia in the mouse carotid ligation injury model. To our knowledge this is the first report of direct adventitial adenoviral-mediated gene transfer achieving altered arterial wall morphometry. 26. Accuracy of Intravascular Ultrasound (IVUS) and Quantitative Angiography (QA) for Diameter Measurement of Phantom Arterial Models. B. Z. Cooper, M.D., M. Cooper, Esq., J. A. Ramirez, M.D., J. G. Najjar, M.D., S. B. Blattman, M.D., M. Song, M.D., J. D. Kirwin, M.D., W. Rodino, M.D., and T. F. Panetta, M.D. Department of Surgery, SUNY Downstate Medical Center, Brooklyn, New York. Objectives. QA in a single plane is the standard method for measuring vessel diameter during surgical and endovascular procedures. IVUS, a relatively new technology, is another means of obtaining this measurement. A comparative analysis of these two measuring modalities, however, has not been reported. This study was designed to validate the accuracy of these two modalities by comparing each to direct caliper measurement, the gold standard, using phantom iliofemoral artery segments (PAS). Methods. Measurement PAS diameter with a 12.5-MHz mechanically rotating IVUS catheter (Boston Scientific Corp.) and QA (OEC Corp.) was compared to direct caliper measurement (Mitutoyo Corp.) at 60 different locations within PAS. At each location measurements were made in two perpendicular planes through the center point. These measurements were averaged. The intraclass correlation coefficient between the caliper and IVUS measurements and the caliper and QA measurements were calculated. Fischer’s Z transformation test was then used to compare the two correlation coefficients. Results. The intraclass correlation coefficient (ICCC) for IVUS and QA in two planes was 0.89 and 0.82, respectively. The ICCC for QA in one plane was 0.73. IVUS in two planes correlated more closely with direct caliper

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS measurement than QA in one plane (P ⫽ 0.00008). IVUS in two planes correlated more closely with direct caliper measurement than QA in two planes (P ⫽ 0.02). QA in two planes correlated more closely with direct caliper measurement than QA in one plane (P ⫽ 0.04). Conclusions. IVUS may be used to more accurately measure lumen diameter than QA. Diameter measurements with QA in two perpendicular planes compared to one plane more accurately measures lumen diameter (see figure).

PARALLEL SESSION I Metabolism, Endocrinology, and Nutrition 27. Granulation Tissue Apoptosis Induced by a Musculocutaneous Flap. M. A. Carlson, M.D., and B. T. Baxter, M.D. Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska. We hypothesized that wound coverage with an MCF would result in regression and apoptosis of the wound’s GT. Wistar rats (350 g; n ⫽ 32) underwent skin excision (4 cm 2 square from the dorsum), an MCF was placed over the developed GT in 16 rats on postwound day 10, and 16 rats (8 MCF ⫹ 8 control) were sacrificed on both day 12 and day 14. Paraffin sections were labeled with TUNEL and propidium iodide, and fluorescent micrographs were analyzed by computer. A wound’s apoptotic rate (⫽No. of TUNEL figures ␹ No. of PI labeled nuclei ⫻ 100) was the mean of four micrograph rates, and a group rate was the mean of eight wound rates. Cross-section area was calculated from H & E micrographs. Qualitatively, the MCFcovered GT had decreased cellularity (H & E) compared to the control GT at 2 and 4 days of coverage. The group rates within each column of the table were different (P ⬍ 0.05, ANOVA). The GT apoptotic rate increased at least fivefold in the MCF rats compared to the control rats after 2 and 4 days of MCF coverage. The GT cell population density decreased nearly 50% in MCF group at 2 and 4 days, and the

TABLE—ABSTRACT 27 Group

n (rats)

Apoptosis (% ⫾ SD)

MCF, 2 days cont, 2 days MCF, 4 days cont, 4 days

8 8 8 8

5.52 ⫾ 0.91* 0.52 ⫾ 0.24 4.89 ⫾ 0.33* 0.89 ⫾ 0.49

Cell pop. dens x-section area (nuc/micrograph) (mm 2) 1977 ⫾ 343* 3947 ⫾ 1275 1938 ⫾ 496* 3009 ⫾ 462

6.35 ⫾ 2.84** 8.05 ⫾ 1.80 NA 9.27 ⫾ 1.66

* P ⬍ 0.001 compared to corresponding control, unpaired t test. **P ⫽ 0.055 compared to control. NA, GT margins indistinct. GT cross-section area decreased marginally in the MCF group compared to the control group at 2 days. These data, along with the qualitative wound histology, support the hypothesis that wound coverage with an MCF is associated with GT regression and apoptosis. The mechanism of GT regression in this model is as of yet unclear. 28. Systemic Heat Shock Attenuates the Neuroendocrine Response to Surgical Stress in Rats. B. B. O’Neill, M.D., S. Sheen-Chen, M.D., W. J. Welch, Ph.D., and H. W. Harris, M.D. UCSF Surgical Research Lab at SFGH, San Francisco, California. Much of the morbidity associated with elective surgery results from activation of the body’s neuroendocrine stress response. The

315

ability to dampen this response could reduce surgical morbidity and thus revolutionize the perioperative management of elective surgical patients. The heat shock response (HSR) is a welldescribed programmed cellular response to stress that can protect cells from an otherwise lethal injury. If the HSR can protect individual cells, we wondered whether this response could protect an entire organism as well. Specifically, we hypothesized that the induction of a systemic HSR would make rats resistant to surgical stress and thus attenuate their neuroendocrine response to surgery. Rats were either preconditioned by whole body hyperthermia (42°C ⫻ 20 min) or maintained at 37°C (controls). Multiple organs from both groups were then assayed for heat shock protein (hsp) expression via Western blot. After thermal preconditioning, the rats underwent a standardized surgical stress (laparotomy, visceral manipulation, 25% hemorrhage). Subsequently, plasma was assayed for total corticosterone and corticosterone-binding globulin (CBG) as a measure of neuroendocrine axis activation. Preconditioned rats showed activation of the HSR as evidenced by widespread systemic expression of hsp72 versus controls (Fig. 1).

Corticosterone (rat equivalent to cortisol) was reduced by 25% (Fig. 2), whereas CBG levels were unchanged (data not shown). In conclusion, preconditioned rats showed systemic activation of the HSR with widespread expression of hsp72 and the subsequent attenuation of the animals’ neuroendocrine response to surgical stress. These data suggest that activation of the heat shock response may protect an entire animal from the stress of surgical procedures and thus potentially reduce perioperative morbidity. 29. Imidazole Blockade of Cortisol Synthesis in Burn Patients. D. W. Hart, M.D., S. E. Wolf, M.D., A. A. Ferrando, Ph.D., C. G. Wigginton, B.S., R. R. Wolfe, Ph.D., and D. N. Herndon, M.D. Department of Surgery, The Shriner’s Hospitals for Children and The University of Texas Medical Branch, Galveston, Texas. Imidazole antifungal agents inhibit synthesis of the sterol ring, thus damaging fungal cell walls. Recently, clinicians have successfully applied this pharmacologic effect to noninfectious human pathology resulting from sterol excess—notably, hypercortisolemic depression and Cushings syndrome. Burn catabolism is also thought to be mediated by excess cortisol production and activity. For this reason, we chose to examine the effect of a common imidazole, itraconazole, on cortisol production and skeletal muscle protein catabolism in burned children. Methods. Five subjects received at least a 1-week course of itraconazole. None were clinically suffering from invasive fungal wound infections, had documented fungemia or bacteremia, or received any other anabolic agents prior to or during study. All subjects were studied in two periods: Baseline (untreated) and Treatment in the itraconazole subjects, and Baseline and Time Control in 12 subjects serving as a control group. Main outcome measures for comparison were resting energy expenditure, urine cortisol levels, and cross-leg protein net protein balance derived from stable isotope tracer methodology. Results. The 5 itraconazole and 12 control subjects were similar in age, sex, burn size, and time after burn at the metabolic studies. Subjects receiving imidazoles experienced a decrease in urinary cortisol excretion and improved skeletal

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 29

Baseline urine cortisol Treatment urine cortisol Baseline protein net balance Treatment protein net balance

teral nutrition may increase inflammatory response through enhanced E-selectin levels after a small dose of LPS.

Time control

Imidazole

33.3 ⫾ 5.7 38.0 ⫾ 7.3 ⫺0.048 ⫾ 0.015 ⫺0.042 ⫾ 0.011

37.0 ⫾ 3.6 19.3 ⫾ 3.7 ⫺0.061 ⫾ 0.017 ⫺0.017 ⫾ 0.010

muscle protein kinetics compared both with their untreated baseline levels and with time control subjects (P ⬍ 0.05). There were no differences in resting energy expenditure over time or between groups. Urine cortisol levels below are measured in micrograms per deciliter and the net balance of protein synthesis and breakdown in ␮mol/min/100 cc leg (see table). Conclusion. Imidazole therapy successfully decreases cortisol production in burned children. As a result, skeletal muscle protein metabolism is improved, with subjects becoming less catabolic. 30. Lack of Enteral Nutrition Increases Gut Expression of E-Selectin but Not ICAM-1 after Lipopolysaccharide (LPS) Challenge. K. Fukatsu, M.D., B. L. Zarzaur, M.D, C. D. Johnson, Ph.D., A. H. Lundberg, M.D., M. K. Hanna, M.D, H. G. Wilcox, Ph.D, and K. A. Kudsk, M.D. Departments of Surgery and Pharmacology, The University of Tennessee, Memphis, Tennessee. Total parenteral nutrition (iv-TPN) increases ICAM-1 expression in the small intestine and E-selectin expression in the lung. Endothelial activation induced by lack of enteral nutrition may change the response to injury or infection. This study examined the influence of nutrition on organ E-selectin and ICAM-1 levels after LPS challenge. Methods. First, 43 mice were injected with saline only or 2, 20, 200, 2000, or 10,000 ␮g/kg LPS ip. After challenge, lung E-selectin and ICAM-1 levels were quantified at 3 and 5 h, respectively, using the dual radiolabeled monoclonal Ab technique. Second, 80 mice were fed chow, intragastric (ig)-TPN, or iv-TPN for 5 days and then received 2 or 200 ␮g/kg LPS ip. E-selectin and ICAM-1 expression in the lung, small intestine, and heart was evaluated at 3 and 5 h, respectively, after challenge. Statistical analysis was performed using ANOVA. Results.

TABLE—ABSTRACT 30 E-Selectin (␮g/g Tissue) Expression after 2-␮g LPS Injection

Lung Intestine Heart

CHOW

IG-TPN

IV-TPN

22.2 ⫾ 5.0 1.3 ⫾ 0.2 1.8 ⫾ 0.5

23.7 ⫾ 1.6 1.7 ⫾ 0.3 3.6 ⫾ 0.6

35.8 ⫾ 9.2 2.9 ⫾ 0.4* ,† 2.9 ⫾ 0.5

Note. Mean ⫾ SEM; *P ⬍ 0.01 vs CHOW; †P ⬍ 0.03 vs IG-TPN.

Lung E-selectin expression increased in an LPS-dose-dependent fashion, while ICAM-1 levels reached plateau at 20 ␮g/kg of LPS (see table). iv-TPN increased intestinal E-selectin, but not ICAM-1 expression after 2 ␮g/kg LPS, compared to chow or igTPN. There was no significant difference in E-selectin or ICAM-1 levels among groups after 200 ␮g/kg LPS. Conclusions. E-selectin rather than ICAM-1 may be a determining factor for the degree of LPS-induced inflammation at early phase. Lack of en-

31. Trophic Feeds Increase Hepatic Glutathione (GSH) Concentration during Parenteral Nutrition. A. Dzakovic, M.D., O. Eshach-Adiv, M.D, A. Rhodes, B.S., P. R. Ling, M.D., Y. M. Yu, M.D., B. Bistrian, M.D., and T. Jaksic, M.D. Children’s Hospital, Beth Israel/Deaconess Hospital, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. Purpose. Hepatic concentrations of the important intracellular antioxidant GSH are decreased during parenteral nutrition. The purpose of this study was to determine whether enteral trophic feeding of GSH precursors would increase the concentration of GSH in the liver during postoperative total parenteral nutrition (TPN). Methods. Male 250-g Sprague–Dawley rats underwent central venous line insertion and surgical postpyloric gastrostomy tube placement. Full TPN (250 kcal/kg/day) was administered for 7 days. Enterally rats received 10 cc/day infusions of: (1) amino acid GSH precursors (60 mg/day cysteine; 86 mg/day glycine; 31 mg/day glutamate) [TROPHIC 1], (2) isonitrogenous alanine (132 mg/day) [TROPHIC 2], or (3) normal saline [TROPHIC 3]. [GSH] concentrations in the ileal mucosa and liver tissue were measured by gas

TABLE—ABSTRACT 31 Glutathione Concentrations

Ileal [GSH], ␮mol/g Hepatic [GSH], ␮mol/g

TROPHIC 1 (n ⫽ 10)

TROPHIC 2 (n ⫽ 10)

TROPHIC 3 (n ⫽ 10)

4.5 ⫾ 0.2

4.8 ⫾ 0.3

4.2 ⫾ 0.4

11.7 ⫾ 0.6**

7.0 ⫾ 0.8

5.0 ⫾ 0.4

** P ⬍ 0.001 vs hepatic [GSH] in TROPHIC 2 and TROPHIC 3. chromatography/mass spectrometry against a stable isotope standard. Results were expressed as means ⫾ SE. Comparisons between trophic groups were made by ANOVA. Results. (See table.) Ileal mucosal volume was similar in all groups (P ⬎ 0.6). Conclusion. Trophic feeding of the amino acid precursors for GSH synthesis, during parenteral nutrition, does not affect intracellular intestinal mucosal GSH but markedly increases hepatic GSH concentration and hence may improve host defenses. 32. Role of Polyamine Biosynthesis in Burn-Induced Small Intestinal Apoptosis. V. L. Chappell, M.D, D. H. Chung, M.D., S. E. Wolf, M.D, and J. C. Thompson, M.D. Department of Surgery, The University of Texas Medical Branch and Shriners Burns Hospital, Galveston, Texas. Intestinal epithelial homeostasis involves a balance of proliferation and apoptosis. We have previously shown that cutaneous burn injury increases small intestinal epithelial apoptosis; the exact cellular mechanisms are not known. Polyamines are essential polycationic compounds that are necessary for normal cell growth and proliferation, and its biosynthesis is specifically inhibited by ␣-difluoromethylornithine (DFMO). The purpose of this study was to examine the effects of DFMO on the small intestinal epithelial apoptosis induced by cutaneous burn injury. Methods. Male C57BL6 mice were randomized to receive either DFMO (2% in drinking water) or control water for 3 days. They were then subdivided to receive 30% TBSA cutaneous scald or sham burn. At 12 h after burns, mice were sacrificed and proximal small intestine was har-

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS vested. They were weighed and analyzed for PCNA and TUNEL assays to determine intestinal epithelial cell proliferation and apoptosis, respectively. Statistical analysis was performed using oneway analysis of variance and t test (*P ⬍ 0.05 vs control; †P ⬍ 0.05 vs 0 h). Results. DFMO decreased proximal small intestinal weight as compared to control mice. Burn injury decreased intestinal weight

at 12 h; there was more marked decrease in DFMO-treated group (Fig. 1). Burn injury induced small intestinal apoptosis at 12 h; this increase was significantly inhibited by DFMO treatment (Fig. 2). There were no changes in proliferation as measured by PCNA assay. Conclusions. Cutaneous burn injury resulted in rapid induction of small intestinal epithelial apoptosis in mice. Inhibition of polyamine biosynthesis decreased burn-induced intestinal apoptosis. These findings suggest a specific role of the polyamine biosynthesis pathway in the regulation of intestinal apoptosis that occurs after burns. 33. Eicosapentanoic Acid (EPA) Inhibits Tumor Growth and Reduces Vascular Endothelial Growth Factor-␣ (VEGF-␣) Expression. R. Tevar, B.S., T. Babcock, M.S., D. Jho, B.S., W. S. Helton, M.D., and N. J. Espat, M.D. Department of Surgery, University of Illinois, Chicago, Illinois. The mechanism(s) whereby EPA, an omega-3 fatty acid, inhibits tumor growth are not well defined, but may be related to alterations in angiogenic growth factors. We hypothesized that oral EPA will decrease tumor growth and down-regulate VEGF-␣ expression in the liver of tumor-bearing rats. Methods. Fischer 344 rats (200 –250 g body wt) underwent flank implantation of the MCA-induced fibrosarcoma on day 0. Rats were randomly divided into three groups on day 13: EPA (1 cc, 5.0 mg/kg/day) ⫹ 10 IU vitamin E (n ⫽ 10); corn oil (1 cc) ⫹ 10 IU vitamin E (n ⫽ 10), and saline (1 cc) ⫹ 10 IU vitamin E (n ⫽ 9). (Vitamin E as mixed tocopherols was used to prevent EPA oxidation and liver vitamin E depletion.) On day 14, gavage feeding was initiated and continued through day 28. On day 29, rats were sacrificed; tumors were removed, weighed, and divided by the carcass weight to obtain percentage of tumor volume (TV); the liver was flash frozen until VEGF-␣ mRNA was measured by RTPCR. Densitometry was performed and band density normalized to GAPDH. Data are means ⫾ SEM, ANOVA, *P ⬍ 0.01 vs saline and corn oil. Results. EPA rats had 33% reduction in TV compared to saline and 25% reduction compared to corn oil (Fig. 1). EPA rats had decreased expression of VEGF-␣ compared to corn oil and saline rats (Fig. 2). Conclusions. These data demonstrate that EPA supple-

317

mentation inhibits tumor growth, potentially through alterations in the expression of the proangiogenic growth factor, VEGF-␣. The mechanism(s) of EPA as an inhibitor of tumor-related angiogenic growth factors merits further study. 34. Antiproliferative Effects of a Novel SERM GW7604 on Breast and Endometrial Cells. D. J. Bentrem, M.D., R. Dardes, M.D., J. MacGregor Schafer, Ph.D., J. Zapf, Ph.D., and V. C. Jordan, Ph.D., D.Sc. Department of Surgery, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois; and Signal Pharmaceuticals, San Diego, California. New agents called selective ER modulators (SERMs) are being sought to improve the safety profile of tamoxifen so they can be used widely as preventives for breast cancer and osteoporosis without elevating the risk of endometrial cancer. GW5638 is a tamoxifen derivative with a novel carboxylic acid side chain. The compound can preserve bone density but is an antiestrogen with no uterotropic activity in the rat and is being considered as an agent to treat breast cancer. The primary goal was to study the interaction of GW5638 with ER␣ to provide insight into the site specific pharmacology of new SERMs. We have compared the actions of 4-hydroxytamoxifen (4-OHT), the active metabolite of tamoxifen, with GW7604, the presumed metabolite of GW5638, in human breast (MCF-7) and endometrial (ECC-1) cell lines in vitro. Northern blot analysis was used to compare the transcriptional activity of the SERMs at the wild-type ER. Computer-assisted molecular modeling was used to observe the surface of antiestrogen ER complexes and to identify the critical interaction of the SERM side chains with individual amino acids. We found that the side chain of 4-OHT weakly interacts with aa351 of the ER but the carboxylic acid of GW7604 causes a strong repulsion of the aspartate amino acid at position 351. GW7604 did not potentiate the growth of ECC-1 uterine cancer cells at any concentration but 4-OHT was weakly estrogen-like at low concentrations. GW7604 (10 ⫺7 M) blocked the growth-promoting action of estradiol (10 ⫺10 M) in both ECC-1 and MCF-7 cells in vitro (P ⬍ 0.05). The compound GW7604 is active at the ER and able to block the agonist effects on transcription of both estradiol and tamoxifen in wild-type ER (P ⬍ 0.05). We conclude that GW7604 is able to inhibit breast cancer cell growth yet is less estrogenic in the ECC-1 human endometrial cell line than 4-OHT.

PARALLEL SESSION I Gastrointestinal I 35. Cerulein-Induced Acute Pancreatitis Is More Severe in Mice Heterozygous for the ⌬F508 Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutation Than Wild-Type Controls. Q. Liu, M.D., H. Fischer, Ph.D., W. J. Welch, Ph.D., and H. W. Harris, M.D. UCSF Surgical Research Lab at SFGH, San Francisco, California. Carriers of a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride transport protein, have recently been shown to be at increased risk for developing chronic pancreatitis. Therefore, we postulated that mutations in CFTR would also confer an increased risk of acute pancreatitis. Specifically, we hypothesized that mice heterozygous for the most common CFTR mutation (⌬F508), while phenotypically normal, would experience a more severe acute pancreatic injury following cerulein-induced hyperstimulation compared to CFTR wild-type controls. To address this question, transgenic mice heterozygous for the ⌬F508 mutation (N ⫽ 33) and their wild-type controls (N ⫽ 12) were injected with cerulein (50 ␮g/kg/h ⫻ 6 h) versus saline and assayed for pancreatic injury via tissue edema, Evans blue dye extravasation, plasma amy-

318

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

lase concentration, and pancreatic histology. The quantitative indicators of acute pancreatitis and organ injury were all greater in the ⌬F508 heterozygous mice than the wild-type controls. The total water content (81.7 ⫾ 1.1 vs 79.9 ⫾ 2.5%, P ⬍ 0.05), Evans blue dye extravasation (300 ⫾ 103 vs 197 ⫾ 64 ng/mg pancreas, P ⬍ 0.05), and plasma amylase concentrations (6351 ⫾ 466 vs 4450 vs 561 U/L, P ⫽ 0.02) were all higher in the cerulein-treated ⌬F508 heterozygotes than the control mice. In addition, there was histologic evidence of severe pancreatic inflammation in the heterozygous mice. In conclusion, mice heterozygous for the ⌬F508 CFTR mutation experience a more severe pancreatic injury following cerulein hyperstimulation than wild-type animals. These data may yield insight into the potential role of chloride transport in the pancreas and the molecular pathogenesis of acute pancreatitis.

(I␬B), regulate gene transcription encoding for various immune and inflammatory proteins including cytokines. We hypothesized that NF-␬B and I␬B are involved in cytokine-mediated pulmonary injury during severe acute pancreatitis. Severe pancreatitis was induced by CDE diet (n ⫽ 160). Bronchoalveolar lavage and lung I␬B␣ and I␬B␤ proteins (Western) and NF-␬B activation (EMSA) were determined at 0, 6, 12, 18, 24, 30, 36, and 48 h. Additional mice (n ⫽ 135) were randomized to CDE diet ⫾ PDTC, an inhibitor of NF-␬B activation. Pulmonary tissue was collected after 0, 24, 36, 48, and 72 h for determination of TNF mRNA (RT-PCR), neutrophil infiltration by myeloperoxidase activity (MPO), and microvascular permeability by Evans blue dye. Results are means ⫾ SEM, P ⬍ 0.05, two-tailed Student’s t test. Degradation of the inhibitory proteins I␬B␣/␤ occurred 24 h after beginning the CDE diet; however, I␬B␣ rapidly

36. Activation of the Capsaicin Receptor, Vanilloid Receptor-1 (VR-1), Promotes Neurogenic Inflammation in Cerulein-Induced Pancreatitis. M. M. Hutter, M.D., J. Maa, M.D., E. C. Zerega, E. F. Grady, Ph.D., S. J. Mulvihill, M.D., N. W. Bunnett, Ph.D., and K. S. Kirkwood, M.D. Departments of Surgery and Physiology, University of California, San Francisco, California. Activation of the VR-1 on sensory nerves stimulates the release of substance P (SP). Prior studies have shown that SP promotes neurogenic pancreatic inflammation via the neurokinin-1 receptor (NK1R). We tested the hypotheses that stimulation of the VR-1 induces pancreatic plasma extravasation (PE) and that blockade of the VR-1 reduces the severity of acute pancreatitis. The VR-1 agonist capsaicin was injected intravenously (iv) in Sprague–Dawley rats (total n ⫽ 37), and pancreatic PE was measured with Evans blue. Rats were pretreated with the VR-1 antagonist capsazepine (CPZ, 1.8 mg/kg), the NK1R antagonist CP 96,345 (1 mg/kg), or carrier. In a second set of experiments (n ⫽ 29), cerulein (10 ␮g/kg/h) was infused iv for 4 h, with simultaneous infusion of either CPZ (0.38 mg/kg/h) or carrier. Endpoints included PE (ng/mg), amylase, wet/dry ratio, lung and pancreatic myeloperoxidase (MPO, abs/min/g), and pancreas histologic score (blinded). Statistical comparisons used ANOVA with SNK or t test (interval data) or rank sum test (ordinal data). Data are means ⫾ SEM, *P ⬍ 0.05. Capsaicin induced a dose-related increase in pancreatic PE [9.0 ⫾ 3 (carrier), 17 ⫾ 3 (1 ␮m/kg), 42 ⫾ 10* (2 ␮m/kg); n ⫽ 4 –7/group]. Capsaicin-induced PE was blocked by CP 96,345 (3.0 ⫾ 0.5* vs 42 ⫾ 10, n ⫽ 3– 4/group) and by CPZ (8 ⫾ 1* vs 17 ⫾ 3, n ⫽ 5–7/group). CPZ reduced the severity of cerulein-induced pancreatitis (n ⫽ 5–10/group) (see table). Other endpoints were not significantly different. We conclude that activation of the VR-1 induces SP-mediated pancreatic plasma extravasation and is an im-

TABLE—ABSTRACT 36

Cerulein CPZ ⫹ cerulein

PE

Pancreas MPO

Histology score

64 ⫾ 15 27 ⫾ 7*

0.089 ⫾ 0.007 0.011 ⫾ 0.004*

18 ⫾ 1 14 ⫾ 1*

portant determinant of severity in cerulein-induced pancreatitis in the rat. 37. The Role of NF-␬B and I␬B in Cytokine-Mediated Pulmonary Injury during Acute Pancreatitis. C. Jaffray, M.D., J. Yang, M.D, M. Murr, M.D., C. Mendez, M.D., and J. Norman, M.D. Department of Surgery, University of South Florida, Tampa, Florida. Nuclear factor ␬B (NF-␬B) and its inhibitor protein, inhibitory ␬B

reconstituted by 30 –36 h. NF-␬B activation followed at 24 –36 h (Fig. 1, P ⬍ 0.05). PDTC markedly attenuated the CDE diet-induced increase in TNF mRNA (Fig. 2, 48 h), neutrophil infiltration (not shown, P ⬍ 0.05), and microvascular leakage (Fig. 3, 72 h). In conclusion, NF-␬B and I␬B␣/␤ proteins play an important role in pancreatitis-associated pulmonary injury via TNF production. 38. Iron Deficiency Transiently Suppresses Biliary Neuronal Nitric Oxide Synthase. M. I. Goldblatt, M.D., D. A. SwartzBasile, Ph.D., S. Choi, M.D., P. Rafiee, Ph.D., A. Nakeeb, M.D., S. K. Sarna, Ph.D., and H. A. Pitt, M.D. Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin. We have previously demonstrated that iron deficiency results in altered gallbladder and sphincter of Oddi (SO) motility and cholesterol crystal formation. In addition, gallbladder neuronal nitric oxide synthase (nNOS) has been shown to be markedly reduced after 8 weeks on an iron-deficient diet. However, the effects of an iron-deficient diet on SO nNOS and the influence of prolonged iron deficiency have not been determined. Therefore, we tested the hypothesis that iron deficiency would downregulate both gallbladder and SO nNOS expression with increased downregulation over time. Thirty-eight adult female prairie dogs were fed either an iron-supplemented (Fe⫹) (200 ppm) (n ⫽ 9) or an iron-deficient (Fe⫺) (8 ppm) (n ⫽ 10) diet for 8 or 16 weeks (Fe⫹ n ⫽ 9, Fe⫺ n ⫽ 10). Blood hemoglobin (HbG) was measured as a percentage of the Fe⫹ HbG. Cholesterol (XOL) crystals per 10 HPFs were measured in the gallbladder bile, and gallbladder and SO smooth muscle were harvested to measure nNOS by Western blot analysis. nNOS protein levels were measured by densitometer. Student’s t test and Mann–Whitney rank sum test were used where appropriate. Results are shown in the table. These data suggest that iron deficiency results in (1) a decrease in HbG at 8 weeks, (2) cholesterol crystal formation that increases over time, and (3) a transient decrease in the gallbladder and sphincter of Oddi levels of nNOS. We conclude that iron deficiency acutely suppresses gallbladder and sphincter of Oddi nNOS. Compensatory mechanisms return nNOS to baseline levels while cholesterol crystal levels increase over time.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 38 ⫹

Fe ⫹ Fe ⫺ Fe ⫹ Fe ⫺

(8 weeks) (8 weeks) (16 weeks) (16 weeks)

HbG (% Fe )

XOL crystals per 10 HPFs

Gallbladder nNOS

Sphincter of Oddi nNOS

100 ⫾ 4 90 ⫾ 3* 100 ⫾ 6 95 ⫾ 4

0.4 ⫾ 0.3 1.6 ⫾ 0.4* 0.0 ⫾ 0.0 52.6 ⫾ 25.3* ,‡

570 ⫾ 296 74 ⫾ 26* 941 ⫾ 312 809 ⫾ 269 ‡

984 ⫾ 397 299 ⫾ 110§ 1027 ⫾ 352 1230 ⫾ 303 ‡

* P ⬍ 0.05 vs Fe ⫹; § P ⫽ 0.09 vs Fe ⫹; ‡P ⬍ 0.05 vs Fe ⫺ (8 weeks).

39. Bicarbonate Secretion by Isolated Pancreatic Duct Cells Is a Metabolically Active Process. J. P. Regan, M.D., and C. Alvarez, M. D. Department of Surgery, University of Maryland, Baltimore, Maryland; and UMDNJ–NJMS and East Orange VAMC, New Jersey 07019. Evaluation of pancreatic HCO 3⫺ production is hampered by a lack of adequate models of duct cell isolation. Here we examined the ability of bovine duct explants to secrete bicarbonate. Methods. Segments of the main pancreatic duct (the epithelium and underlying connective tissue) were harvested fresh and allowed to recover in culture for at least 48 h. They were mounted on Ussing chambers, with modified Kreb’s solution bubbled with 95% O 2/5% CO 2 in the serosal bath while the mucosa was bathed in unbuffered Kreb’s and 100% O 2. HCO 3⫺ output in the mucosal bath was determined by the ␮mol/h/cm 2 of acid added to maintain a stable mucosal pH (pH m). Secretin (SEC, 10 nM) was added after the following inhibitors: ouabain (OUAB, 1 mM), DIDS (500 mM), and dinitrophenol (DNP, 500 ␮M). Results are reported as ratios of the baseline output at pH m

postoperative adhesion formation without affecting wound and anastomotic integrity. After developing a standardized peritoneal injury which resulted in a reproducible adhesion model, 40 CD-1 mice were randomized and treated intraperitoneally with either VEGF mAb (n ⫽ 20) or IgG isotype control mAb (n ⫽ 20) at the time of abdominal closure. Animals (n ⫽ 10/group) were sacrificed on postoperative day 14, and the development of intraabdominal adhesions was determined and graded blindly using a well-established criterion. Animals (n ⫽ 10/group) were also sacrificed 5 days postoperatively and their laparotomy wound and gastrointestinal anastomoses were assessed

TABLE—ABSTRACT 40 Breaking strength (Newtons) Adhesion score Grade:

0

1

2

3

Laparotomy wound

Gastrointestinal anastomosis

VEGF mAb IgG mAb

8 0

1 1

1 3

0 6

0.89 ⫾ 0.18 0.92 ⫾ 0.25

0.68 ⫾ 0.12 0.71 ⫾ 0.14

by tensiometry. Statistical analyses were performed using the Mann–Whitney and Fisher’s exact tests. Treatment with VEGF mAb resulted in a significantly lower incidence of adhesion formation compared to control animals (P ⬍ 0.001). Laparotomy wound and gastrointestinal anastomotic strength were similar between groups. This study demonstrates that the formation of postoperative intraabdominal adhesions is attenuated following the administration of VEGF mAb without adversely affecting wound strength or anastomotic integrity (see table). 7.4 and are given as means ⫾ SEM and compared with ANOVA. Results. A passive HCO 3⫺ output, independent of Cl ⫺/HCO 3⫺ exchange, is present at pH m 7.4 (3.5 ⫾ 0.3 ␮mol/h/cm 2, n ⫽ 32). It is profoundly reduced at pH m 8.0, despite a persistent large HCO 3⫺ gradient toward the lumen. HCO 3⫺ output at pH m 8.0 is increased by SEC and this response can be inhibited (see figure). Conclusions. Duct cells, isolated as explants, produce HCO 3⫺ in response to SEC at physiological pH m. This active process can be blocked by inhibition of the Na-K-ATPase, Cl ⫺/HCO 3⫺ exchange, and oxidative metabolism, confirming in vivo data and validating this model. The novel finding that pH m affects secretion bears further investigation. 40. Anti-Vascular Endothelial Growth Factor Attenuates Peritoneal Adhesion Formation. S. Sookhai, FRCSI, J. Wang, Ph.D., K. Austin, AFRCSI, D. Maguire, FRCSI, R. Cahil, AFRCSI, W. O. Kirwan, FRCSI, and H. P. Redmond, FRCSI. Department of Surgery, Cork University Hospital, Cork, Ireland. Postoperative intraabdominal adhesion formation remains a significant cause of complications in surgical patients. We determined whether a specific VEGF monoclonal antibody (mAb) could prevent

41. Cholestasis Induces Murine Hepatocyte Apoptosis and DNA Synthesis with Preservation of the Immediate Early Gene Response. M. A. Bird, M.D.,* L. W. Schrum, Ph.D.,* R. A. Rippe, Ph.D.,† D. A. Brenner, M.D.,† and K. E. Behrns, M.D.* Departments of *Surgery and †Medicine, CB No. 7210, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Introduction. Major hepatic resection in patients with unrelieved obstructive jaundice carries an increased risk of postoperative liver failure. We hypothesized that cholestasis induces hepatocyte apoptosis and impairs hepatic regeneration by inhibition of known early immediate genes, NF-␬B and AP-1. Aim. To determine if the immediate early gene response in hepatic regeneration remains intact in cholestasis. Methods. Balb/c mice underwent either sham operation (SO) or common bile duct ligation (BDL). A two-thirds partial hepatectomy (PH) was performed 1 week later with remnant liver harvested at 0, 15, 30, or 60 min after PH. TUNEL and PCNA immunohistochemistry for apoptosis and DNA synthesis, respectively, of intact liver was performed. Liver samples from 0, 15, 30, or 60 min PH were analyzed for NF-␬B and AP-1 binding activity using electrophoretic mobility shift assays. Results. Increased serum bil-

320

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

irubin and H&E stains confirmed cholestasis in BDL mice. BDL induced marked hepatocyte apoptosis (0.32% SO vs 6.32% BDL) and DNA synthesis (0.56% SO vs 14.86% BDL) in intact liver. Substantially higher basal levels of both NF-␬B and AP-1 binding activity were present in BDL compared to SO. Fold induction of NF-␬B and AP-1, however, was similar. Summary. Cholestasis induces hepatocyte apoptosis and DNA synthesis. Basal NF-␬B and AP-1 DNAbinding activity are increased in BDL mice, but fold induction of these immediate early genes is not different from controls. Conclusion. Although basal NF-␬B and AP-1 DNA binding are increased in cholestasis, the immediate early gene response to PH remains intact.

42. Bile Acid Stimulates Intestinal Epithelial Cell Migration through Increased TGF␤ Expression. E. D. Strauch, M.D., J. Y. Wang, Ph.D., K. Lally, M.S., and B. L. Bass, M.D. Department of Surgery, University of Maryland, Baltimore, Maryland. Free bile acids may modulate intestinal epithelial growth, differentiation, and other epithelial functions. We hypothesized that bile acids could modulate intestinal repair and investigated a possible role for TGF␤, a widely expressed cytokine in the intestinal villus, in this repair process. Methods. Using a well-established model of epithelial restitution, IEC-6 cells were plated on 60-mm Matrigelcoated plastic dishes and grown to confluence. The epithelium was wounded by scraping with a 6-mm-wide blade to create a smooth denuded edge and cell migration was measured 8 h later. Cells were grown in control DMEM with 5% FBS with or without 0.01–2 mM taurodeoxycholic acid (TDCA). In parallel experiments, cells were harvested for Northern analysis of TGF␤ and GAPDH expression; tritiated thymidine uptake was used to measure proliferation. AntiTGF␤ antibody was added to cells grown in the presence of 0.05 mM TDCA and migration was measured at 8 h. Results. TDCA at physiologic luminal concentrations augments IEC-6 cell migration,

with a maximal effect at 0.05 mM. TDCA inhibited proliferation at these concentrations. TGF␤ expression increased in response to bile acid, while wounding had a minimal effect on TGF␤ expression (figure, left). Blockade of TGF␤ function with TGF␤ antibody eliminated the effect of bile on cell migration (figure, right). Bile acid at physiologic luminal concentrations augments small intestinal epithelial cell migration. The process is dependent on TGF␤ and is independent of cell division. The data further support a role for bile acids and TGF␤ in differentiated intestinal cell function and in preservation of an intact mucosa.

RESIDENT RESEARCH AWARDS 43. Endotoxin (LPS) Stimulates PHAS-1 Phosphorylation in Macrophages. M. W. Potter, M.D., K. K. Elbirt, Ph.D., S. A. Shah, M.D., K. R. Sheth, M.D., and M. P. Callery, M.D., FACS. Department of Surgery, UMASS Medical School, Worcester, Massachusetts. Introduction. Translational control of cytokine gene expression may occur in sepsis. Phosphorylation of cytoplasmic PHAS-1 leads to

its dissociation from eIF-4E, allowing the initiation of translation. We examined whether LPS activates PHAS-1 in macrophages focusing on potential signal transduction mechanisms involved. Methods. A total of 5 ⫻ 10 6 thioglycolate-elicited rat peritoneal macrophages (PM) were stimulated with 100 ng/ml LPS (Escherichia coli 0111:B4) with/without p38-MAPK activator sodium salicylate (NaSal, 1mM), p38 inhibitor SB203580 (20 ␮M), or rapamycin (20 ng/ ml), a FRAP/mTOR inhibitor. PM TNF-␣ production was measured by ELISA and compared by Student’s two-tailed t test (P ⬍ 0.05). PHAS-1 activation was determined by the accumulation over time of its hyperphosphorylated ␥ isoform [Western blot (WB)]. Results. LPS, but not NaSal alone, induced rapid TNF-␣ production in PM (P ⬍ 0.05). Both LPS and NaSal alone activated p38-MAPK (WB not shown). LPS-stimulated phosphorylation of PHAS-1 (␥ isoform accumulation) occurred over 120 min. In contrast, NaSal failed to activate PHAS-1 indicating that p38-MAPK activation alone was insufficient for PHAS-1 activation (Fig. 1). Also, PM treatment with SB203580 did not prevent PHAS-1 activation, while rapamycin did regardless of LPS stimulation (Fig. 2). Conclusion. This is the first demonstration that the translational repressor protein PHAS-1 is activated by LPS in macrophages through a FRAP/mTOR-dependent but p38-independent signaling mechanism. These pathways may reveal an important regulator of cytokine synthesis in sepsis. 44. Mechanism of Inhibition of Smooth Muscle Cell Migration by Dexamethasone. C. Pross, M.D., M. M. Farooq, M.D., N. Angle, M.D., J. A. Freischlag, M.D., and H. A. Gelabert, M.D. Division of Vascular Surgery, Gonda (Goldschmied) Center For Vascular Surgery, UCLA Medical Center, Los Angeles, California. Dexamethasone (DEX) has been shown to inhibit development of neointimal hyperplasia in rats. We hypothesize that DEX inhibits neointimal hyperplasia by altering matrix metalloproteinase (MMP) activity resulting in inhibition of smooth muscle cell migration. Methods. Rat aortic smooth muscle cells (RASMC) were harvested and cultured for two to four passages. A migration assay was performed in a Boyden chamber with chemoattractant (PDGF) and varying concentrations of DEX (10 ⫺9 to 10 ⫺5 M). The number of

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS migrated cells was counted under light microscopy. Zymography was performed on culture medium to assess MMP activity and Western blotting was performed to assay for MMP level. Results. DEX progressively inhibited RASMC migration in a dose-dependent fashion. This effect was statistically significant for concentrations of 10 ⫺7 to 10 ⫺5 M (P ⬍ 0.0005). Zymography showed that DEX inhibits MMP-2 activity in a dose-dependent manner. Western blots indicated total MMP-2 secretion was also inhibited by DEX (see figures). Conclusions. DEX inhibits PDGF-induced migration of RASMCs and MMP-2 activity in vitro. Our data suggest that DEX suppresses MMP activity and secretion resulting in the inhibition of smooth muscle cell migration. This may explain the mechanism by which DEX inhibits neointimal hyperplasia. 45. ␤-Catenin Antisense Oligonucleotide Treatment of Human APC Mutant Colon Cancer Bearing Mice Decreases Tumor Growth Rate. D. W. Green, M.D., H. Roh, Ph.D., J. Pippin, B.S., and J. A. Drebin, M.D., Ph.D. Department of Surgery, The Washington University School of Medicine, St. Louis, Missouri. Loss of the adenomatous polyposis coli (APC) tumor suppressor gene plays a significant role in colorectal carcinogenesis. An important function of the APC gene product is to inhibit the activity of ␤-catenin, a signal transduction protein that forms a complex with multiple transcription factors. To more precisely delineate the role of ␤-catenin in colon cancer growth, we treated SW480 APC mutant tumor-bearing mice with antisense (AS) oligonucleotides (ODNs) against ␤-catenin mRNA and examined the effects on tumor growth. Methods. Groups of five Balb/C-nude mice underwent single, subcutaneous injection of 1 ⫻ 10 6 SW480 cells. The three groups were then begun on daily intraperitoneal injections of either AS ␤-catenin ODN, scrambled sequence (SC) ␤-catenin ODN control, or saline control. Tumor volumes were measured twice weekly beginning day 5 and averaged among groups. Group averages were compared using a Student’s t test. Results. ␤-Catenin AS treatment resulted in a decreased rate of tumor growth compared to both SC ODN and saline control groups (Fig. 1), P ⬍ 0.01 and P ⬍ 0.01, respectively. Notably, 3 of 5 animals in the AS ␤-catenin treatment group failed to develop tumors while 10 of 10 animals in the two control groups developed tumors. Conclusion. Treatment with ␤-catenin AS ODN significantly slows the rate of growth in human colon cancers with APC gene mutations. Strategies targeting ␤-catenin may be useful in the treatment of human colon cancer.

321

PARALLEL SESSION II Oncology I 46. Epithelial Cyclooxygenase-2 (COX-2) Expression in Response to Environmental Stress: A Model for Pathogenesis of Colon Cancer. S. Arbabi, M.D., M. R. Rosengart, M.D., I. Garcia, B.A., and R. V. Maier, M.D. Department of Surgery, University of Washington, Washington, Seattle, Washington. Recent studies indicate a close relationship between COX-2, the inducible COX, and the pathogenesis of colorectal cancer. Overexpression of COX-2 increases growth rate of colorectal tumors, whereas COX-2 inhibitors suppress neoplastic changes. Currently, the stimuli and pathways that control COX-2 expression in this process are not well defined. We studied COX-2 expression in response to environmental stress inducers, such as hyperosmolarity and lipopolysaccharide (LPS), in a human colon cell line. We further questioned whether extracellular signal-regulated kinase (ERK) and

p38, members of the mitogen-activated protein kinase family, might mediate COX expression. Methods. Human colon cancer cells (Caco-2) were stimulated with increasing concentrations of sodium chloride (NaCl) or LPS. Cellular protein was subjected to Western blot analysis using antibodies to COX-2 and COX-1. Results. LPS failed to induce COX-2. Increase in osmolarity induced a dosedependent COX-2 expression within 2 h that peaked by 6 – 8 hours. NaCl at 40 and 100 mM induced an 8- and 50-fold increase in COX-2, respectively (see figure). COX-1 expression was not affected. Specific p38 inhibitors attenuated osmotic-induced COX-2 expression, whereas an inhibitor of ERK activation had no effect. Conclusions. Increase in osmolarity activates p38 and, subsequently, induces COX-2 expression in the colonic epithelium. Lack of response to LPS, in contrast to endothelial cells, is teleologically expected of the colonic epithelium that is in constant contact with the fecal bacteria. This model also predicts that an increase in luminal osmolarity in colon, such as seen with carbohydrate malabsorption, may induce COX-2 and thereby promote neoplastic phenotype. Further identification of other stimuli that induce p38 activation and COX-2 expression may help elucidate the mechanisms for pathogenesis of colon carcinoma. 47. Transcriptional Cofactor P300/CBP-Associated Factor (PCAF) Interacts with Smad3 to Mediate TGF-␤-Induced Transcription. X. H. Feng, Ph.D., and X. Lin, Ph.D. Departments of Surgery and Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas. Resistance to the growth inhibitory actions of TGF-␤ is common in human cancers. Tumor suppressors Smad2, Smad3, and Smad4 are critical transcriptional factors mediating TGF-␤ responses. We previously demonstrated that coactivator p300/CBP is essential for TGF-␤-induced transcription. In this study we were to determine fundamental mechanisms how another transcriptional coactivator, p300/CBP-associated factor (PCAF), participates in Smad3-mediated gene transcription. Methods. Hepatocarcinoma HepG2 cells were transiently transfected to examine the effects of PCAF on TGF-␤and Smad-induced transcription. Transcription assay was performed by luciferase reporter assays. Physical interactions between Smad3

322

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

and PCAF were determined by coimmunoprecipitation and GST pull-down analysis. Results. We observed that overexpression of PCAF induced the promoter activity of the human plasminogen activator inhibitor-1 (PAI-1). In the presence of TGF-␤, PCAF increased the promoter activity to a higher level. Significantly, cotransfection of PCAF and Smad3 in HepG2 cells achieved highest transcription in comparison to PCAF or Smad3 alone, suggesting a functional synergism between the two proteins in TGF-␤-induced transcription. Using Gal4 transcription assay, we found that PCAF stimulated the transcription activities of Smad3 in response to TGF-␤. Finally, coimmunoprecipitation and GST pull-down analysis demonstrated that Smad3 physically interact with PCAF in vivo and in vitro. Conclusion. Our data demonstrate that PCAF physically interacts and functionally synergizes with Smad3 in TGF-␤-induced transcription. This allows us to establish a working model for transcriptional cooperation of Smads and cofactors in TGF-␤-induced growth inhibition. 48. Mechanism of TGF-␤ Growth Inhibition in a Rodent Pancreatic Islet Cell Line. S. S. Guo, M.D., X. Wu, M.D, Ph.D., and M. P. Sawicki, M.D. Department of Surgery, VAMC-WLA and UCLA School of Medicine, Los Angeles, California. Currently, the molecular mechanisms of pancreatic endocrine tumor (PET) growth are poorly understood. Transforming growth factor-␤ (TGF-␤) has been shown to either stimulate or inhibit cell growth, depending on the specific cellular context. Smad4, a key protein in the TGF-␤ signal cascade, was found to be mutated in 55% of nonfunctional PETs, suggesting that TGF-␤ may inhibit islet cell growth. However, the effect and mechanism of TGF-␤ growth regulation have not been studied in islet tumor cells. We hypothesize that TGF-␤ has an important role in islet tumor growth regulation. The rodent islet tumor cell line TGP-61 was grown in recommended media to 70% confluency and treated with TGF-␤ for 2 to 24 h. TGF-␤ exhibited a dose-dependent suppression of growth in rodent pancreatic islet tumor cells as measured by cell counting. TGF-␤ also inhibited growth by arresting cells in G0/G1 as demonstrated by flow cytometry. Furthermore, by using Western blot analysis, we found that this growth inhibition by TGF-␤ is associated with a rapid and dramatic suppression of cyclin D1 expression as well as activation of the JNK (c-Jun N-terminal kinase) signal transduction pathway. In contrast, there was no induction of p15, p21, or p27 or activation of the ERK (extracellular signal-regulated kinase) pathway by TGF-␤ in islet tumor cells, which frequently occurs in other cell types in response to TGF-␤. Likewise, the expression of cyclin A and cyclin E was also not affected. These findings demonstrate for the first time the effects of TGF-␤ in pancreatic islet tumor cells and uncover a possible islet tumor-specific TGF-␤ growth regulatory mechanism. This mechanism involves the JNK pathway and cyclin D1, but not the other proteins that frequently mediate the inhibitory effect of TGF-␤ in other cell types. 49. p63 Compensates for the Functional Loss of the Tumor Suppressor Gene p53. Y. Suliman, M.D., O. Opitz, M.D., N. Williams, M.D., FRCSI, and A. K. Rustgi, M.D. GI & Surgery Department, University of Pennsylvania, Philadelphia, Pennsylvania. Introduction. p63, a p53 homologue, has been shown to be critical in normal squamous epithelial development. p63 can bind to p53-responsive promoters and thus activate p53-dependent genes in vitro, but its biological properties in vivo have not been explored. p63 might compensate for the inactivation of p53 in a tissue-specific fashion as in oral– esophageal cancers. Methods. Immunohistochemistry (IHC) for full-length p63 and N-terminal truncated isoforms of p63 was performed in the tongue and esophagus of wild-type (wt) and p53 null mice. The p63 isoforms were determined by Western blot (WB) analysis of isolated tongue and esophageal epithelia.

As functional target of p53 and p63, p21 expression was determined by IHC. Results. p63 IHC results are summarized in the figure. WB revealed increased expression of p63 in esophageal and tongue epithelia of p53 null mice. p21 expression was comparable in wt and p53 null mice IHC. Conclusions. There appears to be a dynamic equilibrium between proliferation and differentiation that is reflected as well by the localization of the p63 isoforms as the truncated version may act in a dominant-negative manner in differentiated cells. Finally, p21 appears to be induced by overexpressed p63 in the oral– esophageal epithelium. These findings provide important insights into the pathogenesis of head and neck and esophageal cancers. 50. FRAP-p70s6K Signaling Is Required for Pancreatic Cancer Cell Proliferation. S. A. Shah, M.D., M. W. Potter, M.D., R. Ricciardi, M.D., R. A. Perugini, M.D., and M. P. Callery, M.D., FACS. Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts. Introduction. FRAP-p70s6K signaling regulates mitogenic responses to growth factors in eukaryotic cells. Constitutive p70s6K activation occurs in some human malignancies and may contribute to dysregulated growth. We examined whether inhibition of this pathway affects mitogen-induced proliferation and cell cycle progression of human pancreatic cancer cells in vitro. Methods. Quiescent BxPC-3 and PANC-1 human pancreatic cancer cells treated with ⫾20 ng/ml rapamycin (FRAP inhibitor) were repleted with 10% FCS to induce cell cycle entry. Proliferation was measured with MTT assay. Cell cycle and apoptosis were determined by FACS analysis. Phosphorylation of p70s6K, AKT, and cdc2 was evaluated by Western blot. Statistical analysis included a two-tailed t test (P ⬍ 0.05). Results. Rapamycin (Rapa) inhibited the phosphorylation of p70s6K while inducing G0 –G1 cell cycle arrest (P ⬍ 0.005). In both cell lines, Rapa inhibited serum-induced proliferation (P ⬍ 0.05) without affecting apoptosis. Cdc2 phosphorylation was inhibited by 15 min

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS with Rapa (not shown), consistent with cell cycle arrest. AKT phosphorylation was not affected, indicating FRAP specificity of Rapa (see figure). Conclusion. FRAP-p70s6K signaling appears to be necessary for G1- to S-phase progression and proliferation in pancreatic cancer cells. This supports earlier work demonstrating a similar regulatory role for PI-3 kinase, an upstream activator of FRAP-p70s6K. 51. Growth of B16F10 Melanoma Tumors Requires uPAProducing Host Cells. K. A. Joseph, M.D., N. Chuang, M.D., R. Shapiro, M.D., and P. Shamamian, M.D. SA Localio Laboratory for Surgical Research, Department of Surgery, NYU School of Medicine, New York, New York. The serine protease urokinase (uPA) has been implicated in tumor invasion and metastasis. We previously found that wildtype (wt) mice injected subcutaneously (sc) with B16F10 cells rapidly develop invasive tumors. However, B16F10 tumor growth is significantly reduced in uPA-knockout (uPA⫺/⫺) mice. This is despite the observation that mouse B16F10 melanoma cells produce uPA indicating that host-derived uPA is required for tumor growth. We therefore hypothesize that established B16F10 tumors harvested from wt mice and transplanted into uPA⫺/⫺ mice would restore tumor growth as normal wt accessory (inflammatory and/or stromal) cells would be carried over with the transplanted tumor. Methods. B16F10 melanomas were established by sc injection into three wt mice. Tumors were harvested aseptically 20

TABLE—ABSTRACT 51 Group

Weight (g) ⫾ SD

Orig. tumors WT uPA⫺/⫺

0.42 ⫾ 0.33* 0.68 ⫾ 0.41 1.28 ⫾ 0.89*

Growth rate (mm 3/day)

uPA activity (mU/ mg protein) ⫾ SD

19.81 ⫾ 18.05* 538.77 ⫾ 75.3* 39.76 ⫾ 24.02 1033.13 ⫾ 381.67 95.72 ⫾ 72.64* 1257 ⫾ 646.87*

* Not significant vs WT. days later and divided into 2.5-mm 3 tumor fragments that were transplanted sc into wt and uPA⫺/⫺ mice (six/group). After day 15, the tumors were excised, weighed, and assayed for uPA activity. Statistical analysis was performed by two-tailed t test. Results. Transplantation of established wt tumors into uPA ⫺/⫺ mice results in successful restoration of tumor growth (see table). Conclusions. We have shown previously that B16F10-derived uPA is not sufficient for tumor development in uPA ⫺/⫺ mice. These data suggest that wt-derived uPA producing normal host cells present in the tumor explant must be responsible for successful tumor growth. This further confirms the biological relevance of uPA in tumor growth and invasion. 52. NT4 Upregulates HLA, TAP, and HsP-70 Expression in Neuroendocrine Tumors Possibly via the Induction of RFX-B and RBP-J␬: A Potential New Class of Immunomodulatory Drugs. I. Avital, M.D., D. Inderbitzin, M.D., D. Tyan, Ph.D., A. T. Lefor, M.D., and W. Arnaout, M.D. CedarsSinai Medical Center, Department of Surgery and The HLA Immunogenetics Laboratory, UCLA School of Medicine, Los Angeles, California. Carcinoid, thyroid, and pancreatic cancers are tumors of neuroectodermal origin. P75 LNGF and TrK-B belong to a family of tyrosine kinase receptors whose ligand is NT-4. Neurotrophins (NT4) have a pivotal role in regulating differentiation of neural

323

cells. We hypothesized that P75 LNGF and TrK-B localize to neuroendocrine tumors and their ligand, NT-4, may participate in the regulation of HLA, TAP, and HsP-70 expression, potentially via RFX-B and RBP-J␬ (transcription factors) respectively. Tumor cell lines were treated for 4 days with NT-4 (30 ng/ml). HLA class I and II, TAP-1 and -2, HsP-70, RFX-B, and RBP-J␬ expression was analyzed via SSP-PCR, RT-PCR, and immunohistochemistry. Analyses were performed before and after incubation with NT-4 and were compared to incubations with INF-␥. Quantitative analyses were performed by FACS (significance was assessed by the t test). P75 LNGF and TrK-B localized to the surface of the various tumors. NT-4 was found to up-regulate HLA class I and II, on these tumors. NT-4 induced de novo surface expression of HLA class II while HLA class I expression increased by up to 50% (P ⬍ 0.05). TAP expression increased by up to 350% (P ⬍ 0.01) and HsP-70 expression was increased by 45% (P ⬍ 0.05). Dose– response curves were established and demonstrated that NT-4 is most effective at a dose of 30 ng/ml. Preliminary results indicate that NT-4 and INF-␥ act synergistically. Up-regulation of HLA class I and II was paralleled with the up-regulation of RBP-J␬ and RFX-B, respectively. This study indicates that NT-4 is a major up-regulator of HLA expression on various neuroendocrine tumors. This up-regulation may be accentuated by the up-regulation of HsP-70 and TAP. Moreover, NT-4 may operate via RFX-B and RBP-J␬. We present a novel class of immunomodulatory molecules that may be useful in drug therapy to halt tumor progression. 53. Cytosolic Phospholipase A 2 ( CPLA 2)-Mediated ICAM-1 Expression Is Calcium Dependent. C. C. Barnett, Jr., M.D., E. E. Moore, M.D., C. C. Silliman, M.D., Ph.D., E. K. Abdalla, M.D., D. A. Partrick, M.D., and S. A. Curley, M.D. Departments of Surgery, UT MD Anderson Cancer Center, Houston, Texas; and University of Colorado, Denver, Colorado. Human malignancies such as hepatitis-related hepatocellular cancer and squamous carcinoma arise in a setting of chronic inflammation. Upregulation of ICAM-1 is a seminal late event in malignant transformation following chronic inflammation. cPLA 2 is a lipid mediator activated by inflammatory stimuli, which has been shown to mediate ICAM-1 upregulation. Since lipid mediators are known work via calcium-dependent mechanisms, we hypothesize that ICAM-1 upregulation is calcium dependent. c PLA 2 -mediated HUVEC were grown to confluence in T-25 flasks and stimulated with TNF-␣ or LPS for 6 h. Additional groups were preincubated with AACOCF3 (specific cPLA 2 inhibitor) or BAPTA A.M. (specific inhibitor of intracellular Ca 2⫹) before exposure to inflammatory stimuli. ICAM-1 expression was determined by mean fluorescent intensity (MFI) measured using FITC-labeled moAb to ICAM-1 via FACS. The role of intracellular Ca 2⫹ on cPLA 2 activity was determined by thin liquid chromatography. Groups were compared using ANOVA with Scheffe’s post hoc analysis, P ⬍ 0.05 was considered significant, N ⱖ 4 for experimental groups (see figure). Both cPLA 2 and Ca 2⫹ inhibition significantly inhibited inflammatory upregulation of ICAM-1.

324

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

Pretreatment with BAPTA A.M. attenuated HUVEC cPLA 2 activity, in response to LPS (69.7 ⫾ 3.5 vs 98.1 ⫾ 0.4 U, P ⬍ 0.05) and to TNF-␣ (69.9 ⫾ 2.6 U vs 97.5 ⫾ 0.4 U, P ⬍ 0.05). These findings suggest that appropriate molecular target suppression may prevent malignant degeneration in the presence of chronic inflammation.

PARALLEL SESSION II Pediatrics 54. Novel Effect of Leptin on Small Intestinal Absorptive Capacity after Massive Small Bowel Resection. P. Y. Pearson, M.D., and M. Z. Schwartz, M.D. Departments of Surgery, A.I. duPont Hospital for Children, Wilmington, Delaware; and Thomas Jefferson University, Philadelphia, Pennsylvania. This study was designed to examine the effect of systemic leptin administration on small bowel absorptive function after massive small bowel resection (MSBR). Methods. Twenty-one adult male Sprague–Dawley rats underwent an 80% small bowel resection and end-to-end jejunoileal anastomosis. Seven days following resection, all rats had placement of a jugular venous catheter connected to a subcutaneously placed osmotic minipump and were divided into three groups based on the content of each minipump: Group 1 (n ⫽ 7).1% bovine serum albumin (BSA); Group 2 (n ⫽ 7) leptin 2 ␮g/kg/ day; and Group 3 (n ⫽ 7) leptin 4 ␮g/kg/day. Following a 14-day infusion period, [ 14C]galactose absorption was measured using a closed-recirculation technique. Mucosal DNA content was determined for all groups using a standard DNA purification kit. Mucosal RNA was extracted and RT-PCR was performed using the following

TABLE—ABSTRACT 54

Group

Galactose absorption (␮mol/cm 2)

1 2 3

1.63 ⫾ 0.06 2.36 ⫾ 0.11* 1.80 ⫾ 0.42

DNA content (␮g/mg SGLT (band GLUT-5 (band mucosa) intensity) intensity) 8.81 ⫾ 1.2 9.01 ⫾ 0.49 9.59 ⫾ 0.41

1.08 ⫾ 0.03 1.09 ⫾ 0.08 1.13 ⫾ 0.02

0.84 ⫾ 0.02 0.96 ⫾ 0.04** 0.92 ⫾ 0.04

* P ⬍ 0.01. **P ⬍ 0.05. primers: sodium/glucose cotransporter (SGLT-1), fructose transporter (GLUT-5), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—internal standard. PCR products were separated on a 4% agarose gel and relative band intensities were measured. Statistical analysis was performed using ANOVA and is expressed as means ⫾ SEM. Results. (See table.) Conclusions. This study demonstrates for the first time that leptin enhances small intestine carbohydrate absorptive function beyond the normal adaptive response following MSBR. A mechanism for this effect is suggested by increased expression of the GLUT-5 transporter gene. Leptin may be clinically useful in patients with inadequate intestinal function.

55. Epithelial Cell Permeability Is Not Increased by Serum from Mice Following Massive Small Bowel Resection. D. P. O’Brien, M.D., L. E. Stern, M.D., C. R. Erwin, Ph.D., and B. W. Warner, M.D. Division of Pediatric Surgery, Children’s Hospital Medical Center, and Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio. Background. Increased intestinal permeability to bacteria and/or bacterial products is proposed to be a major source of infection and

liver dysfunction in patients with the short bowel syndrome. In previous studies, serum from mice that had undergone small bowel resection (SBR) significantly enhanced cell growth when added to cultured intestinal epithelial cells, suggesting that a serum factor(s) mediates the enterocyte response to SBR. The purpose of this study was to test the hypothesis that SBR serum increases the permeability of intestinal epithelial cell monolayers. Methods. Rat portal vein serum was collected 3 days after either SBR or sham operation (n ⫽ 4 –5 per group). Rat intestinal epithelial cells (RIEC-6) were grown to confluence on the apical chamber of a transwell system and then incubated for 3 days in 10% fetal bovine serum (FBS; control), 1% FBS plus 9% sham serum, or 1% FBS plus 9% SBR serum. Monolayer permeability was determined by measuring the passage of a 10-kDa dextran rhodamine probe. Results. Sham serum-treated monolayers demonstrated the greatest permeability to the dextran rhodamine (see figure). SBR serum reduced permeability to near control levels. Conclusions. During the early phase of intestinal adaptation, a serum factor(s) diminishes the increased epithelial permeability demonstrated by sham serum. These data are inconsistent with the hypothesis that enterocyte permeability is affected by a circulating factor(s) in the serum following massive small bowel resection.

56. Effect of Hypoxia on Fetal Gastrointestinal Motility. M. Sase, M.D., J. J. Lee, M.D., M. J. Ross, M.D., and T. L. Buchmiller-Crair, M.D. Departments of Surgery and Obstetrics and Gynecology, Harbor–UCLA Medical Center, Torrance, California. During periods of fetal hypoxic stress, blood is shunted to lifepreserving organs, such as the heart and the brain. Reduced oxygen supply to the small intestine (SI) induces mucosal injury and perhaps intestinal dysmotility and may contribute to neonatal necrotizing enterocolitis (NEC). As little is known about hypoxia and GI motility, this effect was studied in a rabbit model. Twenty-one rabbits were randomized into two groups: hypoxia (Hyp) and control (Cont). Seven litters were studied at gestational days 24, 27, and 30 of their normal 31-day gestation. Under ultrasound guidance the fetal stomach was percutaneously accessed. Fluorescein, labeled with color-coded microspheres for precise fetal identification, was injected. Hyp rabbits breathed 11% oxygen for 1 h; Cont rabbits breathed room air. Maternal arterial blood gases were measured. Two hours after injection, the fetuses were delivered by Cesarean section. The SI was harvested, the length was recorded, and the distance fluorescein traveled was measured by UV light optical density. Results were analyzed by the unpaired Students’ t test. All fetuses survived the study period (N ⫽ 167). The maternal arterial pO 2 of Hyp rabbits during the hypoxic period was significantly lower than during normoxic periods (Hyp ⫽ 42.7 ⫾ 4.4 vs 139 ⫾ 66.6 mm

325

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Hg). The length fluorescein traveled in Hyp was shorter than Cont at all gestational days (P ⬍ 0.01): Day 24 (Hyp 6.7 ⫾ 2.0 cm vs Cont 8.4 ⫾ 2.1 cm), Day 27 (Hyp 10.1 ⫾ 2.9 cm vs Cont 19.1 ⫾4.4 cm), and Day 30 (Hyp 16.8 ⫾ 3.5 cm vs Cont 23.1 ⫾ 5.2 cm). The percentage of motility, defined as the length fluorescein traveled divided by the total SI length, was also smaller in Hyp vs Cont at all gestational days studied. Fetal rabbit gastrointestinal motility, as assessed by the distance fluorescein traveled after percutaneous gastric injection, was significantly decreased by maternal hypoxia during the last third of gestation. Hypoxic reduction in motility, in concert with the known mucosal injury during hypoxia, may contribute to neonatal NEC. 57. Vascular Endothelial Growth Factor Is Upregulated in Neuroblastoma and Hepatocyte Cocultures. E. A. Beierle, M.D, L. F. Strande, M.S., A. C. Berger, M.D.,* and M. KuangSing Chen, M.D. Division of Pediatric Surgery, UMDNJ, Camden, New Jersey; and *Surgery Branch, NCI, Bethesda, Maryland. Introduction. We hypothesize that angiogenic factors may be altered by the interaction between neuroblastoma and host tissues. Methods. Conditioned media are collected from cultures of human hepatocytes (H) and human neuroblastoma cells (NB) and from these cells grown together in a noncontact, coculture system (CC). ELISA is used to detect vascular endothelial growth factor

59. Repetitive Electrical Stimulation of a Rectus Abdominis Muscle Detrusor Myoplasty Produces More Efficient Bladder Evacuation. G. A. Perez-Abadia, M.D., J. G. Van Savage, M.D., J. W. Bardoel, M.D., T. G. Harralson, B.S., L. G. Palanca, M.D., M. M. Palacio, M.D., C. J. Maldonado, Ph.D., J. I. Harty, M.D., G. R. Tobin, M.D., and J. H. Barker, M.D., Ph.D. Divisions of Plastic & Reconstructive Surgery and Urology, Department of Surgery, University of Louisville, Louisville, Kentucky. Introduction. The rectus abdominis muscle (RAM) has been used in combination with electrical stimulation to treat acontractile bladders and holds promise for children with spina bifida. The purpose of this study was to determine whether repetitive electrical stimulation of the RAM was more effective emptying the bladder than a single period of stimulation. Methods. Eight dogs were divided in two groups. In Group I (n ⫽ 4) the RAM received a single period of electrical stimulation. In Group II (n ⫽ 4) the RAM received four separate periods of electrical stimulation with a 5-s rest between each. The right RAM was dissected free (creating a muscle flap 20 cm in length) and wrapped around the bladder. The inferior epigastric vessels, two lower intercostal nerves, and the pubic insertion were preserved. The RAM was stimulated with two pairs of bipolar electrodes inserted into the muscle near the nerve entry zone. Stimulation frequencies were 40, 60, and 80Hz and the pulse width was 320 ␮s. Bladder evacuations were measured in milliliters and percentage volume of

TABLE—ABSTRACT 57 Time (h) 48 72

Control 60.5 ⫾ 1.8 61.5 ⫾ 2.2

H 56.3 ⫾ 1.7 59.0 ⫾ 2.8

NB 58.7 ⫾ 1.7 62.0 ⫾ 5.8

TABLE—ABSTRACT 59 CC 71.0 ⫾ 2.1* 73.7 ⫾ 1.3*

Note. Mean ⫾ SEM, cell No. ⫻ 10 4, *P ⬍ 0.02. (VEGF), basic fibroblast growth factor, epidermal growth factor, and interleukin-8. Human umbilical vein endothelial cells (HUVEC) are cultured with standard medium (control) and the three conditioned media (H, NB, CC). After 48 and 72 h, viable cells are counted to determine cell proliferation. Student’s t test is used for statistical comparisons with significance determined at P ⬍ 0.05. Results. VEGF is markedly elevated in the coculture medium compared to the media from hepatocytes or neuroblastoma cells grown alone (400 vs 270 and 103 (pg/10 6 cells), P ⬍ 0.05). Other growth factors are undetectable in all media. HUVEC plated with coculture-conditioned medium have a significant increase in cell proliferation compared to the control and the other conditioned media. The proliferative effects of the cocultured medium are significantly decreased at 48 h when anti-VEGF antibody is added to the HUVEC cultures [54 ⫾ 2 vs 40 ⫾ 1 cells ⫻ 10 4 (CC vs CC ⫹ anti-VEGF), P ⬍ 0.02] (see table). Conclusions. The interaction of neuroblastoma with host cells (hepatocytes) results in an increased production of VEGF. This substance stimulates endothelial cell proliferation in vitro and may function to enhance the tumor’s metastatic potential.

58. Role of Cellular Mechanics in Lung Branching Morphogenesis. K. A. Moore, M.D., Y. Kong, M.D., Ph.D., S. Huang, M.D., Ph.D., M. E. Sunday, M.D., Ph.D., and D. E. Ingber, M.D., Ph.D. Departments of Surgery and Pathology, Children’s Hospital and Harvard Medical School, Boston, Massachusetts. Permission to publish abstract not obtained.

Frequency (Hz)

Group I (single), %

Group II (repetitive), %

P value

40 60 80

66 ⫾ 12 70 ⫾ 14 66 ⫾ 11

87 ⫾ 9 85 ⫾ 7 90 ⫾ 3

NS NS ⬍0.05

capacity. Statistical analysis was performed using ANOVA and Dunnett post hoc test. Results. Table shows percent bladder evacuation. Values are means ⫾ SEM (see table). Conclusion. Repetitive electrical stimulation of the RAM detrusor myoplasty was more efficient in evacuating the bladder than a single period of stimulation. Chronic studies are under way to determine whether this method will be useful as an alternative treatment for acontractile bladder in children with neurogenic bladders. 60. Bone Marrow Tissue Engineering for Treatment of Congenital Hematopoietic Disorders. A. S. Krupnick, M.D., A. Shaaban, M.D., S. Hayashi, S. Bouchard, M.D., T. Saydam, M.D., A. Radu, B.S., and A. W. Flake, M.D. The Department of Surgery, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania. Low levels of stem cell engraftment after in utero hematopoietic stem cell transplantation occur due to donor MHC restriction. Concurrent transplantation of a bone marrow environment, native to the donor stem cell, might increase the level of engraftment. Our purpose is to develop a method to engineering a bone marrow environment. Between one and two murine femurs were mechanically crushed to a fine suspension. The suspension was combined with liquid collagen as an injectable scaffolding and nonwoven polyglycolic acid mesh (PGA) as a surgically implantable vehicle. Twelve syngeneic recipient mice were classified based on the type of scaffolding, site of implantation, and time until analysis (see table). No engraftment of bone was evident in animals 1– 4 and only minimal

326

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 60 Site: 2-week time point 5-week time point

Subcutaneous Mouse 1 PGA Mouse 3 PGA

Mouse 2 collagen Mouse 4 collagen

Intramuscular Mouse 5 PGA Mouse 6 PGA

engraftment in animals 5– 8. Although fragments of bone did engraft in animals 9 and 10, no marrow was detectable. The mesenteric site of implantation in animals 11 and 12, however, contained a vascular nodule grossly resembling trabecular bone. Histologically this tissue mimicked bone marrow and contained sinusoids, hematopoietic cells, megakaryocytes, and trabecular bone. We have demonstrated that the small bowel mesentery with a collagen-based delivery system can support bone marrow formation. Future work will focus on determining the factors responsible for this development and cotransplantation of bone marrow with hematopoietic stem cells in order to increase levels of engraftment.

61. Delivery and Expression of Genes at 28 h Postfertilization in a Chick Model of Fetal Alcohol Syndrome. A. Thakur, M.D., J. B. Atkinson, M.D., and S. E. Fraser, Ph.D. Division of Pediatric Surgery, UCLA Medical Center and California Institute of Technology, Los Angeles, California. Fetal alcohol syndrome (FAS) results in significant morbidity, including craniofacial abnormalities. Precise gene transfer may be relevant for the treatment of these craniofacial abnormalities. Current strategies remain difficult and nonfocal and may be confounded by the lack of imaging techniques for precise gene transfer and inadequate markers for in vivo study. This investigation evaluated: (1) strategies for in vivo embryo imaging in a chick model of FAS; (2) efficiency of gene transfer; and 3) precision of embryonic transduction by using direct microscopic visualization with adenoviral injection into chick embryos in vivo. Twenty-four white Leghorn chick eggs were incubated for 24 h postfertilization (hpf) and equal numbers underwent injection either with ethanol (experimental group) or with Tyrode’s solution (controls). At 28 hpf, or until embryos were about 2mm in size, a window was made in the shell and vitelline membrane was deflected using fine tungsten needles, therefore exposing the head. A glass needle was placed at the midbrain/hindbrain level and either 1 ⫻ 10 7 pfu of an adenovirus containing the green fluorescent protein (GFP) reporter gene or normal saline was injected into the lateral head. The eggs were then sealed, incubated at 37°C for 24 h, and reimaged. Images were obtained using fluorescent and confocal laser microscopy. At 24 h postinjection, all embryos were alive and were successfully imaged in vivo. Control and experimental embryos were statistically different with respect to embryo size, head size, and number of malformations. There was no difference between embryos that underwent adenoviral vs normal saline injection. Fluorescent and confocal microscopic imaging demonstrated green fluorescence in the focal region of the injection site in all the embryos. This study validates this chick model in the study of FAS. Embryonic 2-mm chick embryos survive adenoviral transduction in a focal manner in vivo and that this delivery results in production of imageable levels of protein. These results suggest that successful early embryonic transduction is possible and may be used in mammalian systems, including humans, to introduce genes for the treatment of craniofacial abnormalities seen in FAS.

Mesentery

Mouse 7 collagen Mouse 8 collagen

Mouse 9 PGA Mouse 10 PGA

Mouse 11 collagen Mouse 12 collagen

PARALLEL SESSION II Transplant I 62. Facilitating Cells Home to the Thymus after Allogeneic Marrow Transplantation. M. J. Schuchert, M.D., R. D. Wright, and Y. L. Colson, M.D., Ph.D. Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania; and Department of Cardiothoracic Surgery, Brigham & Women’s Hospital, Boston, Massachusetts. Facilitating Cells (FC) comprise a unique, bone-marrow-derived population that promotes allogeneic hematopoietic stem cell (SC) engraftment and donor-specific transplantation tolerance without graft-vs-host disease (GVHD). FC have been shown to express the thymic developmental markers Thy1, c-kit, and CD25. To test the hypothesis that FC home to the recipient thymus after marrow infusion, FC were evaluated for expression of the thymic homing marker CD44. In addition, trafficking studies were performed to elucidate FC migration patterns following allogeneic transplantation. FC were isolated from murine bone marrow by multiparameter rare-event cell sorting. A total of 10,000 female SC (c-kit ⫹ / SCA1 ⫹ /Lin ⫺ ) and 50,000 male FC (CD8 ⫹ /TCR dim/⫺ ) were injected into lethally irradiated (950 cGy) female recipients. Ten days following injection, recipient tissues (bone marrow, spleen, thymus, lung) were harvested and analyzed for presence of male FC via PCR, utilizing primers specific for the murine Y chromosome. CD44 expression was assessed by flow cytometry. Flow cytometric analysis demonstrated that the FC population expresses the thymus homing glycoprotein CD44 (mean channel value 114 ⫾ 35, P ⬍ 0.001 compared to negative control, t test). In addition, PCR analysis of recipient tissues 10 days posttransplantation revealed that male FC localized to the thymus of all recipients tested (n ⫽ 3). In marked contrast, FC were not detected in spleen, bone marrow, or lung from the same recipients. These studies demonstrate for the first time that FC express thymic homing markers and gain entry into the recipient thymus following intravenous infusion. These observations may provide important insights into the mechanism(s) by which the FC promotes allogeneic SC engraftment and donor-specific tolerance without GVHD. 63. Cyclosporine Directly Promotes Epstein–Barr Virus Transformation of Human B Cells. J. C. Merrick, M.D., C. Chen, Ph.D., T. D. Johnston, M.D., K. S. Reddy, M.D., and D. Ranjan, M.D. Transplant Section, University of Kentucky, Lexington, Kentucky. We have previously shown that oxidative stress alone promotes transformation of human B cells infected with Epstein–Barr virus (EBV) in vitro; a model of posttransplant lymphoproliferative disorder (PTLD). Our laboratory has investigated the direct effects of cyclosporine (CyA) as an oxidant stimulating this transformation. In three separate experiments, human B-lymphocytes infected with EBV were cultured in triplicate with CyA (500 ng/ml), vehicle control, H 2O 2, and vitamin E. Oxidative stress was evaluated using a lipoperoxidase (LPO) assay and B cell transformation was evaluated using colony counts and [ 3H]thymidine uptake (see table). At thera-

327

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 63 Lipid peroxidation (mmol/10 6 cells ⫾ SD) 3.62 ⫾ 0.88* 5.71 ⫾ 1.57 2.32 ⫾ 1.67*

Control H 2O 2 H 2O 2 ⫹ Vit. E

5.46 ⫾ 0.48* 8.70 ⫾ 1.24 4.19 ⫾ 1.47*

Control CyA CyA ⫹ Vit. E

B cell transformation (colonies/well or CPM/well ⫾ SD) 23.25 ⫾ 1.25* 48.75 ⫾ 2.06 25.75 ⫾ 0.85* 12,587 ⫾ 744* 21,790 ⫾ 1,961 7,349 ⫾ 632*

Control H 2O 2 H 2O 2 ⫹ Vit. E Control H 2O 2 H 2O 2 ⫹ Vit. E

28.00 ⫾ 2.27* 49.00 ⫾ 2.16 22.50 ⫾ 1.76* 12,481 ⫾ 670* 26,514 ⫾ 2,732 16,146 ⫾ 2,088**

Control CyA CyA ⫹ Vit. E Control CyA CyA ⫹ Vit. E

* P value ⬍ 0.005, **P value ⬍ 0.05 vs Cya or H 2O 2, t test.

peutic concentrations, CyA had an oxidative effect on B cells in vitro similar to H 2O 2, which was abrogated by the addition of vitamin E. Both CyA and H 2O 2 were capable of promoting transformation of B cells infected with EBV suggesting a link between oxidative stress and B cell transformation. This effect was also blocked by vitamin E. These results may have clinical implications in PTLD chemoprophylaxis. 64. Protein Kinase C Abrogates the Protective Effect of Hepatic Ischemic Preconditioning. R. Ricciardi, M.D., R. S. Chari, M.D., R. D. Kim, M.D., B. K. Schaffer, M.D., S. A. Shah, M.D., K. R. Sheth, M.D., M. P. Callery, M.D., S. H. Quarfordt, M.D., and W. C. Meyers, MD. Department of Surgery, UMASS Medical School, Worcester, Massachusetts. Introduction. A short transient episode of warm ischemia prior to prolonged cold ischemia [ischemic preconditioning (IPC)] protects the cold-preserved liver graft. We hypothesized that inhibition of protein kinase C abrogates the protective effect of IPC on hepatic grafts. Methods. Control pig livers underwent routine harvest (n ⫽ 6). IPC livers underwent 15 min ischemia before harvest, with (n ⫽ 5) or without (n ⫽ 5) the protein kinase C inhibitor (chelerythrine). After cold storage and reperfusion, graft function was determined by bile flow, response to bile acid challenge, and graft O 2 consumption. Graft circulatory impairment was estimated with liver tissue blood flow (thermistors) and vascular resistance. Liver damage was deter-

TABLE—ABSTRACT 64

Bile flow (mL/15 min) Bile challenge (mL/15 min) O 2 consump. (mL/100 g/min) Tissue flow (mL/100 g/min) Vascular resistance (mm Hg) LDH (postreperfusion rise) Endothelial preservation * P ⬍ 0.05.

Control

IPC alone

IPC ⫹ Chel

1.1 ⫾ 0.1

2.5 ⫾ 0.5*

1.4 ⫾ 0.4

3.5 ⫾ 0.6

7.6 ⫾ 1.1*

2.5 ⫾ 0.4

0.47 ⫾ 0.07

0.72 ⫾ 0.06*

0.59 ⫾ 0.06

25 ⫾ 4.6

56 ⫾ 3.7*

26 ⫾ 3.1

0.021

0.015*

0.020

3.6 ⫾ 1.0

3.7 ⫾ 0.3

3.1 ⫾ 0.3

0.55 ⫾ 0.08

1.3 ⫾ 0.06*

0.7 ⫾ 0.06

mined with LDH assay. Endothelial preservation was determined by Factor VIII immunostain (graded 0, poor preservation to 2, excellent). Cytoplasmic extracts were analyzed for presence of protein kinase C by Western blot. Statistical significance was confirmed with t tests. Results. (See table.) IPC grafts demonstrated reduced cytoplasmic protein kinase C levels before harvest, which did not occur in chelerythrine or control grafts. Conclusions. Chelerythrine, a specific protein kinase C inhibitor, completely reverses the protective effects of hepatic ischemic preconditioning. Activation of protein kinase C is an important mechanism in the hepatic response to ischemic preconditioning. 65. Protective Role of the L-Arginine–Nitric Oxide Synthase Pathway on Preservation Injury after Rat Liver Transplantation. Y. Takahashi, M.D., S. H. Chia, M.D., G. P. Yagnik, B.S., N. Murase, M.D., and D. A. Geller, M.D. Starzl Transplant Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. Background. A major problem complicating liver transplantation is the preservation injury that results from cold storage and subsequent ischemia/reperfusion injury following organ revascularization. The L-arginine–nitric oxide (NO) pathway has been recognized to play critical roles during infection, inflammation, organ injury, and transplant rejection. Recent data indicate that NO synthesis has beneficial effects in several models of liver injury. The purpose of this study was to examine the role of the L-arginine–NO pathway on preservation injury in a model of rat liver transplantation. Methods. Orthotopic liver transplantation was performed with 18 h preservation in syngeneic (LEW-LEW) rats. Liver preservation injury was determined by measuring serum liver function tests 6 to 48 h after transplantation. In some experiments, rats received L-arginine supplementation 0 to 24 h after transplantation. In other experiments, NO synthase inhibitors (L-NAME or L-NIL) were injected at the time of isograft revascularization. Results. L-Arginine produced a significant improvement in liver preservation injury by 12 h after reperfusion vs control. The NO synthase inhibitor L-NAME caused a significant increase in liver injury 24 h after injection. The iNOS-

TABLE—ABSTRACT 65 Group Control L-Arginine L-NAME L-NIL

AST (U/L)

ALT (U/L)

4,744 ⫾ 667 3,018 ⫾ 213* 11,676 ⫾ 3,990* 4,261 ⫾ 300

3,518 ⫾ 295 2,524 ⫾ 175* 5,106 ⫾ 1,118* 3,229 ⫾ 275

328

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

specific inhibitor L-NIL had no significant effect on liver injury (see table, mean ⫾ SE, n ⫽ 3– 6 animals per group, *P ⬍ 0.05 vs control). Conclusions. The results show that L-arginine supplementation and NO synthesis improve hepatic injury and have a protective role in the transplanted liver graft. The protective effect may be mediated by low-level cNOS-derived NO and provide a useful strategy to improve liver ischemia/reperfusion injury in the transplant setting. 66. The Effects of Bcl-2 Gene Transfer on the Regenerating Rat Liver. K. Taira, M.D., M. Shiraishi, M.D., S. Hiroyasu, M.D., M. Nagahama, M.D., E. Nozato, M.D., M. Toure, M.D., T. Ohshiro, M.D., and Y. Muto, M.D. First Department of Surgery, University of the Ryukyus, Okinawa, Japan. Purpose. Apoptosis-modulating genes such as Bcl-2 have been proved to block programmed cell death and even promote cellular proliferation. We evaluated the effect of bcl-2 after gene transfer on the regenerating liver. Materials and methods. We used 16-weekold Wistar rats, which were partially hepatectomized (70%) (PH) at 0 (in groups 1 and 2) or 24 h (in groups 3 and 4) after gene transfer. Next, Bcl-2 or control marker (LacZ) gene transfection was performed by a systemical injection of 1 ⫻ 10 9pfu/ml of adenovirus vector, which encodes either human Bcl-2 (AxBcl2) in groups 1 and 3 (n ⫽ 39) or Escherichia coli ␤-galactosidase (AxCALacZ) in groups 2 and 4 (n ⫽ 39) with a CAG promotor. The rats were allowed to survive until the scheduled sacrifice until 21days after PH. The body weight and removed liver weight of all rats were measured. The liver tissue and blood samples were periodically obtained in order to perform blood chemistry and histological analysis. Results. In the Bcl-2-transfected groups, the expression of bcl-2 was observed from 0.5 h to 14 days in both groups, and it was predominantly distributed in the periportal area. In the LacZ-transfected groups 2 and 4, the expression of Bcl-2 was seldom detected. On the other hand, the immunoreactivity for Bax was detected during the first 24 h in the Bcl-2-transfected groups. In the LacZ-transfected groups 2 and 4, immunoreactivity for Bax was detected throughout the course. The liver weight/body weight ratio was indicated in three patterns. The early phase, which comprised the period up to 24 h after PH, indicated liver regeneration without any increase in the liver weight. The proliferative phase, which means the time from 24 h to 3 days after PH, rapidly increased. A controlled phase, which shows a recovered liver/body weight ratio from 9 days after PH, equal to the normal rat liver/body weight ratio. Conclusion. Our study demonstrated that bcl-2 after gene transfer appeared to accelerate both cellular proliferation and liver regeneration. 67. Overexpression of Dominant-Negative (⌬N) I␬B␣ Protects the MIN6 Pancreatic ␤ Cell Line from CytokineInduced Apoptosis. J. J. Wu, B.S., M. S. Baker, M.D., J. Chen, M.D., Ph.D., and D. B. Kaufman, M.D., Ph.D. Department of Surgery, Northwestern University Medical School, Chicago, Illinois. Cytokine-mediated injury of pancreatic ␤ cells is relevant in the field of islet transplantation. Immediate islet transplant graft injury, termed primary nonfunction, is directly associated with a host nonspecific macrophage inflammatory response requiring proinflammatory cytokine release. We sought to determine the effect of the combination of cytokines IL-1␤ (200 U/ml), TNF␣ (1000 U/ml), and IFN␥ (750 U/ml) on NO production and rates of apoptosis using the MIN6 mouse pancreatic ␤ cell line. Normal control MIN6 cells (exposed to cytokines for 24 – 48 h) were treated with L-NMMA or ZVAD. To create the MIN6-I␬B␣⌬N cell line, I␬B␣⌬N cDNA was subcloned into the vector SFFV-neo under the transcriptional control of Friend Spleen Focus Virus 5⬘ long terminal repeat. MIN6-I␬B␣⌬N cells were produced by stable transfection of the plasmid using Lipofectamine. Mobility gel-shift assays of stimulated MIN6-I␬B␣⌬N cells showed that NF␬B nuclear

TABLE—ABSTRACT 67 Treatment

Mean nitrite ⫾ SE (pmol/well)

Apoptosis rate (%)

Normal MIN6 control ⫹ Cytokines ⫹ Cytokines ⫹ L-NMMA ⫹ Cytokines ⫹ ZVAD I␬B␣⌬N mutant MIN6 ⫹ Cytokines

47.8 ⫾ 5.2 385.0 ⫾ 10.4 47.8 ⫾ 1.4 212.01 ⫾ 8.0 30.5 ⫾ 15.9 57.0 ⫾ 8.3

5.2 48.2 35.4 11.1 17.4 27.2

translocation was inhibited compared to MIN6 normal controls. NO production was determined by the Greiss reaction. Apoptosis rates were quantified by flow cytometric analysis of sub-G1 phase DNA content. Results are shown in the table. Cytokines induced NO production and increased the rate of apoptosis in normal MIN6 cells. L-NMMA inhibited NO production but did not significantly affect rates of apoptosis. Apoptosis appeared to be caspase dependent since it was inhibited by ZVAD. However, in the MIN6I␬B␣⌬N cell line, cytokine-induced NO production and rates of apoptosis were both reduced by interruption of NF␬B activity. These data help define the mechanisms of cytokine-induced injury of MIN6 cells and offers insight into new strategies to protect islet transplants. 68. 1,25-Dihydroxyvitamin D 3 Alters the TGF␤ Signaling Pathway in Renal Tissue. J. K. Aschenbrenner, M.D., G. J. Malin, B.S., H. W. Sollinger, M.D., Ph.D., B. N. Becker, M.D., and D. A. Hullett, Ph.D. Departments of Surgery & Medicine, University of Wisconsin, Madison, Wisconsin. Background. Chronic rejection (CR) is a leading cause of late graft failure. TGF␤, a known modulator of CR, and vitamin D both interact with Smad proteins, important regulators in the TGF␤ signaling pathway. We noted prolonged allograft survival using 1,25(OH) 2D 3 as a sole immunosuppressant in a rat renal CR model (Transplantation 2000; 69:8,S196), and a deceleration of renal graft loss in patients on calcitriol (Transplantation 2000; 69:8, S229). Given the effects of 1,25-(OH) 2D 3 on CR, we hypothesized that 1,25(OH) 2D 3 would alter TGF␤ 1 signaling in renal tissue. Methods. Renal lysates were prepared from Lewis rats receiving a diet with or without 1,25-(OH) 2D 3 (1000 ng/rat/day). Human renal cortical epithelial cells (HRCE) in culture were treated with 1,25-(OH) 2D 3 (50 – 500 nM) for 3 h. Cell media and renal lysates were examined using ELISA, RT-PCR, and Western blot analysis. Results. Exogenous vitamin D decreased vitamin D receptor (VDR) and Smad3 protein levels (Western blot analysis). Vitamin D-treated rats had a 70% reduction in bioactive renal TGF␤ 1 (ELISA). Vitamin D also decreased total and bioactive TGF␤ 1 production in HRCE (fold increase vs control 1.4 ⫾ 1.9); however, it did not inhibit the TGF␤-stimulated increase in bioactive TGF-␤ 1. RT-PCR demonstrated no significant difference in mRNA expression for VDR or TGF␤ 1 in renal lysates. Vitamin D induced Smad6 mRNA expression in HRCE cells (n ⫽ 4; P ⫽ 0.001) without significantly changing TGF-␤1, Smad2, or Smad7 mRNA expression. Conclusion. Vitamin D significantly affects the regulation of TGF-␤ signaling molecules. This could alter tubulointerstitial fibrosis in renal transplants, although not directly by antagonizing TGF-␤ 1. These observations explain in part our recent data showing improved rat renal allograft survival, as well as improved renal function, in patients on oral calcitriol after renal transplantation. 69. Solid Organ Transplantation of HIV Patients in the Era of Highly Active Antiretroviral Therapy. S. Potdar, M.D., S. F.

329

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 69 Organ

Months followup

Age (years)

Sex

Diagnosis

Liver 1 2 3 4 5

44.0 41.0 41.8 43.1 40.8

M M M M F

HCV HCV HCV HCV Drug induced

14 32 17 0.4 0.43

Kidney 1 2

50.3 48.9

Polycystic Hypertension

21 24

T.Bili

3.9 1 0.7 3.9 BUN

M M

Dodson, V. Scantlebury, R. Shapiro, J. J. Fung, and C. A. Bonham. University of Pittsburgh, Pittsburgh, Pennsylvania. Introduction. The presence of HIV in a patient with end-stage organ failure has been considered a contraindication for transplantation. However, no clinical trials have been conducted to examine the role of HIV in transplant recipients. Our aim is to examine the

impact of solid organ transplantation on progression of HIV and the effect of HIV on outcomes after transplantation from our institution. Methods. From 1997 to present, seven patients with HIV underwent transplantation for end-stage liver disease (n ⫽ 5) or end-stage renal disease (n ⫽ 2). Results. One patient died 10 days after liver transplantation from multisystem organ failure. The remaining six patients are alive with original graft. The primary disease, outcome and biochemical parameter are shown in the table. Patient survival and graft survival for liver and kidney are shown in the figure. No patient has developed opportunistic infections associated with progression to AIDS. Conclusion. With the use of HAART, solid organ transplantation does not appear to be associated with accelerated progression of HIV disease or increased early graft loss.

PARALLEL SESSION II Shock I 70. The Actin-Binding Protein Gelsolin Ameliorates Pulmonary Edema Induced by Lysophosphatidic Acid. J. J. Schwartz, M.D., M. Sambade, M.D., H. Yin, Ph.D., and R. Turnage, M.D. Department of Surgery & Physiology, Dallas VA Medical Center & University of Texas Southwestern Medical School, Dallas, Texas. Lysophosphatidic acid (LPA) has been implicated as an important paracrine mediator that activates several endothelial cell signaling

AST

ALT

GGTP

173 172 79 83 173 25 15 54 Died from multisystem organ failure 373 103 373

Crest. 24 42

ALKP

215 76

1.6 3.1

pathways. Recent studies in our laboratory have found that LPA increases microvascular permeability in an isolated, perfused lung model. Gelsolin is a circulating actin-binding protein that has been shown to bind to and attenuate LPA activity in vitro. This study examines the hypothesis that gelsolin can mitigate the increase in microvascular permeability associated with LPA exposure. The lungs of normal Sprague–Dawley rats were perfused ex vivo with Krebs buffer containing 3% bovine serum albumin (BSA), 3% BSA ⫹ 10 ⫺6 M LPA, or 3% BSA ⫹ 10 ⫺6 M LPA ⫹ 5 ␮M gelsolin. After 30 min of ex vivo perfusion, pulmonary capillary pressure, pulmonary vascular resistance (R t), and microvascular permeability (capillary filtration coefficient, K f) were measured. The data are expressed as means ⫾ SEM and analyzed using the ANOVA and Tukey’s post hoc test. P ⱕ 0.05 was considered a significant difference between groups (see table). The presence of gelsolin within the perfusate significantly attenuated the increase in pulmonary microvascular permeability induced by LPA. These data suggest that gelsolin may serve a protective role during systemic inflammatory states by binding and inactivating LPA.

TABLE—ABSTRACT 70 n ⱖ 4

Control

LPA

LPA ⫹ Gelsolin

Kf Pc Rt

0.009 ⫾ 0.002 6.13 ⫾ 1.27 3.02 ⫾ 0.79

0.30 ⫾ 0.03* 7.33 ⫾ 0.21 3.21 ⫾ 0.51

0.08 ⫾ 0.02* ,# 8.31 ⫾ 0.85 3.76 ⫾ 0.43

Note. K f, g/mm Hg/min/100 g wt; r t, mm Hg/ml/min/100 g wt. *P ⬍ 0.05 vs control; #P ⬍ 0.05 vs LP. 71. TNF Receptor I Mediates Chemokine Production and Neutrophil Accumulation in the Lung Following Systemic LPS. C. M. Calkins, M.D., J. K. Heimbach, M.D., D. D. Bensard, M.D., X. Meng, Ph.D., and R. C. McIntyre, M.D. Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado. TNF-␣ is critical effector of endotoxin (LPS) induced acute lung injury, and its effects are mediated by two structurally related receptors—RI and RII. Chemokines (KC) are peptides responsible for the final step in tissue neutrophil (PMN) accumulation. We hypothesized that TNFRI receptor signaling positively affects PMN accumulation in the lung via regulation of chemokine production. Therefore, the purposes of this study were to (1) delineate LPS-induced lung TNF production and (2) characterize the contribution of both

330

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS resulted in a significant survival advantage for traumatized mice given NS398. Additionally, the increased production of TNF-␣ and NO from splenic macrophages in traumatized mice was normalized after treatment with NS398. These data suggest that daily treatment with a specific COX-2 inhibitor not only suppresses PGE 2, but normalizes key proinflammatory cytokines after trauma. These findings correlate with significantly increased survival in injured mice treated with NS398 and given a subsequent septic challenge and suggests that COX-2 inhibitors may play an important role in modulating the inflammatory response and improving survival after trauma.

TNF receptors on lung chemokine production and neutrophil influx following systemic LPS. Methods. Wild-type (WT), TNFRI and TNFRII knockout (⫺/⫺) mice were injected with saline or LPS (0.5 mg/kg Escherichia coli, ip). After 2 or 4 h, lungs were analyzed for TNF and KC expression (ELISA) and PMN accumulation (MPO assay). Results. There was an increase in lung TNF (saline 5.0 ⫾ 1.2 pg/mg total protein vs LPS 950 ⫾ 318, P ⬍ 0.05) after LPS. Lung KC and PMN accumulation were also increased compared to saline control. Lung KC and PMN accumulation was significantly lower in TNFRI⫺/⫺, but not TNFRII⫺/⫺ mice despite no difference in TNF production (TNFRI⫺/⫺ 925 ⫾ 301 vs TNFRII⫺/⫺ 837 ⫾ 267, P ⫽ 0.82) (see figure). Conclusions. Acute lung injury following endotoxin is characterized by increased lung (1) TNF production, (2) chemokine production, and (3) neutrophil accumulation. The maximal effect of LPS induced lung neutrophil accumulation appears dependent upon the TNFRI receptor but not the TNFRII receptor. 72. NS398 Treatment after Trauma Attenuates Inflammatory Response and Increases Survival. V. E. Mack Strong, M.D., P. J. Mackrell, M.D., E. M. Concannon, B.S., J. R. Mestre, M.D., G. Symth, M.D. H. A. Naama, M.D., P. P. Stapleton, Ph.D., and J. M. Daly, M.D. Department of Surgery, Weill Medical College of Cornell University, New York, New York. Prostaglandin-E 2 (PGE 2) production after trauma contributes to immune alterations that may increase susceptibility to infectious complications. We hypothesize that blocking PGE 2 with NS398, a selective COX-2 inhibitor, will alter this response and improve outcome. This study evaluated the effect of NS398 given over 7 days on proinflammatory cytokines and survival after a septic challenge. BALB/C mice (n ⫽ 8/group) were given 10 mg/kg NS398 intraperitoneally BID for 7 days, starting immediately after sham injury or trauma (femur fracture ⫹ 40% hemorrhage). Four groups, sham ⫹ placebo (C); sham ⫹ NS398 (CN); trauma ⫹ placebo (T); or trauma ⫹ NS398 (TN) were studied. On day 7 after trauma, mice were sacrificed and splenic macrophages were evaluated for PGE 2, TNF-␣ and NO production (via Student’s t test). In a separate study, mice (n ⫽ 10 –11/group) were traumatized and given NS398 for 7 days. On day 7, cecal ligation and puncture was performed, and mice were evaluated for survival over 3 weeks (via log rank test) (see table). NS398 treatment of injured mice (TN) decreased PGE 2 production compared to trauma ⫹ placebo (T) (3.9 ⫾ 0.3 vs 3.1 ⫾ 0.4 pg/␮g protein) and

TABLE—ABSTRACT 72 Groups

Survival (%)

NO (nmol/␮g protein)

TNF-␣ (pg/␮g protein)

C C ⫹ NS398 T T ⫹ NS398

9 0 0 45

52.3 ⫾ 3.1 54.8 ⫾ 2.1 61.2 ⫾ 3.1 48.0 ⫾ 3.3 #

2.5 ⫾ 0.4 2.6 ⫾ 0.3 4.2 ⫾ 0.5 2.6 ⫾ 0.3 #

Note. Data are means ⫾ SEM; *P ⬍ 0.02 by log rank (Mantel– Cox) test (T vs TN); # P ⬍ 0.05 by Student’s t test (T vs TN)/ˆ(C vs T).

73. Major Surgery Drives a Systemic Proinflammatory T-Cell Response. K. J. Sweeney, AFRCSI, C. Coates, Bsc., M. R. Kell, FRCS, and J. V. Reynolds, Mch. Department of Clinical Surgery, St James’s Hospital, Dublin 8, Ireland. We have previously demonstrated that major surgery results in activation of the adaptive immune system. This study investigates the mechanism by which T-cells are activated and their pattern of response to injury. Isolated peripheral blood mononuclear cells (PBMC) and serum samples were obtained from 25 patients preoperatively, 24 h postoperatively, and 1 week following major upper gastrointestinal surgery. Patients’ PBMCs were stimulated with superantigen (staphylococcal enterotoxin B (SEB)) and cytokine ELISAs for interleukin-2 (IL-2) and interleukin-10 (IL-10) were performed on the 48-h supernatants. Healthy volunteer PBMCs were incubated with patients’ serum alone or serum in the presence of CTLA-4 chimera, a potent inhibitor of antigen presentation. CD 69 expression on CD 3⫹ T-cells was then assessed by flow cytometry. There was a significant increase in the production of IL-2 24 h postoperatively and this was sustained 1 week following surgery (P ⬍ 0.05, Kruskal–Wallis). There was no change in IL-10 production throughout the study period (P ⫽ 0.13). Postoperative serum activated healthy volunteer T-cells. This activation was prevented by

TABLE—ABSTRACT 73

PBMCs PBMCs & CTLA-4

Preop

24-h postop

1 week postop

0.6 (0.1) 1.9 (0.5)

4.5 (1.3)* 1.8 (0.4)

5.3 (1)* 1.4 (0.3)

Note. Mean (SEM) % CD3 ⫹ cells expressing CD 69 after serum incubation. *P ⬍ 0.05 vs preoperative serum incubation, ANOVA. CTLA-4 (table). T-cell activation following major surgery occurs via an antigen presentation mechanism. Postoperative T-cell response is proinflammatory, suggesting a role for the adaptive immune system in the development of postoperative systemic inflammatory response syndrome. 74. Pericytes Augment the Capillary Barrier Effect in in Vitro Cocultures. C. J. Dente, M.D., C. P. Steffes, M.D., C. Speyer, Ph.D., and J. Tyburski, M.D. Wayne State University, Detroit, Michigan Most in vitro studies of capillary permeability focus on endothelial cell (MVEC) monolayers and ignore the second cell of the capillary wall: the microvascular pericyte (PC). PC exist in all tissues at various MVEC:PC ratios and may be involved in control of microvascular permeability. We describe a novel model, which more realistically mirrors the in vivo morphology. We investigated the permeability characteristics of MVEC, PC, and MVEC:PC cocultures to large and small molecules. Semipermeable inserts were coated with collagen and plated with early passage bovine pulmonary MVEC. On

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 74 Culture Collagen only MVEC only 10:1 Co Cx 5:1 Co Cx 1:1 Co Cx

ER (Day 4) (ohm/cm 2)

105 ⫾ 35 103 ⫾ 28 116 ⫾ 51 147 ⫾ 77

ER (Day 6) (ohm/cm 2)

Alb. clearance (␮l/min)

124 ⫾ 29 195 ⫾ 52 $ 214 ⫾ 41 $ 302 ⫾ 73 $,%

0.141 ⫾ 0.027 0.064 ⫾ 0.017* 0.043 ⫾ 0.019 $ 0.038 ⫾ 0.013 $ 0.026 ⫾ 0.008 $,%

* P ⬍ 0.05 vs collagen. $ P ⬍ 0.05 vs MVEC. %P ⬍ 0.05 vs 10:1 and 5:1.

day 3, bovine pulmonary PC were added at concentrations to create MVEC:PC ratios of 1:1, 5:1, and 10:1, mimicking various in vivo tissue ratios. Electrical resistance (ER) was measured on subsequent days to assay small molecule permeability and fluorescently labeled (FITC) bovine albumin was used in a permeability assay to calculate an albumin clearance for each culture. The results are summarized in the table. Addition of PC to MVEC monolayers increased the barrier to small and large molecules in cocultures at a 5:1 and 10:1 ratio, mimicking the permeability characteristics of many visceral organs, including the lung. At a ratio of 1:1, mimicking the brain microvasculature, PC addition increased the capillary barrier to even a greater degree. This model is an improvement over past in vitro models as it includes both of the cells present in the microvasculature. Furthermore, it demonstrates the additional barrier effect of PC when added to confluent MVEC monolayers, establishing them as a factor in capillary permeability in the steady state. Further in vitro studies using this model may elucidate the role of PC in the permeability changes of inflammatory and posttraumatic states. 75. Heparin Treatment of Endothelial Cells Decreases Their Levels of Active Stress Kinases. M. Hamel, M.S,* M. D. Cipolle, M.D., Ph.D.,† D. Kanyi, M.D.,* M. D. Pasquale, and L. J. Lowe-Krentz, Ph.D.* *Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania; and †Department Surgery, Division of Trauma, Lehigh Valley Hospital, Allentown, Pennsylvania Introduction. This study was conducted to test the hypothesis that heparin decreases stress kinase levels in wounded or stressed endothelial cells. Methods. Immunofluorescence for cell-specific localization patterns and Western blotting techniques were used to evaluate total activity levels of the stress kinases, JNK and p38, and the phosphatase, MKP-1. Colocalization of the kinases with stress fibers was examined using phalloidin. Conditions tested included: (1) confluent vs subconfluent monolayers, (2) stressed (TNF or LPS) vs nonstressed cells, or (3) and heparin vs no heparin. Results. Data indicated that wounded and subconfluent endothelial cells contain a population of active JNK not observed in confluent endothelial cell cultures. This active JNK was found in a fibrous pattern reminiscent of stress fibers and appeared to colocalize with phalloidin. Exposure of subconfluent endothelial layers to inflammatory agents caused increases in p38 activity similar to the response in confluent endothelial layers; however, these conditions resulted in only minor increases in JNK activity. Heparin decreased stress kinase activity in both subconfluent and confluent endothelial layers (whether induced or constituitive as in the case of subconfluent endothelial JNK). The decrease in kinase activity appeared to be partly due to inactivation of activated JNK and p38, since it was accompanied by an increase in synthesis of the dual activity phosphatase, MKP-1. Conclusions. These data indicate that subconfluent endothelial monolayers may more accurately mimic wounded layers, and heparin treatment of endothelial cells modulates the inflammatory response by decreasing the activity of stress kinases in endothelial cells. Therefore, in addi-

331

tion to its anticoagulant effect, heparin appears to play an important role in limiting the inflammatory response in wounded or stressed endothelial cells. 76. Murine ␤-Defensin-3 Is an Inducible Peptide with Limited Tissue Expression and Broad-Spectrum Antimicrobial Activity. R. S. Burd, M.D., Ph.D., J. Sullivan, M.S., J. L. Furrer, B.S., and A. L. Smith, M.D. Department of Surgery, Molecular Microbiology and Immunology, University of MissouriColumbia, Columbia, Missouri. Introduction. ␤-Defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. Similarities between mammalian ␤-defensins may permit the use of murine models to further define the role of these peptides in innate host defense. Murine ␤-defensin-3 (mBD-3) is a recently identified peptide that exhibits homology at the gene level to human ␤-defensin-2 (hBD-2), one of two ␤-defensins identified in man. The purpose of this study was to determine: (1) the tissue distribution of mBD-3 expression, (2) the effect of gram-negative bacterial infection on mBD-3 expression, and (3) the antimicrobial activity of mBD-3. Methods. mBD-3 mRNA expression was assayed by PCR using cDNA derived from a panel of tissues. Expression of mBD-3 was also evaluated in tissues obtained from mice 24 h after intraperitoneal (ip) infection with 10 6 Escherichia coli using RT-PCR. Based on the sequence deduced from mBD-3 cDNA, a 40-amino-acid peptide was assembled using automated Fmoc solid phase synthesis and purified to homogeneity by RP-HPLC. The antimicrobial activity of synthetic mBD-3 was evaluated in microdilution broth assays using representative bacterial and fungal organisms. Results. Constituitive expression of mBD-3 mRNA was not observed in heart, brain, spleen, lung, liver, esophagus, small intestine, kidney, or testis. Following ip infection, expression of mBD-3 mRNA was observed only in the esophagus. The MICs of synthetic mBD-3 were 50 ⫾ 14 ␮g/ml (mean ⫾ SEM, n ⫽ 4) for E. coli, 47 ⫾ 19 ␮g/ml for Pseudomonas aeruginosa, 75 ⫾ 25 ␮g/ml for Staphyloccus aureus, and 67 ⫾17 ␮g/ml for Candida albicans. Conclusions. mBD-3 is an inducible peptide with limited tissue expression. Because it exhibits broad-spectrum antimicrobial activity, this peptide may serve as an innate defense against microbial invasion at specific mucosal surfaces in the mouse. 77. p38 MAPK: Posttranslational Regulation of TNF-␣ Expression in Human PMN. B. W. Nolan, M.D., J. Friel, A. J. Duffy, M.D., K. Sheth, M.D., H. Collette, R.N., M. De, and P. E. Bankey, M.D., Ph.D. Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts. TNF-␣ mediates local and systemic inflammatory responses. We hypothesize that neutrophil (PMN) TNF-␣ expression is regulated by p38 MAPK. PMN were isolated from healthy volunteers (n ⫽ 8) and then incubated for 1.5 h in 2 ␮M SB202190 (p-38 inhibitor), 10 ␮M PD 98059 (ERK inhibitor), or vehicle. Cells were stimulated with LPS (0 to 1 ␮g/ml) over 18 h. TNF-␣ mRNA was quantified by fluorescence. Secreted and total cell associated TNF-␣ were quantified by L929 bioassay. Expression of membrane TNF-␣ (mTNF-␣) was determined by flow cytometry. MAPK activity was determined by western blot. Means and standard error were determined. Data were analyzed by t test (see figure). LPS activated p38 in a dosedependent fashion (Western). TNF-␣ message was constitutively expressed and steady-state levels were unchanged over 2-h of LPS stimulation. TNF-␣ was secreted from LPS-stimulated cells ( *P ⬍ 0.01) but not unstimulated cells. SB202190 inhibited LPS stimulated secretion ( **P ⬍ 0.05). Membrane TNF-␣ was expressed on unstimulated cells and upregulated by LPS (MnFI, #P ⬍ 0.05). LPS upregulation of mTNF-␣ was inhibited by SB202190 ( ##P ⬍ 0.05). Total cellular TNF-␣ was increased by LPS (P ⬍ 0.05). Inhibitor treatment had no effect on LPS stimulated expression of total TNF-␣. In con-

332

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

clusion: (1) TNF-␣ is constitutively expressed in human neutrophils and (2) p38 MAPK is a posttranslational regulator of TNF-␣ membrane expression and secretion in LPS-stimulated cells.

PARALLEL SESSION III Education 78. Assessment of Knowledge and Attitudes about Melanoma Prevention among Teenagers. A. Lucci, M.D., and H. Citro, M.S., P.A.C. Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston Texas. Introduction. The incidence of melanoma has increased over the past 10 years in the United States at a rate second only to lung cancer in women. Sunburns early in life are associated with an increased lifetime risk of melanoma. The purpose of this study was to assess the knowledge and attitudes of teenagers about melanoma. Methods. Two hundred and 10 true/false quizzes were administered to junior high and high school students ages 12 to 18 years in Dallas and Houston, Texas. Each student was provided with the correct answers, including an explanatory paragraph for each question, after they completed their quiz. Subsequently, a 15-item postquiz questionnaire was administered. Nine questions pertained to hair color, eye color, and current sun exposure practices. The remaining six-question exam utilized the Leikert scale to determine if this educational exercise would affect future behaviors in regards to sun exposure and skin self-examination. Results. The return rate was 100%, with 109 students age 12–15 years and 101 ⱖ 16 years. Seventy-six percent of all respondents sunbathed outdoors, and 18% had used a tanning bed within the past 6 months. Unfortunately, 33% of the students had at least three blistering sunburns in the past. The average score on the knowledge assessment exam was 65% correct for students ⱖ16 years old and 54% correct for those age 12–15 years. Sixty-eight percent of students age 12–15 indicated that they planned to change their behavior after the exercise, versus 38% of students ⱖ 16 years. Conclusion. Adolescents ⱖ16 years of age knew more about melanoma than did younger students, but the majority were not willing to make behavioral changes to prevent melanoma. Conversely, the younger students had less knowledge but were more willing to modify sun exposure behaviors. The data from this study support education aimed at the younger age groups to most effectively achieve risk reduction and prevent future melanomas. 79. Patients’ Attitudes Regarding Involvement and Education of Surgical Residents. R. A. Cowles, M.D., C. A. Moyer, M.P.H., S. S. Sonnad, Ph.D., D. M. Simeone, J. A. Knol, M.D., F. E. Eckhauser, M. W. Mulholland, M.D., Ph.D., and L. M. Colletti. Department of Surgery, University of Michigan, Ann Arbor, Michigan. Introduction. Education is a mission of academic medical cen-

ters. Patients’ attitudes toward the training of surgical residents and their impressions of how residents impact care is unknown. We hypothesized that patients are satisfied with care provided by residents, but that patients do not understand some aspects of resident training. Methods Patients admitted to the gastrointestinal surgery service were evaluated using a 30-item questionnaire that was developed for this study. Patients who underwent surgery and had a postoperative inpatient stay were included. Answers were tabulated, frequencies were calculated, and correlation between answers analyzed by ␹ 2. Results. A total of 200 patients were surveyed between July 1999 and January 2000. The mean age was 53 years and surveys obtained, on average, on POD 7. Seventy-one percent felt that the teaching hospital was not inconvenient and 74% felt that they received extra attention there. Patients were comfortable with resident involvement (86%) and wanted to help educate future surgeons (91%). When patients either expected several physicians to be involved in care (P ⬍ 0.01), felt that they received extra attention (P ⬍ 0.001), or did not feel inconvenienced (P ⬍ 0.001) they more often responded positively to involvement in resident education. Regarding the education process, 83% felt that the staff surgeon performed a majority of the procedure and 87% felt that it was crucial that the staff surgeon be present for the entire operation. Despite a willingness to help with education, 32% answered that they would not want residents to do any of their operation. Gender, education, and experience in health care did not correlate with answers. Conclusions. The educational mission of academic departments is well received by patients. Orientation to the resident education process is vital to patients’ perceptions of care and may render them willing to participate in educational activities involving residents. 80. Surgical Educator Preferences Regarding Clinical Assessment Topics for Residents. G. J. Cerilli, M.D., H. W. Merrick, M.D., and E. D. Staren, M.D. Department of Surgery, Medical College of Ohio, Toledo, Ohio. This study’s objective was to determine which surgical topics were most highly regarded for assessment of competency of surgical residents. Objective structured clinical examinations (OSCEs) are being increasingly utilized for outcome measurement of resident perfor-

TABLE—ABSTRACT 80 History taking Abdominal pain (29.2%) Breast mass (10.8%) GI bleed (8.5%) Bowel obstruction (8.5%) Trauma (7.2%) Physical examination Acute abdomen (28.9%) Trauma (18.2%) Breast exam (15.8%) Peripheral vascular (8.9%) Cardiac exam (7.5%) Data interpretation Chest film (21.0%) Abdominal series (20.2%) CT scan (19.6%) Mammogram (10.1%) C-spine film (5.2%) Technique Central line (16.1%) Basic suture skills (14.2%) Bowel anastomosis (8.0%) Informed consent (7.9%) Intubation (6.4%)

333

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS mance. It is unclear which areas of evaluation might best be included in such an examination so as to optimize its value. In order to discern a consensus of opinion, 674 surgical educators were surveyed nationally. Eighty-four topics were categorized in four OSCE areas listed below. Respondents selected and ranked the five most important topics from each of these areas. Points were distributed to each topic weighted by a Likert scale. A percentage of total awarded points was determined individually for each topic. A total of 243/674 responses (36.1%) were received, with a useable response rate of 218/674 (32.3%). The five highest ranked topics are listed in the table. Although certainly not inclusive of all the skills necessary for trainees to acquire during a surgical residency, this survey offers useful data regarding those skills viewed as most important by experienced surgical educators and offers guidelines for inclusion in design of objective structured clinical examinations. 81. Comparison of Performance 2 Years after the Old and New (Interactive) ATLS Courses. J. Ali, M.D., R. Adam, MBChB, I. Pierre, MBBS, H. Bedaysie, MBBS, D. Josa, MBBS, and J. Winn, R.N. University of West Indies, West Indies; and University of Toronto, Toronto, Ontario, Canada. We previously (1997) demonstrated superior clinical but similar cognitive performance after the new (Interactive, Group A) compared to the old (Group B) ATLS course. The present study is aimed at determining whether this difference was short term or maintained over time (2 years). Methods. Two groups of 13 physicians of the original of 16 each were available for the study which compared

TABLE—ABSTRACT 81

Group A 1997 1999 Group B 1997 1999

MCQ

OSCE

Approach

Priority

87.5 ⫾ 5.1 ⫹ 71.5 ⫾ 2.8

18.8 ⫾ .1* 18.7 ⫾ .2*

4.9 ⫾ .1* 4.9 ⫾ .1*

6.4 ⫾ .2 6.4 ⫾ .2

86.5 ⫾ 5.6 ⫹ 70.2 ⫾ 3.3

15.6 ⫾ .2 ⫹ 15.0 ⫾ .2

4.0 ⫾ .2 4.0 ⫾ .1

6.3 ⫾ .2 6.2 ⫾ .2

performance in a 40-item MCQ examination on trauma topics and clinical performance in four trauma OSCE stations consisting of simulated trauma patients with blunt torso, penetrating torso, thermal, and pregnancy trauma. The tests were randomly applied with the examiners blinded to the grouping of the physicians. paired and unpaired t test were used for analysis with P ⬍ 0.05 being considered statistically significant. Overall score (OSCE, maximum standard 20), adherence to priority score (priority, scale 1–7), and overall approach (approach, scale 1–5) were analyzed. Results. (See table; means ⫾ SD; ⫹P ⬍ 0.05 compared to 1999; *P ⬍ 0.05 compared to Group B.) There was similar attrition of cognitive performance in both groups but the clinical performance (OSCE, approach) continue to be superior in the interactive course group. Adherence to priority scores were maintained at the same level in both groups. Conclusions. Although knowledge base decreases similarly with time after both courses, the new interactive course participants maintain a consistently higher clinical skill performance level.

82. Factors Influencing Outcome on the American Board of Surgery (ABS) Certifying Exam. E. Y. Sako, M.D.,* E. Petrusa, Ph.D.,† and J. Paukert, Ph.D.* *Department of Surgery, University of Texas Health Science Center, San Antonio, Texas; and †Department of Medical Education, Duke University, Durham, North Carolina.

TABLE—ABSTRACT 82 Practice type Private Academic Fellowships Thoracic surgery Plastic surgery Vascular surgery Other Military or research

182 (44%) 24 (6%)

87% 96%

38 (9%) 27 (7%) 38 (9%) 78 (19%) 22 (5%)

89% 85% 100% 90% 95%

Preparation Formal mock oral Informal mock oral Commercial course Combination Other No specialized prep

97 (24%) 38 (9%) 43 (11%) 125 (31%) 7 (2%) 99 (24%)

97% 100% 74% 90% 100% 88%

Total

409 (100%)

90%

The certifying (oral) examination given by the ABS is usually taken in the first 2 years after completion of a general surgery residency. The purpose of this study was to determine if fellowships (cardiothoracic, plastic, vascular surgery), which are remotely related to the examination’s subject content, negatively impact the pass rate on the first attempt. A second goal was to determine if specialized preparation (formal or informal “mock orals,” commercial courses) positively affect the pass rate on the first attempt. We contacted general surgery residency program directors in the United States to distribute a survey to their 1997 and 1998 graduates. The survey covered basic demographics, the type of practice or fellowship with which the individual was involved at the time of examination, and preparation. Our response rate was 65% (464/717). Only 409 respondents had taken the oral exam. &chi 2 analysis of these 409 responses revealed no differences for any type of practice or preparation. The difference in pass rate for those taking a commercial course did not reach statistical significance due to the small numbers in that category (see table). Results from this observational study suggest that the type of practice or fellowship had little effect on the passing rate of the ABS oral examination. Similarly, the type of examination preparation did not affect outcome. 83. A Model for Tracking Student Experiences on Surgical Clerkships. K. W. Millikan, M.D., and L. S. Hauge, Ph.D. Rush Medical College, Chicago, Illinois. Purpose. This study was conducted to identify the range and nature of surgical clerkship experiences regarding patient demographics, surgical content, experiences, and clinical skill practice in three different hospital settings— university, community, and public. Methods. An instrument was developed to track the location and type of learning experience, patient demographics, surgical content, and clinical experiences of students on their surgical clerkship. Following a pilot period, 23 students used the instrument to record the events of their surgical clerkship. Students entered their data into a customized spreadsheet. Data were analyzed to describe the frequency of tasks performed, the nature and location of learning experience, exposure to surgical topics, and patient demographics. Results. Students were involved in an average of 245 common surgical tasks over their 8-week clerkship. The large majority of these tasks included caring for surgical tubes, drains, and wounds; attending to patients’ fluids and electrolytes; taking H & Ps; and writing supervised orders and notes. Of their exposure to common tasks, students had the opportunity to observe 25% and perform 70%

334

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

of those tasks. Sixty-six percent of task work occurred on the patient floor and 23% occurred in the operating room. Students were exposed to a broad range of surgical topics, 71% of which were general surgery topics. Only 25% of these experiences were auditory, whereas 39% involved exposure to a patient, and 36% included participation in an operation. While 13% of surgical topics were received in a lecture format, 34% of surgical topics were taught in the operating room, 36% on the patient floor, and 13% in the patient office. Patient load and characteristics tended to vary across hospital settings, and on average, students worked with 164 patients during their clerkship. The smallest patient load (m ⫽ 113) occurred in the university hospital and the largest patient load (m ⫽ 251) occurred in the public hospital. Thirty-four percent of patients were 65 years or older, and the racial characteristics of patients was, on average, 46% Caucasian, 15% Hispanic, 36% African-American, and 4% Asian. Conclusion. Although surgical services and hospital settings may offer students different clerkship experiences, the common clinical and didactic components of a surgical clerkship can balance a student’s exposure to surgical topics and practice of clinical skills. Tracking surgical clerkship experiences is valuable to identifying the range and nature of medical students’ didactic, clinical, and operative experiences.

84. Future General Surgery Residents: Do Medical School Surgical Rotations Influence Subspecialty Choice? H. Chen, M.D., J. M. Hardacre, M.D., C. Martin, and K. D. Lillemoe, M.D. Department of Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland. A comprehensive exposure to general surgery is essential for medical students pursing careers in surgery. To determine if surgical clerkships influence subspecialty choice, we surveyed medical students who interviewed for general surgery training over 2 years at Johns Hopkins Hospital. The questions focused on the number of index operations seen during medical school and future career fields. Of 211 surveys sent, 146 (66%) were returned. The mean age of the students was 26 ⫾ 0 years, with 21% being female. Students anticipating subspecialization in cardiothoracic (CT), plastic (PLAS), pediatric (PED), and transplant (TX) surgery saw significantly more operations in their respective fields. Similar trends were seen in vascular (VAS) surgery and surgical oncology (ONC). Despite the apparent differences in exposure to subspecialty operations, all students saw equal numbers of hernia repairs (HER) and laparoscopic cholecystectomies (LC). There was no difference among students with regard to time spent on general surgery rotations. While medical students pursuing careers in surgery have equal exposure to general surgery, their anticipated subspecialty field highly correlated with their operative exposure to that field (see table). Thus, medical school surgical rotations appear to highly influence subspecialty choice.

85. General Surgery Resident’s Postgraduate Year Level Does Not Influence Operating Room Time in Laparoscopic Cholecystectomies. W. N. Wang, M.D., R. Marshal, M.D., and R. S. Haluck, M.D. General Surgery, The Milton S. Hershey Medical Center–Penn State, Hershey, Pennsylvania. Regardless of PGY, residents contribute equally to OR time. A retrospective study was performed utilizing all of the lap cholys performed under one attending’s supervision in a 2-year period. Acuity of the case was determined by final pathologic diagnosis (normal/cholelitiasis, acute, chronic, acute on chronic changes). Cases were divided into three groups based on PGY of the operating surgeon. Group A was PGY 1–3. Group B was PGY 4 –5. Group C was fellows in laparoscopy and the attending. Data were analyzed using the GraphPad Instat–the Kruskal–Wallis test (nonparametric ANOVA). To determine if acuity of the case impacted the surgeon or OR time, final pathologic diagnosis given were reviewed. A 3 ⫻ 4 table was created. The groups comprised the rows and the number of each pathologic acuity comprised the columns. This was analyzed using the SAS system, Fisher’s Exact test (two tail). To determine if a difference existed in mean OR times when grouped by level of pathologic acuity, the cases were divided into three groups based on the final pathologic diagnosis. Data were analyzed using the Kruskal–Wallis test. OR time was paired with pathologic acuity and analyzed using GraphPad Instat–the Spearman Rank correlation. Group A had a mean OR time of 59.61 min, SD ⫽ 25.29, n ⫽ 28. Group B had a mean OR time of 55.96 min, SD ⫽ 19.43, n ⫽ 28. Group C had a mean OR time of 71 min, SD ⫽ 37.16, n ⫽ 15. A P of 0.6370 was obtained when comparing mean operating room times by the PGY groups. Comparing the pathologic populations of the three PGY groups yielded a P of 0.830. When evaluating the mean operating room times for each pathologic level of acuity a P of 0.0027 led to a Spearman Rank correlation being performed on the data yielding a positive r of 0.3158 and a two-tailed P value of 0.0092. Resident level does not effect the operating room time in performing lap cholys. Acuity of the gallbladders was the same for all groups suggesting that the population operated on was equal. There was a difference in mean OR time based on pathologic acuity which correlated positively lending additional validity to the study.

PARALLEL SESSION III Peripheral Vascular II 86. Apoptosis in Human Vein Graft Stenosis. A. Westerband, M.D, D. C. James, M.D., C. L. Wixon, M.D., D. Crouse, B.S., R. L. Heimark, Ph.D., J. D. Hughes, M.D., M. L. Aguirre, M.D., W. T. Bellamy, Ph.D., and J. L. Mills, M.D. Departments of Surgery &

TABLE—ABSTRACT 84 Index operations seen (mean ⫾ SEM) Anticipated field

N

CT

PLAS

PED

TX

VAS

ONC

HER

LC

CT surg PLAS surg PED surg TX surg VAS surg ONC Other

29 28 10 9 5 23 36

32 ⫾ 7 3⫾1 7⫾1 5⫾3 2⫾1 7⫾2 4⫾1

5⫾2 36 ⫾ 7 10 ⫾ 4 10 ⫾ 5 6⫾4 7⫾2 7⫾2

7⫾3 4⫾1 31 ⫾ 6 3⫾1 1⫾1 7⫾3 4⫾2

2⫾ 0 0⫾ 0 1⫾ 0 24 ⫾ 19 4⫾ 3 3⫾ 1 1⫾ 0

9⫾2 6⫾5 4⫾3 6⫾2 17 ⫾ 4 6⫾2 5⫾2

18 ⫾ 2 17 ⫾ 5 14 ⫾ 3 16 ⫾ 2 20 ⫾ 3 28 ⫾ 8 19 ⫾ 3

10 ⫾ 1 8⫾2 9⫾2 7⫾1 10 ⫾ 3 10 ⫾ 4 10 ⫾ 1

10 ⫾ 1 8⫾1 10 ⫾ 2 9⫾1 7⫾1 14 ⫾ 3 10 ⫾ 1

P value

—

⬍0.01

⬍0.01

⬍0.01

0.017

0.08

0.13

0.99

0.99

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

335

Pathology, University of Arizona Health Sciences Center, Tucson, Arizona. The extent to which apoptosis, a genetically encoded cell death program, participates in cellularity regulation in myointimal hyperplasia has not been fully evaluated. The ratio of Bcl-2 protein, an inhibitor of apoptosis, to Bax protein, an inducer of apoptosis, determines survival or death after an apoptotic stimulus. We speculated that an alteration of this regulatory mechanism may be present in vein graft stenotic lesions that develop after infrainguinal bypass procedures. Methods. We examined a series of preocclusive stenotic lesions obtained during surgical revision to prevent graft thrombosis. Serial 4-␮m paraffin-embedded sections were cut for histology and immunohistochemistry. Bax expression was determined using a rabbit polyclonal antibody against human Bax protein. Comparison was made with Bcl-2 using a monoclonal mouse anti-human Bcl-2 oncoprotein. Apoptosis was demonstrated by the terminal uridine nick end-labeling (TUNEL) method and morphologic examination. Immunolabeling of Ki67, a marker for cell replication, was also performed. Apoptosis and proliferation indices (number of positive cells/total cells ⫻ 100, respectively) were calculated on representative gridded cross-sections of each lesion. Student’s t test was used to compare TUNEL and proliferation indices (mean ⫾ SD) between stenotic grafts and control veins, respectively. A P value ⬍ 0.05 was considered statistically significant. Results. Eighteen graft stenotic lesions retrieved between 6 and 18 months following primary bypass procedures were analyzed. Ten age-matched greater saphenous veins were used as controls. Stenotic lesions exhibited moderate amounts of Bax in a cytoplasmic distribution; in contrast, there was almost no detectable Bcl-2. Profiles of apoptosis and proliferation were discordant in serial adjacent sections (apoptotic index 0.34 ⫾ 0.26% vs proliferation index 1.24 ⫾ 0.97%). Control veins occasionally showed light immunostaining for either Bcl-2 or Bax. The index differences for both apoptosis and proliferation between stenotic vein grafts and control veins were statistically significant (P ⬍ 0.001). Conclusions Human vascular cells in vein graft stenosis express Bax, which increases the susceptibility of these cells to undergo apoptosis. Apoptosis participates in cellularity regulation but cell replication still predominates in these stenotic lesions. Local enhancement of apoptosis may constitute a novel strategy to maintain stable cell populations in human vein grafts. 87. Proliferation of Human Vascular Smooth Muscle Cells (HVSMC) Is Regulated by Gender-Specific Protein Kinase C (PKC) ␣ Expression. S. A. Miller, M.D., C. H. Selzman, M.D., B. D. Shames, M.D., T. D. Morrell, M.D., F. G. Robertson, Ph.D., A. Banerjee, Ph.D., and A. H. Harken, M.D. Department of Surgery, University of Colorado, Denver, Colorado. Atherosclerotic cardiovascular disease affects men more frequently than women. The cellular mechanism(s) that modulate gender-specific atherogenesis remain unclear. We have previously demonstrated a mitogenic response of HVSMC to monocyte chemotactic protein-1 (MCP-1) which is mediated by PKC. We postulated a gender-specific HVSMC proliferative response would occur due to differences in PKC expression. Methods. HVSMC obtained from male and female transplant donors were exposed to MCP-1. Proliferation was assayed by MTS and cell counting after 72 h. Induction of cell cycle was observed with immunohistochemistry for proliferating cell nuclear antigen (PCNA) after 24-h inhibition of calciumdependent PKC (cPKC) activity in male cells, with Go¨ 6976, was evaluated by cell counting. PKC␣ levels were determined by immunoblotting. Results. MCP-1 induced proliferation in male HVSMC by MTS (figure, A) and by cell counting: male 54,207 vs female 19,257 female and unstimulated control 22,510, P ⬍ 0.05. MCP-1 increased the percentage of PCNA expressed in males vs females (30.2 vs 6.7%, P ⬍ 0.05). Inhibition of cPKC activity blocked the mitogenic response of males to MCP-1 as seen by cell counting (21,521 vs 54,207, P ⬍

0.05). Immunoblotting of unstimulated male and female cells showed increased PKC␣ levels in male cells (figure, B) (n ⫽ 3 donors). Atheroprotection by the female relates to differences cPKC (PKC␣) expression. 88. The Effect of Cotinine on Telomerase Activity in Human Vascular Smooth Muscle Cells. T. Jacob, Ph.D., E. Ascher, M.D., N. Clouden, M.D., A. P. Hingorani, M.D., Y. Gunduz, M.D., M. Ward, M.D., and B. Tsemekhim, M.D. Division of Vascular Surgery, Maimonides Medical Center, 4802 10th Avenue, Brooklyn, New York. Introduction. Cotinine has been shown to be the main stable metabolite of nicotine with biological half-life approximately 10 times longer than that of nicotine. It has also been demonstrated to have a potent effect on vascular smooth muscle cell proliferation. The purpose of our experiment is to evaluate the effect of cotinine on cellular growth and abnormal proliferation of vascular smooth muscle cells via its effects on telomerase activity. Methods. Primary cultures of human vascular smooth muscle cells from patients’ samples of greater saphenous vein established in our lab were used in this experiment. Cells from third to fifth passages were used. Cotinine was added in doses equivalent to plasma levels of cotinine in an active smoker by dissolving, 0.0, 2.88 ⫻ 10 ⫺6 , 5.76 ⫻ 10 ⫺6 , and 1.44 ⫻ 10 ⫺5 mol/L of cotinine in the medium. Cell proliferation and telomerase activity were observed. The TRAP assay (telomeric repeat amplification protocol) was used to detect telomerase activity using PCR and standardized amounts of protein extracts. TRAP products were detected by ELISA. The experiment was performed in duplicate. Results. The cell mitogenic effect of cotinine was first seen after a 2-day incubation period. Telomerase activity was detected in all sets of vascular smooth muscle cell cultures treated with cotinine (P ⬍ 0.01). We observed that the telomerase activity was dose dependent. Conclusion. We have concluded that cotinine causes abnormal cell proliferation as shown by increased levels of telomerase activity. This study demonstrated cotinine’s stimulatory effect on human SMC proliferation in vitro at low doses while high doses of cotinine had a toxic effect. These data correlate and explain the results of other studies concerning the mitogenic effect of cotinine and tobacco use. 89. Dacron Modification Alters Macrophage-Mediated Release of Mitogens Active on Fibroblasts and Smooth Muscle Cells (SMC). T. M. Lam, M.D., C. W. Widenhouse, Ph.D., N. Klingman, B.S., and J. M. Seeger, M.D. Departments of Surgery and Materials Sciences, University of Florida and the Malcom Randall VAMC, Gainesville, Florida. Small-diameter vascular grafts fail due to a pathologic healing response that results in luminal narrowing secondary to intimal hyperplasia. It has been proposed that this response is driven by an unopposed, persistent, inflammatory response to the biomaterial that results in smooth muscle cell and fibroblast proliferation. Thioglycollate-elicited macrophages were harvested by peritoneal

336

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

lavage from NZW rabbits and incubated in 24-well culture plates with Dacron, silicone-coated Dacron (Dac/sil), or dexamethasone impregnated silicone-coated Dacron (Dac/sil⫹dex). The macrophage-conditioned media were then collected at the times of media change, pooled by week, and frozen at ⫺70°C. Conditioned media were then evaluated in mitogenic assays utilizing BALB fibroblasts and rabbit SMC cultured from medial explants. The proliferation results are reported as percentages above quiescence ⫾ SD, *P ⬍ 0.05 ANOVA (see figure). Silicone coating of Dacron did not alter the macrophage-mediated fibroblast response although it led to increased SMC mitogen release. The addition of Dex, however, resulted in inhibition of fibroblast and SMC proliferation as compared to Dac/sil or Dacron alone. (The response was not TNF-␣ mediated as evaluated by WEHI assay.) Conclusions. Dacron modified with dexamethasone-impregnated silicone suppresses the fibroplastic and SMC proliferative response induced by macrophage-mediated interactions with Dacron. This may lead to improved long-term results with decreased intimal hyperplasia.

90. Nicotine Induces Endothelial TNF-␣ Expression, Which Mediates Growth Retardation in Vitro. G. K. Albaugh, D.O., B. Kann, M.D., L. Strande, M.S., P. Vemulapalli, M.D., and J. B. Alexander, M.D. UMDNJ–RWJMS, Cooper Hospital, Camden, New Jersey. We hypothesized that cytokines of endothelial origin are involved with the pathologic changes found in atherosclerosis associated with smoking. We chose to assay for TNF-␣ due to its many

TABLES—ABSTRACT 90 Immunohistochemistry DIA Results Time (h):

0 (control)

1

3

24

Mean ISI % Increased activity

32.10

91.88* 186.2%

64.36* 100.4%

33.36 3.9%

* P ⬍ 0.01 compared to time 0.

Proliferation Study Results (10 4 Cells) Growth day:

T2

T3

T4

T5

HUVEC control HUVEC ⫹ FBN HUVEC ⫹ FBN ⫹ anti-TNF-␣

3.82 2.30§ 5.78

14.9 13.3§ 15.6

24.0 16.5§ 23.8

28.2 21.6§ 26.9

§ P ⱕ 0.05 compared to control.

biologic actions that are similar to those found in peripheral vascular disease. Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM-2) on plastic coverslips until 75% confluent. Free base nicotine (FBN) was diluted in EGM-2 to a concentration of 10 ⫺8 M and added to experimental cells. At 1, 3, and 24 h, coverslips were removed and fixed. Immunohistochemical staining was performed using antiTNF-␣. Digital image analysis (DIA) was performed to quantify expression of TNF-␣. An intensity stain index (ISI) measuring area and intensity of stain/total cellular area was determined for each time point (n ⫽ 5). Additional HUVEC were plated in 12-well plates in EGM-2 at 2 ⫻ 10 3 cells/cm 2 on T ⫺2 day. FBN was diluted in media to 10 ⫺9 M and added to wells with and without 0.9 ␮g/ml anti-TNF-␣ on T 0 day. Cell counts were performed in triplicate on days T 2-5 utilizing hemocytometry. Data were analyzed using Student’s t test, with a 95% confidence interval (see tables). In cells exposed to anti-TNF-␣ and nicotine there was inhibition of the growth retardation seen in the cells containing nicotine alone. These data demonstrate the ability of endothelial cells to secrete TNF-␣ in response to nicotine at levels found in serum after smoking and that endothelial cell growth retardation as a consequence of nicotine exposure is TNF-␣ mediated. 91. Simvastatin Inhibits Mitogen-Induced Proliferation of Human Saphenous Vein Endothelial Cells through S-Phase Reduction. R. B. Claytor, M.D., S. A. Shah, M.D., M. P. Callery, M.D., J. M. Li, M.D., J. B. Herrmann, M.D., R. C. Harland, M.D., and M. J. Rohrer, M.D. Department of Vascular Surgery, University of Massachusetts Medical School, Worcester, Massachusetts. Introduction. Simvastatin inhibits proliferation of human smooth muscle cells and of human umbilical vein endothelial cells. TNF-␣ induces apoptosis and G1 cell-cycle arrest of proliferating endothelial cells. We investigated whether simvastatin inhibits proliferation of human saphenous vein endothelial cells by blocking cell cycle entry. Methods. Proliferating human saphenous vein endothelial cells (HSVEC) were serum starved for 24 h to induce quiescence. They were then repleted with 10% FBS (fetal bovine serum) to initiate cell cycle entry. Concurrently, buffer alone, simvastatin, or TNF-␣ was incubated with the HSVEC for 24 to 48 h. Cell cycle progression was determined by FACS analysis of propidium iodide-stained cells. Expression of p21 Cip1⫺Waf⫺1 , a protein activated during cell cycle arrest, was evaluated by Western blot. Proliferation was determined by MTT assay. Statistical analysis included a two-tailed t test. Results. Proliferating HSVEC incubated with simvastatin or TNF-␣ had a marked increase in G1 compared to buffer alone (n ⫽ 6, P ⬍ 0.05), while S-phase was reduced by simvastatin and TNF-␣ (Fig. 1). Western blots demonstrate increased levels of p21 Cip1⫺Waf⫺1 for HSVEC incubated with simvastatin (data not shown). Simvastatin and TNF-␣ reduced proliferation of HSVEC (Fig. 2). Conclusion. Mitogeninduced proliferation of HSVECs is inhibited by simvastatin and TNF-␣ through S-phase reduction. 92. Effect of Diabetes on Acetylcholine Receptor Quantity and Function in Rat Endothelial Cells. F. Cannizzo, M.D.,

337

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Ph.D.,* R. B. Wait, M.D., Ph.D.,† C. Mueller, B.S.,* V. Nguyen, B.A.,* L. S. Dresner, M.D.* *Department of Surgery, SUNY Downstate Medical Center, Brooklyn, New York; and †Department of Surgery, BayState Medical Center, Springfield, Massachusetts. Streptozotocin (STZ)-induced diabetes causes a defect in acetylcholine (Ach)-mediated vasodilation at the level of Ach receptor–Gprotein complexes. This phenomenon is abrogated with insulinmaintained normoglycemia. The purpose of this study is to characterize the effect of diabetes on the Ach receptor (AchR). Sprague–Dawley rats were divided into control (C), diabetic (D), and treated (T). D and T were made diabetic with STZ (60 mg/kg). T was implanted with controlled-release insulin pellets. Weight and glucose were checked weekly. Aortae were harvested on day 40. Ach binding assays were performed by dividing aortae into 4 ⫻ 4-mm squares and incubating in 3H-Ach. Nonspecific binding was characterized using a 250-fold excess of muscarine. Tissue, filter, and

TABLE—ABSTRACT 92 Ach binding (pmol/10 6 cells)

C D T

TABLE—ABSTRACT 93 EC alone

EC coculture

SMC alone

SMC coculture

Human 6.6 ⫾ 0.31 3.0 ⫾ 0.57* 8.4 ⫾ 0.49 6.1 ⫾ 0.50* Bovine 5.0 ⫾ 0.27 5.8 ⫾ 0.65 10.3 ⫾ 0.42 17.1 ⫾ 0.52* %⌬ Human ⫺55.2 ⫾ 8.7** ⫺27.2 ⫾ 6.0** %⌬ Bovine ⫹14.6 ⫾ 13.0 ⫹66.8 ⫾ 5.0 * P ⬍ 0.05 coculture vs alone; **P ⬍ 0.05 human vs bovine (by ANOVA).

Conclusions. These fundamental differences in cell behavior stress the potential importance of using human cells in studies evaluating cell– cell and cell–material interactions in vitro. Since significant differences in cell–material interactions occur in vivo between species, the finding that in vitro heterotypic cell– cell interactions are species dependent is not surprising.

PARALLEL SESSION III

N

30 nM

100 nM

300 nM

AchR quantity (fmol/10 6 cells)

18 19 19

0.87 ⫾ .05 0.86 ⫾ .15 0.78 ⫾ .04

1.46 ⫾ 0.2* 0.97 ⫾ .11* 1.19 ⫾ .12

2.29 ⫾ .16* 1.73 ⫾ .26* ,␶ 2.50 ⫾ .27 ␶

19.4 ⫾ 9.4 f 31.6 ⫾ 17.3 f 97.2 ⫾ 9.6 f

Note. Mean ⫾ SE: * ,␶P ⬍ 0.013; fP ⬍ 0.02 by ANOVA, SNK. filtrate were centrifuged and counted separately by beta scintillation. Ach receptor quantity was determined by PAGE Western blot, visualized by chemiluminescence, and analyzed using NIH Image. Data were analyzed by ANOVA followed by Student–Newman– Keuls. Group C rats maintained normoglycemia. Glu in D rats was ⬎250 mg/dl throughout the study. Group T maintained Glu ⬍ 200mg/dl (see table). We conclude that impaired binding may cause upregulation of receptor quantity; the presence of insulin and/or the maintenance of normoglycemia may potentiate this upregulation. Diminished binding may result from altered receptor structure or altered association of Ach receptor with G-protein. These findings may account for the impaired endothelium-dependent vasodilation seen in this model. 93. Heterotypic Smooth Muscle Cell/Endothelial Cell Interactions Differ between Species. O. Imegwu, M.D., A. M. Graham, M.D., and G. B. Nackman, M.D. Division of Vascular Surgery, Robert Wood Johnson Medical School, New Brunswick, New Jersey. Objective. In vitro coculture models have been used to study heterotypic cell– cell interactions. This study was performed to determine if species of cell origin affects heterotypic smooth muscle cell (SMC) endothelial cell (EC) interactions in coculture. Methods. To study the effect of ECs on SMC proliferation, SMCs were plated on porous PET membranes for 4 days. ECs were added opposite SMCs on day 4, and 48 h later, cells were harvested for cell counts. To study the effect of SMCs on EC proliferation, ECs were added to bare membranes or on membranes opposite SMCs. After 48 h, cells were harvested for cell counts (n ⫽ 3/condition, experiments repeated ⫻2). Cells of human and bovine origin were used. Results. The effect of coculture on cell counts differed between species. The effect of heterotypic interactions between human cells was coinhibitory. This contrasted with the bovine EC stimulation of SMC proliferation and the failure of bovine SMCs to inhibit EC proliferation (see table).

Gastrointestinal II 94. Ileal Bile Acid Transport Protein Is Expressed in the Jejunum Following Transplantation of Ileal Mucosal Stem Cells. M. G. Stelzner, M.D., V. D. Hoagland, B.S., and S. Somasundaram, Ph.D. Seattle VAMC, University of Washington, Seattle, Washington. Introduction. Ileal stem cell transplantation into the distal jejunum may be a treatment option for patients with bile acid malabsorption following extensive ileal resection. However, transplantation has never attempted since there were no markers to differentiate ileocytes from jejunocytes. We hypothesized that ileal mucosal stem cells could be transplanted into the jejunum and would express the ileal bile acid transporter IBAT as a marker in the new location which would be detectable with antibodies. Methods. We raised antibodies against IBAT and showed its exclusive expression in the ileum by immunohistology. Then Lewis rats (n ⫽ 8) underwent laparotomy and isolation of an 8-cm-long proximal jejunal loop with its blood circulation left intact. The preceding and subsequent jejunal segments were anastomosed to restore continuity. The isolated segment was opened and areas of jejunal mucosa were debrided. Ileal stem cells were harvested from 6-day-old Lewis pups and seeded into the denuded segment. The segment and then the abdomen were closed. Two weeks later, the seeded segments were harvested and examined in H & E stains and by immunohistology using our anti-IBAT antibodies. Results. In all rats, seeded segments were found filled with leukocytes and mucus. Some scarring occurred. In five rats, IBAT could not be detected. In three rats, there were areas of neomucosa with cells expressing IBAT protein. No IBAT signal was seen in adjacent jejunum segments. The epithelial layer appeared intact in all specimens. Conclusion. Ileocytes can be successfully transplanted into the jejunum. They express the specific ileal bile acid transporter in this location. Debridement and seeding techniques will have to be improved. Once refined, this method holds promise for treatment of bile acid malabsorption as well as intestinal stem cell gene therapy. 95. Monochloramine Enhances the Basolateral Ca 2ⴙDependent K ⴙ Conductance in Human Intestinal Epithelial Cells. E. C. Mun, M.D., H. M. Kim, M.D., I. Calvo, M.D., J. Mayol, M.D., M. Prasad, M.D., and J. B. Matthews, M.D. De-

338

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 95 Mechanism

A alone

F⫹A

NH 2Cl ⫹ A

M 3 receptor Ca 2⫹ store release Ca 2⫹ ionophore Direct K ⫹ channel activator

68.6 ⫾ 8.7 31.6 ⫾ 6.6 56.7 ⫾ 11.6 34.4 ⫾ 10.0

129 ⫾ 39.6 (*) 90.3 ⫾ 29.7 (*) 95.2 ⫾ 20.2 (*) 42.1 ⫾ 10.4 (NS)

131 ⫾ 6.47 (*) 88.6 ⫾ 17.7 (*) 124 ⫾ 17.5 (*) 13.8 ⫾ 0.6 (*)

Agonist (A) Carbachol, 100 ␮M Thapsigargin, 5 ␮M Ionomycin, 5 ␮M EBIO, 490 ␮M

partment of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts. Monochloramine (NH 2Cl), a powerful neutrophil-derived oxidant, mediates excess Cl ⫺ secretion in inflamed intestinal mucosa. NH 2Cl activates a Ca 2⫹-dependent large conductance K ⫹ channel in intestinal smooth muscle, but in the intestinal epithelial cells, our preliminary data show that it does not by itself activate the basolateral K ⫹ channels (a key regulatory site for Ca 2⫹-dependent Cl ⫺ secretion). However, pretreatment with NH 2Cl induced a dramatic enhancement of K ⫹ channel activation by the Ca 2⫹ agonist carbachol, similar to the known “priming” action of cAMP on K ⫹ channel regulation. Aim. To define the mechanism of K ⫹ channel “priming” by NH 2Cl and cAMP. Methods. Basolateral K ⫹ currents (I K) were measured in human T84 intestinal epithelial monolayers apically permeabilized with nystatin (500 U/ml) in the presence of apical-to-basolateral (140 to 5.0 mM) K ⫹ gradient. All I K values are in ␮A/cm 2, means ⫾ SEM, each n ⫽ 3-8; *P ⬍ 0.05 compared to control; NS, not significant (see table). Results. Neither the cAMP agonist forskolin (F, 10 ␮M) nor NH 2Cl (100 ␮M) activated I K. The effect of forskolin or NH 2Cl pretreatment (30 min) on the I K response to agonists of the Ca 2⫹sensitive K ⫹ conductance that act via distinct mechanisms was then examined. Both F and NH 2Cl “primed” the basolateral K ⫹ conductance to stimulation by agonists that act via a rise in intracellular Ca 2⫹ but not to EBIO (1-ethyl-2-benzimidazolinone), which directly binds to and activates Ca 2⫹-dependent K ⫹ channels without altering cell Ca 2⫹. The I K response to EBIO was inhibited by NH 2Cl but not F. Moreover, both F and NH 2Cl significantly left-shifted the I K response to carbachol (not shown, n ⬎ 80). Conclusion. The inflammatory oxidant NH 2Cl “primes” the basolateral K ⫹ conductance to activation by Ca 2⫹-dependent agonists by a mechanism that involves enhanced Ca 2⫹ sensitivity. This action parallels that of cAMP, suggesting a common target of action. Because NH 2Cl inhibits EBIO action, we speculate that these agents directly interact with K ⫹ channels on a common site. The results suggest a mechanism whereby oxidant mediators of inflammation may induce pathological dysregulation of epithelial transport pathways, leading to diarrhea. 96. Extrinsic Neural Innervation Modulates Absorption of Water and Electrolytes in Canine Proximal Colon in Vivo. M. L. Kendrick, M.D., T. Meile, C.M., N. J. Zyromski, M.D., T. Tanaka, M.D., J. A. Duenes, B.A., and M. G. Sarr, M.D. Department of Surgery, Mayo Clinic, Rochester, Minnesota. Introduction. Extrinsic innervation mediates a proabsorptive effect in small intestine but does not modulate the postprandial

augmentation in absorption. Our aim was to determine whether extrinsic neural input modulates similar effects in the proximal colon in vivo. Methods. Ten adult dogs underwent enteric isolation of a 50-cm proximal colon loop; 5 each were randomized to undergo extrinsic denervation (DEN) of the isolated colonic segment or to serve as controls (CON). After recovery, a 38°C ileal-like electrolyte solution (125 mEq/L Na ⫹, 9 mEq/L K ⫹, 75 mEq/L Cl ⫺, 65 mEq/L HCO 3⫺) was infused at 4 ml/min into the segment. Effluent was collected in 30-min intervals for 2 h after achieving steady state (determined by 14C marker recovery); 4 fasting and 4 postprandial studies were conducted 1 and 12 weeks postop. Net flux of H 2O, Na ⫹, K ⫹, and Cl ⫺ was determined. Colon morphometry was evaluated at 0 and 14 weeks. Data are presented as x⫾ SEM. Unpaired t test was applied for comparisons. Results. Net absorptive flux of H 2O (␮l/ min) was decreased in DEN vs CON at 1 week during fasting (260 ⫾ 45 vs 390 ⫾ 40, P ⫽ 0.03) and postprandially (215 ⫾ 30 vs 365 ⫾ 40, P ⫽ 0.02), but was not different at 12 weeks (data not shown). Na ⫹ and Cl ⫺ followed the trends in H 2O absorption (P ⱕ 0.05). Net absorptive flux of H 2O or electrolytes did not increase postprandially in either group. Crypt depth (␮m) decreased in CON at 14 vs 0 weeks (750 ⫾ 0.01 vs 900 ⫾ 0.02, P ⫽ 0.01) but was unchanged in DEN. Conclusions. Loss of extrinsic neural input decreases colonic absorption. This suggests that extrinsic neural innervation provides net proabsorptive mechanisms for absorption of water and electrolytes during fasting and postprandial states in the proximal canine colon. The well-described postprandial augmentation in absorption occurring in the small intestine does not occur in the proximal colon. 97. Upregulation of Trail-Induced Apoptosis May Predisose to Pouchitis. J. C. Coffey, M.B., M. W. Bennett, Ph.D., J. H. Wang, Ph.D., P. Neary, J. O’Connell, F. Shanahan, MRCP, H. P. Redmond, FRCSI, and W. O. Kirwan, FRCSI. Department of Academic Surgery, Cork University Hospital, Cork, Ireland. Restorative proctocolectomy remains the gold standard for patients with ulcerative colitis (UC) and familial adenomatous polyposis (FAP). Significant morbidity is incurred in patients who develop pouchitis. We investigated whether increased epithelial apoptosis could account for the relationship between ulcerative colitis and pouchitis. Pouch biopsy specimens taken from patinets with a history of pouchitis (group A; n ⫽ 12) were compared with age-matched controls from those who were pouchitis free (group B; n ⫽ 12). Villous atrophy was assessed histologically. Epithelial cell apoptosis was detected with a monoclonal antibody, M30, and TUNEL immunohistochemistry. M30 detects a caspase-cleaved neoepitope on cytokera-

TABLE—ABSTRACT 97 Group A

TUNEL M30 Atrophy

Group B

EP

LP

EP

LP

0.091 ⫾ 0.048* 0.225 ⫾ 0.05*

0.057 ⫾ 0.027* 0.087 ⫾ 0.02*

0.0357 ⫾ 0.007* 0.082 ⫾ 0.03*

0.032 ⫾ 0.018* 0.09 ⫾ 0.03*

0.035 ⫾ 0.005*

0.10 ⫾ 0.031*

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS tin 18, a feature of TRAIL (TNF-related apoptosis-inducing ligand)induced apoptosis. Means were compared using the Wilcoxon Rank Sum test with *P ⬍ 0.05 (see table) taken as significant. A significant increase in villous atrophy was seen in group A patients (see table). This correlated with increased levels of TUNEL expression and with increased M30 immunoreactivity, a feature of TRAIL-induced caspase activation. Our data suggests a role for TRAIL-induced apoptosis in the etiology of pouchitis and provides a potential therapeutic target for the understanding of the pathogenesis of this condition. 98. Role of Mitochondrial Respiration during Calcium Signaling in Gastric Parietal Cells. S. A. Chamberlain, M.D., A. M. Hofer, Ph.D., and D. I. Soybel, M.D. Department of Surgery, Brigham and Women’s Hospital and the Boston VA Healthcare System, Boston and West Roxbury, Massachusetts. Gastric parietal cells (PCs) are rich in mitochondria (MTC). MTC generate the ATP that supports H⫹ secretion. However, recent work in other cell types has suggested that MTC function may modulate pathways of Ca 2⫹ entry and cellular mechanisms of Ca 2⫹ homeostasis. To evaluate the role of MTC in Ca 2⫹ handling by PCs, gastric glands were prepared by collagenase digestion and loaded with the Ca 2⫹ probe fura-2-AM. Intracellular [Ca 2⫹] ([Ca 2⫹] i) was measured

using digital fluorescence imaging of individual PCs (see figure). To evaluate the impact of MTC function on Ca 2⫹ reentry and peak accumulation, glands were also exposed to oligomycin (OL, 5 ␮g/ml, n ⫽ 7), which inhibits the F 1-F 0-ATP synthase in the MTC and prevents ATP production or the protonophore FCCP (1 ␮M, n ⫽ 6), which inhibits MTC respiration and collapses its electrochemical gradients. In contrast to other cells, Ca 2⫹entry was not reduced by OL or FCCP, indicating that mitochondrial function may not be essential for sustained Ca 2⫹ uptake from outside sources. As hypothesized, however, both inhibitors markedly increased Ca 2⫹ accumulation in the cytoplasm (increase, OL 192 ⫾ 30%, P ⬍ 0.002; FCCP 289⫾79%, P ⬍ 0.01), indicating that mitochondrial function is required for effective clearing of Ca 2⫹ that accumulates in the cytoplasm. 99. p21 Gene Regulation during Enterocyte Differentiation: Identification of Activator and Repressor Elements. S. Y. Archer, M.D., J. J. Johnson, B.S., and R. A. Hodin, M.D. Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. Enterocyte differentiation is associated with a withdrawal from the cell cycle and transcriptional activation of cell cycle inhibitor, p21. In the present studies, we sought to define the molecular mechanisms involved in p21 gene activation in an in vitro system. Methods. Transient transfections were performed in HT-29 cells, using CaPO 4/DNA coprecipitation, with plasmids containing various 5⬘ deletions of the p21 promoter upstream of the luciferase (LUC) reporter. After 24 h, cells were replaced in medium with or without

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5 mM NaBu or another histone hyperacetylating agent, trichostatin A (TSA, 0.3 ␮M) for 24 h. After protein extraction, luciferase activity measured. Acid/urea/triton (AUT) gel electrophoresis was performed to evaluate the state of histone acetylation in cells. Results. NaBu and TSA both caused histone H4 hyperacetylation as indicated by AUT gel analysis. Both NaBu and TSA caused a marked increase in the transactivation of LUC reporter plasmids containing 291 bp of the p21 promoter upstream of the transcriptional start site (15-fold, P ⬍ 0.001), an induction similar to that previously shown in larger constructs containing up to 2.4 kb of promoter. A decrease in reporter gene expression was seen between 173 and 153 bp. This was followed by a marked increase in promoter activity from 143 to 117 bp (⬵10fold, P ⬍ 0.001). Finally, only low basal activity was seen in the case of the 93-bp plasmid. Conclusion. p21 gene activation during HT-29 cell differentiation occurs via two regions of cis-acting elements: one located between 93 and 117 bp and the other between 173 and 291 bp upstream from the transcriptional start site. A repressor element appears to reside between 143 and 153 bp. Histone hyperacetylation likely plays a role in this activation. 100. Integrin Expression Alters Internalization of Enteric Bacteria by Cultured Enterocytes. D. J. Hess, M.D., B. Feltis, M.D., J. Cromwell, M.D., J. Garcia-Aguilar, M.D., and C. Wells, Ph.D. Department of Surgery, University of Minnesota, Minneapolis, Minnesota. There is evidence that normal enteric bacteria can cause systemic infections in high-risk patients such as postsurgical and trauma patients. There is direct evidence that normal enteric flora can cross the intestinal epithelium in experimental models but the mechanism remains unclear. Because specific enterocyte adhesion molecules have been identified as receptors for the enteric pathogens Listeria monocytogenes and Yersinia species, we investigated the role of two integrin subunits in bacterial internalization by cultured enterocytes. Caco-2 enterocytes were transfected with ␣-2 or ␣-3 integrin cDNA that was in either the sense or antisense orientation. Altered integrin expression was confirmed by Western blot. Mature cultures (15 to 18 day) of 10 6 enterocytes were incubated for 1 h with 10 8 Salmonella typhimurium, L. monocytogenes, Proteus mirabilis, or Escherichia coli and internalization was quantified using the gentamicin protection assay. Compared to both untreated and vector control enterocytes, bacterial internalization was consistently decreased (two- to six-fold) using enterocytes that overexpressed the ␣-2 integrin subunit (P ⬍ 0.05, ANOVA with Fisher’s post hoc). Data for P. mirabilis and E. coli are shown in the figure and represent the

average of at least three assays, each assay representing the average of triplicate determinations. These data indicate that specific enterocyte integrin adhesion molecules may play a greater role than heretofore appreciated in the internalization of enteric bacteria by enterocytes. Clarification of the mechanisms by which enteric bacteria disseminate to extraintestinal sites may suggest novel prophylactic and therapeutic modalities to decrease the morbidity associated with systemic infection caused by normal enteric flora. 101. Growth Regulation of Gut Epithelial Cells by TGF-␤. Y. Cao, M.D., C. Xie, B.S., W. Yu, M.D., C. M. Townsend Jr., M.D.,

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS E. A. Thompson, Ph.D., and T. C. Ko, M.D. Departments of Surgery and Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston, Texas.

Maintenance of normal growth control in the gut epithelium is essential for health, and transforming growth factor-␤ (TGF-␤) is an important growth inhibitor of gut epithelial cells. Recently, Smad2, Smad3, and Smad4 proteins have been identified as important intracellular mediators of TGF-␤ signaling. Mutations or deletions of Smad proteins in the gut are associated with the loss of TGF-␤ responsiveness and the development of colorectal cancers. The purpose of this study was to determine whether increased expression of Smad2, Smad3, and Smad4 enhances the growth-inhibitory effects of TGF-␤ in gut epithelial cells. The long-term goal is to develop strategies to restore TGF-␤ responsiveness in colorectal cancer. Methods. Rat intestinal epithelial cells (RIE-1) were infected with retrovirus, encoding human Smad2, Smad3, or Smad4 plus the puromycin-resistant gene. Each Smad retrovirus construct contained a FLAG epitope to facilitate identification of Smad protein. Puromycin-resistant cells were selected and examined for Smad expression by immunoblotting with anti-FLAG antibody. Cells were treated with 3 ng/ml TGF-␤ for 4 days, and cell numbers were determined daily by a Coulter counter. Results. Puromycinresistant cell lines were established using retroviruses for Smad2, Smad3, and Smad4. Control cells were infected with parental retrovirus. Immunoblotting with anti-FLAG antibody demonstrated the expression of appropriate molecular weight Smad protein for each infected cell line. After 4 days of TGF-␤ treatment, cell number decreased by 77% in cells infected with Smad3 compared to 46% in control cells (P ⬍ 0.01 by ANOVA). Cells infected with Smad2 or Smad4 had 39 and 45% growth inhibition by TGF-␤, respectively. Conclusions. Only Smad3 enhanced TGF-␤ suppression of gut epithelial cell growth. These results suggest that Smad3 has distinct function in TGF-␤ regulation of gut epithelial cells. Furthermore, increased Smad3 expression may be a novel therapeutic strategy for restoring TGF-␤ responsiveness in colorectal cancer.

PARALLEL SESSION III Oncology II 102. Soluble Interleukin 6 Receptor (sIL-6R) Mediates Colonic Tumor Cell Adherence to the Vascular Endothelium: A Mechanism for Metastatic Initiation? J. F. Dowdall, FRCSI, D. C. Winter, M.D., E. Andrews, FRCSI, W. E. Laug, M.D.,* J. H. Wang, Ph.D.,and H. P. Redmond, M.Ch. Department of Academic Surgery, Cork University Hospital, Wilton, Cork, Ireland; and *University of Southern California, Los Angeles, California. Introduction. Recent evidence suggests that endothelial adherence of tumor cells may be more important than transmigration in the formation of metastases. Soluble receptors of interleukin-6 (sIL6R) shed by activated neutrophils exert IL-6 effects on endothelial cells, which are unresponsive in normal circumstances. This study examined the hypothesis that sIL-6R released by surgical stresses increases tumor cell adherence to the endothelium. Methods. Neutrophils (PMN) were stimulated with LPS (200 ␮g), C-reactive protein (50 ␮g CRP), or tumor necrosis factor-␣ (20 ␮g TNF). sIL-6R release was measured by enzyme-linked immunosorbent assay (ELISA). Colonic tumor cells transfected with green fluorescent protein and endothelial cells were exposed to sIL-6R and tumor cell adherence and transmigration were measured by fluorescence microscopy. Results are expressed as means ⫾ SEM for n ⫽ 5 (Mann– Whitney U test for statistical analysis). Results. Basal release of sIL-6R from PMN was 44.7 ⫾ 8.2 pg/ml at 60 min. This was significantly increased by endotoxin and C-reactive protein (131 ⫾ 16.8

and 84.1 ⫾ 5.3, respectively; both P ⬍ 0.05). However, tumor necrosis factor did not significantly alter sIL6R release. Endothelial and tumor cell exposure to sIL-6R increased tumor cell adherence by 71.3% within 2 h (P ⫽ 0.04) but did not significantly increase transmigration, even at 6 h (P ⬎ 0.1). Conclusion. Mediators of surgical stress induce neutrophil release of a soluble receptor for interleukin-6 that enhances colon cancer cell– endothelial adherence. As adherence is now considered to be the key event in metastatic genesis, these findings have important implications for colon cancer treatment strategies. 103. A Heat-Labile 10- to 100-kDa Gelatin-Binding Component of Serum Potentiates Pressure-Stimulated Colon Cancer Cell Adhesion. S. M. Kavic, M.D., and M. D. Basson, M.D., Ph.D. Yale University and the CT VA Health Care System, New Haven, Connecticut. Introduction. Tumor implantation in wounds may cause morbidity after laparoscopic or open surgery. Increased pressure activates colon cancer cell adhesion in vitro. Since serum may collect in surgical wounds, we sought to determine if serum factors contribute to this effect. Methods. SW620 colon cancer cells were allowed to adhere to a collagen I matrix for 30 min under ambient or 15 mm Hg pressure in 0 –10% serum and after various treatments of the serum to further characterize adhesion-promoting factors. Nonadherent cells were gently washed away and adherent cells were counted in ⱖ20 random HPF/well in triplicate wells. Data from similar studies were pooled and are presented as ⫻ ⫾ SE. Results. Serum dose dependently potentiated SW620 pressure-stimulated adhesion, with a maximal 128 ⫾ 2% increase in adhesion compared with ambient pressure conditions at 5% serum concentration. (n ⫽ 9, P ⬍ 0.001) Heat-inactivating the serum at 60°C for 30 min ablated the pressurepotentiating activity of the serum, resulting in adhesion 100 ⫾ 1% of control (n ⫽ 12, P ⬍ 0.012 vs untreated 5% serum) Filtration to remove molecules over 10 kDa produced 102 ⫾ 1% adhesion relative to ambient conditions (n ⫽ 9, P ⬍ 0.001), but filtration to 100 kDa permitted adhesion that was 135 ⫾ 4% of control (n ⫽ 6, P ⬍ 0.003). When the serum was passed over a gelatin–Sepharose column, which binds various bioactive proteins, including fibronectin, adhesion was 100 ⫾ 1% of control (n ⫽ 9, P ⬍ 0.017). Addition of fibronectin to serum-free media did not reconstitute the effect (see figure). Conclusions. Pressure stimulation of colon cancer cell ad-

hesion is potentiated by one or more heat-labile serum components with molecular weight between 10 and 100 kDa which bind gelatin– Sepharose and is not fibronectin. Irrigating serum from surgical wounds may decrease tumor implantation in laparoscopic or open environments. 104. FDG–PET-Guided Surgery for Recurrent Colorectal Cancer: A Feasibility Study. E. E. Zervos, M.D., D. C. Desai, M.D.,

341

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS L. DePalatis, Ph.D., D. Soble, R.N., and E. W. Martin, Jr., M.D. Department of Surgery, Ohio State University, Columbus, Ohio. Introduction. PET scanning is an accepted diagnostic tool for detection of colorectal cancer (CRC). The purpose of this study was to determine whether diagnostic information provided by preoperative PET could be used to detect disease intraoperatively using ␤ and ␥ probes. Methods. Two studies were carried out. First, tumor “phantoms” were created using 62 ␮Ci FDG in a saline-filled basin. ␥ and ␤ hand-held probes were used to determine detection characteristics with regard to probe type, distance from source, and isotope half-life. In a second study, probes were used intraoperatively to detect tumor in 10 patients (with recurrent CRC as detected by preoperative PET) injected with 10 mCi FDG 90 min before surgery. Counts relative to background were determined for each probe as was histopathologic correlation of “probe-positive” tissue. Results. Phantom studies confirmed that FDG detection by each probe was linearly related to source proximity and half-life (table). Preoperative PET positive

TABLE—ABSTRACT 104 Counts per 10 s Distance from source ( 18F half-life) 0 2 4 6 8

cm cm cm cm cm

(0) (1) (2) (3)

␥ probe

␤ probe

1673 (1673) 134 (892) 118 (317) 73 (106) 49

2561 (2500) 777 (1341) 203 (1057) 123 (813) 72

tissue was detected by both probes with tumor:normal count ratios of 1.6 (␤) and 1.5 (␥). All probe-positive tissue was histologically confirmed to be recurrent CRC. Conclusions. Intraoperative detection of CRC using an FDG source and ␤ and ␥ probes correlates with preoperative PET. With further improvements in probe technology, successful differentiation of normal and tumor tissue as shown here may allow for more precise localization and directed resection. 105. Reverse Transcriptase-Polymerase Chain Reaction (RTPCR) Analysis of Histologically Negative Nonsentinel Nodes in Melanoma. W. R. Wrightson, M.D., M. J. Edwards, M.D., J. Albrecht, Ph.D., A. J. Conrad, Ph.D., and K. M. McMasters, M.D., Ph.D. Department of Surgery, University of Louisville, Louisville, Kentucky; and National Genetics Institute (NGI), Los Angeles, California. Most patients with histologically positive sentinel lymph nodes (SLN) have no additional positive nodes found upon completion lymph node dissection (CLND). Therefore, it has been suggested that CLND may not be required for all patients with positive SLN. This study was undertaken to determine the frequency with which nonsentinel nodes contain submicroscopic nodal metastases detected by RT-PCR. Methods. Negative control lymph nodes were obtained from patients with breast and colon cancer. Positive control lymph nodes contained histologic evidence of melanoma. Nonsentinel nodes were harvested from patients undergoing CLND. A 2-mm 3 piece of each lymph node was snap frozen on dry ice. Lymph nodes were evaluated by routine histology. RT-PCR analysis for melanoma markers tyrosinase, gp100, Mart1, and MAGE3 was performed at NGI, with Southern blot detection. The RT-PCR test was considered positive for the presence of melanoma cells if expression of tyrosinase and at least one other marker were detected. Results. RT-PCR analysis detected the presence of melanoma cells in 0/100 (0%) of negative control lymph nodes and 9/10 (90%) of positive control lymph nodes. A total of 97 histologically negative nonsentinel nodes

from 10 patients who underwent completion lymph node dissection for positive SLN were evaluated. RT-PCR analysis was positive in 18/97 histologically negative nonsentinel nodes (19%) from 4/10 patients (40%). Conclusions. When the SLN is positive, the remaining nodes in that basin are at risk for metastatic disease, despite the fact that these nonsentinel nodes are infrequently histologically positive. Lymphadenectomy may reduce the risk of local recurrence by removing submicroscopic nodal metastases. 106. Cancer/Testis Antigen Expression May Correlate with Stage of Progression in Melanoma. M. Patel, B.A., and J. S. Goydos, M.D. Department of Surgery, CINJ, UMDNJ– Robert Wood Johnson Medical School, New Brunswick, New Jersey. Finding tumor-specific antigenic markers has many important uses including microstaging. Melanomas produce many markers, most of which are also found in benign cells. The cancer/testis (CT) antigens (NY-ESO-1, LAGE 1, etc.) are more specific markers since they are only produced by normal testis and neoplasms. One CT antigen, CTp11, was shown to be expressed by a metastatic variant cell line but not by the parent, nonmetastasizing, cell line. We checked for CT antigen expression in melanoma samples obtained from patients with different stages of disease to see if a correlation exists between antigen expression and stage. Melanoma specimens were preserved in liquid nitrogen in the OR and then stored at ⫺80°C. RNA was extracted from the samples and first-strand cDNA

TABLE—ABSTRACT 106 Specimen type

NY-ESO-1 positive

CTp11 positive

Primary tumors Nodal metastasis Distant metastasis

1 4 2

4 1 1

synthesis was performed using a standard kit. PCR was performed using primers designed using the cDNA sequences of tyrosinase, NY-ESO-1, and CTp11 obtained from GenBank. The PCR product was separated on an agarose gel and visualized by ethidium bromide staining and UV illumination. Twenty-eight tyrosinase-positive tumors (8 primary tumors, 12 nodal metastases, and 8 metastatic tumors) were tested (see table). All but 1 of the CTp11-positive patients are alive and disease free (mean follow up 24 months) while all but 1 of the NY-ESO-1-positive patients either died or are alive with disease with the same follow-up. About half of our tumors produced CT antigens. Primary tumors were more likely to be positive for CTp11 while more advanced tumors were likely to be positive for NY-ESO-1. NY-ESO-1 may be a better antigen marker of aggressive disease. 107. Prevention of the Growth and Metastasis of Malignant Melanoma: The Role of Supplemental Vitamin A. C. D. Tattini, M.D., S. Lynch, M.D., A. Spangenberger, B.S., R. Zienowicz, M.D., N. Weinzweig, M.D., L. Edstrom, M.D., and J. Weinzweig, M.D. Department of Plastic Surgery, Rhode Island Hospital, Brown University, Providence, Rhode Island. Purpose. Vitamin A possesses both wound healing-promoting and antitumor actions. The common denominator among these actions is the vitamin A-induced fibroplasia which results in subsequent increased collagen production. Increased collagen deposition has been shown to result in the production of collagenous capsules around several murine breast and lung tumor systems. This tumorencapsulation process can potentially convert a systemic disease to a

342

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

local one that can be easily treated by tumor excision. The goal of the present study was to determine if vitamin A can promote the encapsulation of a murine melanoma. Methods. Sixty DBA/2J male mice were inoculated intracutaneously with 1,000,000 Cloudman S91 melanoma cells using a 30-gauge needle. The mice were divided into three groups: (1) Conrol—no further treatment; all mice in this group were fed a commercial chow containing 15,000 IU vitamin A (VA) and 6.4 mg beta carotene/kg diet, considerably more than the NRCs recommended daily allowance of VA for normal mice; the control diet was, therefore, not VA-deficient. (2) Pre-VA—mice were fed the basal chow supplemented with 150,000 IU of vitamin A/kg diet for 10 days prior to inoculation and for the remainder of the study.; (3) PostVA—mice were fed the VA-supplemented diet beginning on the day of inoculation and continued for the remainder of the study. Sixty days following inoculation, the mice in each group were euthanized. Ventral skin at the site of inoculation was harvested for histological assessment of local tumor growth and invasiveness. The liver and lungs of each of these mice were also harvested for histological assessment of tumor metastasis. Survival data and tumor size were also recorded. Results. (See table.) Conclusions. These data indi-

TABLE—ABSTRACT 107 60 Days Postinoculation

Group

Survival (%)

Mean tumor size (mm)

Gross tumor (%)

Metastases (%)

Control Pre-VA Post-VA

60 100 100

26.1 0.0 14.2

100 0 40

25 0 0

cate a potential prophylactic as well as therapeutic role for supplemental vitamin A in the treatment of malignant melanoma. 108. Microsatellite DNA Changes at Chromosome 4 in Benign Breast Epithelium from Risk-Defined Women. D. M. Euhus, M.D., N. Shivapurkar, M.D., S. Milchrub, M.D., G. N. Peters, M.D., A. M. Leitch, M.D., A. R. Richey, B.S., and A. F. Gazdar, M.D. Division of Surgical Oncology, UT Southwestern Medical Center at Dallas, Dallas, Texas. Genetic instability is a hallmark of many malignancies including breast cancer. Whether DNA damage is a cause or consequence of malignant transformation is uncertain. Identification of DNA alterations in “normal” breast epithelium from women at high risk for breast cancer would implicate these changes in the pathogenesis of the disease. Random FNA breast biopsy was performed in 22 women completing a comprehensive computer-assisted breast cancer risk assessment. After cytological evaluation, a total of 66 epithelial cell clusters (1– 6/woman) were microdissected using laser capture and microsatellite DNA evaluated by polymerase chain reaction (PCR) using markers specific for chromosome 4 (D4S404, D4S194, D4S2366, D4S1652, D4S1584, and D4S2397). Loss of heterozygosity was detected in 9 of 22 women (41%). Four of the 5 women (80%) with atypical cell clusters were found to have LOH compared to only 5 of 17 women (42%) without atypia (P ⬍ 0.05). The mean Gail risk index was 2.04 for women with LOH compared to 1.42 for women without LOH (P ⫽ 0.04). The 4p locus D4S404 was the most frequently informative and most frequently altered allele. LOH at chromosome 4 correlates with epidemiological and cytological measures of breast cancer risk. LOH may be a very early event in breast cancer pathogenesis. Evaluation of breast epithelial cell clusters using PCR-based techniques may provide an approach for individual molecular risk assessment and for monitoring interventions designed to decrease risk.

109. Focal Adhesion Kinase (FAK) Is Increased in Human Prostate Cancer Specimens and Has Tissue-Specific Localization. J. D. Rovin, M.D., H. F. Frierson, M.D.,* W. Ledinh, J. T. Parsons, Ph.D.,† and R. B. Adams, M.D. Departments of *Pathology, †Microbiology, and Surgery, University of Virginia, Charlottesville, Virginia. FAK is a principal effector regulating adhesion-mediated signals and cell growth. FAK activity is increased in many human cancers and a positive correlation is observed between increased FAK expression and malignant behavior. Herein, we report FAK distribution, cell specificity, and relative expression in normal and neoplastic human prostate tissue. FAK expression and tissue specificity were determined using FAK-specific antibodies and standard immunohistochemical techniques to evaluate archival formalin-fixed paraffin-embedded human prostate tissue. Antibody specificity was verified by immunoblotting several different cell types and prostate cancer cell lines of varying malignant potential. Normal prostate tissue, benign prostatic hyperplasia (BPH), moderately to poorly differentiated prostatic carcinomas, and prostate cancer metastases (lymph node and bone) were evaluated. Cell blocks of prostate cancer cell lines with increasing malignant potential were evaluated for FAK staining. The basal cell (proliferative) layer stained intensely, while the secretory (differentiated) layer of normal prostate epithelium was negative. The surrounding stroma (smooth muscle cells) stained intensely for FAK. Normal lymphocytes (negative control) within the prostate did not stain for FAK, consistent with Western and Northern blot analysis that lymphocytes contain little or no FAK. BPH stained identically to normal prostate tissue. Prostatic intraepithelial neoplasia and invasive carcinomas of various grades were all positive for FAK. Similarly, metastatic foci of prostate cancer stained intensely for FAK. In the prostate cell lines, there was a positive correlation between levels of FAK expression and their malignant behavior in vivo. Intensity of FAK staining was similar for all neoplastic cells and did not differentiate the grade or stage of the tumors. These results demonstrate a differential tissuespecific distribution of FAK in normal and neoplastic prostate tissue. Elevated FAK expression appears to be an early event in prostate tumorigenesis. The similarity in staining between prostate cancer and the basal epithelial layer suggests a correlation of FAK expression with a less well differentiated or more highly proliferative cellular compartment compared to a relatively quiescent compartment like the secretory layer in normal prostate epithelium.

PARALLEL SESSION IV Cardiothoracic/Transplant II 110. Open-Heart Endocardial Radiofrequency Ablation as an Alternative to Incisions in Creating an Atrial Maze. J. A. Caccitolo, M.D., J. M. Stulak, M.S., D. Francischelli, D. N. Jensen, D.V.M., R. Mehra, Ph.D., and H. V. Schaff, M.D. Mayo Clinic, Rochester, Minnesota, Medtronic, Inc., Minneapolis, Minnesota. Radiofrequency (RF) ablation produces transmural atrial lesions in vitro and may provide advantages over incisions currently used in maze surgery. This study examines the feasibility, safety, and efficacy of open-heart endocardial RF-atrial ablation in surviving animals. Eighteen healthy sheep (42.8 ⫾ 4.4 kg, age ⬍ 2years) underwent left thoracotomy with placement of pacing leads on one of the pulmonary veins and dome of the left atrium. Cardiopulmonary bypass was established and atrial lesions were made using incision and suture or a novel, hand-held RF ablation device. Lesions were created in three sites: PVC, a circle excluding

343

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS the pulmonary veins; IAB, a line across the interatrial bundle; and SVC, a line from the superior to the inferior vena cava. Pacing across the PVC lesions was attempted to assess the completeness of each lesion. Preselected animals (incision n ⫽ 4, RF n ⫽ 5) were recovered and pacing attempts were repeated at 1 month. After sacrifice, all hearts were sectioned and for measurement of lesion size and completeness. RF ablation lesions took less time to create than the incision and suture lesions (total bypass time, RF 51.8 min vs incision 106 min; P ⬍ 0.001). No evidence of thromboembolism, atrial rupture, or coronary sinus thrombosis was seen. In all animals, PVC lesions were complete as demonstrated by the inability to pace across them. Stained sections demonstrated that acutely studied incision lesions were thinner than RF lesions; however, all lesions were transmural and similar in width at 1 month. RF ablation consistently created transmural lesions more quickly than the incision and suture method and without additional complications. Endocardial RF ablation appears to be a simple, quick, and effective alternative to surgical incisions during open-heart atrial maze procedures. 111. Ventricular Dysfunction Following Reoxygenation of Hypoxic Myocardium Is Associated with Peroxynitrite Formation. J. M. Pearl, M.D., C. J. Wagner, B.S., D. P. Nelson, M.D., and J. Y. Duffy, Ph.D. Children’s Hospital Medical Center, Cincinnati, Ohio. The relationship of peroxynitrite formation to myocardial injury and ventricular dysfunction after hypoxia and reoxygenation is controversial. We hypothesized alterations in nitric oxide (NO) levels, related in part to inducible NO synthase (iNOS) upregulation during hypoxia, might result in myocardial peroxynitrite formation associated with depressed ventricular function. Methods. Neonatal piglets underwent 90 min hypoxia and 60 min reoxygenation by CPB (n ⫽ 10) or by ventilating with 100% FIO 2 (vent, n ⫽ 5), and 2 h recovery. Additional animals underwent CPB without hypoxia (control, n ⫽ 6). LV myocardium was stained for NT and blindly scored on a 1–5 scale based on localization and intensity of immunohistochemical staining. Systolic LV function (dP/dt) was monitored throughout the experiment. Myocardial iNOS mRNA was also determined by ribonuclease protection assay. Results. Myocardium from animals subjected to reoxygenation had increased NT scores (CPB 3.3 ⫾ 0.96, vent 3.2 ⫾ 0.82) compared to control animals (0.79 ⫾ 0.27, P ⬍ 0.005). The intensity and localization of NT staining were similar regardless of the method of reoxygenation. There was also a 2.3- and 2.0- fold increase, respectively, in LV iNOS mRNA in CPB and ventilator reoxygenated animals compared with controls (P ⬍ 0.05). LV dP/dt decreased after reoxygenation in CPB (653 vs 1255 mm Hg/s, P ⬍ 0.01) and vent (624 vs 1328 mm Hg/s, P ⬍ 0.005) animals, respectively, but did not differ from baseline in controls (1149 vs 1263 mm Hg/s). Conclusion Reoxygenation of hypoxic myocardium resulted in upregulation of iNOS mRNA and dramatic peroxynitrite formation indicated by NT localization, which was associated with ventricular dysfunction after reoxygenation. Prevention of peroxynitrite formation by blocking NO production or preventing generation of reactive oxygen species might result in improved ventricular recovery following hypoxia and reoxygenation. 112. Effects of Aging on Vascular Response to Injury: Aging Milieu versus Vessel Senescence. M. T. Calfa, M.D., A. Aitouche, Ph.D., C. Gay-Rabinstein, M.D., S. Li, M.D., and Si Mai Pham, M.D. Department of Surgery, University of Miami School of Medicine, Miami, Florida. Background. Cardiovascular disease is the most common cause of death in aging population. Aging has been shown to be associated with exaggerated response to vascular injury. Objective. Our objective is to determine whether the exaggerated vascular response associated with aging is due to the intrinsic property of the vascular

smooth muscle cells or the aging milieu. Methods. Young (2-monthold) and aging (22-month-old) Fischer (F344) rats were used as donors and recipients. Balloon-injured aortas were crosstransplanted between sex-matched, young, and aging rats. Aortas were harvested 30 days after transplantation, and the neointima/ media (I/M) ratio was calculated by computer-aided morphometry. Statistical analysis was performed using analysis of variance and T test with Bonferroni correction. Results. Injured aging aortas developed less neointimal thickening when transplanted (parked) into a young recipient than when transplanted into an aging one (I/M ⫽ 0.26 ⫾ 0.11 vs 0.54 ⫾ 0.17; P ⫽ 0.01). Similarly, injured young aortas parked in an aging recipient developed a more pronounced neointimal hyperplasia compared with those placed in a young one (I/M ⫽ 0.55 ⫾ 0.25 vs 0.25 ⫾ 0.12; P ⫽ 0.005). Conclusions. These data support the hypothesis that the aging milieu, rather than intrinsic vascular senescence, is responsible for the exaggerated vascular response to injury associated with aging. The inference is that by altering the aging milieu, one may be able to modify this exaggerated vascular response.

113. Rat Aortic Smooth Muscle Cell Density Affects Activation of MAP Kinase and Akt by Menadione and PDGF Homodimer BB. X. A. Li, Ph.D., C. Bianchi, M.D., Ph.D., and F. W. Sellke, M.D. Division of Cardiac Surgery, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. MAPK and Akt activities are important in regulating vascular smooth muscle cell (SMC) proliferation, differentiation, and apoptosis, which play critical roles in many pathological conditions such as atherosclerosis and postangioplasty restenosis. We examined whether cell density in culture affects activation of distinct yet cross-talking signal transduction pathways induced by oxidative stress (menadione, M) and growth factor (PDGF), stimuli present in many vascular diseases. Rat aortic SMC were plated sparsely or confluent and incubated with either M (100 ␮M) or PDGF (100 ng/ml) alone and together. Total cell lysates were analyzed by SDS– PAGE and immunoblotting. In sparse cells, M and PDGF synergistically activated ERK 1/2 (measuring phospho-ERK) (see table). In

TABLE—ABSTRACT 113 ERK1/2 activity:

None

2 mM DTT

Treatment:

⫺

M

P

M⫹P

⫺

M

P

M⫹P

Sparse cells (-fold) Confluent cells (-fold)

1 1

44 13

7 4

70 14

1 1

1 4

31 6

16 4

confluent cells, M and PDGF’s activation of ERK 1/2 was, at most, additive (see table). MEK activation (measuring phospho-MEK) correlated with ERK 1/2 activation. M and PDGF also activated p38 MAPK, but without synergism. PDGF induced Akt activation (measuring phospho-Akt), but M completely blocked PDGF-induced Akt activation independent of cell densities. Coincubation with reducing agent DTT inhibited M’s activation of ERK, removed its synergism with PDGF, and reversed its inhibition on PDGF-induced Akt activation. In summary, with SMC the state of cell confluence and/or cell density determine whether distinct pathways of ERK activation (oxidation and growth factors) cross-talk. PDGF functions as a surviving factor by inducing phospho-Akt, whereas oxidative stress may promote apoptosis by inhibiting PDGF-induced Akt activation. Furthermore, effects of oxidative stress on ERK and Akt activation depend on cell’s redox status.

344

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 114 Day 7

MCP-1 MIP-1a MIP-1b RANTES

Day 14

Day 21

Day 35

Day 45

⫹CMV

⫺CMV

⫹CMV

⫺CMV

⫹CMV

⫺CMV

⫹CMV

⫺CMV

⫹CMV

⫺CMV

G

N

G

N

G

N

G

N

G

N

G

N

G

N

G

N

G

N

G

N

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹

⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹

⫺ ⫹⫹ ⫹⫹ ⫺

⫹⫹ ⫹⫹ ⫹ ⫹

⫹⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫹⫹ ⫺

⫺ ⫺ ⫹ ⫺

⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹

⫹ ⫹⫹ ⫹ ⫹

⫹ ⫹⫹ ⫺ ⫺

⫹ ⫺ ⫺ ⫺

⫹⫹⫹ ⫹⫹ ND ⫹⫹

⫹⫹ ⫹ ND ⫺

⫹ ⫹⫹ ⫹⫹ ⫹

⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹

⫹ ⫹ ⫹ ⫺

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹

⫹⫹ ⫹⫹ ⫹ ⫹

Note. G, graft heart; N, native heart; ND, not done. 114. The Role of Chemokines in the Development of CMVAccelerated Transplant Vascular Sclerosis. V. T. De La Melena, M.D., C. N. Kreklywich, D. N. Streblow, Ph.D., Q. Yin, M.D., J. W. Cook, M.D., J. A. Nelson, Ph.D., and S. L. Orloff, M.D. Department of Surgery, Division of Abdominal Transplantation, Hepato-biliary Surgery, Oregon Health Sciences University, Portland, Oregon. The mechanisms by which cytomegalovirus (CMV) accelerates transplant vascular sclerosis (TVS) are unclear. Chemokines are inducible cytokines that promote cellular migration and activation. We aim to determine the role of CMV and chemokine expression in the acceleration of TVS formation in a rat chronic heart transplantation model. Methods. F344 hearts were transplanted into Lewis recipients treated with low-dose CsA (POD 0 –10). Graft recipients were infected with CMV (10 5 PFU, ip, POD 1). Noninfected graft recipients served as controls. Grafts and native hearts were harvested at days 7, 14, 21, 35, and 45. TVS was assessed as the mean percentage of vessel occlusion (neointimal index, NI). RT-PCR and nested-PCR amplification were used to detect chemokines and CMV respectively, in the grafts and native hearts. Results. Graft vessels showed endothelialitis in the CMVinfected, but not in the uninfected recipients at days 7 and 14. TVS was detected at day 21 with no difference between infected and uninfected recipients (NI ⫽ 38 vs NI ⫽ 35, P ⫽ ns). However, at days 35 and 45, CMV-infected graft vessels showed a significant increase in the severity of TVS (NI ⫽ 64 and 82) compared to uninfected grafts (NI ⫽ 30 and 43, P ⬍ 0.001). Viral DNA was detected in the graft and native hearts as early as 7 days postinfection and at all time points. Chemokine profiles in graft and native hearts differ with or without CMV infection (see table). Conclusion. Early events (endothelialitis) as well as increased and accelerated chemokine expression throughout the development of TVS coupled with the presence of virus at all stages leads to the acceleration of this disease. This is likely due to chemokineenhanced graft heart recruitment of inflammatory and smooth muscle cells. 115. Influence of ex Vivo Graft Irradiation on Canine Small Bowel Transplantation. T. Ishikawa, M.D., K. Iwanami,

M.D., T. Okuda, M.D., Y. Zhu, M.D., A. Fukuda, M.D., S. Zhang, B.S., J. Ou, M.D., M. Nalesnik, M.D., N. Murase, M.D., and R. Venkataramanan, Ph.D. Starzl Transplant Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. Background. Despite the recent progress in immunosuppressive protocols, small bowel remains as an immunologically difficult organ for transplant. To improve outcome of small bowel transplantation (SBTx), modulation of posttransplant immune reactions with graft irradiation (GIR) is a simple and reasonable approach. However, tissue injuries may be caused by GIR. Using canine SBTx model, this study investigated the influence of GIR on intestinal absorption, graft histopathology, and clinical outcome. Methods. Outbred hound dogs underwent autotransplantation of the entire small bowel either with (n ⫽ 5) or without (n ⫽ 5) ex vivo 7.5-Gy GIR. Before and after reperfusion, and after sacrifice at day 30, bowel samples were taken for mucosal enzyme assay and histopathology. Pharmacokinetics of oral CsA (10 mg/ kg) or tacrolimus (FK506, 1 mg/kg) was studied at days 1, 7, 14, and 28. Results. One hour after SBTx, epithelial apoptosis slightly increased, but graft bowels were well preserved. GIR did not induce further histopathological abnormalities of bowel grafts. There was also no evidence of arterial changes associated with GIR at sacrifice. Body weight gain was comparable between groups (see table). Interestingly, however, maximal blood concentration (C max , ng/ml) and area under the curve (AUC, ng * h/ml) of CsA significantly decreased for the first 7 days after SBTx, compared to those of normal animals (see table). In contrast, these parameters of tacrolimus markedly increased early after SBTx. Similar changes were also seen after GIR. Pharmacokinetics of CsA and tacrolimus returned to normal in both groups by day 14. Intestinal cytochrome p450 3A activity was similar between groups and was not affected by GIR. Conclusions. Early after SBTx, intestinal absorption of CsA was significantly inhibited, whereas that of tacrolimus dramatically increased. GIR did not induce further pharmacokinetic changes. GIR seemed to be a safe procedure without causing histopathological or nutritional abnormalities.

TABLE—ABSTRACT 115 CsA (days 1–7)

FK506 (days 1–7)

Group

C max

AUC

C max

AUC

% BW day 28

Normal SBTx SBTx/GIR

1592 ⫾ 38 867 ⫾ 346* 811 ⫾ 132*

9245 ⫾ 781 6405 ⫾ 182* 5871 ⫾ 160*

18.4 ⫾ 5.9 60.5 ⫾ 18* NT

98 ⫾ 42 724 ⫾ 328* NT

6.1 ⫾ 5.3 ⫺4.2 ⫾ 9.2 ⫺1.1 ⫾ 8.5

* ⬍ 0.05.

345

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS 116. In Vitro T Cell Proliferation from Human Kidney Transplant Biopsies with Unremarkable Pathology: New Strategies for an Old Problem. C. R. Smith, M.D., N. J. Poindexter, Ph.D., N. S. Steward, K. C. Lu, M.D., D. C. Brennan, M.D., G. G. Singer, M.D., M. D. Jendrisak, M.D., S. Shenoy, M.D., Ph.D., J. A. Lowell, M.D., T. K. Howard, M.D., and T. Mohanakumar, Ph.D. Departments of Surgery and Medicine, Washington University School of Medicine, St. Louis, Missouri. Acute rejection represents an important event determining 1- and 5-year renal allograft survival. The goal of this study was to determine whether in vitro cell growth of biopsy material correlated with clinical pathology. Kidney needle biopsies (n ⫽ 102) from 66 human renal transplant recipients were analyzed over a 6-year period (1992–1997). Biopsies were placed in complete culture medium with 20 U/ml of IL-2. Wells were examined at 4, 24, and 48 h and cell growth was rated accordingly: A ⫽ no growth, B ⫽ scattered cells, C ⫽ occasional contiguous cells, and D ⫽ colonies of confluent cells. Cells were analyzed for phenotype by FACS using fluorescentconjugated antibodies to CD3, CD4, and CD8. A retrospective chart analysis for clinical correlation was subsequently conducted on these patients. The most common clinical presentations included asymptomatic elevations in creatinine (61%) and hypertension (14%). Twenty-six percent of all biopsies (27/102) documented no evidence of rejection. Fifty-six percent of these patients (15/27) had cultures at 4 h demonstrating no cell growth (Grade A, P ⬍ 0.001), while mild growth (Grade B) was demonstrated in 44%. Interestingly, 25% (3/12) of those patients with Grade B growth and unremarkable histology went on to have a pathologically confirmed rejection episode within approximately 1 month (23 ⫾ 16.9 days). No evidence of significant proliferation (C or D rating) was observed for this group. When biopsy reports documented rejection (74%, 75/102), cell growth (B–D) was observed for 93% (70/75) of specimens at 4 h (P ⬍ 0.001). Cyclosporine toxicity was demonstrated in 40% (2/5) of patients with positive biopsies but no in vitro growth. T cell phenotypes of graft infiltrating lymphocytes were 73% CD4 ⫹ and 16% CD8 ⫹, on average, when pathology documented no rejection, and 65% CD4 ⫹ and 22% CD8 ⫹ when rejection was documented. These results suggest that a subgroup of patients with unremarkable biopsies and significant in vitro proliferation may be at risk for impending rejection and may warrant repeat biopsy within 2–3 weeks and/or specific tailoring of immunosuppressive therapy. Additionally, in vitro growth within 4 h correlates well with acute rejection (Grade I–V, r ⫽ 0.551). 117. Tacrolimus-Based Nonlethal Conditioning Leads to Stable Mixed Chimerism and Donor-Specific Tolerance to Lung Allografts in Rats. M. Thanikachalam, M.D.,* S. Li,* A. D. Murdock, M.D.,† Y. Kurimoto, M.D.,* J. S. Gammie, M.D.,† A. Zeevi, M.D.,† S. Yousem, M.D.,† and S. M. Pham, M.D.* †Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania; and *Department of Surgery, University of Miami, Miami, Florida. Previously, we have reported that mixed bone marrow chimerism leads to donor-specific lung transplantation tolerance without long-

term use of immunosuppressive drugs. However, the radiation dose (1000 cGy) required is too toxic for clinical use. This study aids in determining whether radiation dosage can be reduced to a clinically acceptable level. Methods. Fully mismatched ACI and Wistar Furth rats were used as donors and recipients, respectively. Recipients were administered 10 mg of anti-lymphocyte serum on day ⫺5, tacrolimus 1 mg/kg, im, from day ⫺1 to day 10, and 500 cGy of total body irradiation prior to bone marrow transplantation (BMT; day 0) with 100 ⫻ 10 6 of T-cell-depleted bone marrow cells. Levels of donor chimerism were determined 4 weeks after marrow transplantation. Chimeras underwent single left lung transplants at 6 weeks after BMT. Chest X-ray and histology determined lung-graft survival. Long-term bone marrow engraftment was monitored by flow cytometry. To determine in vitro tolerance, mixed lymphocyte reactions (MLR) were performed. Results. Reliable, long-term (⬎16 months) donor bone marrow engraftment was observed. The mean level of donor chimerism in the peripheral blood was 24%. There was no graft-versus-host disease. Chimeras demonstrated long-term donorspecific transplantation tolerance to lung grafts (⬎300 days) (Group 3). Chimeras were immunocompetent to reject third-party lung grafts (Group 4). MLR showed donor-specific hyporeactivity (see table). Conclusions. In this study, we demonstrate that short-term course of tacrolimus-based conditioning regimen leads to stable mixed bone chimerism and donor-specific tolerance to rat lung allografts at a much reduced radiation dose of 500 cGy.

PARALLEL SESSION IV Peripheral Vascular III 118. The Mitochondriopathy Of Claudication Is Produced By Peripheral Arterial Disease (Pad) And Not Associated Comorbidities. I. I. Pipinos, M.D., T. Denson, B.S., A. D. Shepard, M.D., P. V. Anagnostopoulos, M.D., A. Katsamouris, M.D., and M. D. Boska, Ph.D. Departments of Surgery and Neurology, Henry Ford Hospital, Detroit Michigan. Introduction. The etiology of the defective calf skeletal muscle mitochondria seen in patients (pts) with claudication is controversial. Recent reports have ascribed these changes to the frequent comorbidities (e.g., diabetes, hypertension, dyslipidemia) found in these pts and not PAD. To investigate this question we examined calf muscle mitochondrial function in the limbs of pts with unilateral PAD. Methods. Phosphorus magnetic resonance spectroscopy ( 31P MRS) was used to evaluate calf muscle bioenergetics in the legs of 10 pts with multiple comorbidities and unilateral PAD (ankle– brachial index ⬍ 0.8 but ⬎ 0.5 in the symptomatic limb and ⬎ 0.95 in the asymptomatic limb). A 90-s, in-magnet, isometric study exercise (specifically designed to avoid the confounding effects of muscle ischemia) was used. Phosphocreatine (PCr) and adenosinodiphosphate (ADP) recovery rate constants (rrc), two very sensitive measures of oxidative mitochondrial function, as well as intracellular pH, were determined and results from both limbs were compared (paired t test). Results. End exercise intracellular pH (7.10 ⫾ 0.01 vs

TABLE—ABSTRACT 117 Group

n

Treatment

Donor

Recipient

Chimerism

Survival days

Histograde a

1 2 3 4

5 5 8 5

No (No BM) Yes Yes

ACI ACI ACI Lewis

WF WF WF WF

0% 0% 24% (15–55) 18% (10–27)

⬍10 ⬍10 ⬎300 ⬍10

4 4 1# 4

a #

Rejection grade on a scale of 0 (no rejection) to 4 (severe rejection). P ⬍ 0.01 vs groups 1, 2, and 4 (Mann–Whitney U test).

346

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

7.10 ⫾ 0.01) was the same in both legs, confirming that the test exercise did not significantly reduce oxygen supply and induce anaerobic glycolysis. PCr and ADP rrc were significantly slower in the symptomatic extremity (see table). Conclusions. Calf skeletal mus-

120. Chloroquine as an Inhibitor of 3-AminopropanalInduced Tissue Ischemia in the Rat Hindlimb. J. R. Syrek, M.D., S. Ivanova, Ph.D., S. Tabibzadeh, M.D., S. Friedman, M.D., L. Scher, M.D., and K. J. Tracey, M.D. Department of Surgery, North Shore University Hospital, 300 Community Drive, Manhasset, New York.

TABLE—ABSTRACT 118

Ankle–brachial index PCr rrc (s ⫺1) ADP rrc (s ⫺1)

Symptomatic extremity

Asymptomatic extremity

P value

0.67 ⫾ 0.03 0.021 ⫾ 0.003 0.032 ⫾ 0.007

1.01 ⫾ 0.02 0.036 ⫾ 0.003 0.057 ⫾ 0.011

⬍0.001 0.007 0.07

cle mitochondrial function is significantly worse in the symptomatic limbs of pts with unilateral PAD than it is in their asymptomatic limbs. PAD, rather than associated comorbidities, is the primary cause of the mitochondriopathy seen with claudication. 119. Inosine Attenuates Tourniquet-Induced Skeletal Muscle Reperfusion Injury. A. Wakai, M.B., D. C. Winter, M.D., J. T. Street, M.B., R. G. O’Sullivan, M.B., J. H. Wang, Ph.D., and H. P. Redmond, M.Ch. Department of Academic Surgery, Cork University Hospital, Cork, Ireland. Although inosine, a stable metabolite of adenosine, causes mast cell degranulation, it has multiple anti-inflammatory effects. The aim of this study was to ascertain whether inosine modulates skeletal muscle reperfusion injury. Anesthetized male C57BL/6 mice were randomized (n ⫽ 10) to receive an intraperitoneal injection of inosine (100 mg/kg) or drug vehicle (phosphate-buffered saline). Thirty minutes later, animals underwent 2-h bilateral tourniquet hindlimb ischemia followed by 3 h of reperfusion. A control group (n ⫽ 8) was anesthetized for the duration of the study without ischemia or reperfusion. Serum tumor necrosis factor (TNF)-␣ and macrophage inflammatory protein (MIP)-2 were measured before ischemia and at the end of reperfusion, using enzyme-linked immunosorbent assays. The lungs and muscle (right gastrocnemius) were harvested at the end of reperfusion. Wet/dry weight ratios and myeloperoxidase (MPO) content were used to estimate tissue edema and neutrophil sequestration, respectively. At the end of reperfusion, inosine pretreatment resulted in lower MPO levels in muscle (P ⫽ 0.02) and lung (P ⫽ 0.0002) than saline pretreatment. Similarly, muscle (P ⫽ 0.04) and lung (P ⫽ 0.02) wet/dry ratios were significantly reduced with inosine than saline pretreatment. The table

TABLE—ABSTRACT 119 Preischemia

MIP-2 (pg/ml) TNF-␣ (pg/ml)

End of reperfusion

1/R ⫹ saline

I/R ⫹ inosine

I/R ⫹ saline

I/R ⫹ inosine

10.2 ⫾ 2.4 4.6 ⫾ 0.9

11.8 ⫾ 1.5 4.8 ⫾ 1.0

24.8 ⫾ 5.4* 14.8 ⫾ 2.2*

4.4 ⫾ 2.3* 1.2 ⫾ 0.9*

Note. Data are means ⫾ SEM; *P ⬍ 0.05 vs preischemia. Statistical comparison by ANOVA. Significance at P ⬍ 0.05.

shows the results with respect to serum proinflammatory cytokine levels. These findings demonstrate that inosine attenuates local muscle and distant lung leucosequestration and edema associated with skeletal muscle reperfusion injury.

Introduction. 3-Aminopropanal (3-AP) is a cytotoxic end-product of polyamine catabolism, which is produced in excess during tissue ischemia by the enzyme polyamine oxidase (PAO). The PAO inhibitor chloroquine has been shown to be cerebroprotective in an animal model of cerebral ischemia. However, whether chloroquine also confers protection during hindlimb ischemia was previously unknown. Methods. Male Sprague–Dawley rats were subjected to complete left hindlimb ischemia (HLI) by ligation of all branches on the left side of the infrarenal aorta and left iliac artery and ligation of the left common femoral artery. Animals were injected with chloroquine (25 mg/kg, ip) 1 h after onset of HLI (n ⫽ 7). Vehicle-treated animals were injected ip with an equal volume of saline (n ⫽ 7). After 24 h, blood samples were obtained, and the left and right anterior compartment muscles (ACM) were collected. Serum 3-AP levels were measured by immunoblotting using antibodies directed against 3-AP-modified proteins with comparison to a standard curve. Results. Serum 3-AP levels are summarized in the table. Analysis of

TABLE—ABSTRACT 120 Group

3-AP protein equivalents (mg/ml)

SEM

P value

Sham Chloroquine Vehicle

Undetectable Undetectable 59,764

— — 9.016

— — ⬍0.001*

* P value calculated by one-way ANOVA.

the left ACM in the vehicle-treated group demonstrated histopathologic ischemic changes: tissue edema, inflammatory infiltrates, and loss of muscle cross-striations. Muscle architecture in the right ACM was normal. In the chloroquine-treated group, both the right and the left ACM demonstrated normal muscle architecture. Conclusion. These results indicate that chloroquine blocks the formation of the cytotoxin 3-AP and inhibits the histologic muscle changes associated with hindlimb ischemia. Inhibition of PAO with chloroquine may become a therapeutic alternative in patients with tissue ischemia. 121. Exercise-Induced Hyperemia Unmasks Regional Blood Flow Deficit in Experimental Hindlimb Ischemia. L. S. Brevetti, M.D., R. Paek, M.D., S. M. Brady, B.A., J. I. Hoffman, M.D., R. Sarkar, M.D., Ph.D., and L. M. Messina, M.D. Division of Vascular Surgery, UCSF, San Francisco, California. Experimental models of hindlimb ischemia are characterized by rapid normalization of resting blood flow (BF) complicating the longterm evaluation of molecular therapies for limb ischemia. Limbs that remain ischemic by angiographic or histologic criteria often have normal rates of resting BF. In a rat model of severe hindlimb ischemia, peroneal nerves were stimulated to allow measurement of exercise-induced regional BF with fluorescent microspheres. Methods. Ischemia was induced in adult rats by ligation of the left common iliac and femoral arteries and their branches. Fluorescent microspheres were injected into the left ventricle before and after 5 min of bilateral peroneal nerve stimulation 3, 10, and 24 days postischemia. The tibialis anterior (TA) and gastrocnemius (GA) muscles were harvested and fluorescence was measured. BF was calculated as ml/min/g of tissue and compared with unoperated controls. Re-

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS sults. The mean muscle mass of the ischemic TA and GA as a percentage of total body weight decreased over time vs control (TA 0.13 ⫾ 0.05 vs 0.25 ⫾ 0.03%, P ⬍ 0.05; GA 0.51 ⫾ 0.27 vs 0.70 ⫾ 0.07%, P ⫽ 0.07 at day 24). Despite clinical evidence of severe hindlimb ischemia in experimental groups, i.e., delayed capillary refill and pressure sores, resting BF rates were not significantly different from control (see table). However, the exercise-induced

TABLE—ABSTRACT 121

Control 3 days 10 days 24 days

TA at rest

TA after exercise

GA at rest

GA after exercise

0.156 ⫾ 0.11 0.114 ⫾ 0.09 0.137 ⫾ 0.08 0.130 ⫾ 0.08

1.953 ⫾ 1.71 0.118 ⫾ 0.07* 0.255 ⫾ 0.15* 0.601 ⫾ 0.47*

0.115 ⫾ 0.05 0.190 ⫾ 0.11 0.140 ⫾ 0.10 0.146 ⫾ 0.07

0.668 ⫾ 0.33 0.198 ⫾ 0.13† 0.369 ⫾ 0.25† 0.425 ⫾ 0.32†

* P ⬍ 0.05 when compared to control TA after exercise. †P ⬍ 0.05 when compared to control GA after exercise.

hyperemia was dramatically blunted in all of the ischemic groups (see table). Conclusion. Despite findings of muscle atrophy, delayed capillary refill, and pressure sores in this model of hindlimb ischemia, resting BF rates in the ischemic groups were not significantly decreased. Peroneal nerve stimulation resulted in up to 10-fold increase in BF and unmasked a severe deficit in vascular reserve in the ischemic groups. Resting BF is not an accurate reflection of the deficit in models of chronic limb ischemia, and this novel model of exercise-induced hindlimb ischemia will allow better evaluation of molecular therapies to reverse critical limb ischemia.

347

123. Sustecal-Induced Postprandial Hyperemia Is Blocked by Adenosine A 2b Receptor Antagonism in the Rat. P. J. Matheson, Ph.D., N. D. Carricato, B.S., P. D. Harris, Ph.D., R. N. Garrison, M.D., and M. A. Wilson, M.D., Ph.D. Center for Applied Microcirculatory Research & Departments of Physiology & Surgery, University of Louisville & Louisville VAMC, Louisville, Kentucky. Introduction. Postprandial hyperemia or increased gastrointestinal blood flow during nutrient absorption is a complex vascular response that is not completely understood. Prior studies show that Na ⫹-linked secondary active transport of nutrients (D-glucose, L-glutamine) causes vasodilator nitric oxide production via an adenosine (ADO) A 2b receptor-mediated mechanism. We hypothesized that ADO A 2b receptors are involved in the development of postprandial hyperemia during gastrointestinal nutrient exposure (gastric gavage of a nutritionally complete diet). Methods. Anesthetized male Sprague–Dawley rats were cannulated for colorimetric microsphere determination of blood flow using the phantom organ technique. Animals received gastric gavage with Sustecal (2 mL, MeadJohnson). Microspheres were injected at baseline (BL) and 30 and 60 min postgavage. Five minutes prior to and during nutrient gavage, animals received iv infusion of either vehicle alone or alloxazine (ADO A 2b receptor antagonist, 75 ␮g in 1 mL DMSO–saline over 5 min bolus ⫹ 60 min infusion). Differences were determined by ANOVA with Tukey–Kramer HSD (see figure; *P & lt; 0.05 vs BL; †P ⬍ 0.05 vs vehicle). Results. At 30 min, Sustecal gavage increased

122. Growth Hormone Restores Ischemia–ReperfusionInduced Diapragmatic Dysfunction. D. S. Moneley, AFRCSI, M. C. Barry, M.Ch., R. McLaughlin, M.Ch., C. Kelly, M.Ch., and D. J. Bouchier-Hayes, M.Ch., FRCS. Department of Surgery, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland. Previous work has shown that recombinant human growth hormone (rhGH) improved postoperative respiratory function in patients undergoing elective infrarenal aortic aneurysm repair. Our aim was to investigate the effect of short-term ischemia–reperfusion (IR), on diaphragm function and the subsequent role of rhGH in mediating this response. Male Sprague–Dawley rats (n ⫽ 30) were randomized into three groups: (a) control, (b) IR, and (c) IR treated with rhGH for 5 days preoperatively. IR was achieved by placing an infrarenal aortic clamp for 30 min followed by 2 h of reperfusion. Diaphragm muscle contractile function was assessed using electrical field stimulation in a tissue bath. Results are expressed as peak tension and fatigability (see table). We conclude that short-term ischemia–reperfusion causes significant diaphragmatic muscle dysfunction which is prevented by subcutaneous administration of rhGH for 5 days preoperatively.

TABLE—ABSTRACT 122 Group

Twitch (SEM) (g)

Tetanus (SEM) (g)

Control IR IR ⫹ rhGH

242.01 (38.45) 108.55 (7.15) $ 319.14 (30.71)*

605.52 (77.63) 228.12 (14.38) $ 704.39 (45.69)*

* P ⬍ 0.01 vs IR; $ P ⬍ 0.05 vs control. Kruskal–Wallis ANOVA.

blood flow to the antrum, duodenum, jejunum, colon, spleen, and both kidneys. ADO A 2b receptor-blockade significantly reduced or completely prevented all of these effects. At 60 min, blood flow had returned to baseline levels in all organs in each group. Conclusions. These data suggest that ADO A 2b receptors are required for increased gastrointestinal blood flow during nutrient absorption, particularly in the proximal gut (antrum, duodenum, and jejunum). 124. Examination of the Apoptotic Pathway and Proteolysis in the Pathogenesis of Popliteal Artery Aneurysms. T. Jacob, Ph.D., A. P. Hingorani, M.D., E. Ascher, M.D., Y. Gunduz, M.D., and B. Tsemekhim, M.D. Division of Vascular Surgery, Maimonides Medical Center, 4802 10th Avenue, Brooklyn New York. Introduction. Paucity of vascular smooth muscle cells, thinning of the tunica media, and degradation of the extracellular matrix are the characteristics of aneurysmal arteries. We studied the morphology of extracellular matrix and quantified inflammatory cells and elastic lamellae and its fragmentation in popliteal artery aneurysm (PAA). Since programmed cell death plays a major role in determining the cellularity in tissues, we investigated its role in PAA development. Materials and Methods. Ten PAA specimens were obtained from patients undergoing repair. All were males with ages 48

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

to 81 years (mean 67 years). Normal controls were popliteal arteries obtained from patients without PAA undergoing infrainguinal bypass surgery (n ⫽ 8). Standard histochemistry techniques were used to quantitate elastic lamellae, disrupted fragments of elastin, and inflammatory cells in PAA. Proteolytic activity was determined by 10% gelatin gel zymography under nonreducing conditions. Tissue sections were processed by standard immunohistochemistry techniques to detect vascular smooth muscle cells (VSMC), macrophages, T lymphocytes, death-promoting proteins, Bcl-2 family proteins, and PARP. Detection of apoptosis in the arterial tissue was attempted by TUNEL technique. Results. Histochemical studies revealed conspicuous disruption and fragmentation of elastic lamellae in PAA compared to normal arteries. Increased gelatinolytic activity was observed at 92, 72, and 67 kDa in all of the aneurysmal tissues. Anti-␤-actin immunostaining demonstrated a significant decrease of VSMCs in the PAA walls. The control arteries had fewer CD68 ⫹ macrophages and CD3 ⫹ T cells in their media. There was a significant increase in the number of cells undergoing apoptosis in aneurysmal tissue than in the normal vessels, as well as an increased expression of Bax, CPP-32, and Fas. Only aneurysmal arteries showed CD8 ⫹ T cells expressing death-promoting molecules. Conclusions. The results show disruption in the elastic lamellae and increased proteolysis in PAA. VSMCs of PAA show the presence of markers of apoptosis and signaling molecules capable of initiating cell death. Programmed cell death may contribute to the rarefaction of cells in the wall layers of PAA. This loss of VSMCs may contribute to imbalance in the protein profile, causing extracellular matrix degradation. This study indicates that the inflammatory infiltrate expressing death-promoting proteins in the aneurysm tissue may have a key role in the pathogenesis of aneurysmal disease. 125. Analysis of Coagulation Changes Associated with Supraceliac (SC) Aortic Crossclamping (AXC) Using Thromboelastography (TEG). P. V. Anagnostopoulos, M.D., A. D. Shepard, M.D., I. I. Pipinos, M.D., P. A. Chaudhry, M.D., S. B. K. Raman, M.D., T. Mishima, M.D., and H. Morita, M.D. Departments of Surgery & Pathology, Henry Ford Hospital, Detroit, Michigan. Introduction. The etiology of the coagulation changes seen with SC AXC remains controversial; both primary fibrinolysis and clotting factor consumption have been implicated. The cause of these changes was investigated with TEG, a test that measures the vis4coelastic properties of thrombus to dynamically assess coagula-

Increased activity of the intrinsic coagulation cascade during SC clamping was reflected by a lower r value at time b (12.6 ⫾ 3 vs 20.0 ⫾ 3, P ⫽ 0.048) compared to ir AXC. Decreased speed of solid clot formation was noted following unclamping (time c) in the SC group but not the ir group [as defined by an increased k value (ANOVA P ⫽ 0.010) and a decreased ␣ angle value (ANOVA P ⫽ 0.005)]. FBG levels were lower in the SC than in the ir group at times c (P ⫽ 0.013) and d (P ⫽ 0.02), but PT, PTT, and PLT did not differ between the groups at any time points. Conclusions. Thirty minutes of SC AXC does not result in primary fibrinolysis. There is increased clotting activity during SC clamping followed by decreased speed of clot formation and decreased FBG levels after unclamping. These changes are consistent with clotting factor consumption.

PARALLEL SESSION IV Oncology III 126. Antitumor and Antiangiogenic Therapy with Somatostatin Receptor-Mediated in Situ Radiation. S. A. Gulec, M.D., G. J. Drouant, B.S., J. Fuselier, B.S., C. T. Anthony, Ph.D., J. B. Heneghan, Ph.D. J. B. Delcarpio, Ph.D., and E. A. Woltering, M.D. Department of Surgery, Cell Biology and Anatomy, LSUHSC, Peptide Research Laboratories, Tulane University, New Orleans, Louisiana. Expression of somatostatin receptor subtype-2 (sst-2) in angiogenic tumor vessels is homogeneous, while tumor cell expression of this receptor is often heterogeneous. We have developed a novel in vitro three-dimensional tumor angiogenesis model to study the antitumor and antiangiogenic effects of radiolabeled somatostatin analogs (r-SRIF). We hypothesized that targeted in situ radiation with auger electrons from these r-SRIFs would produce receptor-specific cytotoxicity in somatostatin subtype-2-expressing cells. IMR-32 human neuroblastoma (sst-2⫹) and MDA MB-231 human breast cancer (sst-2⫺) xenografts were created in nude mice from monolayer cell cultures. Fragments of these tumors were embedded in threedimensional fibrin gels supplemented with endothelial growth medium. Tumor fragments were treated with 50 ␮Ci/ml of 111In–DTPAJIC 2DL, an sst-2-preferring r-SRIF or an identical volume of medium. Angiogenic initiation (fraction of tumor fragments that developed an angiogenic response), the total angiogenic response (a

TABLE—ABSTRACT 126 Angiogenic initiation

Control group 111 In-DTPA-JIC 2DL

Mean angiogenic score ⫾ SD

Tumor degeneration

IMR

MDA

IMR

MDA

IMR

MDA

24/30 25/30

25/30 24/30

11.9 ⫾ 3.3 Disrupted

12.4 ⫾ 3.9 6.4 ⫾ 2.9

0/30 30/30

0/30 0/30

tion and fibrinolysis. Methods. Eight pigs underwent SC AXC for 30 min; five pigs undergoing 30 min of infrarenal (ir) AXC served as controls. Blood was drawn before AXC (time a), before unclamping (time b), and 5 and 60 min after unclamping (times c and d). TEG and standard coagulation tests [prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen (FBG), and platelet count (PLT)] were performed. Measured TEG parameters included: fibrinolytic index (a measure of fibrinolysis), r value (a reflection of intrinsic coagulation cascade activity), and the k and ␣ angle values (measures of the speed of solid clot formation). Repeated measures ANOVA and t test were used for statistical analysis. Results. There was no difference in the fibrinolytic index at any time point between the two groups.

semiquantitative visual angiogenic index), and the presence or absence of tumor degeneration (vacuolization to tumor lysis) were assessed on Day 14. 111In JIC 2DL induced degeneration in the angiogenic blood vessels arising from both MDA MB-231 and IMR-32 tumor fragments. However, 111In JIC 2DL induced tumor lysis only in the somatostatin receptor-expressing tumor cells (see table). Targeted in situ radiotherapy with radiolabeled somatostatin analogs induces receptor-specific cytotoxicity in both angiogenic blood vessels and somatostatin receptor subtype-2-expressing tumor cells. 127. Blocking NF-␬B Enhances Adriamycin Cytotoxicity. M. E. Murday, M.D., J. Malaty, M.S., S. Rekkas, B.S., P. Hebig,

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS M.S., S. MacKay, Ph.D., L. L. Moldawer, Ph.D., S. Hochwald, M.D., E. M. Copeland, M.D., and D. S. Lind, M.D. Department of Surgery, University of Florida, Gainesville, Florida. Purpose. Chemoresistance may involve the antiapoptotic transcriptional regulator nuclear factor-␬B (NF-␬B). We determined the effect of adriamycin on NF-␬B binding and if inhibition of NF-␬B enhanced adriamycin cytotoxicity in a breast cancer cell line (EMT6). Methods. EMT-6 cells were incubated with adriamycin (Adria) ⫾ the NF-␬B inhibitor MG132 and NF-␬B binding (electrophoretic mobility shift assay, EMSA), DNA synthesis ([ 3H]thymidine uptake), cell viability (MTT assay), and apoptosis (caspase-3 activity) were measured at 24 h. Data represent means ⫾ SEM analyzed by Student’s t test. Results. Adria (0.1 ␮M) upregulated NF-␬B binding, (Fig. 1), while MG132 inhibited NF-␬B binding (data not shown). MG132 also enhanced adriamycin inhibition of DNA synthesis (Fig. 2) and cytotoxicity (Adria ⫹ MG132 25% viability versus Adria (0.1 ␮M) 69% viability, P ⬍ 0.05) while it enhanced caspase-3 activity (Fig. 3). Conclusions. EMT-6 breast cancer cells demonstrate consti-

349

(MH-Lac) were established. mGM-CSF production was measured by ELISA. Cell proliferation in vitro was determined by serial cell counts. Syngeneic mice were inoculated subcutaneously with MH134 parental cells, MH-Lac, MH-GMhi, and MH-GMlo. Tumor size was measured, and tumors were examined histologically. Results. MHGMhi and MH-GMlo produced 505.26 and 93.21 pg/mL of mGMCSF, respectively. Proliferation of mGM-CSF-producing cells in vitro was not different compared to control groups. mGM-CSF-producing tumors were rejected around 2 weeks, and tissue sections demonstrated a significant infiltration of immunoreactive cells (see figure). Conclusion. The expression of mGM-CSF gene by murine hepatoma cells induced an antitumor effect in vivo. This study suggests the potential for gene therapy with GM-CSF as a treatment for hepatocellular carcinoma. 129. Heat Shock Protein 70 Antisense Oligonucleotide Treatment Enhances 5-FU-Mediated Cancer Cell Death. F. Alcocer, M.D., A. Gustin, M.D., J. Salazar, M.D., S. Vickers, M.D., and W. D. Whitley, M.D. Department of Surgery, University of Alabama, Birmingham, Alabama. Introduction. Heat shock protein 70 (hsp70) helps protect the cell during stress. Increased expression may correlate with progression of human cancer. In this study, we hypothesized that treatment with hsp70 oligonucleotide would sensitize pancreatic cancer cells to che-

tutive NF-␬B binding, while inhibition of NF-␬B binding enhances adriamycin cytotoxicity via a caspase-dependent mechanism. Constitutive NF-␬B binding may identify chemoresistant tumors while inhibition of NF-␬B may represent a novel biologically based therapy. 128. Antitumor Effect of Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) Expression by Murine Hepatocellular Carcinoma Cells. A. L. O’Brien, M.D., X. Lin, Ph.D., J. A. Goss, M.D., F. C. Brunicardi, M.D., and P. Seu, M.D. Department of Surgery, Baylor College of Medicine, and The Baylor/Methodist Liver Center, Houston, Texas. Purpose. GM-CSF has been shown to enhance the immunogenicity of tumor cells. In this study, we examined the effect of hepatomaproduced GM-CSF on tumor growth in a murine model. Methods. MH134 murine hepatoma cells were cotransfected with CMV expression plasmids containing cDNA encoding murine GM-CSF or LacZ and neomycin resistance. Two cell lines stably expressing mGM-CSF (MH-GMhi and MH-GMlo) and one cell line stably expressing LacZ

motherapeutic agents. Methods. Pancreatic cancer cell line (ASPC-1) growing in DMEM ⫹ 15% FBS were seeded into 96-well plates and incubated up to 3 days in presence of 12 ␮M hsp70 antisense oligodeoxinucleotides and sixfold dilutions of 5-fluorouracil (5-FU). Proliferation was determined by a colorimetric assay and results are expressed as a percentage of inhibition. Protein extraction and Western blot for hsp70 were performed. Results. ASPC-1 cells constitutively overexpress hsp70 (Fig. 1, thin arrow). Treatment with hsp70 antisense resulted in partial inhibition of hsp70 expression (Fig. 1, thick arrow) and inhibition of proliferation when compared to controls (14,179 ⫾ 655 vs 19,551 ⫾ 267, P ⬍ 0.05, n ⫽ 3). As expected, incubation with 5-FU inhibited cell proliferation in a dosedependent fashion; however, when hsp70 antisense was added, the sensitivity to 5-FU toxicity was markedly increased (Fig. 2). Conclusions. These data provide evidence for a link between overexpression of hsp70 and cancer cell survival. Increased sensitivity to 5-FU toxicity after abrogation of hsp70 should prompt the extension of these studies into in vivo models. 130. Exploitation of Tumor Hypoxia for Targeted Cytokine Expression. N. Hanna, M.D.,* W. Al-Jumaily, Ph.D.,* M. Saunders, M.D.,† R. Salloum, M.D.,‡ H. Mauceri, Ph.D.,‡ R. Weichselbaum, M.D.‡ *University of Kentucky, Lexington, Kentucky; †University of Manchester, Manchester, United Kingdom; and ‡University of Chicago, Chicago, Illinois. Tumor hypoxic fraction is responsible for treatment resistance and

350

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

failures. It has been shown to be an independent prognostic indicator for local recurrence and overall survival. We exploited hypoxic conditions unique to tumors to obtain selective expression of TNF-␣ using a constructed plasmid containing a hypoxia-inducible enhancer (EPO) and a radiation-inducible promoter (EGR-1) linked to TNF-␣. We hypothesize that hypoxic conditions or radiation will result in endogenous production of TNF-␣, which is cytotoxic and may enhances radiation cytotoxicity through generation of reactive oxygen intermediates. MIA PaCa-2 human pancreatic cancer cells were transfected with the Epo.Egr.TNF plasmid using calcium phosphate method. Cells were then exposed to various hypoxic conditions ⫾ irradiation. Conditioned media and cells were harvested at 6, 24, and 36 h after exposure. TNF-␣ levels were measured by ELISA and expressed in picograms per milliliter of medium. No significant levels of TNF-␣ were detected in normal cells or when exposed to hypoxia ⫾ radiation. Cells transfected with the plasmid produced significant levels of TNF when exposed to hypoxia. In early hypoxia, TNF levels at 6, 24, and 48 h were 435, 1097, and 709 pg/ml, respectively. In delayed hypoxia, TNF levels were 908, 526, and 369 pg/ml at the same time points. Escalating doses of irradiation resulted in progressive increase in TNF production. Furthermore, combining radiation with hypoxia resulted in a eightfold increase in TNF production. This gene therapy approach results in significant local production of TNF-␣, which would results in increased tumor cell kill and radiosensitization. Therefore, tumor hypoxia, which exists in almost all solid tumors regardless of their histological origin, can be exploited for targeted tumor specific rather than tissue specific gene expression. Furthermore, spatial and temporal regulation of gene expression can be achieved with radiotherapy by incorporating a radiation inducible promoter in the genetic construct. 131. Chemosensitization of Pancreatic Cancer by Inhibition of the 26S Proteasome. R. J. Bold, M.D., S. Virudachalam, M.S., and D. J. McConkey, Ph.D. Department of Surgery, UCD Medical Center, Sacramento, California; and Department of Cell Biology, MD Anderson Cancer Center, Houston, Texas. Pancreatic cancer is resistant to induction of apoptosis by chemotherapies; agents that regulate sensitivity to apoptosis may lead to chemosensitization of pancreatic cancer. Methods. MIA-PaCa-2 human pancreatic cancer cells were treated in vitro with the 26S proteasome inhibitor PS-341 and levels of the cell cycle regulator p27 KIP1 and apoptosis gene family members (BCL-2, BAX, and BAK) were determined by Western blotting. The cytotoxic effect of gemcitabine was determined by the MTT assay in the presence of various doses of PS-341. MIA-PaCa-2 xenografts (N ⫽ 24) were established in athymic mice and treated with vehicle (control), gemcitabine, PS-341, or the combination of PS-341 and gemcitabine. Tumor area was measured biweekly, and tumor volume at sacrifice. Results. PS-341 increased p27 KIP1 and decreased BCL-2, without effect on BAX or BAK. PS-341 increased the in vitro cytotoxicity of gemcitabine (see below). Gemcitabine treatment resulted in xenografts 41% of control; the addition of PS-341 reduced tumors to 25% of control (see table). Conclusions. Inhibition of the 26S proteasome disrupts cellular content of key regulators of cell cycle progression and apoptotic

TABLE—ABSTRACT 131 PS-341 (nM) Gemcitabine

0

10

100

1000

0 125 ␮M 1000 ␮M

100 ⫾ 10.2 87.7 ⫾ 2.6 39.8 ⫾ 2.8

68.5 ⫾ 4.7 55.3 ⫾ 8.5 25.5 ⫾ 6.5

47.6 ⫾ 4.2 39.5 ⫾ 1.6 32.6 ⫾ 1.9

31.3 ⫾ 3.5 26.2 ⫾ 2.8 18.1 ⫾ 1.4

Note. % Control ⫾ SD.

control leading to increased sensitivity to standard chemotherapeutic agents, such as gemcitabine, in pancreatic cancer. Combination therapy may lead to better response rates. 132. 26S Proteasome Inhibition Limits Growth of Established Pancreatic Cancer Xenografts. S. A. Shah, M.D., M. W. Potter, M.D., R. Ricciardi, M.D., and M. P. Callery, M.D, FACS. Department of Surgery & Cell Biology, UMASS Medical School, Worcester, Massachusetts. Introduction. 26S proteasome inhibition induces apoptosis in some solid tumors (breast, prostate). We have reported similar proapoptotic effects in human pancreatic cancer cells in vitro. We now examine systemic proteasome inhibitor therapy in an in vivo xenograft model of human pancreatic cancer. Methods. BxPC3 human pancreatic cancer cells in log phase of growth were implanted subcutaneously in athymic nu/nu mice. Once tumors became palpable (50 mm 3), grafted mice were randomized to four weekly injections of either vehicle or 1.0 mg/kg of PS-341, a boronic acid proteasome inhibitor. Tumor volumes and body weights were measured biweekly. Forty days later, tumors were harvested and analyzed histologically for apoptosis (TUNEL assay, H & E stain). Tumor levels of p21 Cip1⫺Waf⫺1, a cell-cycle checkpoint protein normally degraded by the 26S proteasome, were determined by Western blot. Statistical analysis included an ANOVA. Results. Systemic 26S proteasome inhibitor delivered either intraperitoneal (ip, 84% reduction) or intravenous (iv, 72% reduction) significantly limited established tumor growth (*P ⬍ 0.005 vs controls) over 40 days (Fig. 1) without causing toxicity (animal weight, activity). Both cellular apoptosis (histology) and p21 Cip1⫺Waf⫺1 protein levels (Fig. 2) were increased in PS-341treated tumors indicating that this systemic therapy was effective at the cancer cell level. Conclusion. Systemic 26S proteasome inhibition induces apoptosis and inhibits the growth of established human pancreatic cancer xenografts. This new biological therapy may have value in the treatment of pancreatic cancer. 133. Human Ductal Pancreatic Adenocarcinoma (PDA) Cells That Express PDX-1 Can Be Targeted with a Rat Insulin Promoter Thymidine Kinase Gene. T. A. Tirone, M.D., T. Lee, B.S., L. Nguyen, B.S., and F.C. Brunicardi, M.D. Department of Surgery, Baylor College of Medicine, Houston, Texas.

TABLE—ABSTRACT 133 LacZ expression (LU)

C-1 M-1

Percent cell death

RIPZ

RSVZ

mRIPZ

RIPtk

Utk

HV

UT

4.2 ⫻ 10 5⌬ 1.2 ⫻ 10 5

9.1 ⫻ 10 5 9.5 ⫻ 10 5

1.4 ⫻ 10 5T

13 ⫾ 0.1* 0 ⫾ 0.1

8 ⫾ 0.1 7 ⫾ 0.1

0 ⫾ 0.1 0 ⫾ 0.1

4 ⫾ 0.1 0 ⫾ 0.1

⌬ P ⬍ 0.05 C-1 RIPlacZ vs M-1 RIPlacZ and TP ⬍ 0.05 C-1 mRIPlacZ vs C-1 RIPlacZ, Student’s t test. *P ⬍ 0.05 C-1 RIPtk vs M-1 RIPtk ANOVA.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 134 Sham

H-VEH

H-DHEA

V max (mg kg ⫺1 min ⫺1) 0.78 ⫾ 0.09 0.38 ⫾ 0.12* 0.67 ⫾ 0.10 # ALT (U/liter) 40.2 ⫾ 6.5 82.5 ⫾ 10.8* 48.9 ⫾ 11.2 # Estradiol (pg/ml) 48.6 ⫾ 12.0 24.4 ⫾ 7.1* 18.8 ⫾ 1.7* Note. Means ⫾ SE, n ⫽ 7/group. *P ⬍ 0.05 vs Sham; # P ⬍ 0.05 vs H-VEH (one-way ANOVA and Student–Newman–Keuls test).

Purpose. PDA cells are hypothesized to arise from a pluripotential stem cell and contain the transcription factor PDX-1 found in mature islets. In this study we investigated if PDA cell lines CAPAN-1 (C-1) and MIA-1 (M-1) could be targeted using the rat insulin promoter (RIP) driving the thymidine kinase gene (tk). Also, we explored which of the transcription factors known to activate RIP (PDX-1 or BETA2) is responsible for RIP activation in human PDA cells. Methods. RIPlacZ was created and transfected into C-1 and M-1 cell lines. RIPtk was created and transfected into C-1 and M-1 cell lines. Tk driven by a ubiquitous promoter (Utk), a hollow vector (HV), and untransfected cells (UT) were used as controls. Cells were treated for 5 days with 15 ␮g/ml of ganciclovir and cell viability was assessed using a MTS assay. RT/PCR was performed on C-1 and M-1 RNA with primers specific for PDX-1 and BETA2. Nuclear extract from C-1 cells was subjected to a gel shift assay with an antibody to PDX-1. A PDX-1 binding site on RIP was mutated (mRIP) with PCR and a mRIPlacZ construct was created and transfected in C-1 cells. Results. (See table.) Neither cell type contained RNA for BETA2; however, C-1 cells contained RNA for PDX-1. The nuclear extract of C-1 cells bound to the PDX-1 sequence of RIP (CTTAAT) and a supershift was observed with the PDX-1 antibody. Conclusions. The data suggest that RIP can drive the expression of a suicide gene in human PDA cells. However, the transcription factor PDX-1 is vital for rat insulin promoter activation in human PDA cells.

PARALLEL SESSION IV Shock II/Gastrointestinal III 134. Dehydroepiandrosterone (DHEA) Attenuates Hepatocellular Dysfunction and Liver Damage in EstrogenDeficient Females Following Trauma-Hemorrhage. J. F. Kuebler, M.D., D. Jarrar, M.D., P. Wang, M.D., K. I. Bland, M.D., and I. H. Chaudry, Ph.D. Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama. Recent studies have shown that administration of the sex steroid DHEA following hemorrhagic shock in males has beneficial effects on the depressed cardiac and hepatic functions. Since the action of sex steroids is gender dependent, the aim of our study was to determine whether administration of DHEA has any salutary effect on the depressed liver function in estrogen-deficient females following hemorrhagic shock. Methods. Ovariectomized (14 days prior to the experiments) female Sprague–Dawley rats (250 –300 g) underwent laparotomy, hemorrhagic shock (40 mm Hg for 90 min), and resuscitation (Ringer’s lactate over 60 min). DHEA (30 mg/kg body wt; H-DHEA) or vehicle (H-VEH) was administered subcutaneously at the end of resuscitation. At 24 h after resuscitation, hepatocellular function, i.e., indocyanine green clearance (V max and K m ) and hepatocyte damage (serum ALT), were measured. Plasma levels of DHEA and 17␤-estradiol were also assayed. Results. (See table.) Conclusions. Administration of DHEA following trauma-hemorrhage restored the depressed hepatocellular function and reduced hepatocyte damage

in estrogen-deficient females. This salutary effect of DHEA appears to be independent of plasma estradiol levels. Therefore, DHEA should be considered a novel and useful adjunct in the treatment of trauma-induced organ dysfunction in ovariectomized and postmenopausal females.

135. Hypertonic Preconditioning Augments Hepatic Heme Oxygenase-1 Following Ischemia–Reperfusion (I/R). G. D. Oreopoulos, M.D, J. Fan, Ph.D., S. B. Rizoli, M.D., Z. Lu, M.D., Y. H. Li, M.D., A. Kapus, Ph.D., and O. D. Rotstein, M.D. Department of Surgery, University of Toronto, Toronto, Ontario, Canada. Hepatic I/R contributes to organ injury and dysfunction after trauma, major surgery, and critical illness. We recently reported on a protective effect of hypertonic saline (HTS) in hepatic I/R (SIS 2000). The mechanisms of HTS’ protection are uncertain. Heme oxygenase-1 (HO-1) is a heat shock protein that is known to protect against hepatic I/R. We hypothesized that HTS might protect against hepatic I/R by inducing HO-1. A rodent model of partial hepatic I/R was used to test our hypothesis. Rats underwent 30 min of hepatic I/R after pretreatment with HTS (H, 7.5% NaCl, 4 cc/kg ia) or normal saline (I, 0.9%). Shams (SM) received normal saline and laparotomy alone. Postischemic lobe HO-1 protein was measured by Western blot. Further, HO-1⬘s role in HTS protection was determined by pretreating rats with the selective HO-1 inhibitor zinc protoporphyrin-IX (ZnPP, 20 mg/kg ip) or vehicle 24 h before hepatic I/R following H or I pretreatment. Results As shown in the figure, HTS augmented hepatic HO-1 protein induction compared to isotonic pretreated rats (blot). HO-1 inhibition with ZnPP augmented liver injury as measured by AST in isotonic rats. This reversal was also observed in HTS protected rats subjected to I/R injury, although not completely. Thus, HTS causes enhanced hepatic HO-1 following I/R. While induction of HO-1 was partly responsible for hepatic protection following HTS preconditioning, other mechanisms appear to be contributory. HTS preconditioning through induction of cytoprotective molecules may represent a novel strategy in the prevention of I/R injury.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

136. Comparison of ERK and JNK Activation Profiles in Mouse Liver during Hemorrhagic Shock Versus Hepatic Ischemia. C. A. McCloskey, M.D., J. J. Baust, D. J. Gallo, and T. R. Billiar, M.D. University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. Introduction. Hemorrhagic shock initiates an inflammatory response, although the earliest signaling pathways remain unelucidated. MAP kinases are known to be involved in inflammation, and we have previously shown the activation of JNK and ERK in mouse liver during hemorrhagic shock. The mechanism of MAP kinase activation in hemorrhagic shock is unknown and we hypothesize that tissue hypoxia is a contributing factor. In order to determine whether the rapid activation of either JNK or ERK could be induced by hypoxia alone, we determined the pattern of JNK and ERK activation in a mouse model of partial hepatic ischemia and compared them to the profiles observed in our mouse model of hemorrhagic shock. Methods. The first group of anesthetized male mice was hemorrhaged to a MAP of 25 mm Hg and sacrificed at 30-min intervals from 0.5 to 2 h (three animals per time point). Sham cannulated animals served as controls. A second group of anesthetized male mice (N ⫽ 6) underwent a laparotomy, and an atraumatic vascular clamp was placed across the left branches of the portal vein and hepatic artery. After 60 min, both the ischemic and the perfused lobes were harvested for analysis. Liver protein extracts from all groups were analyzed by Western blotting for both JNK and ERK phosphorylation. Bands were quantified by densitometry and statistically analyzed by ANOVA. Results. In the hemorrhagic shock model, mean levels of both phoshorylated JNK and phosphorylated ERK were significantly elevated over shams (P ⬍ 0.001 and P ⫽ 0.029, respectively) within 30 min of hemorrhage and remained elevated throughout the shock period. In the partial hepatic ischemia model, the level of phosphorylated JNK in the ischemic liver lobes was also significantly elevated (P ⫽ 0.018) over that of the perfused lobes. The mean level of phosphorylated ERK in the ischemic lobes was less, but not significantly different from that observed in the perfused lobes. Conclusion. JNK is activated early in the liver during both hemorrhagic shock and hepatic ischemia, suggesting that it is secondary to tissue hypoxia. However, ERK is activated during hemorrhagic shock but not in the ischemic liver, suggesting that it is not regulated by tissue oxygenation, and may instead be a response to a circulating mediator(s). 137. FAK and AP-1 Activation Following Hypoosmotic Stress in HepG2 Cells Is Actin Cytoskeleton Dependent. R. D. Kim, M.D., C. E. Darling, M.D., T. P. Roth, M.S., R. Ricciardi, M.D., and R. S. Chari, M.D. Department of Surgery, UMass Medical School, Worcester, Massachusetts. The actin cytoskeleton reorganizes itself immediately following hypoosmotic stress, but its impact on the swelling-activated PI-3-K/ PKB/AP-1 growth cascade is unknown. Focal adhesion kinase (FAK) participates in the cytoskeleton-based activation of PI-3-K. We hypothesized that hypoosmotic stress-induced downstream activation of PKB and AP-1 in HepG2 cells is dependent on an intact actin cytoskeleton and subsequent FAK phosphorylation. Methods. HepG2 cells incubated for 1 h with or without 20 ␮M cytochalasinD (cytD), an actin disruptor, were then exposed for up to 30 min to hypoosmotic medium (200 mOsm/L) to induce swelling. TNF-␣ (1.4 nM) or medium alone served as controls. Western blots measured cytoplasmic phospho- or total FAK and PKB. EMSA measured nuclear AP-1. All experiments were performed in triplicate. Results. [1] Hypoosmotic stress phosphorylated FAK (P-FAK) by 2 min, and this effect was inhibited by cytD (Fig 1). [2] Hypoosmotic stress phosphorylated PKB (P-PKB) by 10 min, and this effect was inhibited by cytD (Fig. 2). [3] Hypoosmotic stress activated AP-1 by 30 min, and this effect was inhibited by cytD (Fig. 3). Conclusion. Actin cytoskeleton integrity following hypoosmotic stress is essential

for the FAK-mediated activation of the PI-3-K/PKB/AP-1 proliferative cascade. These data delineate a possible mechanism by which the cell-swelling-induced cytoskeletal changes can impact proliferative signal transduction in human liver cancer. 138. The Effects of Ischemia on Gene Expression. J. Huang, M.D., E. Dauway, M.D., R. Qi, Ph.D., J. Quackenbush, Ph.D., E. Lazaridis, Ph.D., and T. Yeatman, M.D. Department of Surgery & Biostatistics, University of South Florida, H Lee Moffitt Cancer Center, Tampa, Florida; and The Institute for Genomic Research, Bethesda, Maryland. Microarray gene expression technology has recently made it feasible to characterize the RNA expression of thousands of genes. We hypothesized that warm ischemia associated with the surgical extirpation of human tissues would have significant effects on gene expression profiles. To quantitate the effects of warm ischemia on human tissue, we rapidly dissected normal colonic mucosa and maintained it at room temperature until snap-frozen in liquid nitrogen. Aliquots of tissue were frozen at times 0, 5, 10, 15, 20, 40, and 60 min after extirpation. Microarrays composed of 2400 distinct elements were used to assay mRNA derived from each time point in triplicate. Eisen’s hierarchical clustering methodology and Baysean statistical methods were then used to assay the effect of warm ischemia. Statistical models suggest that three patterns were induced by ischemia, accounting for 68.2, 17.8, and 13.4% of the evaluable genes. Pattern I demonstrated an average change of 27% over 60 min; 63.8% of the genes with at least 80% probability of membership in this pattern showed average increases in expression. The remainder decreased on average. Pattern II genes showed the least ischemiarelated effects, demonstrating an average change of only 12% over 60 min. In contrast to pattern I, we find that 67.5% of the genes with at least 80% probability of membership in this pattern decreased in expression over time. The remaining 32.5% in this pattern increased an average of 12% over 60 min. Finally, pattern III genes (13.4% of the sample) showed the greatest sensitivity to ischemia, changing an average of 50% over 60 min, with about the same number having increased as decreased. Warm ischemia associated with the surgical extirpation of human tissues has significant effects on gene expression profiles. These data support the careful monitoring of ischemic times during tissue harvest. 139. NF-␬B Inhibition Enhances Peroxynitrite (ONOO ⴚ)Induced Enterocyte Apoptosis. D. A. Potoka, M.D., E. P. Nadler, M.D., C. T. Wong, M.S., J. S. Upperman, M.D., and H. R. Ford, M.D. Department of Surgery, University of Pittsburgh and Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania. Introduction. ONOO ⫺ may induce enterocyte apoptosis and gut barrier failure in conditions such as necrotizing enterocolitis and inflammatory bowel disease. NF-␬B is upregulated in the gut during inflammation and, in addition to its proinflammatory effects, may upregulate antiapoptotic factors, such as inhibitor of apoptosis proteins (IAPs). Our goal was to examine the mechanism by which NF-␬B modulates ONOO ⫺-induced enterocyte apoptosis. Methods.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

353

Rat intestinal epithelial cells (IEC-6) were transfected with AdI␬B or control AdlacZ for 6 h at a multiplicity of infection of 1 pfu/cell. AdI␬B contains a mutated superrepressor form of I␬B. After transfection, cells were treated with 50 ␮M ONOO ⫺ or decomposed ONOO ⫺ for 1 h. After a 24-h recovery in medium, apoptosis was determined by flow cytometry with annexin V–FITC and propidium iodide staining. cIAP expression was examined by Western blot, and NF-␬B activation was assessed by gel shift assay. Results. Inhibition of NF-␬B with AdI␬B significantly enhanced ONOO ⫺-induced apoptosis in IEC-6 cells (see table). Western blot analysis showed no change in

TABLE—ABSTRACT 139 Apoptosis (%) ⫺

Treatment

ONOO

No transfection AdlacZ AdI␬B

17.2 ⫾ 1.4 21.4 ⫾ 6.6 36.1 ⫾ 10.8 †

†

Decomposed ONOO

⫺

5.7 ⫾ 1.1 7.3 ⫾ 2.6 8.1 ⫾ 3.4

P ⬍ 0.05 versus no transfection and AdlacZ transfection.

cIAP expression up to 24 h after ONOO ⫺ treatment in nontransfected or AdI␬B-transfected cells. ONOO ⫺ treatment did not activate NF-␬B in IEC-6 cells as determined by gel shift assays. Conclusion. NF-␬B inhibition enhances ONOO ⫺-induced enterocyte apoptosis, suggesting that NF-␬B upregulates a protective factor. This protective factor does not appear to be cIAP, since cIAP expression was not affected by ONOO ⫺ treatment. The protective factor may be expressed constitutively, since ONOO ⫺ did not activate NF-␬B.

Severe burn injury causes alteration of gut epithelial homeostasis. In previous studies bombesin showed beneficial effects on the maintenance of gut mucosal integrity during periods of atrophy or injury. We hypothesize that bombesin reduces gut impairment caused by burn injury. Adult Fischer 344 rats were randomly assigned to five groups: control (group I), sham burn (II), sham burn ⫹ drug (III), burn (IV), and burn ⫹ drug (V). Animals in group IV and V received a 60% total body surface area full thickness scald burn. Treatment groups received bombesin subcutaneously (10 ␮g/kg/day, q8h). Small intestine was harvested at 12 and 72 h after burn, and the proximal and distal small bowels were separately assessed for wet and dry weights, mucosal weights, and mucosal protein content. Data are means ⫾ SEM. Statistical analysis was by unpaired t test (significance at P ⬍ 0.05). In burned animals proximal and distal small bowel dry weights decreased at 12 and 72 h. Mucosal weight was reduced at 72 h. Bombesin restored the dry weight loss (Fig. 1), the mucosal weight (Fig. 2), and protein content in proximal small bowel (Fig. 3) at 72 h after burn. Bombesin showed no effect on distal small

140. Inducible Nitric Oxide Synthase (NOS 2) Mediates Impaired Small Intestinal Transit after Severe Gut Ischemia/Reperfusion (I/R). H. T. Hassoun, M.D., N. W. Weisbrodt, Ph.D., R. A. Kozar, M.D., Ph.D., D. W. Mercer, M.D., F. G. Moody, M.D, and F. A. Moore, M.D. University of Texas/ Houston Medical School, Houston, Texas. We have previously demonstrated impaired small intestinal (SI) transit in a model of gut I/R. We and others have implicated NOS 2 as a potential mediator of ileus during endotoxemia. Therefore, we hypothesized that NOS 2 mediates impaired SI transit after gut I/R. At laparotomy (lap), Sprague–Dawley rats had duodenal catheters placed. SI transit was determined by quantitating the percentage of tracer in 10 segments of small intestine 30 min after catheter injection [expressed as mean geometric center (MGC) of distribution] and was assessed at 6 h reperfusion after 45 or 75 min of superior mesenteric artery occlusion with sham lap as control. Ileal NOS 2 expression was assessed by Western immunoblot [reported as densitometric units (D/U)] and quantitative real-time RT-PCR [reported as a percentage of constitutive housekeeping gene (36B4)]. In additional studies, a selective NOS 2 inhibitor L-NIL (10 mg/kg ip) was administered 1 h prior to gut I/R or sham lap. Data are expressed as means ⫾ SEM (n ⱖ 5 in all groups; ANOVA) (see figure). SI transit was depressed after both 45 and 75 min gut I/R compared to sham controls. NOS 2 protein and mRNA were upregulated in the intestine after 75- but not 45-min gut I/R. In addition, L-NIL improved SI transit after 75- but not 45-min gut I/R. We conclude that NOS 2 mediates impaired SI transit after more severe gut I/R insults.

P1. Relationship of Pulmonary Artery Elasticity and Impedance in Maturing Newborn Pigs. P. W. Domkowski, M.D., Ph.D., R. H. Messier, Jr., M.D., Ph.D., L. H. Diadato, M.D., C. Jordan, B.S., and R. A. Hopkins, M.D. Department of Surgery Brown University School of Medicine Providence, Rhode Island; and Duke University Medical Center, Durham, North Carolina.

141. The Effects of Bombesin on Gut Impairment after Severe Burn Injury. X. W. Wu, M.D, S. E. Wolf, M.D., M. Spies, M.D., V. L. Chappell, M.D., D. N. Herndon, M.D., and J.C. Thompson, M.D. Shriners Burns Hospital and Department of Surgery, University of Texas Medical Branch, Galveston, Texas.

Studies on the behavior of the geometric properties of the pulmonary arterial system have predominately focused on adult humans and animals, while scant information exists concerning the relationship of geometry and elasticity in newborn species of any kind. The current study determined the pulmonary artery characteristic impedance (Z o) and Young’s elastic modulus (E Y) in intact maturing

bowel. Bombesin decreases the burn induced mucosal damage in proximal small bowel. Thus it may exert a potentially therapeutic role in restoring and maintaining mucosal integrity after severe burns.

POSTER SESSION

354

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P1

48 h, n ⫽ 6 2 weeks, n ⫽ 5 3 months, n ⫽ 5

R mean,mm

PVR

Z0

E ␥ (dyne/cm 2)

3.0 ⫾ 0.3 4.9 ⫾ 0.4* 6.9 ⫾ 0.4* ,†

5365 ⫾ 1292 1824 ⫾ 325* 566 ⫾ 97* ,†

1237 ⫾ 251 433 ⫾ 95* 162 ⫾ 17* ,†

3.6E ⫹5 ⫾ 7.9E ⫹4 5.6E ⫹5 ⫾ 3.8E ⫹4* 4.1E ⫹5 ⫾ 3.1E ⫹4,†

* P ⬍ 0.01 vs 48 h; †P ⬍ 0.01 vs 2 weeks ⫾ SEM. PVR and Z 0 , dyne s cm ⫺5. newborn pigs. Through simultaneous measurement of pulmonary artery pressure, flow and diameter, PVR, Z O, and E Y were calculated in 48-h-, 2-week-, and 3-month-old anesthetized, open-chest Yorkshire pigs. Z o reflects proximal artery distensibility while E Y measures elasticity. The current study hypothesized that PVR, Z o, and E Y are elevated at birth and undergo a steady maturational decline (see table). R mean increased with maturation, while Z o decreased. E Y peaked in 2-week-old pigs. These data demonstrate that PVR and Z o underwent a decrease with age, while there was a concomitant elevation in E Y in 2-week-old pigs. Although there is an increase in R mean between 48-h- and 2-week-old pigs, the pulmonary artery becomes stiffer, reflected by the elevated E Y. This finding may help explain the stiff nature of the newborn proximal pulmonary arteries observed clinically in infants with pulmonary hypertension, even when conventional measurements such as PVR are dramatically decreasing. P2. A New Surgical Adhesive (BioGlue) Causes Acute Phrenic Nerve Injury and Diaphragmatic Paralysis. S. A. LeMaire, ¨ ndar, Ph.D., J. S. Coselli, M.D., Z. C. Schmittling, M.D., A. U M.D., C. Ko¨ksoy, M.D., B. A. Deady, B.S., F. J. Clubb, D.V.M., Ph.D., and C. D. Fraser, Jr., MD. Department of Surgery, Baylor College of Medicine, and The Cullen Cardiovascular Laboratories of Texas Heart Institute, Houston, Texas. A new bioadhesive (BioGlue, CryoLife, Inc.) is currently under clinical investigation as a hemostatic adjunct for cardiothoracic operations. Although the proximity of nerves to cardiothoracic structures renders them vulnerable to injury, no studies have evaluated the potential nerve toxicity of BioGlue. The purpose of this study was to determine if BioGlue causes acute nerve injury using a porcine phrenic nerve model. Methods. Via median sternotomy in 12 domestic pigs (age 10 –15 weeks, weight 32.0 ⫾ 4.8 kg), baseline diaphragmatic excursion was measured using cineflouroscopy during direct phrenic nerve stimulation (3 mA, 200-␮s square wave monophasic pulses at 50 Hz). Diaphragmatic excursion was remeasured 3 and 30 min after exposing the nerve to BioGlue (n ⫽ 6), bovine albumin (negative control, n ⫽ 3), or glutaraldehyde (positive control, n ⫽ 3). Fisher exact tests and t tests for independent samples were used for intergroup comparisons for categorical and continuous variables, respectively. Results. All animals exposed to glutaraldehyde had complete diaphragmatic paralysis at 3 min; diaphragmatic paralysis did not occur in any of the albumin-exposed animals. The mean diaphragmatic excursion in the BioGlue group was lower than in the albumin group 3 min (1.7 ⫾ 3.6 vs 18.7 ⫾ 11.1 mm, respectively, P ⫽ 0.008) and 30 min after exposure (0.33 ⫾ 0.8 vs 18.7 ⫾ 10.1 mm, respectively, P ⫽ 0.002). Five of the six animals (83.3%) exposed to BioGlue had complete diaphragmatic paralysis by 30 min (P ⫽ 0.047 vs albumin). Conclusions. BioGlue causes acute phrenic nerve injury with diaphragmatic paralysis. When using this adhesive, contact with nerves must be avoided. Further study is needed to delineate the degree and duration of impaired nerve function and to evaluate possible neuroprotective strategies. P3. Effect of Creatine Monohydrate on Cardiac Function in a Rat Model of Endotoxemia. L. C. Vona-Davis, Ph.D., P. D. Wearden, M.D., Ph.D., N. K. Karne, B.S., and R. C. Hill, M.D.

Department of Surgery, West Virginia University, Morgantown, West Virginia. Background. Creatine and its analogues have been added to heart preparations and shown to be protective agents in ischemic myocardium. This study was undertaken to investigate whether creatine administered during perfusion could alter the recovery of cardiac function in a rat model of endotoxemia. Methods. Acute endotoxemia was induced in rats by bolus injection of Escherichia coli endotoxin (LPS, 4 mg/kg, ip) while control rats were injected with an equal volume of 0.9% normal saline. After 4 h, isolated hearts were perfused via a Langendorff column with Krebs– Henseleit buffer containing different concentrations of creatine monohydrate (1, 3, or 10 mM). Cardiac performance was evaluated via a paced (300 bpm) isovolumetric balloon preparation. Measurements of cardiac function including left ventricular developed pressure (LVDP), maximum rate of ventricular pressure development (LV dP/dt), and coronary flow were made for both LPS and control animals. Results. As anticipated, LVDP and LV dP/dt at a given preload and heart rate were significantly (P ⬍ 0.05) lower at 4 h post-LPS exposure. The addition of creatine monohydrate to the perfusion buffer did not improve the endotoxin-induced myocardial depression. In contrast, creatine administration did produce a significant (P ⬍ 0.05) negative inotropic effect on control hearts. The LVDP of control rats was reduced by 30% at the lowest concentration and by 50% at the highest concentration of creatine monohydrate. Coronary flows were unchanged by the addition of creatine in the buffer of either treatment. Conclusions. The data suggest that cardiac dysfunction in an acute model of endotoxemia was not improved with extracellular creatine. The deleterious effects of exogenous creatine monohydrate in normal hearts should be examined in future experimental studies. P4. Advantages of Positron Emission Tomography over Computed Tomography in Mediastinal Staging of Non-Small Cell Lung Cancer. D. W. von Haag, M.D., D. M. Follette, M.D., P. Roberts, M.D., L. Segel, Ph.D., and T. Taylor, R.N. Department of Cardiothoracic Surgery, University of California, Davis Medical Center, Sacramento, California. New treatment algorithms in early stage non-small cell lung cancer (NSCLC) involving preoperative chemotherapy require accurate clinical staging of the mediastinum. Computed tomography (CT) scanning is usually used to evaluate mediastinal metastases. The role of positron emission tomography (PET) scanning is undefined. We hypothesize PET scanning is more accurate than CT scanning in clinical staging of the mediastinum. Over the past 5 years 52 patients with NSCLC were evaluated with CT and PET scans; all had their mediastinal lymph nodes sampled by mediastinoscopy or during pulmonary resection. Sensitivity, specificity, positive and negative predictive values, and accuracy were calculated for both CT and PET scans. Statistical analysis was performed with Fisher exact and McNemar’s tests. Eighty-eight percent (46/52) of patients had negative pathology. CT scan results were not significantly related to presence or absence of pathology. PET scans were significantly better at detecting metastases than CT scans (PET 4/8, CT 3/16) (P ⫽ 0.01); however, positive predictive values were low (50 and 16%,

355

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS respectively). PET scans were significantly better than CT scans in determining absence of metastases (PET 42/44, CT 30/33) (P ⫽ 0.001); negative predictive values were 95 and 88%, respectively. Combined imaging did not increase predictive values. PET scanning is superior to CT scanning for evaluating mediastinal metastases in NSCLC. Ninety-five percent of patients without metastases on PET scan have negative pathology. Combined modality is not synergistic. P5. Inotropic Support Improves Organ Blood Flow during Simulated Off-Pump Bypass Surgery. J. A. Young, M.D., Y. S. Sun, M.D., L. W. Nifong, M.D., and R. W. Chitwood, Jr., M.D. Department of Surgery, East Carolina University, Greenville, North Carolina. Introduction. Simulated off-pump coronary bypass surgery decreases cardiac output (CO) and systemic blood pressure (BP) which leads to an associated decrease in tissue perfusion. Our objective was to decrease this effect with pharmacologic means. Methods. Revascularization was simulated on six anesthetized male Holstein calves via a sternotomy. After hemodynamic equalization, the heart was positioned to simulate bypass grafting of the circumflex and right coronary artery. Positions were held for 15 min. The sequence of vertical and lateral positioning was alternated between successive animals. At the end of each time point, hemodynamic and blood flow measurements were recorded. Epinephrine (4 ␮g per ml) was given by continuous infusion to maintain a mean arterial blood pressure 60 –70 mm Hg. Blood flow was determined by radiolabeled microsphere techniques. Continuous variables were analyzed with t testing. Results. The results are summarized in the table. Values are

TABLE—ABSTRACT P5 Organ

Base (A)

Vertical (B)

Lateral (C)

Recovery (D)

Brain Heart Kidney

26.5 ⫾ 0.6 45.0 ⫾ 1.3 175.9 ⫾ 9.3

35.3 ⫾ 1.9* 69.1 ⫾ 3.8* 191.9 ⫾ 20.5

23.1 ⫾ 0.8* 42.1 ⫾ 1.4 102.0 ⫾ 7.6*

28.4 ⫾ 1.2 40.6 ⫾ 1.2 108.1 ⫾ 9.8*

means ⫾ standard error. *P ⱕ 0.005 relative to baseline. No significant differences were measured for BP, CO, and mixed venous gas oxygen saturation. Blood flow is in cc/100 g tissue/min. Blood flows were significantly different between groups B and C. Conclusions. Inotropic support titrated to our target mean arterial blood pressure was tolerated well and improved or maintained end organ perfusion. As noted in previous studies, end organ perfusion is limited more by lateral manipulation when compared to vertical. The unknown effects of other chemical and mechanical agents to improve the perfusion of end organs should be studied further. P6. Fracture Locations Influence Likelihood of Rectal Injury in Pelvic Fractures. R. Aihara, M.D., W. W. LaMorte, M.D., Ph.D., M.P.H., E. F. Hirsch, M.D., and F. H. Millham, M.D. Department of Surgery/Section on Trauma, Boston University School of Medicine, Boston Medical Center, Boston, Massachusetts. The management of pelvic fractures is substantially complicated by the presence of rectal injury, which occurs in approximately 2% of patients with pelvic fractures. We sought to determine whether certain fracture locations are associated with a higher risk of rectal injury. We performed a retrospective review of all patients admitted to our Level I trauma center with the diagnosis of blunt pelvic fractures over a 7-year period. Data were collected from hospital records, trauma registry sheets, and radiology reports including plain films and CT scans. Fractures were mapped according to ana-

tomical location along the pelvic ring for each patient. Patients with rectal injuries were also identified. Factors associated with rectal injury were explored initially with &chi 2 or Fisher’s exact test and subsequent analysis used multiple logistic regression to control for confounding. A total of 372 patients (244 male and 128 female with a mean age of 38.8 years) were reviewed. There were 8 patients with associated rectal injuries (2.2%). Fracture locations in these 8 patients were distributed as follows: superior pubic ramus, 4; inferior pubic ramus, 3; widened symphysis, 6; sacrum, 2; SI joint, 6; iliac wing. 2; ischium, 0; and acetabulum, 3. We found that rectal injuries were significantly associated with widening of the symphysis (OR ⫽ 15.82, P ⬍ 0.0001), and fractures of the SI joint (OR ⫽ 7.12, P ⫽ 0.009) and posterior pelvic elements defined as sacrum or SI joint (OR ⫽ 4.4, P ⫽ 0.043). Fractures involving the pubic symphysis, SI joint, and the posterior elements were significantly associated with rectal injuries. Other fractures were not. On the basis of these data, evaluation of the rectum should include further examination with a rigid sigmoidoscope in all patients sustaining pelvic fractures involving the pubic symphysis, SI joint, or sacrum.

P7. The Impact of a Clinical Pathway for Gastric Bypass Surgery on Resource Utilization and Cost of Care. D. P. Bryant, M.D., R. Haluck, M.D., M. P. Rodgers, R.D., M. Lowery, R.N., and R. N. Cooney, M.D. Departments of Surgery, The Milton S. Hershey Medical Center, Hershey, Pennsylvania. Background. Clinical pathways have been hypothesized to improve patient care and reduce costs by utilizing a standard protocol to enhance efficiency. Our hypothesis was that development of a gastric bypass pathway would decrease hospital resource utilization and cost of care. Methods. From June 1998 to March 1999, 16 gastric bypass procedures were performed. These patients constitute a prepathway (Pre-path) group. From April 1999 to December 1999, 12 gastric bypass procedures were performed after institution of a clinical pathway, the postpathway (Post-path) group. The impact of a clinical pathway on hospital length of stay in days (LOS) and resource utilization was examined. Cost of care for room and board (Room$), operating room (OR$), supplies (Sup$), laboratory/ radiology (Lab$), and total cost (total$) were determined using cost/ charge ratios obtained from hospital financial information system. Data are presented as means. (See table. *P ⬍ 0.05 vs Pre-path by Student’s t test). Results. Despite increased obesity and medical acuity of the Post-path group, hospital LOS significantly decreased. Number of medical diagnoses related to severe obesity was increased in the Post-path group (4.8 vs 3.7 dx/pt). Total costs were also decreased by over $1600/case (more than 15%). The cost of supplies as well as the lab/radiology costs were both significantly decreased with the institution of the pathway protocol. Although total operating room time (ORT) was similar, OR costs were increased in the Post-path group. This appeared to be caused by an increase in equipment cost (use of harmonic shears) and anesthesia time, as the mean surgical time (215 min) was decreased by over 20 min.The overall incidence of complications (Pre-path 12%, Post-path 16%) was similar and 2 patients in each group required readmission to the hospital. Conclusions. Institution of a clinical pathway for gastric bypass surgery decreased hospital LOS and resource utilization. The reduction in cost was not associated with an increase in morbidity or hospital readmission.

TABLE—ABSTRACT P7 Group

ORT LOS Room$

Pre-path 5.14 6.9 Post-path 5.06 3.7*

$3641 $2389

OR$

Sup$

$3467 $1,152 $4251 $ 679*

Lab/Rad

Total$

$629 $312*

$10,176 $ 8,511

356

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

P8. Early and Late Outcome of Bedside Percutaneous Tracheostomy in the Intensive Care Unit. E. A. Mittendorf, M.D., C. R. McHenry, M.D., C. M. Smith, R.N., C. J. Yowler, M.D., and J. R. Peerless, M.D. Department of Surgery, MetroHealth Medical Center, Case Western Reserve University, Cleveland, Ohio. In order to simplify long-term airway management in critically ill patients, the feasibility of performing percutaneous tracheostomy (PT) in the intensive care unit (ICU) was investigated. Methods. A protocol for PT in the ICU was initiated at our institution in 1997. Bedside PT was considered for patients with positive and endexpiratory pressure ⬍ 10 cm H 2O, no previous tracheostomy, no thyromegaly, or other anatomic distortion of the tracheal region and no other indication to go to the operating room. Indication for tracheostomy, duration of endotracheal intubation, APACHE II score at the time of procedure, morbidity, and mortality were determined for all patients who underwent PT. Patients were prospectively evaluated for development of late complications until decannulation or for a minimum of 3 months following PT. Whether or not a percutaneous endoscopic gastrostomy (PEG) tube was placed concomitantly was also noted. Results. Since 1997, 67 patients underwent PT in the ICU without incident, including 40 who underwent concomitant PEG. The indication for PT was prolonged ventilatory support (38), airway protection (26), and maxillofacial trauma (3). Mean duration of endotracheal intubation prior to PT was 14 days (range 5–35 days). Average APACHE II score at the time of procedure was 13.7 (range 3–28). Morbidity from PT included wound bleeding (2), early cuff leak (1), and late bleeding from the tracheal granulation tissue (1). Fourteen patients died of causes unrelated to PT, 5 to 208 days post-PT. Thirty-five patients were decannulated after an average of 51 days following PT (range 9 –170 days), 2 of whom noted a minor change in voice. Conclusion. PT can be performed in the ICU with minimal early and late morbidity eliminating the need for an operating room, the risks of patient transport, and the costs associated with each of them. P9. Dedicated Craniofacial/Skull Base Trauma Team Improves Efficiency of Patient Care. R. A. Mathiasen, M.D., R. Jarrahy, M.D., J. B. Eby, M.D, H. K. Shahinian, M.D., and D. R. Margulies, M.D. Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California. Purpose. To study the addition of a dedicated craniofacial and skull base trauma team to the resources of a private Level-I trauma center and its effect on efficiency of patient care. Methods. A retrospective review of the trauma registry and charts of a private Level-I trauma center for all craniofacial and skull base trauma cases for a period of 18 months prior to the establishment of a craniofacial/skull base team (Pre-SBS) and for 18 months after such team was instituted (SBS). Results. From February 1997 to July 1998, 29 craniofacial/skull base trauma cases were performed. Twenty-eight such cases were performed by the Skull Base Surgery team from August 1998 to February 2000. The mean age and ISS for each group were not different (T test). The presence of a dedicated craniofacial/

TABLE—ABSTRACT P9 Pre-SBS, n ⫽ 29 SBS, n ⫽ 28 Age ISS No. consults/pt Time to OR (days) Length of stay (days) Patients transferred (%)

36.8 ⫾ 16.2 14.8 ⫾ 7.9 2.3 ⫾ 0.9 8.3 ⫾ 4.9 11.8 ⫾ 7.3 24.1%

39.8 ⫾ 19.8 14.0 ⫾ 7.5 1.2 ⫾ 0.5 3.0 ⫾ 1.6 6.8 ⫾ 5.4 3.6%

P NS NS ⬍0.001 ⬍0.001 ⬍0.01

skull base trauma team decreased the need for multiple subspecialty consultations, reduced time to operation, and provided for shorter inpatient hospitalizations. Only 1 of 28 SBS patients required transfer to another institution for definitive care (see table). Conclusion. Adding a dedicated craniofacial/skull base trauma team to a Level-I trauma center enhances efficiency of patient care and allowed for definitive care for injuries that previously required transfer to other institutions for further treatment. P10. The Chief Resident Roles as Surgeon, Teacher, Student, and Administrator on University and Public Hospital Services. D. J. Esposito, M.D.,* L. S. Hauge, Ph.D.,* H. Reyes, M.D.,† and R. A. Prinz, M.D.* †Department of Surgery, Cook County Hospital, and *Department of General Surgery, Rush University, Chicago, Illinois. Purpose. The purpose of this study was to examine the time spent on daily teaching, learning, and administrative tasks by a chief surgical resident on service in two different hospital settings. Methods. A case study method was used to record the nature and quantity of the chief resident’s daily work. The subject for this case study was selected based on prior excellent performance reviews by faculty, residents, and students. The resident kept a confidential daily log of time spent in various roles for a period of 4.5 months. During this time, the resident completed general surgery service rotations of approximately equal length in a university hospital and a public hospital. Faculty members on these services were unaware of the resident’s daily logging. Time was logged in the following categories: operating room, administrative duties, outpatient clinics, chief resident rounds, attending rounds, teaching, academic conference attendance, and preparing for teaching activities. Results. On a daily weekday average, the chief resident spent approximately 3.25 h operating, 50 min on chief rounds, 45 min in academic conferences, 40 min teaching, 40 min preparing teaching activities, 35 min doing administrative tasks, 30 min in the outpatient clinic, and 30 min on attendings’ rounds. An ANOVA was used to compare time spent on these tasks in the university and public hospital settings. Results indicated that the resident spent significantly more time at these tasks in the university hospital: administrative (F ⫽ 7.8, df ⫽ 1, P ⫽ 0.007), attending rounds (F ⫽ 52.73, df ⫽ 1, P ⫽ 0.001), chief rounds (F ⫽ 124.68, df ⫽ 1, P ⫽ 0.001), operating (F ⫽ 3.99, df ⫽ 1, P ⫽ 0.05), and teaching (F ⫽ 8.12, df ⫽ 1, P ⫽ 0.006). The resident spent significantly more time in outpatient clinics in the public hospital (F ⫽ 25.34, df ⫽ 1, P ⫽ 0.001). Total time spent at each hospital was not logged. However, the aggregate time spent on all logged tasks was significantly greater at the university hospital (F ⫽ 20.68, df ⫽ 1, P ⫽ 0.001). There were no significant differences on time spent in academic conferences or preparing teaching activities at the two hospitals. Conclusion. There are significant differences in time spent on educational, administrative, and patient care activities by a chief resident in public and university hospitals. Further evaluation of these differences may improve resident education at both settings. P11. Managed Care Pilot Survey. D. S. Fusco, M.D., I. A. Munshi, M.D., L. Volz, M.D., D. W. Page, M.D., J. Garb, M.S., and R. B. Wait, M.D., Ph.D. Baystate Medical Center, Springfield, Massachusetts. Managed care and HCFA rulings have a profound effect on our ability to deliver care and support academic activities. Purpose. This project was designed to determine (1) the level of understanding of basic managed care and health care policy among surgical residents and attendings and (2) whether there are differences in knowledge of these issues between residents, community, or hospital-based surgeons. Methods. Thirty-seven question survey covering basic managed care principles administered to surgical attendings and residents at a University Hospital affiliate. Scores are given as number of correct answers. Statistical analysis was performed using

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS ANOVA and linear regression. Results. Sixty-four questionnaires were distributed and all were completed (see table). Significant dif-

TABLE—ABSTRACT P11

n Mean Range SD

Residents

Community

Hospital

29 16.79 10–24 3.20

18 22.17* 17–28 2.83

17 20.35* 14–26 3.72

Note. ANOVA, P ⬍ 0.0001; *P ⬍ 0.01 vs residents.

ferences in basic managed care knowledge between residents and attending surgeons exists yet, no differences between community or hospital-based surgeons exists. There is a weak correlation between years in practice and knowledge base (r ⫽ 0.4, P ⬍ 0.05), but no correlation exists with PGY level. Conclusions. Knowledge of basic health care issues is generally very poor among all surgeons tested. Formal teaching of managed care principles should become a part of all training programs. P12. Does Training of Specialty Residents Affect Patient Outcome in Vascular Surgery? T. T. T. Huynh, M.D., J. T. Abbas, M.D., H. J. Safi, M.D., and M. J. Reardon, M.D. Department of Surgery, Baylor College of Medicine, The Methodist Hospital, Houston, Texas. The training of surgeons is currently held to standards by the Residency Review Committee. Notwithstanding the importance of training programs, how they affect patient outcome is not well known. This study was undertaken to compare patient outcome in vascular surgery, with or without the training of specialty residents (SR) at one institution. Methods. Records of 117 patients undergoing either carotid endarterectomy (CEA) or aortoiliac reconstruction (AIR), performed by one attending surgeon while in private practice (without SR) and after joining the University Faculty (with SR), were identified from the hospital registry. Under the supervision of the attending surgeon, SR were the primary surgeon in 31 CEA and 29 AIR and managed these patients perioperatively. Twenty-six patients underwent CEA and 31 had AIR without the involvement of SR. Patient outcome with and without SR was compared. Results. Patients in both groups had similar demographics, indications for surgery, mortality, and morbidity. Overall, there was one death after AIR with SR and none in the group without SR. Two complications related to CEA occurred in the group without SR (1 neck hematoma and 1 TIA), compared to none with SR. Following AIR, 6 (21%) patients with SR and 10 (36%) without SR developed complications related to surgery. The length of surgery with or without SR was not significantly different in CEA (94.6 ⫾ 18.3 vs 99.9 ⫾ 20.7 min, respectively; P ⬎ 0.05) or AIR (146 ⫾ 36.7 vs 129.4 ⫾ 43 min, respectively, P ⬎ 0.05). Interestingly, the intraluminal carotid shunt (which was routinely used) time was 20% shorter with SR, compared to without SR (26.5 ⫾ 6.1 vs 33.3 ⫾ 10.1 min, respectively, P & lt; 0.01). The aortic cross-clamp time was similar in AIR with or without SR (33.3 ⫾ 11.1 vs 33.2 ⫾ 10.3 min, respectively; P ⬎ 0.05). Conclusions. The results of this study suggest that patient outcome following carotid and aortic reconstruction is similar, with or without the training of specialty residents. Furthermore, the participation of specialty residents in cases requiring highly specialized skills, such as carotid intraluminal shunting during endarterectomy, may be beneficial. P13. Physician Leadership: A New Mandate in Surgical Training. K. M. F. Itani, M.D., K. Liscum, M.D., and F. C.

357

Brunicardi, M.D. Department of Surgery, Baylor College of Medicine, Houston, Texas. Background. Traditionally, the initial and subsequent development of physician leadership has occurred at random in surgical training. One possible reason is that surgical educators have focused on detailed instruction about critical patient situations, resuscitation, and technical skills, but have provided little formal training in the essential management and leadership skills. Methods. In order to determine resident perceptions about the importance of these skills and individual strengths and weaknesses in these areas, a questionnaire was administered to 43 residents in our general surgery program. In part 1 of the questionnaire the residents ranked 18 leadership topics on a scale of 1– 4 in their importance (not important, minimally, somewhat, very) for career development. The second portion of the questionnaire asked the residents to rate themselves with regard to their personal confidence/competence in these areas on the same scale. Results. Twenty-five residents (58%) completed the entire questionnaire. All 18 leadership and management topics were rated as at least somewhat important for career development by the majority (96%) of the residents. More than 50% of the residents rated themselves as not competent or minimally competent in 10 of the 18 areas. There was no area in which greater than 75% of the residents felt more than minimally competent. Conclusion. We conclude that while residents see these nontraditional topics as an important part of their professional education they do not feel confident or competent in these areas. Establishing a conscious effort to teach these topics and emphasize their importance during training will enhance residents’ self image, performance, and potential as future leaders.

P14. Evidence-Based Criteria for Teaching Surgical Residents Endoscopy. M. J. Page, M.D., D. Carney, M.D., F. Y. Lim, M.D., W. A. Koltun, M.D., and G. O. Maish, III, M.D. Department of Surgery, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania; and Lebanon VA Medical Center, Lebanon, Pennsylvania. Endoscopy experience is often limited during surgical residency. This ongoing study sought to quantitate the learning curve associated with endoscopic procedures (colonoscopy and EGD) with the goal of developing evidence-based guidelines for teaching endoscopy during surgical residency training. Methods. R3 categorical surgery residents with no previous endoscopy training are monitored during all common endoscopic procedures performed at the VA medical center during a 3-month rotation. The procedures are divided into four categories: (1) all colonoscopies, (2) colonoscopy without biopsy, (3) colonoscopy with biopsy, and (4) all EGDs. The procedure times are plotted and analyzed using best-fit nonlinear regression curves. The mathematically derived plateau point, which is the point of flattening in the slope of the curve, is defined as the point at which endoscopic proficiency is achieved. Results. To date, each resident has averaged 80 colonoscopies (range 79 – 81) during their rotation with an average identified plateau point of 17 patients (11–21). An average of 48 colonoscopies without biopsy were performed with a plateau point of 18 patients (16 –19) with one curve having no identifiable plateau point. Thirty colonoscopies with biopsy were done with a plateau point of 22 patients (21–23). Additionally, each resident performed an average of 43 EGDs with a plateau point of 17 patients (17) with one curve having no identifiable plateau point. No complications requiring intervention have occurred. Conclusions. (1) Endoscopic skills must be learned during surgical residency. (2) Interventional colonoscopy requires more procedures for proficiency than diagnostic colonoscopy. (3) The learning curve for EGD is similar to colonoscopy. (4) The number of procedures required to achieve reasonable proficiency for all common endoscopic procedures was between 17 and 22.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

P15. Statistical Methods in the Surgical Literature over the Past 15 Years. S. S. Sonnad, Ph.D., S. Dalal, M.P.H., and L. M. Colletti, M.D. Department of Surgery, University of Michigan, Ann Arbor, Michigan. Introduction. Statistical methodology appears to be increasingly important in medical research. To test this hypothesis, we looked at statistical content of two top surgical journals. Methods. We examined journal articles from the Annals of Surgery and from the Archives of Surgery. We included odd-year issues from 1985 to 1999. From each of these years, we used a random number table to select three issues for abstraction. For all original research articles in each selected issue, we identified and tallied all reported statistical methods. We defined statistics as P values, t tests, mean and standard deviations, &chi 2 tests, ANOVA, and nonparametric statistics. Results. The final number of included research articles was 367 articles from the Archives and 319 articles from the Annals. Overall, the use of statistics became more common over time. In the Archives, 30 – 50% of articles did not include statistics from 1985 to 1989, whereas over 80% of articles did include statistics from 1991 to 1999. In the Annals, approximately 30 – 40% of articles in 1985–1989 did not include statistics compared to ⬍20% of articles from 1991–1999. In addition to broader use of statistics, our findings indicate greater statistical sophistication over time. Nonparametric statistics appeared in ⬍5% of Archives articles prior to 1990, but in 25–30% from 1991 to 1999. In the Annals, use of nonparametrics was seen in 10% of articles in 1985 and 1987, 20% in 1989, ⬃30% from 1991 to 1995, and ⬃50% in 1997 and 1999. Other statistics show similar trends with increasing use of &chi 2 tests and ANOVA. Use of simpler statistics such as t tests and reporting of descriptive statistics peaked in 1993–1995 and are less seen in the most recent years. Conclusions. Statistical methods are becoming more sophisticated and fewer articles are published without statistics in both the Archives of Surgery and the Annals of Surgery. It is increasingly important for the academic surgeon to understand and incorporate statistical analysis into all types of original research intended for publication in surgical journals. P16. Development of an Integrated Scoring System for Virtual Reality Skills Assessment. D. J. Scott, M.D., E. C. Hamilton, M.D., W. H. Frawley, Ph.D., S. T. Tesfay, R.N., and D. B. Jones, M.D. Departments of Surgery and Academic Computing, University of Texas Southwestern Medical Center, Dallas, Texas. MIST (Virtual Presence, London, England) is a virtual reality system that measures laparoscopic skill in multiple performance dimensions. The purpose of this study was to develop an integrated scoring system. First (n ⫽ 12) and second (n ⫽ 5) year surgery residents practiced six standardized tasks on the MIST system for 30 min per day for 10 days. Raw scores, including time (T), errors (E), economy of motion left (EML) and right (EMR), and economy of diathermy (ED), were recorded for all practice sessions. Based on the raw scores of all trainees, summary statistics were generated; a logarithmic transformation was applied to compensate for skewness. Values were transformed to z scores to overcome discrepancies in dimensionality. Values were inverted and scaled to yield a median score of 50, with high scores indicating superior performance. All performance measures were weighted equally, except for EML and EMR, which were combined and together weighted equally with the other measures. Results are shown in the table. The integrated formulas overcome the problems of dimensionality and scaling and generate a single score which reflects all of the available measures of performance. Integrated scores should allow for comparisons between groups in a manner more meaningful and more efficient than using numerous raw scores. The standardized formulas should be useful for ourselves and others who want to train and assess residents using the MIST virtual reality system.

TABLE—ABSTRACT P16 Task No.

No. of observations

Median score

Interquartile range

1 2 3 4 5 6

638 645 634 589 599 596

50.0 50.0 50.0 50.0 50.0 50.0

41.0–60.3 43.2–57.0 43.5–56.4 43.7–57.5 47.1–53.0 45.2–54.6

P17. Circadian Rhythmicity in Intestinal Active Glucose Absorption Is Anticipatory. A. Tavakkolizadeh, M.B., B.S., FRCS, K. R. Shen, M.D., M. J. Zinner, M.D., S. W. Ashley, M.D., and E. E. Whang, M.D. Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Introduction. The value of enteral nutrition in critically ill patients is established. To determine the optimal time of delivery for such feeds, we have been studying the circadian rhythm in intestinal glucose absorptive capacity. The purpose of this study was to determine the mechanisms mediating circadian periodicity in intestinal Na ⫹/glucose cotransporter (SGLT1) activity. Material and Methods. Rats kept in a 12-h light– dark cycle were sacrificed at circadian times (CT) 3, 9, 15, and 21 (CT0 ⫽ beginning of the light cycle). Stripped segments of proximal jejunum were placed in Ussing chambers and kinetic studies were performed by addition of 3-Omethylglucose aliquots, resulting in final chamber concentrations of 2, 4, 8, 16, 32, and 64 mM. ⌬I scmax (transporter maximal velocity) and K m (transporter affinity) were calculated for each of the four time points. The 24-h food intake of animals was recorded. Results. Rats

TABLE—ABSTRACT P17 Time (CT)

K m (mM)

⌬I scmax (␮A/cm 2)

CT3 CT9 CT15 CT21

27.4 ⫾ 5.7 45.2 ⫾ 10.6 28.0 ⫾ 6.2 18.6 ⫾ 5.8

81.6 ⫾ 11.9 258.9 ⫾ 35.1* 172.7 ⫾ 35.0 97.8 ⫾ 20.8

consumed only 6.6 ⫾ 1.9% of their total daily food intake between CT3 and CT9. ⌬I scmax at CT9 was significantly greater than at CT3 (*P ⬍ 0.05 vs CT3 by ANOVA, n ⫽ 6 – 8/group) (see table). Conclusions. Our results suggest that the circadian variation in intestinal SGLT1 activity is due to modulations in ⌬I scmax, rather than any alterations in K m. Because the greatest change in transport capacity occurs between CT3 and CT9, when less than 10% of the total daily food intake is consumed, transporter modulation is anticipatory, preparing the rodents for the nocturnal increase in feeding. Our data suggest that delivery of enteral nutrition can be optimized to account for circadian rhythmicity in intestinal nutrient absorptive capacity. P18. Laparoscopic Repair of Traumatic Diaphragmatic Injuries. R. A. Kozar, M.D., Ph.D., L. J. Kaplan, M.D., J. Cipolla, M.D., J. Meija, M.D., and M. Haber M.D. Departments of Surgery and Pathology, MCP-Hahnemann School of Medicine, Philadelphia, Pennsylvania; and Department of Surgery, University of Texas–Houston, Houston, Texas. Laparoscopy has been proposed as a diagnostic and therapeutic modality for penetrating diaphragmatic injuries. The purpose of

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS this study was to assess the technical feasibility and strength of various laparoscopic techniques for repair of diaphragmatic injuries. Methods. Swine underwent either open suture repair or laparoscopic (Lap) repair of a 2-cm laceration in either the muscular or the tendinous portion of the right and left diaphragmatic leaflets. Repair was by staple (Lap ST), suture (Lap SUT), or patch (Lap PT) technique. Six weeks after surgery, diaphragms were harvested for histologic analysis, electron microscopy, or bursting strength measurement (BSM). An additional control group with no injury (intact) was used for BSM. Results are expressed as mean ⫾ SEM. Comparison between groups was by ANOVA; significance was set at P ⬍ 0.05. Results. (See table.) All methods of

TABLE—ABSTRACT P18 Bursting Strength Measurement (psi) Method of repair Intact (n ⫽ 4) Open (n ⫽ 4) Lap ST (n ⫽ 8) Lap SUT (n ⫽ 8) Lap PT (n ⫽ 8)

Muscular diaphragm

Tendinous diaphragm

22.85 ⫾ 3.11 17.67 ⫾ 7.32 22.34 ⫾ 4.94 22.61 ⫾ 9.37 23.91 ⫾ 6.41

45.58 ⫾ 5.03 43.97 ⫾ 19.67 43.03 ⫾ 10.00 43.26 ⫾ 5.10 41.49 ⫾ 11.66

repair proved technically feasible. There was no significant difference in BSM between groups. Bursting was due to tissue failure either at or adjacent to the repair site. Histologic analysis confirmed healing of all specimens. Conclusions. Laparoscopic repair of diaphragmatic injuries can be accomplished using any of the currently available techniques. All methods provide an equally strong repair. When selecting a particular technique, familiarity of the surgeon should be used as a guideline. P19. The Effect of Total Parenteral Nutrition (TPN) on Cell Cycle Regulator Expression in Rat Jejunum. C. M. Perez, M.D., E. Whang, M.D., J. Rounds, B.S., P. Abraham, B.S., N. Shimoda, M.D., K. Okamoto, M.D., and D. O. Jacobs, M.D., M.P.H. Department of Surgery, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts; and Department of Surgery, Creighton University, Omaha, Nebraska. Intestinal mucosal atrophy occurs in mammals that are fed intravenously. The mechanism may involve the downregulation of cell cycle regulators that increase enterocyte proliferation. We tested the hypothesis that TPN administration is associated with decreased cyclin D1 protein expression in rodents. Male virus-free Wistar rats (190 –215 g) were randomized to receive chow and water ad libitum (n ⫽ 4) or a standard intravenous feeding formula (n ⫽ 4). Both groups were acclimatized prior to study. After 3 days of feeding, the rats were sacrificed and standardized segments of jejunum were harvested for protein extraction. The extracted proteins were analyzed by Western blotting for cyclin D1 expression and compared using an unpaired t test (see table).

TABLE—ABSTRACT P19 Group

Signal density (measured in pixels)

Control TPN

220.7 ⫾ 16.5 155.5 ⫾ 13.9

Note. Mean ⫾ SEM; P ⬍ 0.05 vs control by unpaired t test.

359

Immunoblotting showed that the levels of cyclin D1 in rats fed TPN were significantly decreased compared to chow-fed controls. This study shows that for the first time that TPN administration is associated with decreased cyclin D1 expression in rat jejunum. Agents that modify cellular proliferation or turnover may be beneficial in decreasing the intestinal mucosal atrophy that occurs during intravenous nutrition.

P20. How Surgeon Age Affects Posttreatment Surveillance Strategies for Melanoma Patients. J. A. Margenthaler, M.D., K. S. Virgo, Ph.D., D. Y. Johnson, B. S. Handler, M.D., E. M. Sugarbaker, B.S., and F. E. Johnson, M.D. St. Louis University Health Sciences Center and John Cochran Veterans Affairs Medical Center, St. Louis, Missouri. The intensity of postoperative melanoma patient follow-up varies widely among physicians. We investigated whether physician age accounts for the observed variation in surveillance intensity among plastic surgeons. The 3032 members of the American Society of Plastic and Reconstructive Surgeons (ASPRS) were surveyed using a custom-designed questionnaire to measure how these surgical experts deal with melanoma patient follow-up. Subjects were asked how they use 14 specific follow-up modalities during years 1–5 and 10 following primary surgical treatment for patients with cutaneous melanoma. Repeated-measures analysis of variance was used to compare practice patterns by TNM stage, year postsurgery, and age. Of the 1142 responses (38%), 395 (35%) were considered evaluable. Nonevaluability was usually due to lack of melanoma patient follow-up in surgeons’ practices. Follow-up strategies for most of the 14 modalities were highly correlated across TNM stages and years postsurgery, as expected. Mean follow-up intensity differed significantly by surgeon age for complete blood count, liver function tests, and chest X-ray, with younger (⬍60 years) physicians ordering the tests more frequently than older (ⱖ60 years) physicians (P ⬍ 0.05, P ⬍ 0.001, P ⬍ 0.01, respectively). The absolute differences, however, were very small. The posttreatment surveillance practice patterns of ASPRS members caring for patients with cutaneous melanoma vary only marginally with physician age. The data suggest that postgraduate education is effective in homogenizing practitioner behavior. P21. Innate Immune Response to Adenoviral VectorMediated Acute Pancreatitis. A. L. Shifrin, M.D., N. Chirmule, Ph.D., K. Chapman, and S. E. Raper, M.D. Department of Surgery and Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, Pennsylvania. The role of host immunity in the development of acute pancreatitis is not well understood. The aim of these studies is to characterize the role of the innate immune system—macrophages and

360

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

natural killer (NK) cells—in the generation of an immune response to an antigenic challenge. C57B6 mice (N ⫽ 3/group) received direct intrapancreatic injection of a recombinant adenoviral vector expressing the gene for beta galactosidase. Immunohistochemical staining was performed with F4/80 anti-mouse macrophage antibody (Serotec). The Ly49c/NK1.1 antibody was used for NK/NKT flow cytometry. Serum cytokine levels were detected with an ELISA. Shortly after adenoviral vector administration, macrophages (see figure) enter the pancreas with maximal infiltration at day 4 and then leave as antigen-expressing cells are eliminated. The cytokines IL-6, IL-, and IL-12 were all elevated (see Table 1). NK and NKT cells enter the pan-

TABLE—ABSTRACT P22 No hiatal hernia

GHL PEL GPL

Hiatal hernia

n

Mean Elastin

n

Mean Elastin

P value

6 7 5

0.27 ⫾ 0.03 0.31 ⫾ 0.04 0.19 ⫾ 0.03

4 4 3

0.13 ⫾ 0.02 0.15 ⫾ 0.03 0.16 ⫾ 0.02

0.001 0.001 0.34

significant and selective loss of elastin fibers in two of three supporting ligaments of the GEJ in patients with hiatal hernia. Elastin may play an important role in maintaining the normal relationship of the GEJ to the diaphragm.

TABLE 1—ABSTRACT P21 Cytokine Levels (pg/ml) Hours

IL-6

IL-10

IL-12

0 0.5 4 24 96

0 0 682 65 0

0 0 272 278 323

16.7 223 93.3 150 248

TABLE 2—ABSTRACT P21 NK/NKT Cells in the Pancreas (%) Days

NK1.1

NKT

0 4 10 28

0 1.6 1.55 1.2

0 5.1 3.7 2.3

creas and remain (see Table 2).Control pancreas had essentially no macrophages or NK/NKT cells. In conclusion, macrophages and NKT cells appear to play a major role in the development of acute pancreatitis. P22. Elastin Depletion in The Gastric Ligaments of Patients with Hiatal Hernia. J. A. Curci, M.D., C. G. Davis, B.S., R. W. Thompson, M.D., and N. J. Soper, M.D. Department of Surgery, Washington University School of Medicine, St. Louis, Missouri. Herniation of the stomach through the esophageal hiatus (HH) is associated with dysfunction of the lower esophageal sphincter (LES) and reflux disease. The gastroesophageal junction (GEJ) is maintained in anatomical position in part by several thin ligamentous structures including the phrenoesophageal ligament (PEL), gastrohepatic ligament (GHL) and gastrophrenic ligament (GPL). We hypothesized that loss of elastin within these suspensory ligaments of the GEJ may be associated with HH. Tissue specimens (1 cm 2) of PEL, GHL, and GPL from patients undergoing laparoscopic Nissan fundoplication for reflux disease with or without HH. Fixed sections were stained overnight with the nonregressive Hart’s elastin stain. Three random areas of each section were digitally imaged and elastin content was analyzed as a fraction of total imaged tissue using the digital tool Optimas. Results are summarized in the table and expressed as means ⫾ SE. In patients with HH, two of the ligaments (GHL and PEL) demonstrate an approximately 50% reduction in elastic fibers compared with ligaments of control patients without HH. No significant loss of elastic fibers are seen in the GPL. There is

P23. Effects of Tezosentan, a Dual Endothelin Receptor Antagonist, on the Cardiovascular and Renal Systems of Neonatal Piglets. A. Chin, M.D., J. Radhakrishnan, M.D., L. Fornell, B.S., and E. John, M.D. Divisions of Pediatric Surgery and Nephrology, University of Illinois at Chicago, Chicago, Illinois. Introduction. Endothelin is believed to have a vasoactive role on the cardiovascular and renal systems. In this study we have examined the effects of tezosentan, a dual endothelin receptor antagonist designed for parenteral use, on the cardiovascular and renal systems of healthy neonatal piglets. Methods. Eight, 7- 10-day-old domestic piglets weighing 2.5–3.0 kg were anesthetized, intubated, and ventilated with catheters placed into the jugular veins, left ventricle, and femoral artery. Urine output was monitored via a suprapubic cystostomy. The piglets were bolused with 0.9% NS for surgical losses. Maintenance fluid with sodium [ 125I]iothalamate was infused to aid in calculation of glomerular filtration rate (GFR). Mean arterial pressure (MAP), heart rate (HR), arterial blood gases, and urine output (UV) were continuously monitored and the piglets were allowed to recover for 30 min before baseline values were obtained. Tezosentan was then infused for 1 h at a rate of 1 mg/kg/h. The piglets were then allowed to recover for an additional 1 h. Cardiac index (CI), renal blood flow (RBF), systemic vascular resistance (SVR), and renal vascular resistance (RVR) were obtained at baseline, at the end of tezosentan infusion, and at 1 h postinfusion. CI and RBF were measured by injecting radiolabeled microspheres at timed intervals and obtaining gamma counts. GFR, SVR, and RVR were calculated using standard formulae. After the final injection of microspheres, the piglets were sacrificed and their kidneys were removed, weighed, and counted for radioactivity. Statistical significance was determined by the Student’s t test. Results. (See table.) Conclusions. The inhibition of endothelin receptors with tezosentan produced a statistically significant effect on the piglet cardiovas-

TABLE—ABSTRACT P23 n ⫽ 8 MAP SVR CI HR RBF UV RVR GFR

Baseline

During infusion

1 h postinfusion

94 ⫾ 7 0.14 ⫾ 0.03 278 ⫾ 58 132 ⫾ 17 1.16 ⫾ 0.38 0.57 ⫾ 0.24 4.60 ⫾ 1.47 0.21 ⫾ 0.03

62 ⫾ 4* 0.07 ⫾ 0.02* 367 ⫾ 75* 154 ⫾ 8* 1.86 ⫾ 0.37* 0.64 ⫾ 0.12* 2.03 ⫾ 0.36* 0.20 ⫾ 0.03

58 ⫾ 10* 0.06 ⫾ 0.04* 308 ⫾ 119 149 ⫾ 18* 1.69 ⫾ 0.30* 0.12 ⫾ 0.03* 1.78 ⫾ 0.54* 0.20 ⫾ 0.04

* P ⬍ 0.05 versus baseline.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS cular system with a drop in MAP and SVR and an increase in CI and HR. it also produced, in our piglets, a statistically significant increase in RBF and UV and a decrease in RVR without affecting GFR. P24. A Novel Model for Studying Wound Healing. D. T. Efron, M.D., D. Most, M.D., H. P. Shi, M.D., and A. Barbul, M.D. Departments of Surgery, Sinai Hospital of Baltimore and The Johns Hopkins Medical Institutions, Baltimore, Maryland. In order to study wound healing, it is often necessary administer various wound-active substances by the systemic route. It is unclear whether the observed effects are the result of local or systemic influence of the agent administered. Furthermore, high systemic doses are often required to achieve activity at the wound level. Direct intrawound administration of substances is traumatic and disruptive to the fragile wound environment and increases the risk of infection. We devised a system for continuous atraumatic delivery of substances directly to subcutaneously implanted polyvinyl alcohol sponges, an adaptation of a well-established model of wound healing. Sponge– catheter constructs were fashioned by feeding identical lengths of silicone catheters through two 40-mg sponge disks (on edge). The distal sponge was fixed 0.5 cm from the distal, ligated end of the catheter, and centered over two 1-mm holes in the catheter tubing. The proximal sponge was fixed over nonperforated catheter with its edge 2 cm proximal from the close edge of the distal sponge. Each construct was connected to a miniosmotic pump (infusion rate 1 ␮l/h) loaded with an appropriate infusate and inserted subcutaneously on the dorsum of an anesthetized male Sprague–Dawley rats. Hydroxyproline (OHP) content of sponges, a measure of collagen deposition, was determined at 7 days postwounding. Infusion of India ink confirmed selective delivery to the distal sponge. Saline infusion alone significantly elevated OHP content compared to noninfused sponges (450 ⫾ 43 vs 328 ⫾ 36 ␮g/100-mg sponge, P ⬍ 0.05). Infusion of S-methylisothiourea (a selective iNOS inhibitor, 84 ␮g/ sponge/24 h) successfully inhibited NO production (35.9 ⫾ 3.1 vs 49.6 ⫾ 3.6 ␮M nitrite/nitrates in sponge fluid, P ⬍ 0.05) and decreased sponge OHP content (385 ⫾ 60 vs 568 ⫾ 70 ␮g/100-mg sponge vs saline, P ⬍ 0.05) without the toxic side effect (i.e., weight loss) seen with systemic administration. The data demonstrate that manipulation of wound physiology is possible by local delivery of low doses of wound-active compounds to the wound site. This promises to be a powerful tool for the study of both normal and impaired wound healing. P25. A Reduction of TNF-␣ mRNA Expression in the Brain Attenuated Nitrogen Excretion in the Urine after Laparotomy in Rats. T. Tajiri, M.D., S. Yoshida, M.D., N. Ishibashi, M.D., K. Tanaka, M.D., T. Muraoka, M.D., and K. Shirouzu, M.D. Kurume University School of Medicine, Kurume, Japan. One of target organs for cytokines is the brain, because intracerebroventricular injection of TNF-␣ caused an increase in nitrogen loss via up-regulation of HPA axis. The aim of this study was to deter-

mine whether prophylactic injection of steroid into the intracerebroventricular (ICV) space would attenuate TNF-␣ mRNA expression in the brain after the surgery, resulting in less nitrogen excretion in the urine. Methods. Forty-eight male SD rats (200 –230 g body wt) were catheterized into the ICV space, using stereotaxic coordination, on day 0. On day 4, the rats were assigned into four groups: (1) saline ICV and anesthesia alone (control), (2) saline ICV plus laparotomy (trauma), (3) ICV injection of methylprednisolone (MP) (0.2 mg/kg, ICV) plus lapa. (MCVT), (4) intraperitoneal injection of MP (5 mg/ rat) plus lapa. (MIPT). Either saline or MP was administered 1 h prior to the anesthesia and laparotomy was carried out by 5 cm of mid incision. The animals were decapitated either 3 or 24 h after the surgery. Brain cortex, hypothalamus, pituitary, liver, and gastrocnemius were harvested from the animals sacrificed at 3 h after the surgery. Twenty-four-hour urine was measured by total nitrogen analyzer. Results. The data are expressed significant differences (P ⬍ 0.05) as shown in the table. Conclusion. A prophylactic lowdose ICV injection of steroid caused a decrease in not only brain TNF-␣ mRNA expression but also nitrogen loss after trauma. Thus, it is likely that cytokines in the brain regulate protein balance rather than those in peripheral tissues after trauma. P26. Significance of Fas Ligand as a Prognostic Marker for Recurrence of Duke’s Stage B Colonic Carcinoma. S. Alrawi, M.D., I. Alhamrawy, M.D., A. Shukur, M.D., J. Cunningham, M.D., A. J. Acinapura, M.D., and R. Raju, M.D. Lutheran & Maimonides Medical Centers, Brooklyn, New York. Background. Fas ligand (FasL) belongs to the tumor necrosis factor (TNF) superfamily, which is implicated in apoptosis. Many diverse tumors have been found to express FasL suggesting that “Fas counterattack” against immune effector cells may contribute to tumor immune escape. Objective. To determine whether tumors from patients with Duke’s B colonic carcinoma express FasL, a potential prognostic marker for aggressive behavior and early spread. Materials and Methods. Fifty patients with Duke’s B primary colon carcinoma histopathologically diagnosed during the period 1989 –1995 were included in the study. Tumor differentiation, PCR evaluation and immunohistochemical expression of FasL in the primary tumors were performed in all patients. A staining score of 4⫹ (⬎75%)was considered strongly positive. Results. Thirty-five patients aged 40 – 85 years were included in our study. Ten patients (22.7%) developed local recurrence or distant metastasis. Thirty-five percent of the tumors were well differentiated, 54% were moderately differentiated, and the rest were poorly differentiated. FasL expression was strongly positive in 68%, focal in 15%, weak in 2%, and negative in 15%. All lymph nodes in the studied specimens were negative for FasL, compared to 31.6% from nonrecurrent tumors (OR ⫽ 3.17, ␹ 2 ⫽ 5.02, P ⫽ 0.025). Conclusion. Patients with Duke’s stage B colonic carcinoma and strong positivity for FasL are more prone to immune escape and earlier tumor spread. We recommend measurement of FasL expression in resected Duke’s B tumors for early identification of patients at risk of recurrence.

TABLE—ABSTRACT P25 TNF-␣ mRNA

Control

Trauma

MCVT

MIPT

Cort. Hypo. Pitu. Liver. Muscle Nitrogen

0.56 (0.23) a 0.26 (0.06) a 0.16 (0.02) a 0.64 (0.09) a 0.12 (0.03) a 336.0 (39.3) a

0.84 (0.10) b 0.53 (0.11) b 0.50 (0.14) b 1.12 (0.08) b 0.21 (0.02) b 912.5 (152.8) b

0.43 (0.06) a 0.31 (0.04) a 0.25 (0.05) a 0.98 (0.13) b 0.15 (0.04) a,b 608.4 (74.6) c

0.53 (0.10) a 0.26 (0.06) a 0.23 (0.08) a 0.67 (0.10) a 0.09 (0.02) a,c 688.7 (108.6) c

Note. mg/day.

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P27. Invasiveness and Regulation of MMP-2 and MMP-9 Expression in Pancreatic Tumor Cell Line SUIT-2 and Its Sublines. X. Q. Yang, MSBS, J. E. Bartsch, MSBS, E. D. Staren, M.D., Ph.D., J. Howard, M.D., T. Iwamura, M.D., Ph.D., and H. E. Appert, Ph.D. Department of Surgery, Medical College of Ohio, Toledo, Ohio; and First Department of Surgery, Miyazaki Medical College, Miyazaki, Japan.

gel invasiveness. However, stimulated breast tumor cell lines differ in their expression of MMP-2, MMP-9, and MMP-10 compared with constitutive expression. These three MMPs may contribute to breast tumor cell malignancy as a result of being up-regulated under certain conditions. The MMPs, which do not include the poorly expressed MMP-2, MMP-3, MMP-9, and MMP-12, may contribute to breast tumor cell invasiveness as a result of their constitutive expression.

Background. Previous investigations have suggested that expression of matrix metalloproteinases (MMPs) may be related to increased invasiveness of various tumors. This study evaluated a possible relation between pancreatic tumor cell invasiveness and MMPs. Methods. A Matrigel invasion assay was performed with pancreatic tumor cell line SUIT-2 and its sublines S2-007, S2-013, S2-020, and S2-028. The degree of invasiveness of stimulated and unstimulated cell lines was correlated with MMP gene expression measured by RT-PCR and MMP protein product measured by gelatin zymography. Cell lines were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), concanavalin (Con A), and polymerized collagen type I gel (Vitrogen). Results. For SUIT-2, S2-007, S2-013, S2-020, and S2-028, 3.2, 1.0, 4.1, 6.4, and 0.4%, respectively, of the cells invaded the Matrigel membrane. TPA, Con A, and Vitrogen resulted in the up-regulation of MMP-2 in S2-020. TPA up-regulated MMP-9 expression in all cell lines, but the effect on SUIT-2, S2-020, and S2-013 was significantly stronger than on the S2-028 and S2-007 sublines. Vitrogen resulted in MMP-9 up-regulation in each of the cell lines, while Con A could up-regulate MMP-9 expression in only the SUIT-2. There was no constitutive expression of either MMP-2 or MMP-9 in SUIT-2 or its sublines. Gelatin zymography of MMP protein product was found to be consistent with the results from RT-PCR. There was a positive relationship between Matrigel invasiveness and up-regulation of MMP-2 and MMP-9 expression. Conclusions. These data show that MMP-2 and MMP-9 are not constitutively expressed in the pancreatic tumor cell lines but may be up-regulated by TPA, Con A, and Vitrogen. MMP-2 and MMP-9 expression correlated with degree of tumor cell invasiveness. The ability to up-regulate MMP-2 and MMP-9 expression may play a role in facilitating pancreatic tumor cell invasion.

P29. Doxycycline Inhibits Canine Osteosarcoma Cell Proliferation in Vitro by Inducing Cell Cycle Arrest in G1 Phase and Apoptosis. A. M. Shoieb, Ph.D., J. L. Bell, M.D., and P. S. Dudrick, M.D. Department of Surgery, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee.

P28. Regulation of MMP Expression in Breast Tumor Cell Lines. J. E. Bartsch, MSBS, X. Q. Yang, MSBS, E. D. Staren, M.D., Ph.D., and H. E. Appert, Ph.D. Department of Surgery, Medical College of Ohio, Toledo, Ohio. Background. Previous studies have shown that matrix metalloproteinase (MMP) production is related to the ability of breast tumors to invade through the extracellular matrix (ECM). The goal of this study was to assess the regulatory mechanisms controlling MMP expression. Methods. The mRNA expression levels of MMPs 1–16 were evaluated by means of semiquantitative RT-PCR on breast tumor cell lines MDA-MB-231, T47D, and MCF 7. Cell lines were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), concanavalin (Con A), and collagen type I (Vitrogen), and the differential regulation of MMP expression was evaluated. Results. Matrigel invasion assays were performed with the cell lines and the degree of invasiveness was correlated with MMP expression. Constitutive expression of all MMPs occurred to varying levels in one or more cell lines. Upon stimulation with TPA, MMP-9 expression was increased in each of the cell lines but there was no increase in the mRNA levels of MMP-2. Administration of Con A had no significant effect on MMP-2 and MMP-9 expression in any of the cell lines. Both Con A and TPA caused an increase in the mRNA expression of MMP-10 in the MDA-MB-231 cell line, but had no effect in the other cell lines. Vitrogen stimulation resulted in an increased expression of MMP-2 and MMP-9 in the MDA-MB-231 cell line. MDA-MB-231, T47D, and MCF-7 were found to have high, intermediate, and low Matrigel invasion, respectively. Conclusions. These studies demonstrate no correlation between constitutive MMP expression and Matri-

Doxycycline (DOX), a chemically modified tetracycline, inhibits proliferation of canine osteosarcoma cells (COS31) in vitro. Induction of apoptosis by DOX has been demonstrated in various cancer cell lines. The aim of this study was to define the mechanisms of inhibition of COS31 cell proliferation by DOX in vitro. Methods. The IC 50 of COS31 cells was determined after exposure to varying [DOX] (0.025–100 ␮g/mL, quadruplicate samples) using a calorimetric cell proliferation assay. Survival curves were generated using log-probit iterative least-squares method. Cell cycle analysis identifying stage of arrested proliferation was determined by flow cytometry. DNA fragmentation (gel electrophoresis) and flow cytometry were the two methods used to demonstrate apoptosis. Results. DOX inhibited proliferation of COS31 cells with an IC 50 of 8.3 ␮g/mL. COS31 cell proliferation was arrested in G1 phase at [DOX] ⫽ 10 ␮g/mL (Table 1). Apoptosis was demonstrated by DNA fragmentation at [DOX] ⫽

TABLE 1—ABSTRACT P29 Percentage of Cells in Phase of Cell Cycle, 0 – 48 h

G1 S G2/M

0h

24 h

48 h

27 60 13

51 43 6

74 23 3

TABLE 2—ABSTRACT P29 Apoptotic Index [DOX] (␮g/mL)

AI (%)

5 10 20

11 21 48

10 and 20 ␮g/mL at 48 –72 and 24 – 48 h, respectively. Apoptotic index (AI) at 12–24 h determined by flow cytometry is shown in Table 2.Conclusions. DOX inhibits proliferation of COS31 cells in vitro by arresting cells in the G1 phase of the cell cycle and inducing apoptosis. This is the first study to suggest a link between G1 cell cycle arrest and induction of apoptosis in osteosarcoma cells in vitro. P30. Sentinel Lymph Node Biopsy for Melanoma: Metastases vs Benign Intracapsular Nevus in Sentinel Nodes. J. E. Duncan, M.D., D. Corbett, M.D., K. Hoffmeister, M.D., B. Knollmann-Ritschel, M.D., D. Turton, M.D., J. Williams, M.D., and R. C. Jones, M.D. Departments of Surgery, Dermatology, Medical Oncology, Nuclear Medicine, and Pathology, National Naval Medical Center, Bethesda, Maryland.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS We review the experience and results of sentinel lymph node biopsy for melanoma at our institution under the auspices of a qualified sentinel lymph node surgeon over a 3-year period. Each melanoma patient had sentinel nodes (SNs) localized by lymphoscintigraphy in the presence of the operating team. SLNB was performed using intraoperative gamma detection and 1% isosulfan blue dye followed by wide local excision of the primary site. Sentinel nodes were evaluated for metastatic disease by standard H & E staining as well as by immunohistochemistry using S-100 and HMB-45 murine monoclonal antibodies. Patients with confirmed sentinel node metastases proceeded to therapeutic lymph node dissection. Thirtyseven patients underwent a total of 46 SLNB procedures from May 1997 to January 2000. One-hundred percent of procedures identified sentinel nodes, with 2.4 ⫾ 1.1 SNs per lymphatic basin. In four patients (10.8%), sentinel node metastases were observed. In these patients, 4/10 (40%) of at-risk basin sentinel nodes were positive while none of the nonsentinel nodes (0/85) from therapeutic lymph node dissection were positive for metastases. In an additional 4 patients (10.8%), 4/15 (26%) sentinel nodes were found on immunohistochemistry to have focal intracapsular nevus cell aggregates, a benign finding that was viewed histologically distinct from metastatic melanoma. Failure to distinguish benign findings from metastases on SLNB could subject a significant number of patients to additional unnecessary surgery, adjuvant therapy, and morbidity. This finding of benign immunohistochemistry-positive melanocytes provokes questions that merit additional scrutiny in analysis of SLNB and survival and of studies that ascribe significance to new adjuvant regimens in patients accrued from SLNB patients. P31. Genistein Enhances Radiation Sensitivity of Human MCF-7 Breast Cancer Cells in Vivo. J. L. Jones, M.D.,*,† J. R. Zhou,* Ph.D., and G. L. Blackburn, M.D., Ph.D.* *Department of Surgery, Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; and †Department of Surgery, University of Tennessee Medical Center, Knoxville, Tennessee. Genistein is the dominant isoflavone in soybeans. It has been shown to arrest cell cycle progression in the G2/M phase in multiple cell lines, including MCF-7 human breast cancer cells (Shao, 1998). Since G2/M is the most radiosensitive phase of the cell cycle, we hypothesized that treatment with genistein prior to radiation would enhance the radiosensitivity of these tumors in mice. Thirty-two female SCID-beige mice were orthotopically injected with 5 ⫻ 10 6 MCF-7 cells. Once tumors developed the mice were assigned to four groups (n ⫽ 8); two received a control diet and two were fed the same diet supplemented with 0.14% genistein by weight. After a week of dietary treatment they were irradiated with 0.75 Gy/day of 137Cs gamma irradiation for five consecutive days. Tumor growth rates were calculated by linear regression of tumor volumes (L*W 2*␲/6) and the slopes were compared with ANOVA. Both the radiation (0.048%/day) and the genistein ⫹ radiation (0.037%/day) groups showed significantly decreased tumor growth rates compared to either the control (0.064%/d) or the genistein only (0.069%/day) groups (P ⬍ 0.05). Further, the genistein ⫹ radiation group showed a lower

363

tumor growth rate compared to the radiation group, though this comparison did not reach statistical significance. These data suggest a synergistic effect between genistein and gamma radiation on the growth of MCF-7 breast tumors in mice (see figure). P32. Peptide YY Augments the Inhibitory Effects of ␣-Tocopherol Succinate on Prostate Cancer Cell Growth. A. T. Rose, M.D., and D. W. McFadden, M.D. Department of Surgery, UCLA Medical Center, Los Angeles, California.

␣-Tocopherol succinate (ATS) has been shown to induce apoptosis of hormone-refractory prostate cancer cells in vitro and inhibit cell growth in vivo. Peptide YY (PYY) has growth inhibitory activity against multiple cancer cell lines and is synergistic with ATS against breast cancer cells. No data exist on the impact of PYY and ATS against prostate cancer cell lines, cells similar to breast cancer in their hormonal regulation. Methods. A hormone-refractory human prostate cancer cell line, PC-3, was treated with ATS alone at doses ranging from 10 to 100 ␮g/cc, PYY alone at doses of 50 to 500 pmol, or a combination of the agents. MTT assay was performed at 24, 48, and 72 h to determine cell viability. Statistical analysis was performed with the Student’s t test. Results. Both ATS and PYY exhibited a growth inhibitory effect on the prostate cancer cells compared to controls. ATS caused decreased cell viability with 50 or 100 ␮g/cc by 24 h (P ⬍ 0.001), with 25 ␮g/cc by 48 h (P ⬍ 0.001), and with 10 ␮g/cc by 72 h (P ⬍ 0.05). The growth inhibitory effects of the 100, 50, and 25 ␮g/cc doses were more pronounced at longer time points. PYY at a dose of 500 pmol caused growth inhibition at all time points (P ⬍ 0.05), with no inhibition noted at lower doses. When the two agents were used in combination, PYY augmented the growth inhibitory effects of ATS. Decreased cell viability was seen with cells treated with either 25 or 10 ␮g/cc of ATS and all three doses of PYY, with 500 pmol PYY exerting a stronger growth-inhibitory effect than either 250 or 25 pmol (P ⬍ 0.05). No additive effect of PYY was seen with higher doses of ATS. Conclusion. PYY and ATS both exert a growth-inhibitory effect on hormone-resistant prostate cancer cells, with enhancement of the effect when the two agents are used in combination. This inhibition is both time and dose dependent. Further in vivo and clinical studies of the combination of PYY and ATS in patients with hormone-refractory prostate cancer are warranted. P33. Thyroid Tumor Metastasis Is Associated with Heparanase Gene Expression. A. W. Kim, M.D., X. Xu, Ph.D., M. Olson, M.D., P. Gatusso, M.D., and R. A. Prinz, M.D. Department of General Surgery, Rush Presbyterian St. Luke’s Medical Center, Chicago, Illinois. Introduction. Heparanase (HPR) is an endoglycosidase that specifically degrades the heparan sulfate of proteoglycan, a chief component of the extracellular matrix (ECM). ECM degradation is essential for metastatic tumors to penetrate a tissue barrier. This study tested whether HPR was differentially expressed in thyroid tumors and whether HPR gene expression correlated with thyroid tumor metastasis. Methods. Sixty thyroid tumor samples collected from surgical specimens were examined for HPR gene expression by in situ hybridization. HPR expression was compared with clinicopathological features of thyroid neoplasms and statistically analyzed with SuperAnova Turkey test. Ten thyroid tumor cell lines were examined for HPR expression by using RT-PCR. Results. Twentyfour of 33 papillary carcinomas, 2 of 2 medullary carcinomas, and 4 of 5 follicular carcinomas scored HPR positive. However, 15 of 17 follicular adenomas and 3 of 3 Hu¨rthle cell adenoma scored negative. Statistical analyses revealed that HPR is differentially expressed between thyroid carcinomas and follicular adenoma or Hu¨rthle neoplasm (P ⬍ 0.05). Clinicopathological analysis revealed that 20 patients had metastases in local lymph nodes at the time of operation; 16 of them scored HPR positive. In contrast, HPR expression did not correlate with patient age, gender, and tumor size. RT-PCR analysis

364

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

of HPR expression revealed that HPR was not expressed in one follicular adenoma (KAK-1) and three anaplastic (KAT-4, KAT-18, SW1736) carcinoma cell lines. However, HPR was expressed in two follicular (WRO82 and MRO87), two anaplastic (ARO81 and DRO90), and two papillary (NPA87 and KAT-10) carcinoma cell lines. Conclusions. HPR is differentially expressed in thyroid neoplasms, and thyroid tumor metastasis is associated with HPR gene expression. P34. Predictors of Nonsentinel Lymph Node Metastasis in Breast Cancer Patients. U. Sachdev, M.D., K. Murphy, B.A., A. Derzie, M.D., I. Bleiweiss, M.D., and S. Brower, M.D. Departments of Surgery and Pathology, Mount Sinai Medical Center, New York, New York. Background. In order to define a future subset of breast cancer patients in whom the axilla may be staged by sentinel lymph node biopsy alone, the conditions under which nonsentinel axillary lymph node metastasis occur must be delineated. We propose that predictors of nonsentinel lymph node metastasis can be found. Methods. A prospective database including 212 breast cancer patients who underwent sentinel lymph node biopsy followed by completion axillary dissection at our institution between July 23, 1997, and December 15, 1999, was reviewed. The sentinel node was identified using 1% isosulfan blue dye either alone or in conjunction with preoperative administration of Technetium-99m sulfur colloid. In each case, pathologic analysis of each sentinel node included standard H & E as well as immunohistochemical staining for cytokeratin in cases where the sentinel node was negative by H & E. For each patient, age, date of surgery, method of sentinel node identification, primary tumor size, number of sentinel nodes identified, presence or absence of tumor in the sentinel node (SLN⫹, SLN⫺, respectively), presence of macrometastasis (⬎1 mm) or micrometastasis (⬍1 mm) in the sentinel node, and presence or absence of tumor in the nonsentinel nodes (NSLN⫹, NSLN⫺, respectively) were recorded. The identification and false negative rates were calculated. A multivariate logistic stepwise regression was performed to evaluate the relationship between nonsentinel lymph node metastasis and the following parameters: patient age, primary tumor size, presence of lymphatic invasion, use of radioisotope to identify the sentinel node, and the presence of macrometastasis (⬎1 mm) or micrometastasis (⬍1 mm) in the sentinel node. Results. One-hundred ninety patients underwent successful SLN biopsy. The mean age of the patients was 57 years. In 131 patients, the sentinel node was identified with blue dye alone and in 59 patients, the sentinel node was identified using both dye and radioisotope. Onehundred sixty patients had T1 tumors (⬍2 cm), 27 had T2 (2–5 cm), tumors and 1 had a T4 tumor. A mean of 1.75 sentinel nodes per patient were identified in both the radioisotope and dye and the dye alone groups. The identification rate was calculated to be 90% overall. Twelve patients were SLN⫺/NSLN⫹ giving a false negative rate of 18% overall, 22.7% for the blue dye alone group, and 8.7% for the blue dye combined with radioisotope group. The pathologic type of tumor was not found to be related to metastasis in the nonsentinel node. However, tumor size greater than 2 cm, lymphatic invasion of the primary tumor, macrometastasis in the sentinel node, and use of radioisotope all positively correlated independently with metastasis in the NSLN (P ⫽ 0.0001, P ⫽ 0.0483, P ⫽ 0.0008, P ⫽ 0.0271, respectively). Conclusion. Breast cancer metastases in the nonsentinel axillary lymph nodes are more likely to be seen in patients with tumors greater than 2 cm, tumors with evidence of lymphatic invasion, and macrometastasis in the sentinel node. Predictors of nonsentinel axillary node metastasis identify patients in whom the suspicion of metastasis beyond the sentinel node is relatively high and in whom the finding of a negative sentinel lymph node should be closely examined. P35. Identification of Differential DNA Methylation in Esophageal Adenocarcinoma (EAC). S. K. Kurumboor, Ph.D., R. V. Lord, M.D., K. Wickramasinghe, M.D, and K. A. Skinner, M.D. Department of Surgery, University of Southern California, Los Angeles, California.

Changes in DNA methylation patterns can lead to activation or inactivation of genes. The objective of this study was to determine the methylation patterns in genomic DNA from normal, metaplastic, and malignant esophageal tissue. Multiple tissue samples were collected from three patients undergoing esophagectomy for EAC (n ⫽ 2) or Barrett’s esophagus with high-grade dysplasia (n ⫽ 1). Genomic DNA was extracted and methylation-sensitive arbitrarily primed PCR (APPCR) was performed. This technique relies of the different abilities of restriction enzymes HpaII and MspI to cut the CCGG sequence. MspI cuts regardless of the methylation status, whereas HpaII cuts only unmethylated sequences. Genomic DNA was digested with RsaI alone, RsaI ⫹ HpaII, or RsaI ⫹ MspI. A P-PCR was performed on digested DNA using three sets of CG-rich 10-mer primers under low stringency conditions and the PCR products were separated on a high-resolution polyacrylamide gel. Bands were evaluable if present in the RsaI alone lane, but not in the MspI lane. Bands were methylated if there was a band in the HpaII lane. Band patterns from Barrett’s or tumor tissue were compared to that from normal tissue from the same patient. Of 107 evaluable bands identified in DNA from normal esophageal tissue, 29% were consistently unmethylated, and 68% were consistently methylated. Three bands (3%) were differentially methylated in Barrett’s and/or tumor tissue. Two bands were hypermethylated in all metaplastic or malignant tissues. The third band was hypomethylated in tumor samples from one patient. DNA from breast cancer tissue showed a completely different banding pattern. DNA methylation changes in EAC are not global, but are restricted to specific CpG sites. These differentially methylated sequences can be isolated and characterized. These methylation changes are not ubiquitous in all tumors, but appear to be specific to EAC. P36. Inhibition of Constitutive RelA Activity Suppresses Matrix Metalloproteinase-9 (MMP-9) Production in Human Pancreatic Cancer. T. N. Wang, M.D., Ph.D., W. A. I. Frederick, M.D., T. Wu, D. B. Evans, M.D., and P. J. Chiao, Ph.D. Department of Surgical Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas. Introduction. RelA is a member of the Rel/NF-␬B transcription factor family and is constitutively activated in pancreatic adenocarcinoma cell lines. The downstream target genes induced by activated RelA are predominantly unknown. We hypothesize that NF-␬B inhibitors which abolish constitutive RelA activity will suppress MMP-9 production necessary for pancreatic tumor cell invasion. Methods. Two human pancreatic cancer cell lines (Panc-1 and Panc28) were treated with either an NF-␬B activator (TNF) or an NF-␬B inhibitor [curcumin (Cur), n-tosylphenylalanine chloromethyl ketone (TPCK), or pyrrolidine derivative of dithiocarbamate (PDTC)]. Western immunoblot analysis for MMP-9 was performed on control (untreated) and treated cytoplasmic extracts. MMP-9 activity in the cell lines was measured by an ELISA gelatinase activity assay. Each experiment was performed in triplicate. Results. Cur, TPCK, and PDTC suppressed MMP-9 production in both cell lines as demonstrated by Western immunoblot analysis. TPCK and PDTC significantly inhibited MMP-9 activity in both cell lines as demonstrated by the gelatinase activity assay (see table). Conclusion. Constitutively

TABLE—ABSTRACT P36 Cells

Control

TNF

TPCK

PDTC

Panc-1 Panc-28

676 ⫾ 110 877 ⫾ 135

833 ⫾ 177 1231 ⫾ 189

159 ⫾ 145* 75 ⫾ 126*

203 ⫾ 106* 113 ⫾ 92*

Note. Numbers represent gelatinase activity produced by an equivalent concentration of MMP-9 (ng/ml) as measured by ELISA. *P ⬍ 0.005 vs control. Data were analyzed by Student’s unpaired t test.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

365

activated RelA may promote tumor cell invasion by inducing downstream target genes responsible for MMP-9 overproduction. Inhibition of RelA activity may prevent MMP-9-regulated cellular proteolysis necessary for pancreatic cancer cell invasion and metastasis. P37. Inactivation of Rho in Lung Carcinoma Cell Lines Expressing Mutant Ras Proteins Inhibits Proliferation and Induces Neurite Formation. K. A. Varker, M.D., J. S. Wiseman, M.D., and C. L. Williams, Ph.D. Department of Surgery, Guthrie Clinic, and Molecular Pharmacology Laboratory, Guthrie Research Institute, Sayre, Pennsylvania. The expression of activated mutant Ras proteins (Ras⫹) in non-small cell lung carcinoma (NSCLC) is associated with a negative prognosis (Graziano, 1997, Lung Cancer, 17, S37–S58). The oncogenic properties of Ras ⫹ depend in part on the expression of active Rho proteins (Olson et al., 1998, Nature 394, 295–299). To investigate our hypothesis that Rho regulates the proliferation of lung carcinomas expressing oncogenic Ras ⫹, we measured the effects of Rho inactivation on cell proliferation in eight lung carcinoma cell lines. Two of these lines express oncogenic Ras ⫹ (the NSCLC lines NCI-H23 and NCI-H157); the other lines express normal Ras (the NSCLC lines NCI-H522 and NCI-H520 and the small cell lung carcinoma lines NCI-H69, NCI-H146, NCI-H345, and SCC-9). The cells were electroporated with or without cDNA for C3 exoenzyme, which ADP-ribosylates and inactivates Rho. The fold increase in [3H]thymidine uptake by the cells over 48 h was determined by liquid scintillation counting and statistically analyzed by the matched-pairs Student’s t test. Results were obtained from at least three independent experiments for each cell line. Inactivation of Rho significantly inhibits the proliferation of NCI-H23 cells (16.9 ⫾ 3.6- vs 10.8 ⫾ 0.5-fold increase in control cells vs C3 exoenzyme-treated cells, respectively, P ⬍ 0.05). This effect also occurs in NCI-H157 cells (38.7 ⫾ 8.3- vs 24.8 ⫾ 6.1-fold increase in control cells vs C3 exoenzyme-treated cells, respectively, P ⬍ 0.05). In contrast, Rho inactivation does not detectably inhibit cell proliferation in the other cell lines. Inactivation of Rho also induces neurite formation, suggesting differentiation, in the NCI-H23 and NCI-H157 cell lines but not in the other cell lines. These results show that Rho plays an important role in regulating the proliferation and differentiation of lung carcinoma lines expressing oncogenic Ras⫹. P38. v-Src Transformation Is Mediated through Farnesylated Proteins. S. C. Teng, M.D., J. Sun, Ph.D., R. Irby, Ph.D., S. Sebti, Ph.D., and T. J. Yeatman, M.D. Departments of Surgery and Drug Discovery Program at the H. Lee Moffitt Cancer Center, University of South Florida, Tampa, Florida. Src is an oncoprotein which has been implicated in a number of human malignancies. The precise mechanism of Src transformation, however, is poorly understood. We hypothesized that farneslyated proteins downstream of Src may mediate transformation. To test this hypothesis v-Src-transfected rat fibroblasts (3Y1) were treated every 72 h with a 15 ␮M concentration of a farnesyl-transferase inhibitor (FTI). At 2weeks, a focus formation assay was performed to assess transformation. The mean numbers of foci per well were calculated. Statistical significance was determined with an unpaired, one-tailed Student’s t test. Western blots for H-Ras and Rap1A were performed to assess for inhibition of farnesylation. Untreated and FTI-treated v-Src-transfected 3Y1 cells formed a mean of 39.0 (⫾ 2.6) and 29.8 (⫾ 2.9) foci per well, respectively. This 24% decrease was statistically significant (P ⫽ 0.02). Foci in the treated wells were also significantly smaller than foci in the untreated wells. Western blots with antibody directed toward H-Ras confirmed complete inhibition of Ras farnesylation in the treated cell lines (see figure). The specificity of this inhibition was verified by a Western blot using antibody specific for Rap1A. The transforming potential of v-Src is inhibited, but not eliminated, by FTI treatment. This suggests that v-Src transformation is mediated in part by farnesylated proteins.

P39. A Molecular Hypothesis for the Phenomenon of Generalized Arteriomegaly and/or Peritoneal Inflammation in Some Patients with Abdominal Aortic Aneurysms. D. Syn, M.D., K. Hardy, B.A., S. Xia, M.D., and M. D. Tilson, M.D. Department of Surgery, Columbia University and St. Luke’s/ Roosevelt Hospital, New York, New York. Background. Generalized arteriomegaly is commonly observed in patients with abdominal aortic aneurysm (AAA), and inflammation of the peritoneum occurs in some. Self-antigens have been described in AAA, and the following experiments were performed to determine whether three of these antigens [aneurysm-associated antigenic protein– 40 KDa (AAAP-40) and recombinant clones 1 and 5 (rCL-1 and rCL-5)] are detectable by immunohistochemical methods in peripheral arteries and other human tissues. Methods. Antibodies were raised in sheep against synthetic polypeptides encoding unique sequences of AAAP-40, rCL-1, and rCL-5, which are not known to occur in any other proteins. Serial sections were cut of numerous peripheral arteries and tissues from autopsy specimens, and the antibodies were used to probe for immunoreactive proteins. Results. rCL-1 was the most highly specific for adventitial microfibrils of the aortoiliac segment. AAAP-40 was also most prominent in the aortoiliac segment, but antigen was detected in other vessels, including carotid, subclavian, thoracic aorta, coronary, femoral, and popliteal. rCL-5 was widely distributed, not only in arteries but also in other tissues, associated with fibroblasts in lung, dermis, and peritoneum. Conclusions. AAAP-40 and rCL-1 appear to be artery-specific antigenic proteins (ASAPs). If a patient with AAA is primarily reacting to rCL-1, the consequences may be limited to the aortoiliac segment, but if a patient is primarily reacting to AAAP-40, one would predict generalized arteriomegaly. If rCL-5 were the primary antigen in a given case, extravascular manifestations might also occur, like COPD or peritoneal inflammation, e.g., inflammatory aneurysm. There may be a molecular explanation for the clinical heterogeneity of AAA disease. P40. Homocysteine (Hcy) Promotes Chemotaxis and Activates P38 in Bovine Aortic Smooth Muscle Cells (SMC). K. Akasaka, M.D., G. DiLuozzo, M.D, N. Akasaka, M.D., and B. E. Sumpio, M.D., Ph.D. Section of Vasular Surgery, Yale University School of Medicine, New Haven, Connecticut. Purposes. Elevated levels of Hcy have been identified as a risk factor for atherosclerotic disease. However, the Hcy effects on SMC migration are unknown. The purposes of this study are to examine the effects of Hcy on SMC migration, and activation of P38 that is one of the mitogen-activated protein kinases. Methods. The effect of 0.5, 1.0, and 2.0 mmol/L D,L-Hcy as a chemoattractant was assayed using a modified Boyden chamber with SMC. Phosphorylated p38 was detected by immunoblotting, after exposing the SMC to 0.5, 1.0, or 2.0 mmol/L D,L-Hcy in serum-free medium (SFM) for 15 min. Results. The number of migrated cells was increased 8.4 ⫾ 1.9-fold by 2.0 mmol/L D,L-Hcy (n ⫽ 11, P ⬍ 0.05) (see figure, left). There was a p38 activation after exposure to 2.0 mmol/L D,L-Hcy 4.6 ⫾ 1.2-fold (n ⫽ 4, P ⬍ 0.05) (see figure, right). Conclusions. These results

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suggest that Hcy is a chemoattractant for SMC and may involve p38 activation. This finding may be an important mechanism for atherogenesis induced by Hcy. P41. Neointimal Hyperplasia in a Vein Graft Model. N. Angle, M.D., M. M. Farooq, M.D., C. Pross, M.D., H. Gelabert, M.D., M. C. Fishbein, M.D., and J. A. Freischlag, M.D. UCLA Gonda (Goldschmied) Vascular Center, Los Angeles, California. Neointimal hyperplasia (NH) has been primarily described in arterial injury models. The mechanism underlying NH in the vein graft may be different from that in the artery undergoing angioplasty. A vein bypass graft represents a more relevant model for investigating the mechanism of vein graft NH. The objective of the study was to develop a reproducible vein graft model and to characterize the NH. Adult male Sprague–Dawley rats (3 months) were anesthetized with intraperitoneal ketamine and xylazine. The right external jugular vein was harvested using minimal periadventitial dissection. The abdominal aorta was dissected and clamped below the left renal vein and above the first iliolumbar branch. No systemic heparin was used. The aorta was divided and the jugular vein anastomosed in an end-to-end fashion using interrupted 10-O nylon. Vein grafts were explanted at 4 weeks following implantation. Immunohistochemical analysis was performed using hematoxylin and eosin, ␣- actin antibody for smooth muscle cells (SMC), and ED-2 antibody against macrophages. Grafts were compared with nongrafted veins. Mortality of this model was 0% and all grafts remained patent. All grafts developed NH. Mean neointimal thickness was 4.8 ⫾ 0.3 mm (lowpower field) vs 0 in normal vein (P ⬍ 0.001). NH was most prominent at both anastomoses but the entire vein graft showed neointimal thickening. A thick neointima with abundant cellularity and extracellular matrix was demonstrated in all grafts. At 4 weeks, the neointima stained strongly for ␣-actin, suggesting that the predominant cell type was smooth muscle. Macrophages comprised ⬍10% of all cells in the neointima, as manifested by ED-2 staining. Occasional mast cells were also present in the vein wall. Vein graft interposition in the rat aorta results in a prominent and reproducible neointima that, at 4 weeks, is composed predominantly of SMC and extracellular matrix, with a relatively small population of monocyte/ macrophages. This vein graft model is technically simple, clinically relevant, and offers opportunities for a more in-depth understanding of the mechanism underlying NH in the vein graft. P42. Cyclic Strain Induces RhoA Activation in Bovine Aortic Endothelial Cells. A. H. Chen, M.D., S. Li, Ph.D., S. G. Frangos, M.D., G. Di Luozzo, M.D., A. Dhadwal, M.D., and B. E. Sumpio, M.D., Ph.D. Section of Vascular Surgery, Yale University School of Medicine, 333 Cedar Street, FMB 137, New Haven, Connecticut. The purpose of this study is to determine whether cyclic strain activates RhoA in bovine endothelial cells. The repetitive pulsatile pressure induced by the beating heart causes cyclic strain on vascular endothelial cells (ECs). Studies have shown that shear stress

induces biological changes in the ECs by activating key small GTPase proteins in the Ras superfamily such as RhoA. This study will determine whether cyclic strain has similar effects on RhoA. RhoA activation is assessed by detecting translocation of RhoA from the cytosol to the membrane. Cultured bovine aortic ECs were placed in a monolayer fashion on flexible-bottomed membranes. These cells were then serum starved for 18 to 24 h and exposed to 150 mm Hg repetitive deformation, which created an average of 10% strain on the ECs at a frequency of 60 cycles/min for 0 (negative control), 5, and 15 min. Serum (10% fetal bovine serum)-stimulated ECs were used as positive controls. Lysed cell proteins were separated into cytosol and membrane fractions by ultracentrifugation. The translocation of the RhoA is assessed by measuring the absolute quantity of RhoA in the cytosol and in the membrane fractions via Western blot analysis. Statistical significance was determined using one-way ANOVA. Translocation of RhoA to the membrane in ECs is significantly increased over negative control (1.85 ⫾ 0.31-fold, n ⫽ 4, P ⬍ 0.05) after 5 min of cyclic strain and in the 10% FBS group (see figure). Exposure of bovine endothelial cells to cyclic strain activates

the small GTPase protein, RhoA. Preliminary data also show that Rock, an effector protein of RhoA, also translocates to the membrane, indicating that it too may be activated along with RhoA following cyclic strain exposure. These results provide a better understanding of the mechanism by which hemodynamic forces alter the cytoskeleton of endothelial cells. P43. Characterization of Gene Expression in Human Smooth Muscle Cells Following Thrombolysis. H. Dosluoglu, M.D., B. Radolinski, B.S., R. Saadeh, M.S., L. Harris, M.D., and M. Dryjski, M.D., Ph.D. Department of Surgery State University of New York at Buffalo, Buffalo, New York. Introduction. Dysfunction of endothelial cells and smooth muscle cells (SMC) is likely to be involved in early and late vein graft failure following thrombolysis. The aim of this study was to define the gene expression in SMC in an in vitro thrombolysis model using largescale nucleic acid arrays. Methods. Cultured human SMC were exposed to fresh whole blood clot for 2, 6, 12, and 24 h. Thrombus was then lysed using 10,000 U of urokinase and the cells were evaluated for viability and morphology. ATLAS human cDNA expression array membrane containing 588 genes were used to screen genetic response at 6 h of clot exposure. Quantitative PCR was used to define the temporal relationship of gene expression in selected adhesion molecules and cytokines. Results. SMC exposed to blood clot remained viable. A total of 45 genes were expressed or suppressed at least twice as much as the control group in the array membrane.

TABLE—ABSTRACT P43 Percent change

ICAM VCAM TNF␣ IL-6 MMP-2

2h

6h

12 h

24 h

15,349 77 6,100 1,127 ⫺21

24,461 179 3,918 1,304 ⫺23

30,558 108 1,678 370 ⫺34

18,303 80 1,386 112 27

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Twenty-six selected genes were study using PCR method. The results of PCR analysis of 5 genes involved in vascular wall remodeling are summarized in the table. The other genes significantly expressed included cytokines, transcription factors, cell cycle regulators, antioxidants, and aptosis proteins. Conclusions. This model show the magnitude of stress SMC may undergo following thrombolysis. The use of ATLAS technique may help to develop potential therapeutic interventions to improve graft patency following thrombolysis. P44. External Beam Radiation Reduces Venous Neointimal Hyperplasia in an Animal Model of PTFE Dialysis Grafts. B. S. Kelly, M.D., S. C. Heffelfinger, M.D., A. Narayana, M.D., M. A. Miller, M.S., W. A. Karle, B.S., J. F. Whiting, M.D., J. W. Alexander, M.D., and P. Roy-Chaudhury, M.D. Departments of Surgery and Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio. Venous neointimal hyperplasia (VNH) is the most identified pathology resulting in PTFE vascular access dysfunction. Using an animal model that reproduces the pathognomonic features of this lesion, we have demonstrated that perioperative external beam radiation (EBR) reduces the magnitude of neointimal hyperplasia at the graft–vessel anastomosis. Goretex loop grafts were placed bilaterally from the femoral artery to the femoral vein in 12 male, neutered, Yorkshire cross pigs. The right infrainguinal region was irradiated (16 Gy) at either 24 h before or 24 h after insertion of PTFE grafts. The left graft received no intervention (control). Grafts were harvested on POD 28, and histomorphometric analysis of H and E-stained specimens was conducted to calculate the percentage of luminal stenosis (LS) at the graft–vessel anastomosis. Immunohistochemistry was performed to identify smooth muscle cells, endothelial cells, and specific proangiogenic cytokines. Cell proliferation indices were determined using BrdU. External beam radiation significantly reduced venous neointimal hyperplasia (%LS) at the graft–vein anastomosis when compared to control specimens (38.4 ⫾ 5.20 vs 50.5 ⫾ 5.07%; P ⫽ 0.0212). Likewise, EBR significantly reduced arterial intimal hyperplasia (%LS) at the graft–artery anastomosis compared to control specimens (42.4 ⫾ 5.21 vs 19.7 ⫾ 4.70%; P ⫽ 0.0311). Timing of EBR administration did not significantly affect the %LS. These results demonstrate for the first time that perioperative EBR can significantly reduce venous neointimal hyperplasia and, further support the use of EBR as a novel prophylactic therapy to deter the progression of this costly and morbid clinical problem. P45. Pravastatin Preserves Endothelial Vasomotor Function in an Experimental Model of Diabetes Mellitus. D. S. Moneley, AFRCSI,* C. Gang, M.D.,* E. Kay, FRCPath,† C. Thompson, M.D.,‡ C. J. Kelly, M.Ch.,* and D. J. BouchierHayes, M.Ch.* Departments of *Surgery, †Pathology, and ‡Endocrinology, RCSI, Beaumont Hospital, Dublin 9, Ireland. Endothelial dysfunction, characterized by impairment of endothelial-dependant vasodilatation, is a key event in the pathogenesis of most vascular diseases. The aim of this study was to investigate the effect of pravastatin on ischemia–reperfusion (IR)induced vasomotor dysfunction in the diabetic microcirculation. Sprague–Dawley rats (n ⫽ 18) were randomized into three groups: (a) control; (b) diabetic; and (c) diabetic treated with pravastatin. A mesenteric arteriole was examined pre- and post-IR using intravital microscopy. Endothelial-dependent and -independent vasodilatation, measured by response to acetylcholine (Ach) and sodium nitroprusside (SNP), respectively, was measured in mesenteric arterioles preconstricted with noradrenaline. The arteriolar constriction:dilatation ratio (C:DR) was then calculated. Results are expressed as mean ⫾ SEM following 60 min of reperfusion (see table). These data

TABLE—ABSTRACT P45 Group

Ach (C:DR)

SNP (C:DR)

Control Diabetic Diabetic/pravastatin

1.6 ⫾ 0.085 1.16 ⫾ 0.049$ 1.78 ⫾ 0.04*

1.98 ⫾ 0.01 1.91 ⫾ 0.03 1.96 ⫾ 0.07

* P ⬍ 0.01 vs diabetic; $ P ⬍ 0.01 vs control ANOVA, Tukey– Kramer post hoc test.

demonstrate a restoration of endothelial-dependent vasomotor function in the treated diabetic group compared with untreated. We have shown that pravastatin in an experimental model of diabetes mellitus preserves endothelial function and protects endothelial integrity following IR. P46. IL-10 and Low Shear Stress-Induced Neointimal Hyperplasia. J. E. Rectenwald, M.D., R. M. Minter, M.D., L. L. Moldawer, Ph.D., T. S. Huber, M.D., J. M. Seeger, M.D., and C. K. Ozaki, M.D. Department of Surgery, University of Florida College of Medicine and the Malcom Randall VAMC, Gainesville, Florida. Overexpression of the anti-inflammatory cytokine interleukin-10 (IL-10) blocks atherosclerotic events in vivo, and IL-10 has been recently hailed as an “immunologic scalpel” for atherosclerosis. Alternatively, mice lacking IL-10 receiving atherogenic diets have increased occlusive lesions. It is unclear whether these findings apply to other forms of occlusive arterial remodeling. We hypothesized that lack of IL-10 would exacerbate, and exogenous or overexpression of IL-10 would abrogate, low shear stressinduced neointimal hyperplasia (NH). Methods. Wild type (WT) and IL-10-deficient (IL10⫺/⫺) mice underwent unilateral common carotid artery (CA) ligation. Low shear stress in the patent ligated artery results in remodeling and formation of neointima containing BrdU and SMC ␣-actin-positive cells. Additional groups of WT mice underwent CA ligation and were treated daily with ip saline or 1 ng of human IL-10. In separate experiments, 1 ⫻ 10 10 adenovirus/empty cassette (ad/e) or adenovirus/viral IL-10 (ad/ IL10) was injected im or applied directly to the adventitia (Advnt) of the CA (Buffer, ad/e, ad/IL-10) immediately following ligation. CAs were perfusion fixed 28 days postligation, the time at which NH is near maximum. Results. IL-10⫺/⫺ mice developed identical NH areas compared to WT controls. Mice receiving IL-10 demonstrated NH equivalent to saline controls. Mice receiving im or Advnt ad/IL-10 developed high serum levels of IL-10, yet formed NH areas similar to controls. No antibody to IL-10 was found in the sera of IL-10-treated mice, which failed to attenuate NH. Conclusions. Lack of IL-10 does not exacerbate low shear stress induced-NH, nor does exogenous administration or overexpression of IL-10 attenuate it. Discrepancies between these findings and previous research may be related to IL-10 dosing, inflammation induced by the adenoviral vector, or disparities between the NH models. P47. Early Vascular Lab Screening Can Increase the Number of Arteriovenous Fistulae and Reduce the Need for Catheters to Initiate Dialysis. V. J. Teodorescu, M.D.,* K. Frasier, R.V.T.,* A. Falk, M.D.,† J. Uribarri, M.D.,‡ J. Vassalotti, M.D.,‡ M. A. DiNucci, R.N.,‡ and M. Kerstein, M.D.* Departments of *Surgery, †Radiology, and ‡Nephrology, Mount Sinai Medical Center, New York, New York. Introduction. A screening protocol was implemented so that dialysis access placement could be planned in advance with the

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goal of increasing the number of AVF and decreasing the number of catheters at time of dialysis initiation. Methods. From October 1998 to March 2000, 42 Renal Clinic patients with a serum creatinine of 4 mg/dl were referred for bilateral vein and artery evaluation in the Vascular Laboratory. An AVF was created when the creatinine rose to 6 mg/dl, if the following criteria were met: (1) cephalic or basilic vein greater than 2 mm in diameter in continuity with the deep system, (2) patent ipsilateral subclavian vein, and (3) peak systolic velocities of at least 50 cm/s in the appropriate artery of at least 2 mm in diameter. When these criteria were not met, an arteriovenous graft (AVG) was placed. These patients were compared to a similar group of clinic patients (n ⫽ 37) who began dialysis in 1997, prior to the initiation of the screening program. Results. Two of the 42 patients were treated with peritoneal dialysis. The remaining 40 had either AVF (n ⫽ 29; 72.5%) or AVG (n ⫽ 11; 27.5%). In 1997, only 2 (5%) patients had AVF (P ⬍ 0.05). Currently, 29 patients are being dialyzed. Of these, 14 (35%) required a catheter to initiate HD, a decrease from 78% of patients (n ⫽ 29) requiring a catheter to initiate HD in 1997 (P ⬍ 0.05). Conclusions. The use of this screening protocol has significantly increased the percentage of AVF as well as significantly reduced the number of patients requiring catheter placement for initiation of HD at our institution. Ten patients with AVF still required catheter placement for initiation, suggesting that AVF should be placed even earlier in this population of pre-ESRD patients to allow enough time for adequate maturation. P48. Supplemental Oxygen Alters Arterial Wall Oxygen Concentration after Stent Deployment. A. S. Tretinyak, M.D., E. S. Lee, M.D., S. M. Santilli, M.D., Ph.D. Department of Surgery, University of Minnesota, Minneapolis, Minnesota. We have previously demonstrated the ability of supplemental oxygen (O 2) to reverse arterial wall hypoxia and decrease smooth muscle proliferation and intimal hyperplasia at an arterial anastomosis. We hypothesized that supplemental O 2 would increase the arterial wall O 2 concentration following intraarterial stent (IAS) deployment. Thirty-two New Zealand White rabbits underwent placement of a 3-mm infrarenal IAS and were placed in either a room air (control) or a 40% O 2 environment. The arterial wall O 2 gradient was measured at the stent deployment site using a microelectrode at 0, 7, and 28 days (n ⫽ 5– 6 animals/group). Statistical significance was determined by Student’s t test. Arterial wall O 2 gradients from adventitia (0%) to intima (99%) are displayed as means ⫾ standard

deviations (see figure). O 2 tensions in all supplemental O 2 groups were significantly higher than control groups (P ⬍ 0.001) at similar time points. Supplemental O 2 significantly increases arterial wall O 2 content after IAS deployment when compared to control groups. Supplemental O 2 may provide an effective means of controlling intimal hyperplasia after deployment of an IAS. Further studies are necessary to determine whether decreased cellular proliferation is seen after treatment with supplemental O 2 following deployment of an IAS.

P49. Quality Control of Resident Experience: Compliance with RRC Criteria. H. C. Veldenz, M.D., J. W. Dennis, M.D., and P. S. Dovgan, M.D. Division of Vascular Surgery, University of Florida Health Sciences Center, Jacksonville, Florida. Purpose. Process capability analysis (PCA) is a quality control tool that measures how close a measured output comes to its target (C p) and the location (⫾) from the target (K). The statistics of C p and K could be used to measure variability in reported surgical trainee operative experience (OE). The application of PCA to RRC OEs could improve uniformity of training. Methods. A review of 6 years of vascular surgery cases (1994 –99) using PG5 RRC case logs and the departmental quality monitoring database was undertaken. PCA was used to investigate patterns in resident case numbers. RRC guidelines based on National Program Data from 1997 to 1998 were used to define quality control limits with the 30th, 50th, and 70th percentiles for chiefs in vascular procedures serving as lower, nominal, and upper control limits. Results. Cases were grouped into aortic (AO), cerebral (CER), peripheral (PER), and visceral (VSC), to meet RRC defined major reconstructions. PCA analysis of PG5 submitted data and case totals are in the tables.Conclusions. Statistical control exists when C p ⬎ 1.0, with a small K, i.e., centered tightly and near the desired nominal limit. Vascular surgery PG5 case numbers are not in statistical control, but instead exceed the defined control limits (RRC 50th percentile levels), implying excess case numbers. Potential error is partially explained by the data of total case volume and resident underreporting (as shown in Table 2). The

TABLE 1—ABSTRACT P49 Process Capability Analysis for PG5 OE in Vascular Categories Case class

AO

CER

PER

VSC

Total

Nominal limit 9 15 17 1 42 Mean ⫾ SD 13.7 ⫾ 4.2 15.6 ⫾ 6.4 23.1 ⫾ 10.5 2.5 ⫾ 1.5 55.1 ⫾ 20.2 0.279 0.365 0.237 0.189 0.305 Cp K (%) ⫹70.5 ⫹78.6 ⫹80.1 ⫹175.0 ⫹70.5

TABLE 2—ABSTRACT P49 Total Vascular Surgery Case Reporting for 6 Years 1994 –1999 Case class

AO

CER

PER

VSC

Total

Cases reported by PG5 Departmental log reporting error (%)

199 239 16.7

288 356 19.1

470 615 23.6

39 50 22.0

996 1260 20.9

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS PCA technique demonstrates the utility of quality control analysis in surgical training programs, by identifying that more accurate experience reporting by residents could account for up to 20% more cases, potentially offsetting individual case allocation differences within a particular resident year. P50. Expression of MMP-1 (Active Form) and MMP-13 (Latent Form) Varies by Location in Incompetant Greater Saphenous Veins. D. L. Gillespie, M.D, A. Patel, B.S., B. Fileta, B.S., A. Flagg, B.S., S. Barnes, B.S., A. Chang, Ph.D., M. Kidwell, B.S., J. M. Goff, M.D., S. D. O’Donnell, M.D., J. L. Villavicencio, M.D., and N. M. Rich, M.D. Uniformed Services University, Bethesda, Maryland. Recent investigations have shown that ambulatory venous pressure of the lower extremity varies between proximal and distal venous segments. Venous ulceration, however, tends only to occur in the distal extremity. We hypothesized that these regional differences in venous hemodynamics may result in regional alterations in extracellular matrix-remodeling enzyme activity. Methods. One-centimeter segments of the proximal and distal greater saphenous vein (GSV) were obtained from patients undergoing ligation and stripping for venous insufficiency (n ⫽ 15). All patients had incompetence of the GSV by continuous wave Doppler. Vein specimens were analyzed for MMP-1, -3, and –13; tryptase; and GADPH mRNA using RT-PCR. Quantification of MMP-1, MMP-13 (active/latent forms), and tryptase was performed using Western blot analysis. Western blots were analyzed using scanning densitometry and values were expressed as the median densitometric index. Wilcoxon signed rank test was used for statistical analysis. Results. MMP-1, MMP-13, and tryptase mRNA was expressed in both proximal and distal segments of GSV. MMP-3 mRNA, however, was not found in either segment. The quantity of MMP-1 protein (active form) was higher in proximal vein segments compared to distal segments (69.27 vs 54.18) (P ⫽ 0.006). Similarly we found a significant difference in the quantity of MMP-13 protein (latent form) in proximal segments of the GSV compared to the distal segments (177.84 vs 137.64) (P ⫽ 0.012). We found no difference, however, in the quantity of tryptase between proximal and distal segments of GSV. Conclusions. This study lends supportive evidence that expression of the matrixremodeling proteins MMP-1 and MMP-13 varies by location in the lower extremity in patients with incompetence of the greater saphenous vein. P51. The Role of Pretransplant Recipient Dialysis in Early Renal Allograft Dysfunction. M. A. Beneke, M.D., G. A. Kaysen, M.D., Ph.D., J. P. McVicar, M.D., B. J. Gallay, M.D., and R. V. Perez, M.D. Departments of Surgery and Nephrology, UC Davis Medical Center, Sacramento, California. Early renal allograft dysfunction has been shown to be independently predictive of decreased allograft survival. Identification of donor and recipient factors that correlate with poor early postoperative function might help risk stratify candidates and allow for intervention to maximize allograft survival. A retrospective review of 112 cadaveric renal transplants between 1991 and 1995 was conducted. Donor and recipient variables were evaluated for their correlation with early renal allograft dysfunction. As a serum marker of inflammation, C-reactive protein (CRP) levels were measured from stored pretransplant serum. A univariate analysis by contingency table (␹ 2 or Fisher’s exact test where appropriate) was used to identify nominal covariates of potential significance in predicting delayed graft function (DGF). Donor age ⬎ 50 years, HLA mismatching ⬎ 4, high recipient CRP level, cold ischemia time (CIT) ⬎ 24 h, and recipient histories of dialysis and smoking were identified and then further evaluated by multivariate analysis. Donor age (P ⫽ 0.0064), CIT (P ⫽ 0.0288), pretransplant

369

recipient dialysis (P ⫽ 0.0315), and HLA mismatching (P ⫽ 0.0404) were found to be independent predictors of DGF. Mean preoperative creatinine clearances were similar for recipients developing DGF as for those who did not (9.7 vs 10.2, P ⫽ 0.5164). Creatinine clearance was decreased (P ⬍ 0.05) during the first postoperative week with a recipient history of dialysis (7 of 7 days) and smoking (7 of 7 days), donor age ⬎ 50 years (6 of 7 days), CIT ⬎ 24 h (5 of 7 days), and HLA mismatching ⬎ 4 (1 of 7 days). Prolonged cold ischemia time and advanced donor age are wellknown independent predictors of DGF. Recipient pretransplant dialysis history, however, is a novel predictor of DGF in the face of identical preoperative renal function. Further investigation is warranted to assess whether earlier transplantation with avoidance of dialysis will minimize early postoperative renal allograft dysfunction and prolong allograft survival. P52. Regression of Early- and Late-Stage Mammary Tumors after Adoptive Transfer of Bryostatin- and IonomycinActivated Lymphocytes Is Mediated by CD8ⴙ T-Cells Necessary for Effect. C. S. Chin, M.D., L. Graham, G. G. Hamad, M.D., K. R. Creasy, and H. D. Bear, M.D. Department of Surgery, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia. We have shown that adoptive transfer of tumor-sensitized lymphocytes activated in vitro with bryostatin-1 and ionomycin (B/I) can induce regression of small established tumors. We set out to determine whether similar treatment would be effective against larger tumors and what cells mediate this effect. BALB/c mice were injected in one footpad with IL-2-transfected 4T07 mammary tumor cells. Ten days later, draining popliteal lymph nodes (DLN) were harvested and activated with B/I for 18 h (“pulsed”). Mice with either 4- or 10-day 4T07 flank tumors were treated with cyclophosphamide (100 mg/kg ip, CYP) alone or CYP followed by infusion of either B/I-pulsed lymphocytes transferred immediately or pulsed cells expanded in vitro for 10 days. In some experiments, mice were also treated with rat anti-mouse CD4 monoclonal antibody (GK1.5) or anti-CD8 antibody (2.43). All mice receiving CYP alone or CYP ⫹ sensitized but nonactivated DLN cells demonstrated progressive tumor growth. One-hundred percent (6/6) of mice treated with CYP and immediately transferred pulsed cells or 10-day expanded cells had complete regression of 4-day flank tumors. Treatment of 10-day tumors with CYP and expanded DLN cells induced regression in 4/6 mice. CYP and pulsed cells induced regression in 100% of mice. In vivo depletion of CD4⫹ cells had no effect on regression of 4-day tumors, but treatment with anti-CD8 antibody completely abrogated the effect of immunotherapy. Adoptive transfer of B/I-activated cells, with and without expansion, induced regression of early- and late-stage 4T07 tumors and depends on CD8⫹ but not CD4⫹ T-cells. P53. Neointimal Hyperplasia Following Carotid Artery Balloon Angioplasty Is Accelerated by Hyperhomocyst(e)inemia. J. W. Cook, M.D., M. R. Malinow, M.D., and S. L. Orloff, M.D. Department of Surgery, Division of Abdominal Organ Transplantation, Oregon Health Sciences University, and Oregon Regional Primate Research Center, Portland, Oregon. Neointimal hyperplasia (NH) frequently complicates balloon angioplasty. Hyperhomocyst(e)inemia (hH(e)) is a risk factor for atherosclerosis; however, its role in NH following balloon angioplasty is unknown. This study investigates the effects of hH(e) on balloon angioplasty-induced intimal injury. Methods. Lewis rats were given a hH(e)-inducing (1 methionine, 2 folate) diet (n ⫽ 5) or a normal diet (n ⫽ 6) for 6 months. The right common carotid artery (CCA) was injured by three successive inflations of a 2-Fr embolectomy catheter. The left CCA had a sham operation (con-

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P53

H(e) (day 75, ␮mol/liter) Neointimal index N area (mean, ␮m 2) N thickness (max., ␮m) N thickness (mean, ␮m) M thickness (mean, ␮m)

donor specific heart (Gp 2) and kidney (Gp 3) but not skin (Gp 1) grafts. However, skin grafted simultaneously with kidney, but not heart, is accepted and protects a subsequent donor specific skin graft.

HH(e) diet (n ⫽ 5)

Normal diet (n ⫽ 6)

P*

71 ⫾ 6 7.7 ⫾ 1.8 35,318 ⫾ 11,720 64 ⫾ 24 31 ⫾ 10 62 ⫾ 7

4 ⫾ 0.6 2.5 ⫾ 1.3 6210 ⫾ 1726 17 ⫾ 4 7⫾1 54 ⫾ 3

⬍0.001 ⫽0.01 ⫽0.02 ⬍0.05 ⫽0.01 NS

P55. Genistein Delays Rejection of Rat Cardiac Allografts Treated with or without Cyclosporine. A. J. Gruneiro, M.D., I. B. Cetindag, M.D., A. D. Weberg, M.S., and T. P. O’Connor, M.D. Department of Surgery, SIU School of Medicine, Springfield, Illinois. Organ transplantation requires long-term use of immunosuppressive drugs, which have serious side-effect profiles and costs. This study examines the in vivo effects of genistein, an isoflavone found in soybeans, on acute allograft rejection. Acute allograft rejection is initiated by ligation of the T-cell receptor (TCR) and subsequent T cell activation, which are inhibited in vitro by genistein. The five groups studied were: (1) control (no treatment); (2) dissolvent only (per osmotic pump); (3) genistein (20 mg/kg/day); (4) cyclosporine (5 mg/kg/day for 7 days); and (5) Genistein combined with cyclosporine. Animals treated with both cyclosporine and genistein had a delayed rejection with a mean of 30.8, compared to those animals treated with cyclosporine alone which had a mean duration of 23.2 days (P ⬍ 0.05 for both comparisons by ANOVA with Duncan’s multiple range test, P ⬍ 0.05 by life table analysis; see table). Genistein, a compo-

* Student’s t tests, P value ⬍ 0.05 is significant. trol). Two weeks after injury, CCAs were formalin perfusion fixed, sectioned, and stained for elastin. Computer-interfaced microscopy was used to measure neointimal index (NI ⫽ % vessel occlusion), total neointima (N) area, maximum and mean N thickness, and mean media (M) thickness. Results. Plasma homocyst(e)ine (H(e)) was increased by the hH(e) diet (see table). The hH(e) diet, compared to the normal diet, increased the NI, N area, and N thickness, but not media thickness, of CCAs following balloon expansion (see table). NH did not develop in the noninjured control CCAs (data not shown). Conclusions. Hyperhomocyst(e)inemia accelerates NH following angioplasty of the rat CCA and may be a risk factor for NH following arterial injury. Reduction of hyperhomocyst(e)inemia by dietary modification may decrease NH formation after arterial injury.

TABLE—ABSTRACT P55

P54. Organ Transplant Specificity of Tolerance with Nondepleting Anti-CD4 Monoclonal Antibody RIB5/2 and Intravenous Donor Alloantigen. N. Otomo, M.D., Y. Shimizu, M.D., J. Margenthaler, M.D., S. Yu, M.S., M. Lehmann, M.D., Ph.D., and M. W. Flye, M.D., Ph.D. Washington University School of Medicine, St. Louis, MO. Background. The effect of a single dose of anti-CD4 mAb, RIB5/2, plus donor splenocytes (sc) on tolerance to donor specific heart, kidney, and the usually more resistant skin grafts was examined. Methods. BUF (RT1 b ) rats were given a single dose of RIB5/2 ip plus 25 ⫻ 10 6 LEW (RT1 l ) sc intravenously (iv) 21 days before a first graft of LEW kidney, heart, or skin. In addition, LEW heart or skin was grafted ⬎40 days after acceptance of the first LEW heart or kidney grafts. Results. While skin grafts alone are not accepted, a LEW kidney transplant (Gp 6) protects both simultaneously and subsequently transplanted LEW skin grafts. In contrast, neither a single (Gp 4) or double heart transplant is protective (Gp 5) (see table). Conclusion. RIB5/2 ⫹ sc tolerize for

Group

N

Mean survival (SD)

Range

Control Dissolvent Genistein Cyclosporine Genistein and cyclosporine

7 7 6 5 5

8.4 (1.3) 11.4 (3.6) 23.2 (7.4) 23.2 (2.4) 30.8 (2.3)

7–11 7–16 14–31 21–26 28–33

nent of soybeans, significantly prolongs survival or rat cardiac allografts and has an additive effect to cyclosporine treatment. Further studies will be needed to determine which property of genistein contributes to this effect and to determine if either oral genistein or a diet rich in soy proteins have similar effects. P56. High-Lipid Diets Alter in Vivo and in Vitro Immune Reactivity and Lymphocyte Membrane Lipids. R. A. IglesiasMa´rquez, M.D., E. Santiago-Delpı´n, M.D., A. A. Roma´n-Franco, C. Estrada, and D. Roma´n. Departments of Surgery and Pa-

TABLE—ABSTRACT P54 Group

First graft

Mean survival

1 2

Skin Heart

9⫾ 0 ⬎100 ⫾ 34

(n ⫽ 5) (n ⫽ 18)

3

Kidney

⬎100 ⫾ 37

(n ⫽ 14)

4

Skin Heart Skin Heart ⫻2 Skin Kidney

12 ⬎100 16 ⬎70 ⬎67 ⬎100

5 6

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

2.6 (n 0 (n 6.8 (n 52 (n 35 (n 0 (n

⫽ ⫽ ⫽ ⫽ ⫽ ⫽

5) 5) 3) 3) 6) 6)

Second graft

Heart Kidney Skin Heart Skin Skin

Mean survival

⬎50 ⬎50 16 ⬎50 21 17

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0 (n 0 (n 4.2 (n 0 (n 2.1 (n 1.5 (n

⫽ ⫽ ⫽ ⫽ ⫽ ⫽

3) 3) 4) 4) 3) 3)

Skin

12 ⫾ 0

(n ⫽ 2)

Skin

⬎50 ⫾ 0

(n ⫽ 4)

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS thology, University of Puerto Rico and VA Hospital, San Juan, Puerto Rico. Mice fed lipid-enriched diets show increased skin graft survival and incidence of tumors. This study evaluated the effect of high-lipid diets on cell-mediated immunity in vivo and lymphocyte reactivity in vitro and its relation to serum and membrane lipids. Twenty-one S/D rats (175–200 g) were fed regular diet (RD), corn oil (CO), or coconut oil (CCO) for 2 weeks. Two milligrams DNCB was injected on day 0 and day 10 in foot pads, and leg volumes were measured. In vitro, lymphocyte reactivity was tested using PHA lymphocyte transformation ([ 3H]thymidine) in 42 Balb/C, 36 C3H, and 27 CBA mice fed RD, 15% CO, or 15% CCO for 2– 4 months, with and without PHA incubation (25 ␮g); and migration inhibition was tested using spleen cells with and without PHA (0, 5, and 25 ␮g) utilizing Sykes–Moore chamber and agar plate methods. Blood and serum from cardiac puncture and purified spleen lymphocyte membranes were studied with gas chromatography in S/D rats on high fat diets. Results. (1) Leg volumes (RD 1.098, CO 0.939, CCO 0.956; P ⬍ 0.05) and ratios (RD 1.228, CO 1.053, CCO 1.054; P ⬍ 0.05) were decreased by lipid diets. (2) PHA-induced blast transformation (Balb/C, RD 14.3 ⫾ 1.2 vs CO 15.5 ⫾ 1.6 (P ⫽ ns) and CCO 9.03 ⫾ 1.9 (P ⬍ 0.05); C3H, RD 11.4 ⫾ 3.89 vs CO 6.3 ⫾ 1.06 (P ⫽ ns); CBA, RD 10.54 ⫾ 1.3 vs CCO 4.67 ⫾ 0.78 (P ⬍ 0.001)) and migration inhibition (Balb/C, RD 19.1 ⫾ 2.4 vs CO 51 ⫾ 13.8 (P ⬍ 0.05) and CCO 17.6 ⫾ 2.96 (P ⫽ ns); C3H, RD 81.25 ⫾ 13.4 vs CO 109 ⫾ 13 (P ⬍ 0.02)) were also diminished by fat diets. (3) Membrane lipids were altered: 18:0 and 18:1 were decreased (P ⫽ 0.012, 0.0005), and 18:2 increased (P ⫽ 1 ⫻ 10 ⫺8) in CO vs CCO and RD. Regression analysis showed a positive correlation between 18:0 and PHA(0.83; P ⫽ 0.02) and negative for 18:2 (⫺0.82; P ⫽ 0.05). Lipid diets induce a depression of cell mediated immunity both in vivo and in vitro. A change in membrane lipids is associated with this dysimmunity. P57. Cold Ischemic Injury Accelerates Allograft Vasculopathy via Enhanced Proinflammatory Cytokine Expression. E. Reiss, M.D., E. Fishman, M.D., H. Liu M.D., Ph.D., and R. Knight, M.D. Department of Surgery, Mount Sinai Medical Center, New York, New York. The aim of this study was to understand the role of ischemic preservation injury and proinflammatory cytokine expression in the progression of allograft vasculopathy. Methods. The rat aortic allograft model using ACI and LEW strains as donors and the LEW strain for recipients was utilized. Ischemic injury was induced by storing grafts at 4°C for either 1 or 24 h prior to transplantation. Graft vasculopathy was determined by computerized morphometry. Three sections from each graft were studied for degree of luminal narrowing assessed by calculation of the area of neointimal hyperplasia. Cytokine expression was assessed at days 1, 3, and 7 as well as weeks 4 and 8 posttransplantation using a semiquantitative RT-PCR method in which absolute amounts of cytokine PCR product were corrected by dividing by ␤-actin product. Results. At 4 weeks posttransplant the degree of vascular injury was equivalent among all four groups. At 8 weeks posttransplantation LEW allografts preserved for 24 h demonstrated a greater degree of vasculopathy (0.197 ⫾ 0.059 mm2, P ⬍ 0.05) compared to LEW isografts (0.115 ⫾ 0.006 mm 2) preserved for 24 h or allografts preserved for 1 h (0.068 ⫾ 0.017 mm 2). Neointimal hyperplasia in allografts was generally accompanied by marked neutrophil infiltration of the media in contrast to isografts which were relatively devoid of such infiltrates. In all four groups cytokine expression peaked at days 3 and 7 posttransplantation, with negligible amounts detected at weeks 4 and 8. Relative expression of the cytokines TNF-␣, IL-1, and IL-2 was similar in both allograft groups and 2–3⫻ greater than that expressed in either isograft group. INF-␥ and IL-6 expression was similar in all four groups. TGF␤ expression was 2⫻ greater in allografts preserved for 24 h compared to any other group. Conclusion. Prolonged ischemic storage injury can induce vascular injury in both isografts and allografts. The degree of

371

injury correlated with preservation time and was greater in allografts than isografts. Allograft vasculopathy was associated with proinflammatory/fibrogenic cytokine expression in the early posttransplant period, followed by cellular infiltration at later time points. P58. Mechanisms of Tolerance Induction in the Heart/Kidney Model in Miniature Swine. J. D. Mezrich, M.D., K. Mawulawde, M.D., K. Yamada, M.D., R. S. Lee, M.D., M. L. Schwarze, M.D., M. E. Maloney, B.A., E. P. Pillsbury, B.A., S. L. Houser, M.D., D. H. Sachs, M.D., and J. C. Madsen, M.D. Division of Cardiac Surgery and the Transplantation Biology Research Center, Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts. Introduction. We have previously shown that cotransplantation of a kidney and a heart from the same class I disparate donor into a recipient treated with a 12-day course of cyclosporine (CyA) induced tolerance to both organs and prevented chronic rejection. In contrast, isolated heart transplantation resulted in allograft rejection by POD 55. This study investigated the mechanisms whereby kidney cotransplantation induced tolerance to heart grafts. Methods. In group 1, donor renal parenchymal cells were infused into CyA-treated class I-disparate recipients at the time of heterotopic heart transplantation (n ⫽ 3). In group 2, leukocytes from pigs rendered tolerant to class I-disparate kidneys were adoptively transferred to CyA-treated recipients of class I-disparate hearts (n ⫽ 2). In group 3, donors animals were lethally irradiated to eliminate passenger leukocytes from donor kidneys prior cotransplantation into CyA-treated class I-disparate recipients with donor hearts (n ⫽ 3). Results. Parenchymal kidney cell infusion was not effective in prolonging the survival of cardiac allografts. Adoptive transfer of leukocytes from a kidneytolerant pig into the recipients of isolated hearts did prolong cardiac graft survival up to 83 days but did not prevent the development of chronic rejection which lead to eventually graft loss. Irradiation of the kidney allografts resulted in restoration of the acute and chronic rejection responses. Conclusions. These data suggest that donor passenger leukocytes from the kidney may be required to prevent acute cardiac allograft rejection in combined heart/kidney recipients. This mechanism may explain the beneficial effects of heart/kidney cotransplantation on acute rejection in humans. P59. Hepatic Sinusoidal Endothelium, Compared to Aortic Endothelium, Exhibits a Unique Pattern of IL-1␣, TNF-␣, and IFN-␥ Upregulation in Response to Xenogeneic Islets in an in Vitro Dog-to-Pig Model of Discordant Islet Xenotransplant. M. Tan, M.D., A. DiCarlo, M.D., J. I. Tchervenkov, M.D., and P. Metrakos, M.D. Department of Surgery, McGill University Health Centre, Montreal, Quebec, Canada. The proinflammatory mediators IL-1, TNF-␣, and IFN-␥ are associated with destruction of islets. Their role in early injury of islet xenografts is poorly understood. The endothelial response may be important in mediating the early injury seen in islet xenografts. The purpose of this study is to characterize the early cytokine response of hepatic endothelium to discordant xenogeneic islets. Methods. Dog islets were isolated using a modification of the Ricordi technique. Five-hundred islet equivalents were cocultured with porcine sinusoidal hepatic endothelium (PSHEC) and porcine aortic endothelial (PAEC) monolayers. RNA was extracted at 0, 1, 2, 4, 6, 8, 12, and 24 h using Trizol reagent. Gene upregulation was assessed by RT PCR. Products were analyzed using band densitometry and semiquantitated by comparison to a housekeeping gene GAPDH. Results. PSHEC exhibited a prominent IL-1␣ response with a band appearing at 1 h and peaking in intensity at 2 h. Transcript was detectable for 24 h. PAEC showed a delayed, attenuated response with the first band appearing at 2 h with a peak approximately sevenfold weaker. IL-1␣ transcript was no longer detectable after 8 h. TNF-␣ transcript was not detectable in PSHEC. PAEC showed

372

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TNF up-regulation beginning at 1 h with a peak at 2 h and detectable for 24 h. There was early IFN-␥ up-regulation in PSHEC starting at 1 h with a peak at 12 h and detectable for 24 h. IFN-␥ was not detectable in PAEC. Conclusion. We have demonstrated that endothelial cytokine up-regulation cannot be generalized across all types of endothelial cells. By demonstrating that the early PSHEC response to xenogeneic islets is a predominant IL-1␣ and IFN-␥ up-regulation, we characterize possible mediators for early xenoislet injury and potential targets for therapeutic intervention. P60. Increased Expression Connective Tissue Growth Factor (CTGF) in Posttransplant Obliterative Arteriopathy. M. Thanikachalam, M.D.,* J. Fukada M.D.,* M. Calfa, M.D.,* M. Perez, M.D.,† G. R. Grotendorst, Ph.D.,‡ and S. M. Pham, M.D.* Departments of †Pathology, ‡Cell Biology, and Surgery, University of Miami, Miami, Florida. Currently, the major limitation in transplantation is chronic rejection, which is characterized by fibrointimal hyperplasia of arteries, obliterative arteriopathy. In a posttransplant milieu, Type-1transforming growth factor-␤ (TGF-␤ 1) has a beneficial effect as an immunosuppressive cytokine. At the same time, TGF-␤ 1 has been implicated as one of the primary mediator of chronic allograft rejection because of its fibroproliferative properties. Recently, it has been established that fibroproliferative properties of TGF-␤ 1 are mediated through CTGF, a cysteine-rich peptide. This study was to determine whether CTGF is expressed in an established model of obliterative arteriopathy—the rat aortic transplant model. Method. Histocompatible antigens fully mismatched 2-month-old Wistar Furth and Lewis rats were used as donors and recipients, respectively. Thoracic aortas of the donors were transplanted at the abdominal position in the recipients (n ⫽ 6). Grafts were harvested at 2 months posttransplantation. As a control, syngeneic grafts (2-month-old, Lewis3 Lewis; n ⫽ 6) harvested 2 months posttransplant and 4-month-old native thoracic aortas (Lewis; n ⫽ 6), were used. Immunostaining was performed using primary anti-CTGF goat antibody. As a negative control, same sections were stained with nonimmune goat antibody. Sections from rat soft tissue wound chamber tissue were used as positive controls. Results. In comparison to syngeneic grafts, allogeneic grafts showed intimal hyperplasia (average 30% increase) and marked fibroproliferative changes in the media and adventitia. Expression of CTGF in syngeneic grafts was similar to native vessels, except for mild increase in the adventitia. In comparison, all the layers of allogeneic grafts showed marked expression of CTGF (see table). Conclusion. For the first time we report that

TABLE—ABSTRACT P60

Native Syngeneic Allogeneic

Intima

Media

Adventitia

⫺ ⫺ ⫹

⫹ ⫹ ⫹⫹

⫺ ⫺/⫹ ⫹⫹⫹

Note. ⫹/⫺, faint staining in scattered cells; ⫹, frequent cells with cytoplasmic staining; ⫹⫹, abundant cells with cytoplasmic staining; ⫹⫹⫹, diffuse cytoplasmic staining. CTGF may play a role in obliterative arteriopathy. Further studies are needed to document the time course of CTGF mRNA expression. In future, these data may lead to the exciting possibility of blocking CTGF pathway while maintaining immunosuppressive function of TGF-␤ 1 P61. Human Anti-Porcine Xenogeneic Responses: Release of Human Th1 Cytokines and Induction of Porcine Endo-

thelial Surface Molecules. T. S. Coleman, B.S., H. K. Pittman, B.S., S. M. Purser, MAED, C. E. Haisch, M.D., and K. M. Verbanac, Ph.D. Department of Surgery, East Carolina University, Greenville, North Carolina. The objective of this study is to use human anti-porcine mixed lymphocyte endothelial cell culture (MLEC) to investigate the induction of human cytokines and porcine cell adhesion molecules which may be characteristic of in vivo xenogeneic responses. Human peripheral blood mononuclear cells (PBMC) or enriched CD4⫹ T cells depleted of professional antigen-presenting cells (APC) were cultured with resting pig aortic endothelial cells (PAEC line AG08472A) in the absence of exogenous cytokines. After 7 days, [ 3H]thymidine incorporation was determined in triplicate. At culture initiation and throughout the MLEC, PAEC were monitored for cell surface markers by flow cytometry, and supernatants were assayed for human TNF-␣ and IFN-␥ by ELISA. Human T cells (from 11 different individuals) proliferated strongly in response to PAEC (median stimulation index ⫽ 75). High levels of the human Th1 cytokines TNF-␣ (17–352 pg/ml) and IFN-␥ (202–3834 pg/ml) were detected in cultures containing PAEC, with levels peaking on day 4. CD4⫹ T-cellenriched, APC-depleted responders maintained proliferative antiPAEC responses and cytokine release. By Day 3, SLA Class II and VCAM expression was induced in 92–96% PAEC: mean fluorescence intensity (MFI) increased from 5 to 83 ⫾ 12 and 166 ⫾ 74, respectively, and SLA Class I was increased from MFI 31 to 965 ⫾ 269. Thus, both human T cells and PAEC are activated in this system. These results indicate that MLEC is a valid, perhaps superior, model in which to study human anti-porcine cellular responses. Specific cytokines, receptors, and adhesion molecules appear to cross the xenograft barrier and play a critical role in T cell–PAEC interactions. Such interactions are likely to affect VEC activation and immune responses to porcine xenografts in vivo.

P62. High Lung Inflation Pressures Restrict Acellular Hemoglobin Entry into Alveolar Septa Less Than Red Cell Entry. M. J. Schurr, M.D., R. Conhaim, Ph.D., L. Rodenkirch, B.S., K. Watson, B.S., and B. Harms, M.D. Department of Surgery, University of Wisconsin Hospital, Madison, Wisconsin. Introduction. High lung inflation pressures compress alveolar septal capillaries and impede red cell transit. Recently introduced acellular hemoglobin solutions may enter compressed lung capillaries more easily than red cells. To test this hypothesis, we compared acellular hemoglobin entry into alveolar septa with that delivered by red cells under zone 1 conditions. Methods. Isolated rat lungs were perfused with fluorescence-labeled diaspirin crosslinked hemoglobin (DCLHb; 10%) and/or autologous red cells (hematocrit, 20). Septal capillaries were compressed by setting lung inflation pressure above vascular pressures (zone I). After perfusion, the lungs were rapidly frozen, dehydrated, embedded, and sectioned. Acellular and cellular hemoglobin entry into septa was measured using confocal fluorescence microscopy. Results were compared using ANOVA. Results. DCLHb was distributed nearly uniformly throughout alveolar septa. Furthermore, adding red cells to the perfusate did not restrict DCLHb entry into septa, suggesting that cells did not obstruct entrances to septal capillaries. From the confocal images, we estimated the maximum acellular hemoglobin mass within each septum to be equivalent to that of 15 red cells. By comparison, we found an average of 2.7 ⫾ 4.6 red cells per septum in lungs perfused in zone 1 with fluorescence-labeled red cells (P ⬍ 0.05). In zones 2 and 3, these values increased to 30.4 ⫾ 25.8 and 50.4 ⫾ 22.1 red cells per septum, respectively (P ⬍ 0.05 vs zone 1). Conclusions. Septal hemoglobin perfusion in zone 1 using a 10% acellular solution was significantly more uniform than red cell perfusion. Furthermore, the acellular solution increased the hemoglobin concentration per septum by up to fivefold over that obtained with red cell perfusion.

373

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS P63. Elevated Serum Sulfite Concentration in Septic Patients. K. Koike, M.D., H. Kajiyama, M.D.,* H. Mitsuhashi, M.D.,* Y. Nojima, M.D.,* T. Mochizuki, M.D., T. Masuno, M.D., N. Sato, M.D., S. Kushimoto, M.D., Y. Koido, M.D., M. Kawai, M.D., and Y. Yamamoto, M.D. Department of Emergency and Critical Care Medicine, Nippon Medical School, Tokyo, Japan; and *Third Department of Internal Medicine, Gunma University of Medicine, Gunma, Japan. Sulfite is a well-known air pollutant that is toxic for human. It has recently been shown that in vivo administration of lipopolysaccharide (LPS) increases serum sulfite level in rats and that human and rabbit neutrophils produce sulfite in response to LPS. In this study we measured serum sulfite concentrations in patients who suffered from sepsis. Methods. Blood samples were collected from 20 septic patients (male 19, female 5; age 29 –92 years; survivors 11) on days 1, 3, 7, 10, and 14 after diagnosis. The serum sulfite concentration on the day of the worst APACHE II score was determined in each patient by reversed-phase HPLC. Correlations between those values and APACHE II score, sequential organ failure assessment (SOFA), PaO 2/FiO 2, serum thrombomodulin (TM) levels, WBC, and CRP were also analyzed. (unpaired Student’s t test, Spearman rank correlation test, *P ⬍ 0.05) (see table). Results. The median serum sulfite level

TABLE—ABSTRACT P63

APACHE II SOFA PaO 2/FiO 2 TM WBC CRP

R2

P

0.01 0.01 0.02 ⬍0.01 0.12 ⬍0.01

0.96 0.73 0.51 0.46 0.48 0.68

in septic patients (2.6 ␮mol/L; 95% CI, 2.6 to 7.0) was significantly higher than that of normal volunteers (1.6 ␮mol/L; 95% CI, 1.3 to 1.8). Conclusion. Serum sulfite concentrations appear to increase in septic patients. However, no correlation with the severity of illness, the magnitude of multiple organ dysfunction, or the extent of endothelial cell injury was observed. Further studies will be needed to delineate the role of sulfite in the pathophysiology of sepsis. P64. Demonstration of Hsp 70 and Hsp 60 in Bioengineered Living Skin Equivalents Undergoing Thermal Injury. P. Vemulapalli, M.D.,J. L. Van Tran, B. Kann, M.D., G. Albaugh, D.O., L. Strande, E. Doolin, M.D., and C. W. Hewitt, Ph.D. UMDNJ–RWJMS, Cooper Hospital, Camden, New Jersey. Hsps 70 and 60 have been shown by multiple investigators to have a protective effect on cells. Our laboratory has demonstrated that a bioengineered living skin equivalent (LSE), when burned, shows morphologic histopathology similar to burned human skin. In order to further validate this injury model, we examined thermally injured LSEs for the presence of Hsps. LSE is constructed from human keratinocytes grown on a dermal equivalent composed of type I collagen gel populated with human fibroblasts. LSEs were injured by scalding with phosphate-buffered saline (PBS) at 70°C for 6 s and then cooled with room temperature PBS to create a burned (BRN) group. Leukocytes were added to some of the BRN to create a burned, inflammatory group (BINF). Leukocytes were added to nonburned LSEs (CON) to create a nonburned inflammatory group (INF). Supernatants from each of these groups were collected at specific intervals (10, 24, 48, 72 h) posttreatment. Each tissue culture supernatant (CON, INF, BRN, BINF) was analyzed via polyacrylamide gel

electrophoresis (SDS–PAGE) for protein expression. Silver staining was utilized for visualization of the proteins and molecular weight determination. Keratinocyte growth medium (KGM) served as a control. Immunoblotting was performed with polyclonal antibodies (Santa Cruz) to identify specific Hsp70 and Hsp 60 proteins. Immunoblot detected bands for Hsp70 (70 kDa) in both BRN and BINF groups but not in the CON or INF groups. Hsp 60 (60 kDa) was detected by immunoblot in BRN, BINF, and INF groups, but not in the CON group (see table). The cellular ability to express Hsp 60 and

TABLE—ABSTRACT P64

Hsp 60 Hsp 70

CON

INF

BRN

BINF

— —

⫹ —

⫹ ⫹

⫹ ⫹

Hsp 70 is maintained when incorporated into bioengineered tissue constructs. These data suggest that bioengineered tissue constructs are a reliable model to evaluate burn pathology and mechanisms of tissue injury. P65. Low-Volume Resuscitation Exacerbates Hepatic Vascular Stress Gene-Dependent Microvascular Dysfunction in Sepsis. R. Baveja, M.D., N. Kresge, B.A., Y. Yokoyama, M.D., N. Sonin, Ph.D., J. X. Zhang, Ph.D., M. G. Clemens, Ph.D., and T. Huynh, M.D.* Department of Surgery, Carolinas Medical Center and Biology, University of North Carolina, Charlotte, North Carolina. Introduction. The purpose of this study was to elucidate the role of fluid resuscitation in preserving liver microcirculation during sepsis. Microcirculatory dysfunction results from imbalance in vasoconstrictors and vasodilators: an increase in vasoconstrictor endothelin-1 (ET-1) that is counterbalanced by nitric oxide (NO). Previously, we demonstrated that vascular stress gene expression was dependent upon resuscitation state with an increase in eNOS and ET-1 mRNA levels in livers in septic rats that was higher in high-volume resuscitation (HVR) compared to low-volume resuscitation (LVR). It is unknown if the increase in eNOS expression contributes to preservation of microcirculation in sepsis. Methods. Rats were subjected to abdominal sepsis by cecal ligation and puncture (CLP, n ⫽ 6 to 8). These and sham rats received either 2.5 or 5 ml/100g body wt normal saline sq. At 24 h after CLP, the livers were isolated and perfused to study the vascular response to ET-1. Results. The hematocrit was increased in CLP with a higher increase LVR compared to HVR (56 ⫾ 1.9 HVR; 61.6 ⫾ 0.5 LVR). In response to exogenous ET-1, the portal pressure increased to lesser degree in CLP animals with HVR compared to LVR (8.6 ⫾ 0.6 HVR; 9.9 ⫾ 0.5 LVR mm Hg). The increase in portal pressure was accompanied by a decrease in flow rate to the liver that was significantly attenuated in CLP animals with HVR compared to LVR (17 ⫾ 1.1 HVR; 14 ⫾ 1 LVR ml/min). The hyporesponse to the ET-1 was compounded in the calculated portal resistance that showed a significantly lower response livers from CLP animals with HVR compared to CLP with LVR and sham animals ET-1 (0.5 ⫾ 0.05 HVR; 0.8 ⫾ 0.9 LVR mm Hg * min/ml at 10 min, P ⬍ 0.05). Discussion. The results suggest that an increase in NO through eNOS functionally antagonizes the vasconstrictive action of ET-1. We propose that the alteration in the shear stress due to the changes in the hematocrit contributes to the increase in eNOS expression. HVR unlike LVR contributes to an increase in eNOS that counterbalances the vasoconstriction due to ET-1 and preserves the microcirculation in sepsis. P66. Bacterial Wall Components Enhance Tumor Cell Metastatic Potential. M. Doyle, J. H. Wang, and H. P. Redmond.

374

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Department of Academic Surgery, University College Cork, Cork, Ireland.

Aims. Bacterial translocation and endotoxemia may enhance dormant metastatic growth following surgery for malignant disease. Endotoxin (LPS), a bacterial wall component, enhances tumor metastasis in vivo. It is unknown if LPS or another bacterial wall component bacterial lipoprotein (BLP) has any direct effect on tumor growth in vitro. We hypothesized that BLP could enhance tumor growth and metastasis in vivo and in vitro. Methods. BD-9 rats (n ⫽ 24) were randomized into two groups. All animals had a laparotomy and intraperitioneal administration of DHD/K12 rat colorectal carcinoma cells (0.5 ⫻ 10 6 cells/rat) on day zero. Group 1 received phosphate-buffered saline (1 ml, ip). Group 2 received 1 ml BLP (50 ␮g/ml, ip). Animals were sacrificed on day 30 postop and tumor burden was assessed. Human colorectal tumor cell line SW480 was incubated with LPS (0.1–1 ␮g/ml) or BLP (0.1–1 ␮g/ml) and 24-h cell proliferation was determined using 5-bromo-2-deoxyuridine DNA labeling. NF-␬B activation was measured with an NF-␬B plasmid using a transfection technique after 6 h of incubation with BLP and LPS. Data represent means ⫾ SEM in each case. ANOVA was employed to determine statistical significance; *P ⬍ 0.05 was considered significant. Results. (See tables.) There was significant in-

lymph nodes, termed bacterial translocation. Antibiotics were continued for the duration of the experiment. Using cultured human foreskin fibroblasts, the presence of C. difficile toxin B in cecal contents was assayed as actinomorphic changes that were neutralized with specific C. difficile antitoxin. Mice orally inoculated with saline rather than viable C. difficile (n ⫽ 29) had no detectable cecal toxin, while mice orally inoculated with C. difficile (n ⫽ 30) had toxin B-positive cecal contents. All mice had intestinal overgrowth with Enterococcus sp., i.e., 10 9⫺10/g cecum. In mice orally inoculated with saline followed by oral Enterococcus sp., Enterococcus sp. was cultured from the mesenteric lymph nodes of 28 of 29 (97%) mice. In mice orally inoculated with C. difficile followed by oral Enterococcus sp., Enterococcus sp. was cultured from the mesenteric lymph nodes of only 11 of 30 (37%) mice. Thus, surprisingly, in this model, the presence of cecal C. difficile toxin appeared to be protective and was associated with decreased (P ⬍ 0.01, ␹ 2 with continuity correction) incidence of enterococcal translocation to mesenteric lymph nodes. These results suggested that previous observations with cultured enterocytes, demonstrating that C. difficile toxins decreased the integrity of the intestinal epithelial barrier and facilitated bacterial migration across the intestinal epithelium, might have little in vivo relevance. In addition, although C. difficile toxin is well known for its role in inducing a spectrum of disease localized to the intestinal tract, this enterotoxin may have a paradoxical role in inhibiting extraintestinal dissemination of enteric bacteria.

TABLES—ABSTRACT P66 In vivo

Tumor nodules

PBS (1 ml) BLP (50 ␮ g/ml)

142 ⫾ 42 320* ⫾ 47

Cell proliferation

Medium

LPS (10 ng/ml)

BLP (10 ng/ml)

24 h

100⫾3.7

124*⫾1.6

128*⫾1.7

crease in tumor growth in the group who received BLP. Tumor cell proliferation was significantly enhanced at 24 h. NF-␬B was activated in response to LPS and BLP. Conclusion. Bacterial lipoprotein and LPS activate tumor cells directly and significantly enhance tumor cell proliferation in vitro and tumor burden in vivo. Thus it appears that other components of the bacterial cell wall may be as important as LPS in the stimulation of tumor growth following surgery. P67. Paradoxical in Vitro and in Vivo Effects of Clostridium difficile Toxins. B. A. Feltis, M.D., D. Sahar, M.D., and C. L. Wells, Ph.D. Department of Surgery, University of Minnesota, Minneapolis, Minnesota. Clostridium difficile toxins A and B are the widely recognized etiologic agents of a spectrum of antibiotic-associated intestinal diseases ranging from diarrhea to pseudomembranous colitis. Using two relevant, differentiated, and polarized human enterocyte cell lines, namely Caco-2 and HT-29, we recently reported that purified toxins A and B decreased barrier integrity and facilitated bacterial paracellular migration (between epithelial cells) across confluent enterocyte cultures. These results suggested that C. difficile toxins might alter intestinal epithelial permeability and facilitate extraintestinal dissemination of enteric bacteria in vivo. We developed a mouse model to test this hypothesis. Mice were given drinking water containing 2 mg/ml streptomycin and 2 ␮g/ml cefoxitin for 3 days and then orally inoculated with 10 9 toxigenic C. difficile or saline. All mice were orally inoculated 24 h later with 10 9 Enterococcus sp. (resistant to the antibiotic cocktail). After an additional 24 h, mice were sacrificed for analysis of cecal aerobic bacteria, cecal C. difficile toxin, and extraintestinal bacterial dissemination to mesenteric

P68. Induction of TNF-␣ Expression and Apoptosis in the Thymus of Mice after Burn Injury. K. Cho, Ph.D., L. K. Adamson, B.S., and D. G. Greenhalgh, M.D. Shriners Hospitals for Children Northern California and Department of Surgery, University of California at Davis, Sacramento, CA. Burn injury causes distant organ failure in addition to skin damage. Systemic inflammatory response syndrome plays an important role in distant organ failure after skin burn. The cellular and molecular mechanisms underlying the pathogenesis of distant organ failure are not well characterized. We hypothesized that apoptosis of lymphoid tissues (e.g., spleen, thymus) in response to burn injury contributes to the pathologic changes in distant organs. Spleen and thymus tissues of mice (three mice per group) collected at different time points (no burn control and 3 h to 29 days) after 18% TBSA burn were subjected to RT-PCR analysis of TNF-␣ mRNA expression and flow cytometric apoptosis assay using annexin-V. TNF-␣ mRNA was clearly up-regulated in the thymus, but not in the spleen, 1 day after burn injury. In addition, flow cytometric annexin V assay revealed that apoptosis was induced in the thymus at day 1 after injury, but not in the spleen (see figure). These findings suggest that burn injury

may induce TNF-␣-mediated apoptosis in the thymus. In addition, lymphoid tissues respond differentially to burn injury in a tissueand/or cell-type-specific manner. Identification of specific subtype(s) of lymphocytes participating in the apoptosis process will help explain the pathogenesis of distant organ failure.

375

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P69

MAP (mm Hg) C M Cerebral BF (ml/100 g/min) C M Coronary BF (ml/100 g/min) C M Renal BF (ml/100 g/min) C M Sphlanchnic BF (ml/100 g/min) C M

Baseline

Pre-Rx

1 h post

2 h post

3 h post

79.6 ⫾ 5.1 82.5 ⫾ 4.0

57.8 ⫾ 2.5 # 49.6 ⫾ 1.0 #,*

51.2 ⫾ 3.4 # 74.5 ⫾ 2.4*

54.7 ⫾ 1.5 # 91.3 ⫾ 10.2*

46.2 ⫾ 4.1 # 83.6 ⫾ 6.8*

24.4 ⫾ 0.8 25.7 ⫾ 0.9

28.8 ⫾ 1.2 # 30.4 ⫾ 1.2 #

30.0 ⫾ 1.3 # 23.3 ⫾ 0.8*

31.9 ⫾ 1.2 # 25.3 ⫾ 0.9*

34.3 ⫾ 1.4 # 27.1 ⫾ 1.4*

67.7 ⫾ 2.9 62.2 ⫾ 2.5

58.6 ⫾ 5.3 # 58.8 ⫾ 3.3

75.7 ⫾ 4.4 # 63.1 ⫾ 3.8*

75.0 ⫾ 4.4 # 68.2 ⫾ 4.1 #

66.3 ⫾ 4.8 56.3 ⫾ 4.9*

164.3 ⫾ 10.8 199.4 ⫾ 8.5

187.8 ⫾ 13.2 182.5 ⫾ 8.4 #

150.0 ⫾ 8.0 119.0 ⫾ 5.5 #,*

136.8 ⫾ 8.5 # 136.5 ⫾ 3.0 #

126.8 ⫾ 5.8 # 120.4 ⫾ 3.6 #

24.3 ⫾ 2.6 26.5 ⫾ 3.4

5.4 ⫾ 0.8 # 9.6 ⫾ 1.8 #

13.1 ⫾ 2.3 # 11.7 ⫾ 2.2 #

20.6 ⫾ 3.3 14.8 ⫾ 2.6 #

P69. Effects of Methylene Blue (MB) on Tissue Perfusion during Septic Shock in Dogs. V. B. Kim, M.D., R. J. Albrecht, M.D., L. W. Nifong, M.D., Y. S. Sun, M.D., and W. R. Chitwood, Jr., M.D. Department of Surgery, Brody School of Medicine at East Carolina University, Greenville, North Carolina. We investigated the precise impact of MB on hemodynamic profile and tissue perfusion during septic shock in dogs. Anesthetized ventilated mongrel dogs were made septic by partially occluding the superior mesenteric artery for 2 h and infusing 2 mg/kg LPS. Crystalloid was infused to maintain a wedge pressure of 10 mm Hg. MB dogs (M ⫽ 5) received a bolus (2 mg/kg) and an infusion (0.7 mg/kg/h) of MB. Control dogs (C ⫽ 5) received saline. Measurements, including blood flow (BF, radiolabeled microspheres), were taken at baseline, pretreatment, and 1, 2, and 3 h posttreatment. Significance was determined using t test and ANOVA. Data are listed as means ⫾ SE (see table; *P ⬍ 0.05 vs control, # P ⬍ 0.05 vs baseline). Septic shock was achieved in 2 h with a decline in MAP. Following treatment, MB dogs became normotensive, but controls remained hypotensive. SVR was higher in the MB group versus controls (3275 ⫾ 365 vs 1831 ⫾ 160, P ⬍ 0.05), but cardiac index (71.5 ⫾ 5.6 vs78.8 ⫾ 9.8 ml/kg/min) did not change significantly. Cerebral and coronary BF were elevated in controls, while sphlanchnic BF declined precipitously with MB. Renal BF worsened regardless of treatment. Despite improvements in MAP and SVR during sepsis with MB, perfusion of the brain, heart, and sphlanchnic circulation appears to be attenuated. MB appears to act as a potent vasopressor at the expense of end organ perfusion, which may have an adverse effect on patients who are already at risk for multiple organ failure.

20.9 ⫾ 3.3 12.9 ⫾ 5.1 #,*

the following classes: monoclonal antibodies and endogenous LPSbinding proteins— bactericidal/permeability increasing protein (BPI), Limulus anti-LPS factor (LALF), synthetic peptides, and polymixin B. We found disparate effects on internalization of FITC–LPS by different classes of antagonist. Anti-LPS IgG rapidly accelerated internalization as did BPI to a lesser degree. In contrast, LALF, polymixin B, and synthetic peptide antagonists decreased RAW cell internalization of LPS compared to FITC–LPS alone (see figure). We

conclude that different classes of endotoxin antagonist exert disparate effects on internalization of LPS by macrophages. These data suggest that internalization of LPS at the level of the monocyte may not be required for effective neutralization of gram-negative endotoxin.

P70. Endotoxin Antagonists of Different Classes Alter Macrophage Uptake of Lipopolysaccharide in Disparate Manners. V. Lazaron, M.D., Ph.D., K. Wasiluk, Ph.D., T. Kellogg, M.D., and D. L. Dunn, M.D., Ph.D. Department of Surgery. University of Minnesota, Minneapolis, Minnesota.

P71. Multiple Trauma Is Proangiogenic. R. G. O’Sullivan, M.B., J. T. Street, M.B., A. Wakai, M.B., J. H. Wang, Ph.D., and H. P. Redmond M.Ch. Department of Surgery, Cork University Hospital, Wilton, Cork, Ireland.

Neutralization of gram-negative endotoxin (lipopolysaccharide, LPS) by IgG antibody antagonists has previously been shown to accelerate internalization of LPS by macrophages. The present study tests the hypothesis that other classes of LPS antagonists will similarly accelerate macrophage internalization of LPS. FITC–LPS internalization by RAW 264.7 cells was assayed in vitro by measuring total bound and internalized signal using flow cytometry. Assays were carried out in the presence and absence of LPS antagonists of

The presence of biologically active angiogenic factors in the systemic circulation following multiple trauma has not been demonstrated. This study sought to assess serum angiogenic cytokine levels in multiple trauma. Venous blood samples were serially collected from multiple trauma patients (ISS ⬎ 16, n ⫽ 10). Levels of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) were determined by enzyme-linked immunosorbent assay. Human umbilical vein endothelial cells (HUVEC) were cultured in

376

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLES—ABSTRACT P71

VEGF (pg/ml) PDGF (pg/ml)

Control

3h

6h

24 h

48 h

7 days

27.8 ⫾ 1.5 349.6 ⫾ 54.7

235.8 ⫾ 47.1* 8756 ⫾ 2528*

193.7 ⫾ 42.8* 6643 ⫾ 2475*

170.5 ⫾ 42.8* 2762 ⫾ 1682*

214.5 ⫾ 31.6* 1340 ⫾ 236*

1061.8 ⫾ 89.2* 8342 ⫾ 2690*

Proliferation (% control)

6h 48 h 7 days

Plasma

⫹mAbVEGF

⫹mAbPDGF

⫹mAbBoth

502 ⫾ 28* 81 ⫾ 10 161 ⫾ 17*

502 ⫾ 28* 98 ⫾ 9 120 ⫾ 10 ⬁

462 ⫾ 31* ,⬁ 85 ⫾ 8 125 ⫾ 8 ⬁

394 ⫾ 33* ,⬁ 90 ⫾ 5 124 ⫾ 6 ⬁

control medium or medium supplemented with patient plasma in the presence or absence of neutralizing antibodies to VEGF and PDGF or both. HUVEC proliferation was measured by bromodeoxyuridine DNA labeling and capillary tube formation on Matrigel was assessed. Data are means ⫾ SEM of triplicate measures (see tables; ANOVA; *P ⬍ 0.05 vs control; ⬁P ⬍ 0.05 vs plasma).Capillary tube formation was promoted by subject plasma (380 ⫾ 46% of control, P ⫽ 0.0004) and attenuated in the presence of mAbPDGF or mAbVEGF⫹PDGF (68 ⫾ 8% and 51 ⫾ 7% of plasma, respectively, P ⬍ 0.05) at the 6-h time point. These findings demonstrate a biphasic pattern of biologically active angiogenic cytokine release in the posttraumatic period and mandates further investigation.

P72. Bacterial Wall Products Attenuate VEGF-Mediated Angiogenesis: A Putative Mechanism for Delayed Wound Healing in Sepsis? C. Power, M.D., J. Wang, Ph.D., J. Street, M.D., and H. P. Redmond, M.Ch. Academic Surgical Unit, Cork University Hospital, Wilton, Cork, Ireland. Introduction. Angiogenesis plays a crucial role in tissue reparative processes, particularly wound healing. Systemic and localized bacterial infection are associated with delayed wound healing. Vascular endothelial growth factor (VEGF) is a potent endothelial cell (EC) mitogen specifically acting through EC receptors KDR and Flt-1 which are upregulated under hypoxic conditions. We investigated the effects of physiologically relevant quantities of the gram negative products lipopolysaccharide (LPS) and bacterial lipoprotein (BLP) on EC VEGF receptor expression in normoxia and hypoxia and subsequently on VEGF-mediated EC proliferation. Methods. Human umbilical vein endothelial cells (HUVEC) were cultured and suspended at a concentration of 4 ⫻ 10 6 cell/ml. They were exposed to LPS and BLP in ascending doses (max ⫽ 1000 ng) for 4 h in normoxia and hypoxia (O 2 ⫽ 2.5%) and receptor expression measured was by flow cytometry. VEGF-mediated proliferation was evaluated using bromodeoxyuridine DNA labeling assay. Directly cytotoxic effects of LPS and BLP were outruled by assessing HUVEC apoptosis, viability, and lactate dehydrogenase (LDH) release. Results. LPS and BLP caused a significant (P ⬍ 0.01) reduction in HUVEC VEGF receptor expression under both normoxic and hypoxic conditions. VEGF-directed EC proliferation was markedly (P ⬍ 0.01) attenuated by concomitant exposure to LPS or BLP. EC proliferation in the absence of VEGF was unaffected by LPS or BLP and did not differ from control values. LPS or BLP did not adversely affect EC apoptosis, viability, or LDH release. Conclusion. Gram-negative bacterial wall products functionally downregulate VEGF receptors on endothelial cells. This renders EC hyporesponsive to the cytokine VEGF and may account for the severely attenuated proliferation seen in vitro. Collectively these data illustrate a potential mechanism by which localized or systemic bacterial infection adversely affects surgical wound healing.

P73. The Actin Cytoskeleton: Opposing Roles in Adherent and Nonadherent Monocytes. M. R. Rosengart, M.D., S. Arbabi, M.D., I. Garcia, B.A., R. Winn, Ph.D., and R. Maier, M.D. Department of Surgery, University of Washington, Seattle, Washington. Numerous studies have demonstrated the importance of the actin cytoskeleton during integrin-mediated formation of focal adhesion complexes and in activation of signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade. In macrophages the actin cytoskeleton is essential in maintaining the primed state of the adherent cell. However, little is known of the role of the actin cytoskeleton when cells are not adherent. Methods. Human monocytes were isolated by negative selection using magnetic beads. Nonadherent cells were pretreated with cytochalasin D (CD) to disrupt the actin cytoskeleton and stimulated with LPS. Activation of ERK 1/2 and p38 MAPK was evaluated by Western blot. mRNA and protein production of IL-8 and TNF were evaluated by Northern blot and ELISA, respectively. Results. LPS activated both p38 and ERK 1/2. CD induced small but reproducible increases in p38 and ERK 1/2 activity. However, CD pretreatment doubled LPS-induced p38 and ERK 1/2 activation. LPS alone resulted in 7 ng/mL IL-8 and 2.9 ng/mL TNF production. CD alone did not stimulate either TNF or IL-8. LPS and CD together produced 31 ng/mL IL-8 and 7 ng/mL TNF. CD alone did not induce IL-8 or TNF transcription; however, subsequent LPS stimulation resulted in significant production of IL-8 and TNF mRNA (see figure). Conclusion. Disrupting the actin

cytoskeleton in nonadherent monocytes activates p38 and ERK 1/2, potentiates LPS-induced signal transduction, and enables LPSinduced cytokine transcription and production. These results are in contradistinction to those obtained in adherent monocytes in which the actin cytoskeleton maintains the primed state. P74. Heat Shock Protein 27 Inhibits Apoptosis in Human Neutrophils. K. Sheth, M.D., A. Duffy, M.D., A. De, Ph.D., R. Ricciardi, M.D., B. Nolan, M.D., and P. E. Bankey, M.D. Department of Surgery, UMASS Medical School, Worcester, Massachusetts.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Prolonged neutrophil(PMN) survival has been implicated in tissue injury following sepsis. A variety of signals have been identified which inhibit neutrophil apoptosis including lipopolysaccharide (LPS) and cytokines. Extracellular release of cellular heat shock proteins (Hsp), such as Hsp60 during necrosis, has been identified as a potent proinflammatory signal for the innate immune system. The role of smaller Hsp, such as Hsp27, remains largely undefined. Therefore, we tested the hypothesis that Hsp27 signals prolonged neutrophil survival. Methods. PMN were isolated from peripheral blood of healthy human volunteers by red blood cell sedimentation and gradient centrifugation. Cells were then placed in medium and cultured for 18 h either alone or with varying concentrations of Hsp27. LPS (Escherichia coli 0114:B4, 1 ␮g/mL) stimulation was used for comparison. Polymixin B (20 u/mL) was added to all medium with the exception of that used for LPS stimulation. Apoptosis was measured using annexin V and propidium iodide staining with flow cytometric analysis. Data are expressed as means ⫾ SEM, and statistical analysis was performed using analysis of variance.(n ⫽ 6). Results. The data demonstrate that Hsp27 significantly inhibits apoptosis compared to unstimulated PMN in a dose-dependent fashion (Fig. 1). LPS also significantly reduces apoptosis, but Hsp27 and LPS costimulation does not have an additive effect (Fig. 2). Conclu-

sion. These data demonstrate that Hsp27 signals prolonged PMN survival, and this may play a role in PMN-mediated immune dysfunction following sepsis.

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4 h. Maximal decrease in ⌬⌿m occurred between 8 and 12 h. Bcl-2 expression was decreased with increased apoptosis. When DC were treated with zVAD, the pancaspase inhibitor, caspase-3 activity and apoptosis were diminished. Our results demonstrate that NO triggers DC apoptosis via inducing cytoC release, mitochondrial disruption, and caspase-9 and -3 activation. Elucidation of the apoptosis pathway in the DC is essential for developing treatments to protect the host’s cells for local defense (see figure). P76. Caspase-3 Inhibition Partially Restores Oxidant Production in Apoptotic Human Neutrophils (PMN). J. F. Sweeney, M.D., P. K. Nguyen, M.S., and D. B. Hinshaw, M.D. Department of Surgery, University of Michigan and Surgery Service, Ann Arbor VAMC, Ann Arbor, Michigan. Caspase-3 activation is the first step in the execution phase of apoptosis. Apoptotic PMN lose functional activity, which emphasizes the tissue injury limiting potential of PMN apoptosis. We hypothesized that PMN functional activity, as evidenced by oxidant production, can be restored in apoptotic PMN by inhibition of caspase-3. Methods. PMN were UV irradiated for 15 min to accelerate apoptosis. PMN were pretreated with the caspase-3 inhibitor DEVD-fmk (100 ␮M) for 30 min prior to UV. At 4 h post-UV, PMN apoptosis was quantitated by flow cytometry with CD16 staining; oxidant production in response to 10 ␮M PMA and caspase-3 activity was quantitated fluorometrically. Data represent means ⫾ SEM (N ⫽ 4) (see figure, *different from control, #different from UV, P ⬍ 0.05 ANOVA).

P75. Caspase-3, Caspase-9, and Mitochondria Mediate Nitric Oxide-Induced Dendritic Cell Apoptosis. A. Stanford, M.D., Y. Chen, Ph.D., X. Zhou, Ph.D., X. Zhang, and H. R. Ford, M.D. Department of Pediatric Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania. Dendritic cells (DC) are critical regulators of the immune response to an invading pathogen or tumor cell. Dysfunction or death of DCs has been implicated in immunosuppression, autoimmune diseases, and malignancy. Nitric oxide (NO), an important proinflammatory mediator released in the tumor microenvironment and in sepsis, may not only exacerbate the disease but also induce DC apoptosis. We examined the effect of an exogenous NO donor, S-nitroso-N-acetyl penicillamine (SNAP), on DCs in vitro. DC2.4 apoptosis was measured by flow cytometry (FACS) with annexin V/propidium iodide staining or immunohistochemistry (Hoescht and TUNEL). Expression of the antiapoptotic protein Bcl-2 was also evaluated. Caspase-1 and -3 enzyme activities were measured. Cytochrome c (cytoC) release and caspase-3 and -9 activation were confirmed by Western blot. Changes in mitochondrial membrane potential (⌬⌿m) were measured using a fluorescent dye, DIOC 6. SNAP induced apoptosis in a time- and dose-dependent manner. CytoC was released as early as 4 h and degraded by 12 h. Concurrently, caspase-9 was detected at

Results. At 4 h, UV-treated PMN demonstrated a threefold increase in caspase-3 activity. This was associated with a significant increase in apoptotic PMN and a 10-fold decrease in oxidant production compared to control PMN. DEVD-fmk blocked increases in caspase-3 activity and significantly reduced PMN apoptosis. Oxidant production was increased fivefold compared to UV-treated PMN but was still significantly less than control PMN. Conclusions. In UVaccelerated PMN apoptosis, inhibition of caspase-3 activity partially restores oxidant production in apoptotic PMN. This suggests that signaling events in the initiation phase of PMN apoptosis, which are proximal to caspase-3 activation, may in part be responsible for loss of oxidant production in apoptotic PMN independent of caspase-3 activity.