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

Journal of Surgical Research 86, 241–307 (1999) Article ID jsre.1999.5739, available online at http://www.idealibrary.com on

ABSTRACTS Abstracts Presented for the Thirty-Third Annual Meeting of the Association for Academic Surgery The Wyndham Frankin Plaza Hotel, Philadelphia, Pennsylvania, November 18 –20, 1999

PLENARY SESSION 1. Gelatinase B (MMP-9) Deficiency Reduces Aneurysmal Degeneration in a Mouse Model of Abdominal Aortic Aneurysms (AAA). R. Pyo, M.D., J. K. Lee, M.D., D. Mao, M.D., J. A. Curci, M.D., J. M. Shipley, Ph.D., S. D. Shapiro, M.D., and R. W. Thompson, M.D. Departments of Surgery and Cell Biology & Physiology, Washington University School of Medicine, St. Louis, Missouri. Purpose. Chronic inflammation, elastin degradation and matrix metalloproteinases (MMPs) are implicated in pathophysiology of AAA. The aim of this study was to determine if one of two elastolytic MMPs, gelatinase B (MMP-9) or macrophage elastase (MMP-12), might be necessary for the development of aneurysmal degeneration. Methods. Adult 129 Sv-J mice underwent transient elastase perfusion (EP) of the isolated abdominal aorta to induce a chronic inflammatory response. A control group was perfused with inactive elastase. Aortic diameter (AD) was measured before EP and on day 14 and expressed as a % increase (AAA . 100%), and aortic elastin was evaluated by light microscopy. The response of wild-type mice to EP was compared to that of genetically-altered mice with homozygous deficiencies (KO) in either MMP-9, MMP-12, or both enzymes. Results. Aneurysm development and aortic elastin degradation were observed in 93% of wild-type EP mice but not in controls. These changes were significantly reduced in MMP-9 deficient mice (MMP-9 KO and MMP-9/MMP-12 double KO), but not in those with an isolated deficiency of MMP-12 (see table). Conclusions. MMP-9 (but not MMP-12) plays a critical role in aneurysmal degeneration. Inhibition of MMP-9 may provide a promising new strategy for limiting the expansion of small AAA.

TABLE—ABSTRACT 1 Mouse strain

n

Wild-type, inactive EP Wild-type, EP MMP-9 KO EP MMP-12 KO, EP MMP-9 & -12 KO, EP

11 15 10 10 10

No. AAA Increase in AD Ao elastin a 0** 14 4** 10 2**

43.5 6 10% 131.1 6 10% 87.4 6 10%** 134.0 6 6.6% 79.3 6 8.8%**

111 0 111 1 111

a Morphologic grading, 0 to 111 (normal). **P , 0.05 vs wildtype, EP.

2. Activin and Its Inhibitor, Follistatin, Play a Role in Embryonic Pancreas Lineage Selection. T. S. Maldonado, M.D., C. A. Crisera, M.D., A. S. Kadison, M.D., S. A. Alkasab, B.A., J. B. Grau, M.D., M. T. Longaker, M.D., and G. K. Gittes, M.D. Department of Surgery, New York Medical Center, New York, New York. Activin, a member of the TGF-b superfamily, and its inhibitor, follistatin, have been implicated in endocrine and exocrine pancreas differentiation. We have previously shown that during pancreatic development activin localized to the epithelium, whereas follistatin localized primarily to the mesenchyme. The purpose of this study was to examine the role of activin and follistatin in mesenchymal– epithelial interactions and lineage selection during pancreas organogenesis by adding them exogenously or blocking their expression in vitro. Mouse embryonic pancreases were dissected at gestational age 11.5 (E11.5). Epithelium was separated from mesenchyme after brief trypsin (1%) digestion. Isolated epithelium and whole pancreas (with intact mesenchyme) were cultured in a collagen matrix in media containing either follistatin (100nM) or activin (10nM) for 7 days. In addition, E11.5 whole pancreas was grown for 5 days in media containing antisense oligonucleotides against either the activin receptor (actRIIb) or follistatin. Mis-sense oligonucleotides were used as controls. All specimens were processed for immunostaining for endocrine and exocrine markers. All pancreases grown in follistatin resulted in a marked increase in exocrine expression compared to controls. Conversely, E11.5 whole pancreas grown in exogenous activin resulted in exclusively endocrine epithelium, staining for glucagon but not insulin. Pancreases treated with antisense to follistatin resulted in epithelium that was exclusively endocrine. Mis-sense-treated pancreases showed normal exocrine and endocrine development. Finally, antisense to actRIIb resulted in an increase in acini and ducts. Based on these data, we conclude that mesenchyme-derived follistatin may inhibit epithelium-derived activin early in pancreatic development, allowing for unopposed exocrine differentiation, and the relative suppression of endocrine differentiation. At later ages (E12-E15) the natural decrease in follistatin and resulting loss of follistatin may liberate epithelial activin to induce progression of endocrine cells to form mature islets by the time of birth. These data show not only that the activin– follistatin system plays an important role in pancreatic development, but also that we have the power to greatly change cell fate in the developing pancreas through exogenous manipulation of this system. 3. Cloning and Analysis of the Mouse Somatostatin Receptor Subtype 5 Promoter: Gene Transcription Regulation by the E-Box Binding Factor, BETA2. M. K. Ray, Ph.D., D. Bundman, B.S., S. Moldovan, M.D., L. Poer, M.S., and F. C. Brunicardi,

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M.D. Department of Surgery, Baylor College of Medicine, Houston, Texas. We recently cloned the mSSTR5 gene, a regulator of islet hormone secretion. The beta cell E box transactivator 2, BETA2, belongs to the basic helix-loop-helix family of transcription factors and has been shown to be a potent regulator of islet hormone gene transcription. The purpose of this study was to clone the mSSTR 5 promoter and to analyze the transcription regulation of mSSTR 5 gene by BETA2. 1.4kb promoter fragment was sequenced and the sequence information was analyzed by computer search to identify the potential transcription factor binding sites. The data revealed the presence of ten E-box binding sites, CANNTG, within this region and three of which are identical to the BETA2 binding site, CACCTG on the insulin promoter. Computer analysis also identified GATA and E47 binding sites adjacent to the E-boxes. To further investigate, the isolated promoter was ligated with restriction enzyme digested pGL2-basic vector having the promoter less luciferase (Luc) coding sequence (Fig. 1). This reporter gene construct, SSTR 5-Luc, was used for transfection into the CV1 cells using lipofectamine in the presence

and absence of BETA2 and the hollow vector, pGL2-basic, was used as control. As shown in Fig. 2, there is a ten-fold transactivation of the reporter gene expression in presence of BETA2 from the SSTR 5-Luc. This data demonstrate that 1.4kb of mSSTR 5 promoter sequence contains ten E-box binding sites and cotransfection with BETA2 results in transactivation of the Luc gene expression. We conclude that BETA2, a potent regulator of islet hormone gene transcription regulates transcription of mSSTR 5. 4. Poorer Outcome in Older Donor Kidneys with Prolonged Cold Ischemia Times (CITs): National Allocation of Older Cadaveric Renal Allografts (CRAs) Is Not Recommended. C. M. Lee, M.D., J. T. Carter, B.S., H. B. Randall, M.D., R. Hirose,

M.D., P. G. Stock, M.D., Ph.D., J. S. Melzer, M.D., D. C. Dafoe, M.D., C. E. Freise, M.D., and E. J. Alfrey, M.D. Departments of Surgery, UCSF Medical School, San Francisco, California; and Stanford University Medical School, Palo Alto, California. In order to ameliorate significant regional differences in waiting time national CRA allocation has been proposed. National allocation results in prolonged CITs so currently only 6 HLA-matched kidneys are allocated in this way. Concomitantly, centers are increasingly using older CRAs to increase organ utilization. Our aim was to examine the effect of average (,24 h, Avg) versus prolonged (.24 h, Lng) CITs in CRAs from older (.55 years, O) versus younger (,55 years, Y) donors. Methods. We reviewed CRA transplants reported to the United Network of Organ Sharing Scientific Registry from 1/1/90 to 7/31/98. We compared 201 variables including donor age (years), CIT (hours), calculated creatinine clearance (C Cr, ml/min), incidence of patients with 6 HLA matches, delayed graft function (DGF) and graft loss (G.Loss). Results. Of the 64,046 CRAs, 35,061 (55%) were Y-Avg, 21,264 (33%) were Y-Lng, 4,308 (7%) were O- Avg, and 3,414 (5%) were O-Lng CRAs. Three-year posttransplant serum creatinine was 1.70 6 0.83, 1.73 6 0.91, 2.31 6 1.04 and 2.42 6 1.09 mg/dl, respectively (p , 0.0001 between all groups). Average group follow-up ranged from 750 to 1055 days (see table and figure). Conclusions. In summary, (1) the negative impact of prolonged CIT is

decreased renal function, increased DGF and graft loss, (2) transplantation of older CRAs, particularly those with prolonged CITs, is associated with worse outcome versus that of younger CRAs. In conclusion, attempts to minimize CITs are more important with older CRAs. Based on these data we recommend against sharing older donor CRAs nationally. 5. Elastase Mimics Pancreatitis-Associated Hepatic Injury via Hepatic TNF Production. C. Murphy-Jaffray, M.D., J. Yang., M.D., and J. Norman, M.D. Department of Surgery, University of South Florida, Tampa, Florida. Recent evidence suggests that pancreatitis-associated hepatic in-

TABLE—ABSTRACT 4

Y-Avg Y-Lng O-Avg O-Lng Note.

1,2,3,etc.

Age

CITs

C Cr

6 HLA

DGF

G.Loss

29 6 13 1,2 29 6 14 3,4 61 6 5 1,3 61 6 5 2,4

15 6 5 1,2 32 6 7 3,4 16 6 5 1,3 32 6 7 2,4

112 6 63 1,2,3 108 6 65 1,4,5 83 6 41 2,4 79 6 34 3,5

4.2% 1,2,3 5.2% 1 4.8% 2 5.1% 3

18.1% 1,2,3 28.7% 1,4,5 32.7% 2,4,6 42.0% 3,5,6

16.5% 1,2,3 20.4% 1,4,5 24.1% 2,4,6 29.9% 3,5,6

Paired numbers are significantly different, p , 0.05.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

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jury is regulated by inflammatory mediator production. Our laboratory has demonstrated in vitro that pancreatic elastase induces inflammatory cell cytokine production and therefore we now explore the in vivo effects of elastase on the liver. Elastase (1.5 U) was administered intraperitoneally (IP, q4h) to mice (n 5 25), while control animals received saline (n 5 11). Animals were further randomized to receive CNI-1493 which attenuates TNF production through p38 MAP kinase inhibition, or saline, one hour prior to elastase. Acute pancreatitis (AP) was induced in a 4 th group of animals (n 5 15) by CDE diet. After 24 h, mice were sacrificed, serum collected, and the liver harvested. Serum hepatic enzymes, serum TNF protein (ELISA), and hepatic TNF mRNA (RT-PCR) were measured. AP induced a significant increase in hepatic enzymes (AST, ALT, and LDH) which was mirrored by IP elastase (Fig. 1, p , 0.05).

was stimulated in quiescent human pancreatic cancer cell lines (BxPC3 and Panc-1) by 10% serum (FCS). In certain samples, PD98059 (an ERK inhibitor) or LY294002 (a PI3K inhibitor) were also added. Viability was determined by MTT assay (n 5 4; two tailed t-test; *, # denote p , 0.05 vs 10% FCS) (see figure). Cell cycle and apoptosis were determined by FACS. p70s6-kinase phosphorylation (indicating PI3K activity) and ERK activation (phospho-ERK) were determined by Western blot. Results. LY294002 inhibited the PI3K pathway (decreased phospho-p70s6kinase), without affecting ERK activation (phospho-ERK) in response to serum. In both BxPC-3 and PANC-1 cell lines, LY294002 inhibited serum-induced proliferation. This was associated with G1 cell cycle arrest and with an increase in the rate of apoptosis. PD98059 inhibited proliferation only in BxPC3 cells, and to a lesser degree than did LY294002. Conclusions. Mitogen-induced proliferation in human pancreatic cancer cells is dependent upon PI3Kinase signaling. Pharmacologic inhibition of PI3K may decrease proliferation, increase apoptosis and potentially confer therapeutic benefit in pancreatic cancer.

Both types of liver injury resulted in a near identical elevation in hepatic TNF mRNA (Fig. 2, p , 0.05) and serum TNF protein (not shown). Elastase-treated animals rendered TNF deficient (CNI1493) had attenuated hepatic enzymes, serum TNF protein, and hepatic TNF mRNA (p , 0.05). In conclusion, the hepatic injury seen with simple IP elastase injection is similar to that which accompanies AP. This data supports recent ARDS studies and in vitro data suggesting that elastase is the factor which propagates pancreatic inflammation into a systemic illness through direct activation of systemic inflammatory cells. 6. Human Pancreatic Cancer Cell Proliferation Is Phosphatidylinositol 3-Kinase (PI3K)-Dependent. R. A. Perugini, M.D., F. J. Vittimberga, Jr., M.D., T. P. McDade, M.D., and M. P. Callery, M.D. Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts. Mutations found in pancreatic cancer (ras, p16, p53) lead to inappropriate cellular proliferation. Mitogens stimulate proliferation via the PI3K and/or the p44/42-MAPK (ERK) signaling pathways. We examined whether inhibition of either PI3K or ERK could limit proliferation in human pancreatic cancer. Methods. Proliferation

7. Platelet-Derived Growth Factor Upregulates VEGF mRNA Expression in Rat Osteoblast-Like Cells. Douglas S. Steinbrech, M.D., Babak J. Mehrara, M.D., Pierre B. Saadeh, M.D., Jonathan S. Luchs, M.D., Joshua A. Greenwald, M.D., Gyu S.Chin, M.D., George K. Gittes, M.D., and Michael T. Longaker, M.D. Departments of Surgery and Pediatrics, NYU Medical Center, Room H-169, 550 First Avenue, New York, New York 10016. Introduction. The release of platelet-derived growth factor (PDGF) after fracture increases osteoblast proliferation, chemotaxis, and collagen synthesis. Vascular endothelial growth factor (VEGF), a potent angiogenic peptide enhances angiogenesis in fracture repair. The purpose of these experiments was to investigate the effect of PDGF on the synthesis of VEGF, and to determine if it may work in concert with other known inducers of VEGF such as hypoxia. Methods. To assess the time and dose-dependence of VEGF expression to PDGF, MC3T3-E1 osteoblast-like cell cultures were placed in varying concentrations of PDGF. VEGF protein production (ELISA) and RNA expression (northern blot analysis) were quantified at 0, 3, 6, 24, and 48 h. To determine if this increase was transciptionallymediated or involves de novo protein synthesis, actinomycin D, cycloheximide, and half-life studies were performed. Statistical analysis performed using ANOVA (*p , 0.05). Results. By ELISA, VEGF protein of osteoblasts exposed to PDGF (100 ng/ml) increased 33

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

(*p , 0.05). Northern analysis demonstrated a 63 increase in VEGF mRNA at 3 h (*p , 0.03). PDGF caused a dose-dependent increase of VEGF mRNA to 43 at 50ng (*p , 0.01). PDGF 1 hypoxia had an additive effect. Cycloheximide did not alter VEGF mRNA, suggesting production is independent of de novo protein synthesis. Actinomycin D decreased VEGF mRNA after PDGF stimulation, implicating primarily transcriptional control. This was further supported by mRNA half-life experiments that demonstrated no enhanced stabilization with PDGF. Conclusions. We have shown that PDGF increases VEGF mRNA and protein synthesis in rat osteoblasts. VEGF upregulation is time and dose-dependent, driven primarily by transcription. Also, PDGF and hypoxia increase VEGF additively. These studies suggest PDGF released during injury contributes to VEGF production by osteoblasts for angiogenesis and subsequent bony wound healing. 8. Enhancement of SGLT-1 and GLUT-5 Gene Expression by Interleukin-11 Following Intestinal Adaptation. K. Alavi, M.D.,* R. Prasad, M.D., and M. Z. Schwartz, M.D. Departments of Surgery, AI duPont Hospital for Children, Wilmington, Delaware; Thomas Jefferson University, Philadelphia, Pennsylvania; and *Washington Hospital Center, Washington, DC. This study was designed to elucidate the mechanism by which interleukin-11 (IL-11) enhances carbohydrate absorption following massive small bowel resection (MSBR). Methods. Ten adult Sprague-Dawley rats underwent an 80% small bowel resection. Seven days later, a 14-day systemic infusion (via the jugular vein) was initiated using subcutaneous osmotic pumps. Rats were then randomized: Group1-0.1% bovine serum albumin (n 5 5); and Group 2-IL-11 at 750mg/kg/d (n 5 5). Following infusion, mucosal samples were obtained and total RNA was extracted. One microgram of total RNA was reverse transcribed and the cDNA sequence was amplified by a polymerase chain reaction (PCR) using the following primers: sodium/glucose co-transporter (SGLT-1), fructose transporter (GLUT-5), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—internal standard. PCR products were separated on a 4%agarose gel and stained with ethidium bromide. Relative band intensities were analyzed and expressed as mean 6 SEM. Statistical analysis was performed using unpaired Student’s t-test. Results. (See figure.) Conclusions. IL-11 significantly enhanced SGLT-1 (107% increase, p , 0.01) and GLUT-5 (176% increase, p , 0.01) gene expression following MSBR. These data demonstrate for the first time that the mechanism by which IL-11 enhances mucosal

carbohydrate absorption following MSBR is, at least in part, through upregulation of carbohydrate transporters.

9. Mechanism of Delayed Rejection in Transgenic Pig-toPrimate Cardiac Xenotransplantation. R. H. Chen, M.D., Ph.D.,* R. N. Mitchell, M.D., Ph.D.,† A. Kadner, M.D.,* and D. H. Adams, M.D.* *Primate Xenotransplantation Laboratory and †Department of Pathology, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts.

Introduction. Transgenic pigs bearing human complement regulatory proteins (CRP) were created to modulate the IgM-activated complement attacks of cardiac xenografts. Nevertheless, transgenic xenografts invariably fail days to weeks after transplantation into primates. We sought to investigate the mechanism behind delayed xenograft failure. Methods. Transgenic pig hearts (n 5 3 for CD59/ DAF, n 5 1 for MCP) were heterotopically transplanted into baboons. Biopsies were taken every three days for histology and immunohistochemistry of vWF, CRP, IgM, and the membrane attack complex (MAC). Results. Transgenic xenografts survived for 5, 6, 7, and 11 days. Serial biopsies revealed gradually increasing microvascular thrombosis and IgM/MAC deposition, despite preserved microvascular endothelium and transgenic human CRP expression. In vitro incubation of naı¨ve pig heart sections with sera from primed baboons, as compared to that from virgin baboons, showed increased IgM deposition. The pattern of in vitro microvascular IgM deposition is similar to that seen on delayed xenograft rejection in vivo. The increased IgM binding appeared to be specific to the porcine endothelial sugar, galactose a1,3 galactose (Gal), since IB4 lectin binding to Gal on the heart sections is blocked if the sections are preincubated with primed baboon sera. Sera from naı¨ve baboons do not appreciably block the binding of Gal by IB4 lectin. Conclusion. Transgenic products are expressed by porcine endothelium, even at the time of terminal graft failure in primates. Xenograft rejection appears to be closely linked to increasing microvascular anti-Gal IgM binding.

PARALLEL SESSION I Clinical Trials/Outcomes 10. Inguinal Herniorrhaphy (IH) Improved Results by Prothesis Utilization. M. Deysine, M.D. Deptartments of Surgery, SUNY at Stony Brook, Stony Brook, New York; and Mercy Medical Center, Rockville Centre, New York. Since 1980 we performed 4,000 IH: the first 2500 IH utilizing the Shouldice technique (group A patients) and the last 1,500 IH with the aid of mesh prothesis and pre-shaped plugs (group B patients). Group B patients underwent intraoperative wound irrigation with a solution of 80 mg of Gentamycin diluted in 250 ml of Normal Saline Solution. 99.8 % of all patients were operated under local anesthesia plus intravenous sedation. Follow up for group A was at a Hernia Clinic at a Teaching Institution (80%, 8 years. Recurrence Rate (RR): 1.7%). Group B was operated at a private Hospital and followed at a private office by yearly appointment and letter survey (10 years 70%. RR: 0.133%). Testicular atrophy and Wound Infection rates for both groups were 0.2% and 0% respectively. In our hands, the introduction of tension free mesh or plug repairs has significantly improved the results of IH by decreasing the complications rate and by seemingly diminishing postoperative discomfort. The introduction of a prothesis demands focused attention towards infection prevention.

11. Clinical Experience with the Axial Flow DeBakey/NASA Ventricular Assist Device. G. P. Noon, M.D., M. E. DeBakey, M.D., D. Morley, R. Benkowski, S. Irwin, and B. A. Bruckner, M.D. Department of Surgery, Baylor College of Medicine, Houston, Texas. The DeBakey/NASA ventricular assist device is a miniaturized axial flow blood pump intended for use in patients with end-stage heart failure. The purpose of this study was to determine the safety and performance of the DeBakey/NASA assist device in patients with end-stage heart failure awaiting transplantation. Currently,

245

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS the assist device is indicated for use as a bridge to transplantation. European clinical trials started in November of 1998. The pump consists of three sub-systems: a pump system, a controller system, and a clinical data acquisition system. A titanium inflow cannula connects the pump to the ventricular apex and a Dacron vascular graft connects the pump to the ascending aorta. Pump hemodynamics include a continuous flow that does produce pulsatility depending on the change in pressure. Pulse pressures up to 20 mm Hg with a closed aortic valve have been generated. This axial flow device is almost noiseless and patient awareness of the device is minimal. Although the pump is in a continuous flow mode, it does exhibit pulsatility depending on the strength of the heart’s native contractility and the resulting pressure difference between pump inflow and outflow. Ten subjects (8M/2F) weighing at least 40 kg and who were at least 19 years of age and NYHA Functional Class IV have been implanted with the DeBakey VAD. Duration of support has ranged from 8 to 115 days with two patients ongoing. Five patients were successfully transplanted. End organ function after implant either showed improvement or remained stable at baseline. The patients achieved normal low level physical activity, at times with reduced pulsatile flow, while being supported by the continuous flow device. Results of the first 10 patients receiving the DeBakey VAD support the device’s safety and ability to perform as a left ventricular support device in patients awaiting transplantation. 12. MRI Interstitial Laser Ablation of Breast Cancer. R. Henry-Tillman, M.D., S. E. Harms, M.D., K. C. Westbrook, M.D., S. Korourian, M.D., and V. S. Klimberg, M.D. Departments of Radiology, Pathology and Surgery, Arkansas Cancer Research Center, UAMS, Little Rock, Arkansas. Rotating Delivery of Excitation Off-resonance (RODEO) breast MRI has been shown by serial section mastectomy to accurately predict the extent of disease in breast cancer patients. We hypothesized that interstitial laser therapy would result in a predictable ablation zone via MRI. Methods. Twenty-six patients with needle biopsy proven breast cancer underwent a 18-g MRI needle placement with stereotaxic guidance through a bare tip laser fiber for interstitial laser ablation. 1000 J of heat was applied using a 805-nm diode laser (Diomed) to the mass. Interactive control of the ablation was provided with dynamic RODEO images. Between 1 and 5 treatment zones were performed in each patient. The final size of the ablation zone was determined by high-resolution post-contrast RODEO images and the patients underwent lumpectomy or mastectomy. The specimens were serially sectioned and compared to the ablation zone on MRI. PCNA stains was used to determine lack of active DNA synthesis in the ablation zone. Results. The size of the ablation zone varied from 0.8 to 1.2 cm. The MRI depiction of the ablation zone correlated with histopathologic size in all cases. H & E sections demonstrated marked degeneration and PCNA-stained cells in all treatment zones. However, in five cases inadequate cell death was demonstrated and may be attributed to stopping the laser during treatment due to poor local anesthesia (4) or the laser tip slipping within the needle (1). Conclusion. RODEO MRI allows accurate visualization of tumor margin, a method for stereotactic placement of a needle into the tumor, and a control system for determining effective laser treatment of the tumor. These results suggest RODEO MRI may be a potential alternative to surgical lumpectomy in patients with breast cancer. 13. Treatment of Metastatic Breast Cancer with Somatostatin Analogues—A Meta-analysis. J. T. Dolan, M.D., D. M. Miltenberg, M.D., T. S. Granchi, M.D., C. C. Miller, Ph.D., and F.C. Brunicardi, M.D. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas. Purpose. Somatostatin analogues (SSA) are known to have antiproliferative effects on breast cancer in vivo and in vitro. This

effect results directly by binding of cell surface somatostatin receptors and indirectly by modulation of growth hormones. Several clinical trials have looked at the efficacy of SSA in the treatment of breast cancer with varying results. This study used the technique of meta-analysis to combine results from all clinical trials and determine toxicity, anti-tumor effects and hormonal effects of SSA on metastatic breast cancer, and to ascertain which treatment regimen was most efficacious. Methods. Data from all available clinical trials were reanalyzed. Meta-analysis was performed by best linear unbiased estimate regression with observations weighted inversely to their variance. Statistical significance was considered p , 0.05. Results. Fourteen studies (n 5 277) met the inclusion criteria. Mild side effects occurred in 47/179 (26%) of patients. Tumor response was seen in 87/210 (41%) of patients: complete response (n 5 9), partial response (n 5 31), and stabilization of disease (n 5 47). Mean duration of response was 3.9 months. Insulin-like Growth Factor-1 (IGF-1) decreased in 56/77 (72.7%) of patients. Prolactin decreased in 34/39 (87.2%) of patients. When SSA was given as first line therapy, 69.5% of patients responded, compared to 28.5% when other drugs were used first (p , 0.006). Patients with ,2 metastases had a 45% response rate compared to 5.6% in those with .2 metastases (p 5 0.3). Conclusion. Even in patients with metastatic breast cancer, treatment with SSA was associated with a tumor response of over 40% and few side effects. Best results were achieved when SSA was given as first line therapy. Most patients showed a decrease in IGF-1 supporting one proposed mechanism of SSA’s antitumor effects.

14. Institutional Validation of Breast Cancer Treatment Guidelines. R. M. Minter, M.D., K. K. Spengler, B.L.S., D. P. Topping, M.D., R. Flug, R.N., E. M. Copeland III, M.D., and D. S. Lind, M.D. Department of Surgery, University of Florida COM, Gainesville, Florida. Several groups have developed clinical guidelines for the management of breast cancer, yet little data exists regarding their validation. Therefore, we examined the effect of published National Comprehensive Cancer Network (NCCN) guidelines for invasive breast cancer on survival, quality of life (QOL), and hospital cost. From 260 consecutive breast cancer pts., 129 pts. were identified for analysis: 93 pts.(72%) followed guidelines (NCCN1) and 36 pts. (28%) with similar stage distribution deviated from guidelines (NCCN2). Patients were excluded for carcinoma in situ, inflammatory cancer, stage IV, and comorbid conditions which affected treatment. QOL and cost data are shown as mean 6 sem and p , 0.05 considered significant (see table). Although deviation from NCCN breast cancer guidelines had no effect on perceived quality of life, there was a trend towards improved survival and a significant decrease in cost in the NCCN1 group. These findings suggest that adherence to NCCN guidelines can significantly reduce the cost of medical delivery without adversely affecting either survival or quality of life.

TABLE—ABSTRACT 14

NCCN 1 NCCN 2 P value a

5-year survival a

QOL b

Hospital cost c

87.6% 83.3% P 5 0.319

4.18 6 0.08 4.24 6 0.14 P 5 0.745

$20.3 6 $1.8 $59.7 6 $25.2 *P 5 0.016

Kaplan–Meier survival analysis. Twelve QOL parameters evaluated using a Likert type scale (1–5). c Hospital costs (in thousands of dollars) compared using a t test. b

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TABLE—ABSTRACT 15 Liquid

Transit, s % of transit at EGJ IBP, mm Hg Perist. Amp., mm Hg % Retrograde flow

Solid

Normal

Fundoplic

Normal

Fundoplic

33 6 31 47 6 22% 20 6 11 88 6 63 47%

71 6 54* 74 6 24%* 18 6 6 79 6 38 89%*

45 6 61 40 6 21% 18 6 9 104 6 67 26%

266 6 240* 73 6 33%* 25 6 8 74 6 32 78%*

Note. Values are means 6 SD (except % retrograde); * P , 0.05 vs normal.

15. Bolus Transit Assessed by an Esophageal Stress Test in Postfundoplication Dysphagia. R. P. Tatum, M.D., G. Shi, M.D., M. A. Manka, B.S., J. Brasseur, Ph.D., R. J. Joehl, M.D., and P.J. Kahrilas, M.D. Departments of Surgery and Medicine, Northwestern University Medical School, Chicago, Illinois. Introduction. Dysphagia is common after Nissen fundoplication but the relationship between dysphagia and bolus transit is poorly defined. This study compared liquid and solid bolus transit of fundoplication patients to normal individuals. Methods. Ten fundoplication patients and 19 healthy volunteers rated their ability to swallow 8 bolus consistencies (from water to meat) from no difficulty (0) to extreme difficulty (3) to compute a dysphagia score (range 5 0-24). A 16-lumen manometric assembly was positioned across the esophagogastric junction (EGJ) and subjects were imaged fluoroscopically while swallowing 5 cc liquid barium and a 5cc marshmallow-like viscoelastic barium bolus in the supine position. Videofluoroscopic images were analyzed for total esophageal transit time and the fraction of time required to cross the EGJ. Manometric tracings were analyzed for the intrabolus pressure (IBP) proximal to the EGJ, intragastric pressure, and distal peristaltic amplitude for each bolus. Results. Dysphagia scores for fundoplication patients were significantly higher (7.3 6 5.1, range 5 1-17) than normals (0.4 6 0.6, range 5 0-2). This correlated with longer total transit times for both liquids and solids in both groups (r 5 .57, p , 0.01) and a greater percentage of transit time attributable to the EGJ (r 5 .64, p , 0.01). Late retrograde flow at the EGJ (escape of bolus proximally up the esophagus) was more frequent in fundoplication patients, as was peristaltic dysfunction. However, no differences existed in manometric parameters between groups (see table). Conclusions. Fundoplication impairs both liquid and solid esophageal bolus transit. Dysphagia perceived by fundoplication patients correlated with increased transit time, particularly across the EGJ. Combined quantitative evaluation with manometry and fluoroscopy reveals functional defects in fundoplication subjects which are not evident by either modality alone. 16. An Objective Analysis of Process Errors in Trauma Resuscitations. J. R. Clarke, M.D., B. Spejewski, Ph.D., A. Gertner, Ph.D., B. L. Webber, Ph.D., C. Z. Hayward, M.D., T. A. Santora, M.D., D. K. Wagner, M.D., C. C. Baker, M.D., H. R. Champion, M.D., T. C. Fabian, M.D., F. R. Lewis, M.D., E. E. Moore, M.D., J. A. Weigelt, M.D., A. B. Eastman, M.D., and C. A. Blank-Reid, M.S.N. Department of Surgery, MCP-Hahnemann Univ., and Univ. of Pennsylvania, Philadelphia, Pennsylvania; Univ. of Pittsburgh, Pittsburgh, Pennsylvania; Univ. of North Carolina, Chapel Hill, North Carolina; Univ. of Maryland, College Park, Maryland; Univ. of Tennessee, Knoxville, Tennessee; Wayne State Univ., Detroit, Michigan; Univ. of Colorado, Boulder, Colorado; Univ. of Minnesota, Minneapolis, Minnesota; and UCSD, La Jolla, California. Purpose. A computer-based system to apply trauma resuscitation protocols to patients with penetrating thoraco-abdominal trauma was previously validated for 96 consecutive patients at a level I

trauma center by both a panel of the trauma attendings and a panel of national trauma experts. This system is now used to objectively critique the actual care given to those patients for process errors in reasoning, independent of outcome. Methods. A chronological narrative of the care of each patient was presented to the computer program. The actual care was compared to the validated computer protocols at each decision point and differences were classified by a predetermined scoring system from 0-100, based on the potential impact on outcome, as critical/non-critical/no errors of commission or omission. Results. Errors in reasoning occurred in 100% of the 96 cases studied, averaging 7.7/patient (range 2-17). Errors of commission (1.6/pt.) involved actions that were irrelevant (33%), unjustified (33%), or premature (30%). Errors of omission (2.4/pt.) were scored more severely than errors of commission (22.0 vs 3.8 for tests and 16.7 vs 4.8 for treatments). The largest number of errors were the failure to record, and perhaps observe, bedside information relevant to the reasoning process, an average of 3.6 missing pieces/patient. Because only 2 of the 10 adverse outcomes were judged to be potentially related to errors in reasoning, no comparisons were made in process errors based on outcome. Conclusions. Process errors occurred in every case. Reducing errors may require an even more systematic approach during trauma resuscitations. The most common error was failure to consider, or document, available relevant information in the selection of appropriate care. A trauma surgeon should treat by protocol, but not by rote.

17. The Efficacy of Thyroidectomy for Graves’ Disease: A Meta-analysis. T. K. Palit, B.S., C. C. Miller Ph.D., and D. M. Miltenburg, M.D. Department of Surgery, Baylor College of Medicine, Houston, Texas. Introduction. Surgery for Graves’ disease has been largely supplanted by anti-thyroid drugs and radioiodine, due to the belief that they are safer and more effective than surgery. However, the reported efficacy, complication rates, and ideal size of thyroid remnant vary considerably, often due to inadequate patient samples. The purpose of this study is to use meta-analysis to determine the efficacy of subtotal thyroidectomy (ST) and total thyroidectomy (TT) in Graves’ disease. Methods. Meta-analysis was performed on published studies in which patients underwent ST or TT for Graves’ disease. Postoperative thyroid status and complication rates were evaluated versus type of procedure and thyroid remnant size. Metaanalysis was performed by weighted least squares linear regression. Results. There were 35 studies comprising 7,241 patients (see table). There was an 8.9% decline in the rate of hypothyroidism for each additional gram of thyroid remnant (p , 0.0001), and a 6.9% increase in the rate of euthyroidism per gram of thyroid remnant (p , 0.0001). Conclusions. Both ST and TT have high cure rates for Graves’ hyperthyroidism, with no significant difference in complication rates. ST achieves a euthyroid state in 60% of patients with a low rate of persistent hyperthyroidism, which may make ST more advantageous.

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TABLE—ABSTRACT 17

Euthyroid Hypothyroid Persistent hyperthyroid Laryngeal nerve injury, perm. Laryngeal nerve injury, temp. Hypoparathyroidism, perm. Hypoparathyroidism, temp. Coexistent thyroid cancer

ST (n 5 6703)

TT (n 5 538)

59.7% 25.6% 7.9% 0.7% 3.0% 1.0% 8.4% 3.8%

0 100% 0 1.0% (NS) 7.0% (NS) 1.6% (NS) 13.5% (NS) 1.1% (NS)

PARALLEL SESSION I Gastrointestinal I 18. The Extracellular Matrix Regulates the Hepatocellular Stress Response. J. E. Gosnell, M.D., W. J. Welch, Ph.D., and H. W. Harris, M.D., M.P.H. UCSF Surgical Research Laboratory at SFGH, San Francisco, California; and the Department of Surgery, UC Davis-East Bay, Oakland, California. Recent studies have shown that the extracellular matrix (ECM) can modulate a variety of cellular functions. Thus, we hypothesized that the ECM would also regulate the viability and response of hepatocytes to various stressors. To address this question, we studied hepatocytes cultured on collagen versus cells cultured on a proteoglycan, laminin-rich basement membrane. Cells were assayed for viability, their response to pro-inflammatory cytokines (as measured by nitric oxide (NO) production) and their capacity to execute a heat shock response. Hepatocytes cultured on collagen showed decreased

thermal stress, only those on the composite ECM exhibited thermotolerance (data not shown). Our data show that the ECM can regulate viability and hepatocellular stress responses in vitro. Furthermore, these data may yield insight into the pathogenesis of hepatic fibrosis and the liver failure that it portends. 19. Does Upregulation of Inducible Nitric Oxide Synthase (iNOS) Play a Role in Hepatic Injury? D. Mercer, M.D., A. Castaneda, M.D., T. Liu, M.D., L. Chang, B.S., and G. Shipley, Ph.D. Department of Surgery, University of Texas Medical School, Houston, Texas. Upregulation of iNOS has deleterious effects in some tissue beds. This study was done to assess its role in two different models of hepatic injury. Conscious rats were given either saline or lipopolysaccharide (LPS; 20 mg/kg IP) and rats killed 5 h later. Other rats were anesthetized and gut ischemia/reperfusion (I/R) injury induced by clamping the superior mesenteric artery (SMA) at its origin for 45 min. Control rats underwent laparotomy and SMA isolation. Rats were allowed to awaken and were killed after 6 h of reperfusion. In both studies serum and liver were prepared for liver enzyme (AST, ALT) and iNOS analysis. Expression of iNOS was assessed by Western Immunoblot and by Quantitative Real Time RT-PCR. Western data is reported in densitometric units (DU). PCR data are reported as number of iNOS transcripts per number of 36B4 molecules (constitutive housekeeping gene). Data are reported as mean 6 SEM (n $5 per group; ANOVA) (see table). Both LPS and gut I/R significantly increased serum transaminase levels, but only LPS caused upregulation of hepatic iNOS. In additional studies, the selective iNOS inhibitor aminoguanidine (45mg/kg IP) significantly reduced

TABLE—ABSTRACT 19 Group

AST (U/L)

ALT (U/L)

iNOS (D/U)

iNOS/36B4

Control LPS Gut I/R

114 6 20 1214 6 154* 1116 6 161*

47 6 4 356 6 85* 368 6 92*

0 6 6 0.4* 0

0 120 6 22 0

* P , 0.05 versus control group. LPS (356 6 85 vs 79 6 14 u/l; p 5 0.02), but not gut I/R (368 6 92 vs 344 6 81 u/l; p 5 NS) induced increases in serum ALT levels. The effect on serum AST levels was similar. These data indicate that LPS and gut I/R elicit hepatic injury through different mechanisms. The deleterious effects of LPS appear to involve induction of hepatic iNOS whereas gut I/R does not. 20. Iron Deficiency Diminishes Gallbladder Neuronal Nitric Oxide Synthase. D. Swartz-Basile, Ph.D., M. Goldblatt, M.D., C. Blaser, P. Decker, M.D., S. Ahrendt, M.D., S. Sarna, Ph.D., and H. Pitt, M.D. Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin.

viability (Fig. 1), spontaneous apoptosis, and attenuated cytokineinduced NO production (Fig. 2), as compared to cells on the composite ECM. Whereas all cells produced heat shock proteins in response to

We have previously demonstrated that iron deficiency results in gallbladder stasis and cholesterol crystal formation. Gallbladder filling and emptying are influenced by both inhibitory and excitatory stimuli with nitric oxide (NO) playing a role in normal relaxation. Iron is known to be a cofactor for nitric oxide synthase, the enzyme controlling NO production. Therefore, we tested the hypothesis that iron deficiency would result in diminished gallbladder levels of neuronal nitric oxide synthase (nNOS) but would not influence the gallbladder’s response to excitatory stimuli. Twenty adult female prairie dogs were fed either an iron supplemented (Fe1) (200ppm) control diet (n 5 10) or an iron deficient (Fe2) (8ppm) diet (n 5 10)

248

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 20

Fe1 Fe2

GBV (ml)

nNOS (protein)

CCK (10 26 M)

ACh (10 24 M)

EFS (5 pps)

0.89 6 .12 1.31 6 .12*

54.9 6 4.6 3.4 6 2.0**

14.3 6 2.7 13.7 6 2.5

17.5 6 2.9 16.4 6 3.5

2.37 6 .48 2.94 6 .85

* P , 0.05; ** P , 0.01 vs Fe 1.

for eight weeks. After an overnight fast, gallbladder bile was aspirated, and volume (GBV) was measured. GB muscle strips were harvested for measurement of nNOS and response to excitatory stimuli. nNOS levels were determined by Western blotting with densitometric quantification of protein bands. Muscle strip response to a spectrum of doses of cholecystokinin (CCK), acetylcholine (ACh) and electrical field stimuli (EFS) was determined, and the area under the response curves were calculated. See table for results. These data suggest that iron deficiency results in 1) increased gallbladder volume, 2) reduced gallbladder nNOS, and 3) a normal response to excitatory stimuli. We conclude that diminished gallbladder neuronal nitric oxide synthase contributes to the gallbladder stasis that occurs with iron deficiency. 21. Hypo-osmotic Stress Activates P38, Erk1/2 and SAPK/JNK in Primary Rat Hepatocytes: Implications in Cell Cycle Progression. R. D. Kim, M.D., and Ravi S. Chari, M.D. Department of Surgery, UMass Medical School, Worcester, Massachusetts. Following hepatocyte injury, changes in the peri-hepatocyte milieu modulate cell volume and influence growth. The link between cell volume and growth has not been defined. We hypothesize that hypo-osmolar stress activates NF-kB and the MAP Kinase cascade. These pathways may serve as links between cell volume alteration and nuclear gene transcription. Methods. Quiescent primary hepatocytes were exposed to hypo-osmotic serum free WE (hWE) medium (210 mOsm/L) over a fixed time course. HGF (0.1 mg/mL) and iso-osmotic WE (iWE) medium (305 mOsm/L) served as positive and negative controls, respectively. Relative densitometry of Western blots measured phosphorylated cytoplasmic p38, ERK1/2, and SAPK/JNK. Electromobility shift assay (EMSA) examined nuclear NF-kB activation. Results. 1) hWE stimulated quiescent hepatocytes to phosphorylate p38, ERK1/2, and SAPK/JNK by 5 min (Fig. 1). 2) hWE stimulated the

nuclear translocation and activation of NF-kB by 60 min (Fig. 2). 3) HGF induced the phosphorylation of all three MAP kinases and the activation of NF-kB with profiles similar to those of hWE. 4) iWE did not induce MAP Kinase or NF-kB activation. Conclusion. These data demonstrate that, in primary hepatocytes, hypoosmotic stress activates both NF-kB and the MAP Kinase cascade, similar to a potent mitogen. Thus, cell volume alone may initiate cell cycle progression. Alteration of cell volume may allow modulation of cell growth.

22. Luminal Trypsin Inhibits Pancreatic Duct Cell (PDC) Bicarbonate (HCO 32) Secretion in Vitro. J. P. Regan, M.D., and C. Alvarez, M.D. Department of Surgery, University of Maryland, and Baltimore VAMC, Baltimore, Maryland. PDCs are responsible for HCO 32 production by the pancreas. Recently, high levels of expression of trypsin-sensitive protease activated receptor (PAR-2) have been noted along the pancreatic ductal system. The role of PAR-2 here is unknown, but since it is believed that pancreatic juice trypsinogen is activated to trypsin at low rates under normal conditions and in greater amounts during pancreatitis, we hypothesized that activation of PDC PAR-2 may inhibit ductal HCO 32 secretion. Methods. PDCs were isolated as explants from bovine main pancreatic ducts, maintained in culture, then mounted on Ussing chambers. A pH Stat technique was used to determine HCO 32 output as mmol HCl/hr/cm 2 required to maintain luminal pH at 7.4. Short circuit current (I sc) and tissue resistance (R t) were also measured. Trypsin (10 mM) was added either to the luminal or serosal baths. Comparisons were made by t-test. Results. Serosal trypsin failed to elicit a significant response in the PDC epithelium. Mucosal trypsin exposure led to a prompt and reversible inhibition of HCO 32 secretion (see figure). A lack of epithelial injury at this trypsin dose was suggested by a stable R t. Conclusions. This is the first functional evidence favoring a regulatory role for PAR-2 in the pancreas and a physiological role for luminal proteases in exocrine secretion. Activation of luminal PAR-2 receptors in PDCs by trypsin could be the mechanism behind the decreased production of pancreatic fluid and HCO 32 observed in pancreatitis.

23. Is Adenocarcinoma Following Esophagoduodenostomy ¨ berg, without Carcinogen in Rats Reflux Induced? S. O M.D., R. V. Lord, M.D., J. H. Peters, M.D., P. Chandrasoma,

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

249

M.D., J. Theisen, M.D., C. G. Bremner, M.D., and T. R. DeMeester, M.D. Departments of Surgery and Pathology, University of Southern California, Los Angeles, California. The aim of this study was to test the hypothesis that acid reflux reduces the incidence of adenocarcinoma in this animal model. 190 male 8 week old Sprague Dawley rats underwent 4 different reflux operations: 1. Esophagoduodenostomy (ED) for duodenogastroesophageal reflux (n559). 2. ED and total gastrectomy for duodenoesophageal reflux (EDTG, n554). 3. EDTG with acid substitution with acidified water (EDTGA, n550). 4. Roux-en-Y no reflux contros (n525). 188 surviving animals were sacrificed at 36 weeks of age and the resected esophagus was examined. All animals except the Roux-en-Y control group had sever reflux esophagitis. The frequency of tumor development was similar in all groups. All tumors were well differentiated adenocarcinomas and were located on the external surface of the bowel either at or immediately distal to the enteroesophageal anastomosis. The mucosa overlying the tumors was not involved. Although an admix-

TABLE—ABSTRACT 23 ED Length of 66.6 6 17.4 esophagus inflamed (%) Ulceration 61.0 (%) Adenoca (%) 20.3

EDTG

EDTGA

Roux

70.5 6 16.1

82.4 6 10.7

0.8 6 0.0*

66.7

72.0

0.5*

27.8

30.0

36.0

Note. Values expressed as means 6 SD. * P , 0.001 vs. all other groups. ture of squamous and columnar epithelium was found microscopically in some cases, this finding was unrelated to reflux category, and there was no definite evidence of columnar lining of the esophagus (see table.) These results suggest that the adenocarcinomas in this animal model are not reflux induced and do not arise from the mucosa. Despite previous reports to the contrary, we suggest that this model may not be useful for the study of reflux induced esophageal adenocarcinoma. 24. Development of Targeted Embryonic Stem Cells with Floxed Somatostatin Receptor Subtype 5 (SSTR 5). M. K. Ray, Ph.D., D. Bundman, B.S., and F. C. Brunicardi, M.D. Department of Surgery, Baylor College of Medicine, Houston, Texas. Cre-loxP system has been used to confine the inactivation of target gene(s) in a particular cell type. Cell specific expression of Cre recombinase, driven by a cell specific promoter, recognizes the floxed-gene (target gene is flanked by the loxP sequences) and excises in a particular cell type. The purpose of this study was to determine whether embryonic stem (ES) cells with floxed-SSTR 5 gene could be developed. LoxP sequence was introduced into the BglII restriction site located at 450 nucleotides upstream of the translation start site, ATG. Neomycin resistance gene (neo) and thymidine kinase gene (tk), flanked by the loxP sequences were isolated from the plasmid pNTL by digestion with BstxI, klenow/ SalI and inserted into the MluI, klenow/XhoI sites (generated by site specific mutagenesis) at the 39 end of SSTR 5 coding sequence (Fig. 1). Neo and tk were used as selection markers to identify the ES cells having gone through the correct event of homologous

recombination. The linearized targeting construct was used for electroporation into the ES cells. The ES cells were grown in neomycin containing media and transfected with Cre expressing vector driven by the cytomegalovirus promoter (CMV). The CMVCre transfectred ES cells were grown in FIAU-containing media and the targeted clone, floxed-SSTR 5, was identified by Southern blot analysis (figure 2) using suitable probe outside the target sequence. We conclude that SSTR 5 gene has been floxed in vitro. We have demonstrated that ES cells with floxed-SSTR 5 gene can be developed. These ES cells can be used for developing beta cell specific SSTR 5 gene inactivation mouse model.

25. Isolated Hepatic Cholinergic Denervation (IHCD) Impairs Glucose and Glycogen Metabolism. C. Xue, M.D., G. Aspelund, M.D., K. Sritharan, B.A., L. A. Slezak, M.D., and D. K. Andersen, M.D. Department of Surgery, Yale University School of Medicine, New Haven, Connecticut. Hepatic innervation plays an essential role in protein synthesis, insulin extraction, and glucose production, but the specific role of hepatic cholinergic innervation remains unclear. We sought to establish a model of IHCD, and to assess whether glycogen storage or the control of net hepatic glucose production (HGP) was altered by IHCD. Sprague-Dawley rats underwent either hepatic vagotomy, or sham operation. Liver was stained for anti-vesicular acetylcholine transporter (VAChT) and (non-specific neural-) protein gene product 9.5 (PGP), at 1, 2, 3, and 4 wks. Uniform staining of PGP and absence of VAChT staining in truncal or hepatic vagotomized rats after 2 wks demonstrated the validity of our model. Liver glycogen content was quantified in fed and fasted IHCD or sham animals by enzyme hydrolysis. Glycogen content of sham rats increased from 6.0 6 1.7 in the fasting state to 10 6 1.8 mg/g after feeding. In contrast IHCD rats showed no comparable increase (3.5 6 0.6 vs 4.0 6 0.9 mg/g). The influence of IHCD on HGP was determined by single-pass isolated liver perfusion on fed IHCD or sham rats. After equilibration, a 30 min 12 ng/ml glucagon infusion was begun and after 10 min, a 200 mU/ml insulin infusion was added. Statistical comparisons were made using Student’s t-test (see table). Our studies show that glycogen accumulation after feeding and the net HGP responses to glucagon and insulin are severely impaired after IHCD. We conclude that hepatic cholinergic integrity is essential to normal hepatic glucose metabolism.

250

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 25 Effect of Insulin on Glucagon-Stimulated HGP (mg/g liver/10 min) Period (min)

Sham

IHCD

I (0–10) II (10–20) III (20–30) III/I 3 100#

4.90 6 0.64 3.71 6 0.47 2.31 6 0.28 47.5 6 8.6%

2.71 6 0.61** 2.07 6 0.44** 1.86 6 0.33* 69.9 6 7.1%**

Note. Mean 6 SEM, n 5 8. a Ratio of glucose output. * P , 0.05 vs sham. ** P , 0.01 vs sham.

shift, was confirmed by supershift using anti-p65 and p50 antibodies. RT-PCR was used to detect transcription of TNF-a mRNA. NF-kB was maximally activated (Fig. 1) and TNF-a mRNA upregulated (Fig. 2) at 60 min following LPS stimulation, compared to control. Both were ablated by ALLN pretreatment. The data demonstrate endotoxin activation of dermal microvascular endothelial cells stimulates an inflammatory response and suggests that the dermal endothelium may represent a non-leukocyte source of inflammatory cytokines in response to sepsis. 27. PEEP Corrects the Alveolar Expiratory to Inspiratory Area Change Seen in Acute Lung Injury. U. G. McCann II, M.D., H. J. Schiller, M.D., D. E. Carney, M.D., L. A. Gatto, Ph.D., and G. F. Nieman, B.S. Department of Surgery, SUNY Health Science Center, Syracuse, New York.

Both thermally injured and normal skin generate inflammatory cytokines (TNF-a) in response to localized and systemic injuries, although the source of these mediators in normal skin is unclear. NF-kB is a ubiquitous transcription factor that binds to the promoter regions of multiple cytokines, such as TNF-a. The present study examines the hypothesis that dermal endothelial cell activation in response to stress is characterized by the expression of TNF-a mRNA, and that endothelial TNF-a transcription is regu-

Introduction. Positive end expiratory pressure (PEEP) reduces ventilator induced lung injury (VILI) presumably by maintaining alveolar patency and decreasing shear stress. We have observed that surfactant deactivation causes shear stress by exaggerating the changes in alveolar area from expiration to inspiration. We hypothesized that PEEP would decrease this alveolar expiratory to inspiratory area change (I-E D) seen in a model of acute lung injury. Methods. Yorkshire pigs were placed into three groups: control (n 5 5), Tween (surfactant-deactivating detergent given by lavage) (n 5 5), and Tween 1 PEEP (7 cmH 2O) (n 5 4). Using in vivo video microscopy and computer image analysis, individual alveolar areas (n'35 per group) were measured at end-inspiration and end-expiration over consecutive increases in tidal volume (7cc/kg to 30 cc/kg.) Data are I-E D means 6 SE. * 5 p , 0.05 vs all other groups/volume; ANOVA. Results. Surfactant deactivation significantly increases I-E D. PEEP prevents this change, returning I-E D to control levels even at supra-physiologic tidal volumes (see figure). Conclusion. PEEP corrects the increased I-E D seen in the acutely injured lung. These data provide an additional mechanism by which PEEP reduces shear stress during mechanical ventilation.

lated by the nuclear translocation of NF-kB. Human dermal microvascular endothelial cells were stimulated with LPS (100 ng/ ml) for 15, 30, 60, 120, and 240 min. Separate cultures were treated with 20 mg/ml ALLN (NF-kB inhibitor) prior to LPS stimulation. NF-kB activation, assessed by electrophoretic mobility

28. Alteration in Vascular Endothelial Cadherin Expression Following TNF-a Stimulation. F. E. Nwariaku, M.D., N. Halaihel, Ph.D., S. L. Duffy, B.S., L. T. Kim, M.D., and R. H. Turnage, M.D. Department of Surgery, UTSWMC, and VA Medical Center, Dallas, Texas.

PARALLEL SESSION I Shock I 26. Endotoxin Stimulates Human Dermal Microvascular Endothelial NF-kB and TNF-a mRNA Expression. E. L. Chan, M.D., S. B. Haudek, Ph.D., B. P. Giroir, M.D., and J. T. Murphy, M.D. Departments of Surgery and Pediatrics. University of Texas Southwestern Medical Center, Dallas, Texas.

251

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

Endothelial cell dysfunction is a major component of systemic inflammation and TNF-a is a potent mediator of endothelial dysfunction. Signal transduction mechanisms by which TNF causes microvascular permeability are unclear. Since adherens junctions are crucial in the maintenance of intercellular integrity and vascular endothelial cadherin (VE cadherin) is an integral molecule in the complex of adherens junction proteins, we postulated that TNF mediated endothelial dysfunction is associated with decreased expression of VE-cadherin. Methods. Cultured human umbilical vein endothelial cells (HUVEC) grown to confluence on Transwell culture chambers were incubated for 24 h in the presence or absence of 100mg/ml of TNF-a (n 5 6 per group). Endothelial monolayer (EC) dysfunction was assessed by measuring the movement of Evans Blue Dye-labeled albumin (EBD) across the EC monolayer over time. In separate experiments, monolayers (n 5 4 per group) were incubated for 8 h in the presence or absence of 100mg/ml of TNF-a and evaluated for VE cadherin expression by immunofluorescence (FITC labeled IgG), confocal microscopy and Western blot. Densitometry was performed with a fluor-S multi-imager. HUVEC viability was assessed by trypan blue exclusion. Statistical comparisons were made with ANOVA and Student’s t-test. Results. Compared to untreated controls TNF-a increased HUVEC permeability to EBD at 6, 9 and 24 h, p , 0.05. Confluent EC monolayers demonstrated marked expression of VE cadherin. Exposure of monolayers to TNF-a for 8 h resulted in significant loss of VE cadherin expression as determined by Western blot, p , 0.05 (see figure), and immunofluorescence. Cell viability was similar in both groups after 24 h, 96% vs 98%. Conclusion. TNF-a mediated endothelial cell dysfunction is associated with decreased expression of vascular endothelial cadherin, a major component of adherens junctions. Inhibition of the signal tranduction pathways culminating in the loss of VE-cadherin expression may prevent cytokine-induced endothelial dysfunction. 29. The Critical Role of Oxygen Radicals in the Initiation of Hepatic Dyfunction Following Trauma Hemorrhage. D. Jarrar, M.D., P. Wang, M.D., W. G. Cioffi, M.D., K. I. Bland, M.D., and I. H. Chaudry, Ph.D. Center for Surgical Research and Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, Rhode Island. Although hepatic dysfunction occurs early following trauma and severe hemorrhage, and persists despite fluid resuscitation, it remains unknown whether reactive oxygen species (ROS) play any role in the initiation of hepatocellular dysfunction and damage. To

test this, male Sprague-Dawley (275-325g) rats underwent laparotomy (i.e., induction of soft tissue trauma) and were then bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the maximal bleed-out (MB) volume was returned in the form of Ringer’s lactate (RL). The animals were then resuscitated with 4 times the volume of MB with RL over 60 min. The ROS scavenger 2-mercaptopropionyl-glycine (10 mg/rat; H-MPG) or vehicle (HVEH) was administered i.v. as a bolus at the beginning of resuscitation. At 2 h after resuscitation, cardiac index (CI; ml/min/100 g) was measured by a dye dilution technique. Hepatocellular function, i.e., the maximum velocity of indocyanine green clearance (V max ; mg/kg/min) and the efficiency of the active transport (K m ; mg/kg), was determined using an in vivo hemoreflectometer. Serum levels of alanine aminotransferase (ALT; SF U/ml) and TNF-a (pg/ml) were determined colorimetrically and with ELISA, respectively. The values (means 6 SE, n 5 5-7/group) are shown in the table. The results indicate that there is a significant depression in cardiac and hepatocellular function with concomitantly increased levels of ALT and TNF-a following trauma hemorrhage and resuscitation. Administration of 2-MPG, however, restored the depressed cardiac and hepatic function, attenuated liver enzyme release and serum levels of TNF-a. Thus, administration of the reactive oxygen scavengers 2-MPG appears to be a useful adjunct for improving the depressed cardiac and hepatocellular functions following trauma-hemorrhage and resuscitation. 30. Protein Kinase A Mediates Cyclic GMP Induced Apoptosis in Vascular Endothelial Cells. M. K. Bullard, M.D., J. D. Smith, B.S., and D. K. Nakayama, M.D. Department of Surgery, University of North Carolina, Chapel Hill, North Carolina. Apoptosis occurs in pulmonary vascular endothelial cells after acute lung injury and contributes to the pathophysiology of adult respiratory distress syndrome. In previous studies, we have shown that nitric oxide induced apoptosis in pulmonary vascular smooth muscle cells appears to be mediated by cyclic guanosine monophosphate (cGMP). We hypothesized that cGMP induces apoptosis in vascular endothelial cells, and involves cyclic nucleotide activation of protein kinase G (PKG), or “crossover” activation of protein kinase A (PKA). Methods. Rabbit vascular endothelial cells (RVECs) were exposed for 12 h to 10 26 M, 8-bromo-cGMP in RVEC media containing 5% fetal calf serum (FCS). Effects on PKG were tested with 12hr treatments of the inhibitor KT5823 (10 26 M). Effects on PKA were assessed with the inhibitor KT5720 (10 26 M). DNA strand breaks were analyzed with flow cytometry after TUNEL staining. Results. Cyclic GMP caused an increase in DNA strand breaks over basal levels. PKA inhibition with KT5720 significantly reduced the number of DNA fragments in both cGMP treated cells and controls. PKG inhibition by KT5823 did not decrease the percentage of DNA strand breaks in basal or treated groups (see figure). Conclusions. We treated vascular endothelial cell cultures with cGMP in an effort to mimic the in vivo milieu, where cyclic nucleotides secreted by arterial smooth muscle cells exert a paracrine effect on adjacent endothelium. Inhibition of PKA, as opposed to PKG, resulted in significant decreases of DNA strand breaks, suggesting that PKA plays a role in the

TABLE—ABSTRACT 29 CI Sham H-VEH H-MPG

38 6 1.8 30 6 1* 36.7 6 3**

V max

Km

ALT

1.2 6 0.3 0.4 6 0.06* 1 6 0.2**

2.6 6 0.4 0.98 6 0.2* 2.58 6 0.7**

23 6 1 47 6 4* 30 6 5**

* P , 0.05 vs SHAM. ** P , 0.05 vs H-VEH by one-way ANOVA and Tukey’s test.

TNF-a 0.14 6 0.1 33 6 14* 2.5 6 1.9**

252

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS ble, suggesting the absence of renal oxidant injury. Lazaroid pretreatment prevents increased AA products after E. coli, suggesting a key role of membrane lipid peroxidation. PTXF probably prevents renal vasoconstriction via adenosine receptor-mediated inhibition or phosphodiesterase inhibition and not altered AA metabolites. Selective effects on P-LT may indicate that LT increases are secondary to impaired blood flow. In sum, these data suggest that cell membrane injury results in altered AA metabolism, thereby altering renal blood flow. 32. Actin Stress Fiber Formation in Pulmonary Endothelial Cells Following Thermal Injury. J. T. Murphy, M.D., H. Wheeler, B.S., and S. Duffy, B.S. Department of Surgery, University of Texas Southwestern Medical Center, Dallas, Texas.

nuclear fragmentation seen in apoptosis. PKA “crossover” activation by cGMP appears to be an early event in the cellular commitment to programmed cell death. 31. Lazaroid Prevents Sepsis-Induced Increases in Renovascular Resistance via Altered Arachidonic Acid Metabolism. P. J. Matheson, Ph.D., R. J. Krysztopik, FRCS, R. N. Garrison, M.D., and M. A. Wilson, M.D., Ph.D. Department of Surgery and Center for Applied Microcirculatory Research, University of Louisville and Louisville VAMC, Louisville, Kentucky. Sepsis initiates a cascade of events in the renal circulation resulting in vasoconstriction and altered flow distribution. The etiology is unknown, but arachidonic acid (AA) metabolites are implicated. We hypothesized that E. coli increases vasoactive renal-origin AA metabolites and that stabilization of the endothelial cell membrane against lipid peroxidation protects against these changes. Methods. Rats were cannulated for hemodynamic monitoring and received vehicle, lazaroid (U74389G), or pentoxifylline (PTXF) 15 min before BL. At 60 min after E. coli or saline infusion, the left renal artery and vein were cannulated for in situ perfusion. Perfusion pressure and flow were continuously monitored, and renal vein effluent was collected in 15-min intervals for 1 h. Thromboxane B 2 (TX B 2), peptido-leukotriene (P-LT), 8-isoprostane (8-isoP), and 6-keto-prostaglandin F 1a (PG F 1a) levels were measured by ELISA. Results. E. coli increased renal vascular resistance and all AA metabolites for the 60-min perfusion period. Lazaroid prevented these responses, while PTXF prevented only the increase in renal vascular resistance and P-LT levels. Data were analyzed by ANOVA (see figures). Conclusions. Metabolite production in saline controls was sta-

Endothelial actin cytoskeletal rearrangement (morphological change from cortical to stress fiber distribution) is associated with increased monolayer permeability in response to inflammatory stress. We hypothesized that pulmonary endothelial cell activation, characterized by actin stress fiber formation, contributes to capillary leak following severe thermal injury. Human pulmonary microvascular endothelial cells, grown on semi-permeable filters, were treated for up to 24 h with 20% serum isolated from burn patients (.45%TBSA; “Early” isolated at 4 h; “Late” isolated at 24 h postburn) or healthy volunteers. Migration of biotinylated-BSA across cell monolayers was assessed as a measure of endothelial permeability. Similarly treated cells were fixed and stained with rhodaminephalloidin. Actin fibers were detected spectrophotometrically and cells with stress fiber morphology were counted. Endothelial permeability was minimal with Control or Early Burn serum stimulation, although Late Burn serum significantly enhanced albumin migration (Fig. 1). Cells in Control serum demonstrated a cortical arrange-

ment of the actin cytoskeleton (Fig. 2), while cells in Burn serum (Fig. 3) showed an increase in the number of cells with actin stressfiber rearrangements (9 6 1.5% vs 69 6 9.5%, p . 0.05). These data demonstrate that increased pulmonary endothelial cell permeability

253

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS in response to human burn serum is associated with endothelial cytoskeletal stress fiber formation. Post-burn endothelial activation may contribute to burn-related pulmonary capillary leak. 33. Cutaneous Burn Injury Causes a Systemic Heat Shock Response. A. L. McMurtry, M.D., K. Cho, Ph.D., D.V.M., M. D. De Wing, M.D., L. J.-T. Young, and D. G. Greenhalgh, M.D. Department of Surgery, University of California, Davis, Medical Center, Davis, California; and Shriners Hospital for Children, Northern California, Sacramento, California. The heat shock response (HSR) is a nonspecific cytoprotective response to physiologic stress, leading to the production of proteins (HSP) that stabilize intracellular processes and facilitate survival under adverse conditions. Most past research in this area has used in vitro or whole-body in vivo insults. We hypothesized that a localized cutaneous burn wound would lead to the HSR in distant organs, and that the HSR would persist while the burn wound remained open. Mice were anesthetized, shaved, and subjected to an ethanol flame burn (;15% TBSA) on the dorsum of their bodies. This did not produce a rise in core temperature as measured by a rectal probe. At sequential timepoints from 3 h to 29 days after the burn mice were sacrificed and tissues (liver, lung, thymus, spleen, and lymph nodes) were harvested. Total RNA was extracted and RT-PCR analysis for mRNA for inducible HSP 27 and HSP 70 performed. All tissues showed a low-level baseline expression of both HSP 27 and HSP 70 in unburned animals. Cutaneous burning caused a consistent increased expression of both HSPs examined in all tissues. mRNA levels peaked at 24 h, and remained elevated to at least 14 days. Animals sacrificed at 29 days had nearly completely healed their burns, and HSP mRNA levels had fallen to near baseline. A moderate sized cutaneous burn induces a systemic HSR in distant organs that persists while the burn wound is open. Most in-vitro models of the HSR demonstrate a more rapid peak (6 – 8 h) than seen in our mouse model, implying that the HSR seen here may be due to the inflammatory focus of the wound rather than to the burning itself. The persistent elevation of HSP 27 and 70 in the inflammatory phase of wound healing further supports this idea.

PARALLEL SESSION I Peripheral Vascular I 34. Endoluminal Arterial Injury in Plasminogen Deficient Mice. Steven J. Busuttil, M.D.,* Carla Drumm, B.S.,* Victoria A. Ploplis, Ph.D.,† and Edward F. Plow, Ph.D.‡ *Case Western Reserve University and Cleveland VAMC, Cleveland, Ohio; †Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana; and ‡Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation, Cleveland, Ohio. Vascular remodeling following arterial injury is characterized by an initial inflammatory reaction. Prior experiments using peritoneal inflammatory models have shown that the plasminogen system plays a role in the intensity of the inflammatory response. This study was undertaken to assess whether the attenuated inflammatory response would correlate into a decrease in vascular remodeling. An endoluminal carotid arterial injury was created in both wild type (Plg (1/1)) and plasminogen deficient (Plg (2/2)) mice utilizing a flexible guide wire. At three weeks post injury the mice were sacrificed and perfusion fixed, and the bilateral carotid arteries were sectioned for histological examination and collection of morphometric data. After arterial injury, electron microscopy of the acutely injured artery revealed that the endothelium is denuded, that there are breaks in the internal elastic membrane, and that there is disruption of the normal circular layer of smooth muscle cells. The intimal area and medial areas were significantly increased between the right and left

carotid arteries of both Plg (1/1)[180% intimal, 153% medial p , 0.05] and Plg (2/2)[148% intimal, 120% medial p , 0.05] mice. In contrast although there was a significant increase in the adventitial area of Plg (1/1) mice (118% p , 0.05) there was no difference in Plg (2/2) mice (13.5%). Interestingly, even after three weeks, four of six injured arteries in Plg (2/2) mice had persistent thrombus within the medial layer, whereas this finding was not found in any of the nine Plg (1/1) mouse arteries. Plasminogen deficiency inhibited the increase in adventitial area seen after injury in Plg (1/1), but not the increase in intimal or medial areas. Not surprisingly, plasminogen deficient mice demonstrated a severe alteration in intramural thrombus clearance. 35. Collagenase-3 (MMP-13) Is Expressed by Vascular Smooth Muscle Cells (SMC) in Human Abdominal Aortic Aneurysms (AAA). J. K. Lee, M.D., D. Mao, M.D., S. J. VanVickle, B.S., and R. W. Thompson, M.D. Departments of Surgery and Cell Biology & Physiology, Washington University School of Medicine, St. Louis, Missouri. Purpose. Collagen degradation is an important step in the pathophysiology of AAA but the specific collagenase(s) are undefined. MMP-13 is one of three mammalian collagenases and has previously been considered to have a limited tissue distribution. The aim of this study was to determine if MMP-13 is expressed in human AAA and if it might be produced by vascular SMC in the aortic media. Methods. Total RNA extracted from human AAA tissue was examined by RT-PCR/Southern blots using oligonucleotide primers and probes specific to MMP-1, MMP-8, MMP-13 and b-actin. Specimens of normal aorta (NA) and atherosclerotic occlusive disease (AOD) were used as controls. Immunohistology (IMH) was used to localize MMP-13 in AAA using anti-human MMP-13 Ab. MMP-13 production was examined in cultural human vascular SMC by RT-PCR and immunocytology. Results. MMP-13 mRNA was abundantly and consistently expressed in diseased aorta (AAA . AOD) but not in NA (see table). MMP-13 was localized to medial SMC in AAA by IMH. Vascular SMC in culture consistently expressed MMP-13 mRNA and protein. Conclusions. These results demonstrate for the first time that MMP-13 is expressed by vascular SMC. MMP-13 is also more abundant in diseased aortic tissue than other known collagenases and it may play an important role in the collagen degradation observed in AAA.

TABLE—ABSTRACT 35 Densitometric Ratio to b-Actin (Mean 6 SE) Tissue

n

MMP-1

MMP-8

MMP-13

NA AOD AAA

4 4 4

Undetected 5.51 6 2.45 3.03 6 1.57

Undetected 0.75 6 0.40 Undetected

Undetected 3.75 6 0.59 6.69 6 0.59

* P , 0.05 vs AOD. 36. Retinoids Decrease Intimal Hyperplasia and Matrix Metalloproteinase Activity in Vivo. C. D. Leville, M.D., R. A. Cambria, M.D., M. S. Dassow, B.S., G. R. Seabrook, M.D., J. M. Jean-Claude, M.D., and J. B. Towne, M.D. Division of Vascular Surgery, MCW, Milwaukee, Wisconsin. The hypothesis tested in this study is that treatment with all-trans-retinoic acid (atRA) decreases intimal hyperplasia and matrix metalloproteinase (MMP) activity in vein grafts. Unilateral reversed jugular vein grafts were placed in the carotid artery of 40 male New Zealand white rabbits. Oral atRA (10mg/kg/day)

254

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

or vehicle (corn oil) was fed 4 days prior to and 10 days after surgery. Serum retinoid levels were obtained 7 days after graft placement. Grafts were harvested on post-operative days 7 and 28. One set of grafts (n 5 5 at each time point) was perfusion fixed and mounted on slides for planimetry. Cross-sectional areas of grafts were measured, and intimal hyperplasia was defined as a ratio of intimal to medial areas (IMR). Another set of grafts was flash frozen and processed for protein and RNA content. Substrate gel zymography determined MMP proteolytic activity, and Northern analysis measured expression of MMP-2 and MMP-9. Non-grafted contralateral jugular veins were used as control tissue. Oral administration of atRA resulted in increased serum retinoic acid levels (173ng/ml 6 43 vs 6ng/ml 6 1, p , 0.01). At 28 days, intimal hyperplasia was decreased in the atRA group compared to corn oil group (IMR 5 0.60 6 0.04 vs 0.87 6 0.1, p , 0.001). Zymography demonstrated decreased proteolytic activity at 68 kDa (active MMP-2) at 7 and 28 days in atRA vein grafts. MMP-2 and MMP-9 expression was decreased at 7 and 28 days in atRA vein grafts on Northern analysis. Non-grafted veins expressed little MMP mRNA with no differences seen between atRA and vehicle groups. All-trans-retinoic acid decreases intimal hyperplasia in an in vivo model of vein bypass grafting. Retinoic acid decreases expression and activity of MMP enzymes in the vein grafts which likely contributes to the noted decrease in intimal hyperplasia.

37. Chlamydia Pneumoniae Activates NFkB and AP-1 in Human Vascular Smooth Muscle and Induces Cellular Proliferation. S. A. Miller, M.D., C. H. Selzman, M.D., B. D. Shames, M.D., H. A. Barton, Ph.D., S. M. Johnson, B.S., and A. H. Harken, M.D. Department of Surgery, University of Colorado, Denver, Colorado. Chlamydia pneumoniae has been epidemiologically linked to atherosclerotic cardiovascular disease. However, few studies have directly implicated C. pneumoniae infection in vascular remodeling. We and others previously demonstrated that nuclear factor kappa B (NFkB), a regulator of the inflammatory response, is essential for vascular smooth muscle cell (VSMC) proliferation. AP-1 activation, a composite of the c-fos and c-jun transcription factors, is also linked to VSMC proliferation. We hypothesized that C. pneumoniae activates NFkB and AP-1, thereby promoting cellular proliferation in vitro. Methods. C. pneumoniae was grown and isolated from Hep2 cells. Human aortic VSMCs were infected with C. pneumoniae in the presence and absence of antibiotics (azithromycin, Az. 100 ng/ml). Two hours following infection nuclear extracts were isolated, and NFkB and AP-1 activation were assessed by electrophoretic mobility shift assay. Corresponding cell proliferation was assayed by direct cell counting 48 h following infection. Results. C. pneumoniae infection induced NFkB and AP-1 DNA binding activity (see figure A) which corresponded to VSMC proliferation (see figure B). These effects were eliminated by azithromycin. Conclusions. We demonstrate a causal effect for the activation of human VSMC proliferation by C. pneumoniae infection, which is abrogated by antibiotic treatment. We therefore implicate C. pneumoniae as a potential therapeutic target in the treatment of atherosclerotic disease.

38. Artery Wall Hypoxia Causes Cellular Proliferation at an Artery Anastomosis. E. S. Lee, M.D., G. E. Bauer, Ph.D., M. P. Caldwell, B.S., and S. M. Santilli, M.D., Ph.D. Departments of Surgery and Cell Biology & Neuroanatomy, University of Minnesota, Minneapolis, Minnesota. We hypothesize that arterial wall hypoxia incites the pathologic formation of intimal hyperplasia at an artery anastomosis. The purpose of this study is to determine the rate of cellular proliferation at an artery anastomosis when the artery wall is most hypoxic. Polytetrafluoroethylene (PTFE) grafts were placed end to end in the infrarenal aorta of 17 New Zealand white rabbits. The anastomotic aortic wall oxygen (O 2) tensions were measured with and O 2 microelectrode in rabbits 0, 7, and 42 days after surgery. Oxygen tensions were also measured in 4 control rabbits for comparison. Bromodeoxyuridine (BrDU) was injected intraperitoneally 24 h prior to rabbit sacrifice. After O 2 tension measurements, the rabbits were sacrificed and the aortic grafts were harvested. Bioquant computer morphometrics was used to measure cells with BrDU counterstaining, and intimal thickness, Student’s t-test was used to compare O 2 tensions, cellular proliferation, and intimal hyperplasia between days. Aortic grafts 7 day after placement revealed a nadir in transarterial wall O 2 tensions (see table, **P,0.0001) when compared to control and active cellular proliferation (*P,0.0002) when compared to aortic grafts 42 days after placement. We conclude that cellular proliferation is highest when the artery wall is most hypoxic and returns to baseline as O 2 tensions normalize. Further studies to determine whether supplemental oxygen will decrease cellular proliferation and subsequent intimal hyperplasia are necessary. 39. Pentoxifylline (PTX) Reverses Primary Oxidative Mitochondrial (MC) Defect in Claudication. I. I. Pipinos, M.D., A. D. Shepard, M.D., A. Katsamouris, M.D., P. V. Anagnostopoulos, M.D., J. A. Nelson, Ph.D., and M. D. Boska, Ph.D. Department of Surgery, Henry Ford Hospital, Detroit, Michigan. Previous morphologic studies and 31P Nuclear Magnetic Resonance Spectroscopy (NMRS) have suggested a primary MC defect in claudicating skeletal muscle. We hypothesized that PTX may alleviate this defect. Pathobiologic response of claudicating muscle to PTX was evaluated in 10 male, non-diabetic claudicants (ankle-brachial index $0.5 and #0.8) with NMRS obtained before and after 12 weeks of PTX. Calf muscles were evaluated with a short duration, submaximal, isometric plantar flexion exercise designed to allow in-magnet testing under conditions of normal blood flow. Phosphocreatine (PCr)

TABLE—ABSTRACT 38

Artery pO 2 (mm Hg) BrDU media (%) BrDU intima (%) Intimal thick (mm)

Control (n 5 4)

Day 0 (n 5 4)

Day 7 (n 5 5)

Day 42 (n 5 4)

61.0 6 2** — — —

25.0 6 1 1.0 6 0.5% — —

19.8 6 1** 5.2 6 2% 28.6 6 3%* 17.7 6 3

58.5 6 1 1.0 6 0.4% 1.4 6 0.9%* 43.2 6 1.3

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 40

TABLE—ABSTRACT 39

PCr t.c. in pts with normal baseline (n 5 3) ADP t.c. in pts with normal baseline (n 5 3) PCr t.c. in pts with abnormal baseline (n 5 7) ADP t.c. in pts with abnormal baseline (n 5 7)

Pre-PTX (s)

Post-PTX (s)

45 6 8

56 6 8

(P 5 0.18)

24 6 5

30 6 3

(P 5 0.36)

188 6 58

97 6 20

(P 5 0.03)

76 6 10

48 6 6

(P 5 0.02)

and adenosinodiphosphate (ADP) recovery time constants (t.c.), two very sensitive measures of oxidative MC function, as well as intracellular pH and ATP production via anaerobic glycolysis were measured. Cellular pH and anaerobic ATP levels remained in the normal range, confirming that the study exercise did not significantly reduce arterial inflow/oxygen supply. Seven of 10 patients (pts) had abnormal baseline PCr and ADP t.c.(PCr t.c. . 66 sec and ADP t.c. . 41 sec). All 7 had significant improvement in MC function with PTX (see table). The majority of pts with claudication have intrinsic oxidative MC dysfunction. This dysfunction is significantly improved by PTX in a manner unrelated to the hemorrheologic effects of PTX. This data points to a potential new mode of action for PTX in the treatment of intermittent claudication. 40. Thermotolerance Preserves Endothelial Vasomotor Function after Ischemia/Reperfusion in Vivo. G. Chen, M.D., C. J. Kelly, FRCSI, H. Chen, M.D., A. Leahy, FRCSI, and D. J. Bouchier-Hayes, FRCSI. Royal College of Surgeons in Ireland, Department of Surgery, Beaumont Hospital, Dublin, Ireland. Ischaemia/reperfusion (I/R) results in loss of endotheliumdependent vasodilatation. Thermotolerance (T) attenuates I/Rinduced microvascular injury. The aim of this study was to investigate the effect of T on I/R-induced vasomotor changes. Endothelial dependent (ED) and -independent vasodilatation (EID), measured by response to acetylcholine and sodium nitroprusside respectively, was measured in mesenteric arteriole (27.5–38 mm) by intravital microscopy. Sprague-Dawley rats were randomized into an untreated group (IR group, n 5 28) and a group in which thermotolerance was induced (TIR group, n 5 16). The vasomotor function was evaluated by arteriolar constriction:dilatation Ratio (C:DR). T was induced by elevating core body temperature to 41 1 0.5°C for 15 min 18 h prior to I/R injury, which was in turn established by occlusion of the superior mesenteric and celiac vascular pedicle for 30 min, followed by 60 min of reperfusion. The “no-reflow” phenomenon was also measured by intravital microscopy (see table). I/R caused a significant decrease in ED vasodilatation, as measured by response to acetylcholine. EID dilatation was preserved. T prevented this decrease in ED dilatation. In this model, I/R caused no-reflow phenom-

Baseline IR group TIR group

ED-C:DR

EID-C:DR

2.06 6 0.20 1.37 6 0.31* ,** 1.95 6 0.19

2.08 6 0.22 2.06 6 0.20*** 2.04 6 0.24

Note. Results, mean 6 SD. Statistics, ANOVA with Scheffe post hoc correction and x 2 test. * P , 0.01 vs baseline; ** P , 0.01 vs TIR; ***P , 0.01 vs endothelial-dependent C:DR.

enon in 16/28 unheated rats. T significantly reduced this no-reflow to 4/16 rats (P , 0.05 vs IR). This study demonstrates that thermotolerance preserves endothelial vasomotor function and markedly reduced “no-reflow” in arterioles. Further investigation of the precise mechanism by which thermotolerance preserve endothelial function is necessary.

41. Mycophenolate Mofetil (MMF) Treatment Reduces Atherosclerosis in the Cholesterol-Fed Rabbit. S. M. Greenstein, M.D., S. C. Sun, M.D., T. M. Calderon, Ph.D., D. K. Kim, M.D., T. C. Schreiber, B.S., R. S. Schechner, M.D., J. W. Berman, Ph.D., and V. Tellis, M.D. Albert Einstein College of Medicine/ Montefiore Medical Center, New York, New York. Atherosclerosis may be caused by an immune/inflammatory response of the artery to injury, similar to that in transplant rejection. MMF, an antiproliferative agent in clinical use, has been shown experimentally to inhibit the vascular changes typical of chronic allograft rejection. This study was conducted to determine the effect of MMF on atherosclerotic plaque formation. Methods. New Zealand white rabbits were fed a high cholesterol diet (0.5% cholesterol in 8% peanut oil). The experimental group (n 5 10) was given MMF (80 mg/kg/day subcutaneously); the control group (n 5 10) received injections of equal volume of 5% dextrose. The aortas were harvested at 3 months for histologic and immunohistochemical analyses. Smooth muscle cells were identified by reactivity with mAb to a-smooth muscle actin. Macrophages and monocytes were detected with mAb RAM 11. Plaque and media layer areas were quantified by computerized morphometric analysis. Results. The animals tolerated the diet and medication. Weight gain was no different between the MMF and control groups. Total cholesterol, HDL, LDL, and triglyceride were significantly increased compared with baseline, but there was no statistically significant difference between the two groups. Histologically, atherosclerotic plaque area was reduced by 52% in the MMF group as compared to the control group (p , 0.025). There was less foamy macrophage accumulation and smooth muscle migration in the plaques in the MMF-treated group (see table). Conclusions. Our results demonstrate that MMF reduces inflammatory response and plaque development in a cholesterol-fed rabbit model, suggesting that it may have an antiatherosclerotic effect.

TABLE—ABSTRACT 41 Cholesterol

Control MMF

Triglycerides

HDL

Plaque/media area ratio, %

Pre-fed

After-fed

Pre-fed

After-fed

Pre-fed

After-fed

0.38 6 0.21 0.21 6 0.20*

70 6 20 75 6 17

1231 6 142 220 6 138

66 6 19 81 6 25

150 6 81 169 6 69

37 6 10 35 6 8

95 6 27 93 6 28

* P , 0.025 compared to control.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

42. Virus Infection Abrogates CD8 1 T Cell Deletion and Skin Allograft Survival Induced by Donor-Specific Transfusion and Anti-CD154 Monoclonal Antibody. N. A. Turgeon, M.D., N. N. Iwakoshi, W. C. Meyers, M.D., R. M. Welsh, Ph.D., D. L. Greiner, Ph.D., J. P. Mordes, M.D., and A. A. Rossini, M.D. Departments of Surgery and Medicine, University of Massachusetts Medical School, Worcester, Massachusetts. Treatment with donor-specific transfusion (DST) plus anti-CD154 monoclonal antibody (mAb) prolongs skin allograft survival in mice. We have documented: 1) that induction of skin allograft survival is abrogated by infection with lymphocytic choriomeningitis virus (LCMV) and 2) that, using a T cell receptor (TcR) alloantigen-specific transgenic model, the mechanism of allograft survival in part depends on deletion of alloreactive CD8 1 T cells. Based on these data, we hypothesized that LCMV infection abrogates graft survival by interfering with deletion of alloreactive CD8 1 T cells. To test this hypothesis, CBA mice were given 2.5-3 x 10 6 CBA-KB5 CD8 1 transgenic (anti-H2-K b) T cells. Two days later, the CBA-KB5 transgenic chimeras received no further treatment (Group 1), or 10 7 C57BL/6 (H2 b) splenocytes (DST) plus anti-CD154 mAb (0.5 mg i.p.) and either no virus (Group 2) or 5 x 10 4 plaque forming units of LCMV, Armstrong strain (Group 3). Three days later, spleen and lymph node cells were analyzed by flow cytometry for quantification of KB5 1 CD8 1 T cells. Results. The number of KB5 1CD8 1 allospecific transgenic cells in chimeras treated with DST plus anti-CD154 mAb was ;5-10-fold less than in untreated controls. Consistent with our hypothesis, the number of KB5 1CD8 1 transgenic T cells was greatly increased in LCMV-infected chimeras treated with DST plus antiCD154 mAb (see table). The KB5 1 transgenic T cells displayed an

TABLE—ABSTRACT 42 Group

Splenic KB5 1 CD8 1 cells

Lymph node KB5 1 CD8 1 cells

1. Untreated 2. DST 1 mAb 3. DST 1 mAb 1 LCMV

33 6 4 3 10 6 3 6 1 3 10 6 474 6 151 3 10 6

6 6 1 3 10 6 1 6 0 3 10 6 372 6 98 3 10 6

tus, an indicator of its downstream signaling potential. FAK expression was increased in adenomas from Min /1 mice (see figure, left), and the amount of tyrosine phosphorylated FAK was increased in both histologically normal Min /1 mucosa and adenomas (see figure, right). The increase in phosphorylated FAK was associated with lowered rates of migration and apoptosis in the histologically normal Min /1 muscosa. Thus, these results show that FAK activity is altered in pre-malignant intestinal mucosa, and suggest that integrinmediated signal transduction is important in early Apc-associated carcinogenesis. 44. Metalloproteinase Inhibition Prevents Acute Lung Injury. D. E. Carney, M.D., U. G. McCann, M.D., H. J. Schiller, M.D., A. L. Picone, M.D., L. M. Golub, Ph.D., N. S. Ramamurthy, Ph.D., Y. Liu, M.D., L. A. Gatto, Ph.D., and G. F. Nieman, B.A. State University of New York Health Science Center, Syracuse, New York. Treatment of acute lung injury (ALI) in critically ill patients has proven difficult, yet it occurs in patients with clearly identifiable risk factors. We postulate that patients at risk can be protected by prophylactic treatment strategies which target neutrophil-derived mediators such as matrix metalloproteinases (MMPs). The purpose of this study was to establish that chemically modified tetracycline (CMT), a potent inhibitor of MMPs, would prevent ALI in our porcine model. Yorkshire pigs were anesthetized, intubated, and then randomized to: Control (n 5 4), LPS (n 5 4) infused with 100 mg/kg of Escherichia coli lipopolysaccharide or CMT 1 LPS (n 5 5) fed CMT (100 mg/kg) prior to infusion of LPS. Bronchoalveolar lavage samples were analyzed for MMP activity by gel zymography. A 92kDA Gelatinase (Gel B) was the positive control. Metalloproteinase inhibition

activated phenotype (FSC hi, CD44 hi). Conclusions. The data demonstrate that LCMV interferes with the deletion of alloreactive CD8 1 T cells mediated by DST and anti-CD154 mAb and may in part be the mechanism by which infection abrogates subsequent allograft survival. The results suggest that virus infection can adversely affect the outcome of tolerance-based allotransplantation. 43. Increased Tyrosine Phosphorylation of Focal Adhesion Kinase (FAK) Correlatates with Altered Intestinal Cell Growth in a Murine Model of APC. M. J. Weyant, M.D., A. M. Carothers, Ph.D., R. T. Bilinski, B.S., and M. M. Bertagnolli, M.D. The New York Presbyterian Hospital and Weill Cornell Medical College, New York, New York. The Min /1 mouse carries a germline mutation of Apc and is a model of familial adenomatous polyposis and sporadic colorectal cancer. Min /1 enterocytes exhibit decreased migration and apoptosis, suggesting defective integrin-mediated growth regulation. FAK is an effector of integrin signaling that can be elevated in cancers to enhance metastasis. Phosphorylation of FAK is integral to its activity as a modulator of cell adhesion, migration, and apoptosis. To determine whether integrin-mediated signaling plays a role in early intestinal carcinogensis, adenomas and enterocytes from Min /1 and wild-type mice were analyzed by immunoblot (IB) and immunoprecipitation (IP) to determine FAK amount and phosphorylation sta-

by CMT (see figure) was associated with preserved physiologic lung function (see table). Physiologic data at 6 h are presented as means 6 SEM (*P , 0.05 vs all groups; ANOVA). Chemically modified tetracyclines are a safe therapy which target terminal neutrophil effectors and attenuate acute lung injury. Similar to the universal practice

TABLE—ABSTRACT 44 Group

PaO 2 (mm Hg)

Airway pressure (mm Hg)

Venous admixture (Qs/Qt 2 %)

Control LPS CMT 1 LPS

227 6 34 81 6 12* 192 6 42

13.3 6 2.4 20.0 6 0.1* 14.2 6 1.6

4.3 6 1.2 29.3 6 9.3* 5.2 6 4.6

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS ofprophylaxis against gastric stress ulceration and deep venous thromboses, CMTs may likewise be applied for preventing acute lung injury in critically injured patients at risk.

PARALLEL SESSION II Education 45. Difficulty with Negative Feedback: Face-to-Face Evaluation of Junior Medical Student Clinical Performance Results in Grade Inflation. L. M. Colletti, M.D., and D. A. Campbell, Jr., M.D. Department of Surgery, University of Michigan, Ann Arbor, Michigan. Objective. Medical students commonly express a desire for more immediate and direct feedback about their clinical performance. We piloted a new clinical evaluation system during the required Junior Year Surgery Clerkship in which students were given direct face-toface feedback regarding their clinical performance. This was compared this to our standard “anonymous” grading system currently in use. Methods. Our new evaluation system (NS) was used concurrently with our standard clinical evaluation/grading system (OS). The two methods were directly compared using a standard t-test. The OS is a subjective evaluation of clinical performance, with a summary grade of 1-6 given as a final grade, with 1 5 fail and 6 5 honors. The NS retains the 1-6 grading scale, however, students were allowed to choose their evaluators, rather than having all of the faculty and all of the house officers on a given service evaluate each of the students on the service, as was done in the OS. Students were instructed to meet with two faculty and one senior resident of their choice. Faculty were instructed to give students direct feed back on their performance, with citation of specific strengths, weaknesses, and areas for improvement; they were also instructed to give each student a final, 1-6 grade on their overall performance. Results. 24 students participated in this study. There was a significant degree of grade inflation with the NS, particularly for students with poorer performances. The average grade using the OS was 5.11 6 0.11; in contrast, with the NS, the average grade was 5.62 6 0.07 (p , 0.001). Further, if students with grades of 5.0 or less in the OS are studied, then the average grade using the OS is 4.24 6 0.32, in contrast to 5.47 6 0.14 with NS (p , 0.005). An additional interesting finding was noted: of the students who failed to participate in the face-to-face interviews (n 5 4), the average grade of these students using the OS was 4.36 6 0.29 (P , 0.05 vs OS total), suggesting that these students may have been aware of their poor performance, were reticent to receive negative feedback, and therefore opted not to schedule an interview. Conclusions. While students desired more timely, direct feedback on their clinical performances, unfortunately, faculty are somewhat poor at giving direct, objective face-to-face feedback, particularly when it involves negative feedback. Increasing faculty awareness of this problem and finding objective, constructive ways to give negative feedback may improve this problem and enhance overall medical student learning in the clinical environment. 46. Identification of High-Risk Residents. P. C. Bergen, M.D.,* J. H. Littlefield, Ph.D.,† G. E. O’Keefe, M.D.,* R. V. Rege, M.D.,* T. A. Anthony, M.D.,* L. T. Kim, M.D.,* and R. H. Turnage, M.D.* *UT Southwestern Medical Center, Dallas, Texas; and †UT San Antonio Health Science Center, San Antonio, Texas. Background. Identification of high-risk residents allows remediation and support for administrative action when necessary. This study characterizes differences in documentation of marginally performing residents in a general surgery residency. Methods. High-risk residents were identified by the former program director. Twenty-four of 115 residents over a 10-year period had

257

1-4 problematic areas: cognitive, synthetic, family/health, and interpersonal skills. Outcomes included: finished (18), voluntary withdrawal (1), and involuntary withdrawal (5). A case control study matching controls to cases by date of entry into the training program was utilized. Records were reviewed for demographics, pre-entry qualifications, ABSITE scores, letters of complaint or praise, events of counseling and monthly ratings. The records of 48 residents were reviewed. Ward evaluations were on 8 performance categories by a 5-point Leikert scale (3-unacceptable to 7-outstanding). The Evaluation Score assigns points only to low ratings. High scores represent progressively poorer performance. Wilcoxon signed ranks test was used to compare the cases and controls for continuous variables. The McNemear test was used in comparisons of categorical data with binary out comes. Exact p-values are reported. Results. Objective data were similar for both groups. Cases tended to score higher on monthly evaluations at year two and by year three this achieved significance (.026). Cases were more likely to have negative faculty letters (.016), events of counseling by a faculty member (.017) and the program director (.005). Conclusions. Identification of residents at risk should begin as early as possible during training. A combination of faculty evaluations and evidence of letters or counseling can detect high-risk residents. Programs may use such indicators to support decisions regarding remedial work or administrative action. 47. Improving the Surgeon’s Participation in Research: Is It a Problem of Training or Priority? C. Y. Ko, M.D., E. E. Whang, M.D., W. P. Longmire, Jr., M.D., and D. W. McFadden, M.D. Departments of Surgery, UCLA Medical Center, Los Angeles, California; and Brigham and Women’s Hospital, Boston, Massachusetts. Although important contributions have originated from basic research performed by surgeons, the future of such work is in jeopardy. In an effort to develop strategies for sustaining and improving surgical research productivity, we characterized the natural history of research participation by present day academic surgeons. Methods. A 25-item survey was sent to 850 senior academic surgeons. Results. 377 (41%) completed surveys were received. 72% of respondents had performed basic science research during training; of these individuals, 99% continued research as junior faculty. However, by age 39 years, 38% had stopped their participation. An additional 17% and 23% stopped basic research between ages 40-49 and 50-59 years, respectively. The most prevalent factors, as perceived by respondents, causing them to stop research participation were increased clinical load (40%) and increased administrative duties (38%). For the subset of respondents who had stopped research prior to age 40 years, 73% cited increased clinical load as the primary reason for stopping research. 85% of respondents stated that research should continue to be an integral part of surgical training. Conclusion. The diminishing participation of surgeons in research appears to be a problem of priority, not of training. These results suggest that faculty, especially at the junior level, need to be better focused towards research, rather than clinical, productivity. The challenge to the leadership of academic surgery will be to enhance such research productivity in the context of the increased clinical productivity demanded by managed care. 48. The Outcome of Research Training during Surgical Residency. A. Thakur, M.D., V. Thakur, M.D., T. L. Buchmiller, M.D., and E. W. Fonkalsrud, M.D. Division of Pediatric Surgery, UCLA Medical Center, Los Angeles, California. Graduates of a university surgical residency program were surveyed to identify the timing of specialty selection and the impact that studying in a research laboratory has on the subsequent acceptance into a fellowship program. Between 1975 and 1990, 86

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residents completed general surgical training at UCLA Medical Center. A survey was sent to each graduate to determine the focus of their previous laboratory research and when they selected their eventual surgical specialty. Responses were received from 67 graduates (78%). Fellowship training was selected by 55 residents (82%); 50 applied to less than five programs, 45 of whom received one of their top three choices. Sixty percent of residents with one or more years of research (N 5 31) selected non-plastic surgery fellowships compared to 27% of those who had no laboratory experience (N 5 4), p 5 0.04. Only 52 % (N 5 35) had selected a specialty after two years of clinical training; 25 more made the selection after the third year of residency. All residents who selected cardiac (N 5 12) or plastic surgery (N 5 4) prior to research were accepted into fellowships in those specialties, whereas only 37% (N 5 7) who had interests in other fields pursued the same specialty (P , 0.0001). Residents performing research in general surgery (N 5 9), surgical oncology (N 5 18), cardiac surgery (N 5 14), and plastic surgery (N 5 3) were more likely to practice in that specialty than those doing research in other specialty laboratories, 61%, 39%, 86%, and 100%, respectively (p , 0.05). Surgical residents who have research experience are likely to apply for specialty training. Residents who select a career in cardiac or plastic surgery prior to research are likely to pursue these fields as their eventual specialty and appear to benefit from research in their specialty. Residents who remain undecided about their field of specialization by the end of the second clinical year may benefit from the option to perform research after the third year. Residents doing research in a specialized laboratory are very likely to pursue fellowship training related to that field.

49. Factors Affecting Improvement on the American Board of Surgery In-Service Training Exam (ABSITE). L. S. Hauge, Ph.D.,* Constantine V. Godellas, M.D.,* and Raywin Huang, Ph.D.† *Department of General Surgery and †Department of Preventive Medicine, Rush University, Chicago, Illinois. ABSITE performance can be used to assess resident knowledge and to evaluate surgical curriculum. To determine factors that lead to improved resident ABSITE performance, a prospective study was performed. Thirty-four surgical residents in program years 2-5 completed a pre- and a post-ABSITE questionnaire about their anxiety, self-efficacy, study practices, physical preparation, and academic preparation for the ABSITE. Department records were used to determine resident probationary status and conference attendance. A preliminary analysis of ABSITE scores indicated a significant improvement between 1998 and 1999 percentile scores (paired t 5 22.25, p 5 .03; m 5 11.9, sd 5 30.5, median 5 41). An improvement in percentile rank score was calculated and used as the dependent variable in a step-wise regression analysis. The following served as independent variables: previous exam performance (1998 percentile score), anxiety (pre- and post-exam Likert scale), probationary status, amount of sleep before exam (2 previous nights), confidence to score in the 25 th and the 50 th percentiles (pre- and post-exam Likert scale), and attendance at the 3 conferences rated most valuable by the residents. Results of the regression analysis demonstrate that all factors account for 62.3% of the variance in improvement scores. A stepwise analysis indicated that the combination of amount of study (36.8%), previous performance (12.2%), and attendance (8.7%) were significant in explaining 57.7% of the variance in improvement scores. Furthermore, Pearson’s correlations indicated that probationary status (1.58, p 5 .001), higher anxiety (1.53, p 5 .001), amount of study (1.61, p 5 .001), past ABSITE performance (2.60, p 5 .001), and conference attendance (1.56, p 5 .001) were correlated with improvement in percentile rank. This study demonstrates that resident individual effort, past AB-

SITE performance, and academic conference attendance have led to resident ABSITE improvement. 50. Resident Education about End-of-Life Issues. R. J. Bold, M.D., P. D. Schneider, M.D., Ph.D., and J. E. Goodnight, M.D., Ph.D. Division of Surgical Oncology, Department of Surgery, University of California, Davis, Sacramento, California. Recently, legislation has been brought forward in several states that allows a physician to perform assisted death for a patient with a terminal illness, such as cancer. The purpose of this study was to determine the experience and opinion on this issue among surgical residents and staff oncologists (surgical, medical and radiation) as a guide to ascertain how residents are educated about this process. Methods. A questionnaire was developed that presented five case scenarios of patients with metastatic malignancies and estimated survival of less than 12 months; subjects were queried regarding previous experience and willingness to participate (either directly or indirectly) in assisted death. Surgical residents (N 5 56) and staff oncologists (N 5 24) at a University Medical Center were surveyed. Results. Response rates were 39% (22/56) for the residents and 87% (21/24) for the oncologists. 86% (19/22) of the residents would aid any one of the hypothetical patients with assisted death while only 19% (4/21) of the staff oncologists expressed a willingness to aid any of the hypothetical patients with assisted death. Furthermore, 32% (7/22) of the residents reported previous involvement in a case of assisted death from any disease process, while only 19% (4/21) of the staff oncologists reported previous direct experience with assisted death in the terminal cancer patient. Conclusions. Although similar incidences of previous experience with assisted death, surgical residents are much more willing than staff oncologists to aid terminal cancer patients with assisted death. These opinions and practices are likely not the result of medical education but are developed from personal values. 51. Perceived Obstacles to Success of Women in Academic Surgery. S. Sonnad, Ph.D., M. W. Mulholland, M.D., Ph.D., and L. M. Colletti, M.D. Department of Surgery, University of Michigan, Ann Arbor, Michigan. Objective. Rising numbers of female medical students have led to expectations of parallel increases in female academic physicians and leaders, which has not occurred. This study examines possible reasons through a survey of both male and female surgeons at one academic center. Methods. We administered to our entire faculty a survey including demographics, responsibilities, perceptions of obstacles, mentoring experiences, career expectations, and family issues. We used t-tests to compare answers between genders. Results. 45/99 male faculty (45%) and 9/16 female faculty (56%) responded. Women reported similar weekly hours of housework to men (11 vs 8), but nearly twice as many hours parenting (34 vs 18). Men and women reported similar perceptions of workload, similar numbers of patients, and similar educational responsibilities. Women have more extramural funding (89% vs 52%, p 5 .006), but fewer publications (4.7 vs 8.8, p 5 .004). Women also perceived obstacles to career success for women not reported by men. 56% of female faculty felt women are held to higher academic standards for promotion (5% of men agreed, p , 0.0001). 89% of women vs 16% (p , 0.0001) of men reported that women had fewer professional opportunities and less mentoring available than did men, and that this inhibited career success. 56% of women reported that men are more often selected as research collaborators than are women with comparable expertise (11% of men agreed, p , 0.0001), and 78% of female faculty felt that informal networks often exclude women (20% of men agreed, p , 0.0001). Two-thirds of both men and women reported having or having had a mentor. Women more often felt mentors used their work to advance the career of the mentor rather than the advisee (50% vs 24%, p 5 .07). 71% of men vs 14% (p , 0.0001) of women

259

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS agreed that there are good role models available. Two-thirds of both male and female faculty want to be in academic medicine and expect to be promoted. However, 70% of men vs 56% of women (p 5 .26) expect to be in academic medicine in 10 years, related to 19% of men vs 33% of women (p 5 .20) who seriously were considering leaving academic medicine. Conclusions. Our study points to areas for improvement, many of which have been undertaken or proposed elsewhere. While formal structures and mechanisms can be changed, many obstacles to women’s advancement in academic medicine and in our department appear to be rooted in societal attitudes and behaviors that are harder to change. However, active attempts by academic leadership toward addressing obstacles for women faculty, many of which also affect men, will move us toward environments where the best physicians can be attracted and retained in academic settings, regardless of gender.

PARALLEL SESSION II Oncology I 52. Comparison of Intradermal (ID) and Subcutaneous (SC) Injections in Lymphatic Mapping. T. Kersey, M.D., J. Van Eyk, B.S., D. Lannin, M.D., and L. Tafra, M.D. Department of Surgery, ECU School of Medicine, Greenville, North Carolina. Introduction. Sentinel node biopsy (SNB) for melanoma, with its ID injection, has a higher success rate than SNB for breast cancer, which is typically performed with a SC, pertitumor injection. No study has investigated differences in transit time of agents used in lymphatic mapping from injection site to the sentinel node, that could account for this clinical observation. Goal. To compare transit time between ID and SC injections with common agents used in lymphatic mapping. Methods. Forty injection sites on five domestic pigs were used. These sites included bilateral, cervical, forelimb, hindlimb and flank areas. Agents included technetium sulfur colloid (Tc99, filtered and unfiltered), isosulfan blue dye (IB) and fluorescein dye (F). At each site both ID and SC injections were made and the transit time to reach the sentinel node was recorded. Results. Sentinel nodes were identified draining all sites, and found to be either hot, blue, or fluorescent (using a Wood’s lamp for identification). Time differences were calculated per centimeter distance from the draining lymph node basin. The table summarizes the results (sec/

TABLE—ABSTRACT 52 Injection technique

Tc99 (filtered)

Tc99 (unfiltered)

IB

F

Dermal Subcutaneous P value

4.8 6 2.5 59.0 6 18.8 0.02

2.7 6 0.54 249 6 14.7 0.008

10.5 6 3.6 6.3 6 2.3 0.3

6.98 6 1.45 8.27 6 2.3 0.4

ments of Surgery and Medical Oncology, Yale University School of Medicine, New Haven, Connecticut. Introduction. CPT11, a camptothecin derivative, has shown significant promise in 5-fluorouracil refractory colon cancer. To date this agent has not been used by direct hepatic artery infusion. This study aims to investigate the pharmacokinetics of CPT11 and its active metabolite SN38 when given by hepatic artery infusion. Methods. CPT11 was administered via the hepatic artery, at 90mg/m 2 in 6 pigs and 125mg/m 2 in 8 pigs. The aorta and common bile duct were cannulated to obtain samples of blood and bile. CPT11 and SN38 concentrations were measured in plasma, bile and liver by reverse phase HPLC. Results. Peak plasma concentrations were 10.02 6 2.58 mM, and 8.33 6 3.74 mM for 90mg/m 2 and 125 mg/m 2 doses of CPT11 respectively. Half-life was 139 6 16 min and 144 6 30 min respectively. Liver extraction rates were 34.5% and 65.8% respectively. Biliary excretion of CPT11 was 6.5 6 4.3% of the original dosage. Hepatic concentration of CPT11 was 9.5 times higher than that in plasma, and hepatic concentration of SN38 was 342.3 times higher than plasma. Conclusion: High liver/plasma ratios of CPT11 and SN38 are seen with hepatic arterial infusion of CPT11. This may improve efficacy and limit toxicity of this agent and suggests that further evaluation of this system is warranted. 54. Do Molecular Methods Add to Immunohistochemistry in the Staging of Pancreatic Cancer? H. M. Brown, M.D., S. A. Ahrendt, M.D., R. A. Komorowski, M.D., K. Doffek, B.A., S. D. Wilson, M.D., and M. J. Demeure, M.D. Departments of Pathology and Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin. Molecular assays based on the polymerase chain reaction (PCR) have demonstrated K-ras mutations in the regional lymph nodes (LN) of 70% of patients with stage I (node-negative) pancreatic cancer. Immunohistochemistry (IHC) is also being used more frequently to identify LN metastases in patients with cancer. We examined the role of both IHC and PCR analysis in the staging of patients with pancreatic cancer. Regional lymph nodes (LN) were examined from thirty patients with Stage I, K-ras mutant pancreatic cancer after standard one-level histopathological examination (HPE). K-ras codon 12 mutations were detected using PCR and restriction digest with BstN1. Step sections were taken at four levels through each LN for hematoxylin and eosin staining and immunohistochemistry with the epithelial cell marker AE3/ AE1. The percentage of patients with LN metastases and the median survival among LN positive (1) and LN negative (2) patients is shown in the table. Standard one-level HPE overestimated the percentage of node-negative patients with pancreatic cancer. The addition of PCR/RFLP to step-sectioning and IHC significantly increased the number of patients with micrometastases detected. However, no significant differences in survival were observed among LN-negative and LN-positive patients using either or both staging approaches. The presence of LN microme-

TABLE—ABSTRACT 54 cm 6 SEM). Conclusions. Tc99 dermal injections were significantly faster compared to a SC injection. The slowest and fastest SC injection agent was unfiltered Tc99 and IB, respectively. Dermal injections provide faster transit of lymphatic agents and may improve the success rate when applied to patients with breast cancer.

53. Evaluation of the Pharmacokinetics of CPT11 (Irinotecan Hydrochloride) and Its Metabolite SN38 When Given by Infusion into the Hepatic Artery. S. Bayar, M.D., G. Pizzorno, Ph.D., J. Leffert, B.S., and R. R. Salem, M.D. Depart-

Median survival (months) Staging method

LN metastases

LN2

LN1

Standard HPE Detailed HPE/IHC K-ras PCR/RFLP Either IHC or PCR

0/30 (0%) 13/27 (48%)* 18/29 (62%)* 25/29 (86%)* ,**

20 23 18 24

— 16 20 19

* P , 0.001 vs standard HPE; ** P , 0.01 vs detailed HPE/IHC.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

tastases does not predict systemic tumor cell dissemination and survival in patients with pancreatic cancer. 55. Vitamin E Succinate Promotes Breast Cancer Tumor Dormancy. M. P. Malafa, M.D., and L. T. Neitzel, M.S. Department of Surgery, Southern Illinois University School of Medicine, Springfield, Illinois. Vitamin E succinate (VES), an ester analogue of vitamin E, has growth inhibitory effects on breast and a variety of cell lines in vitro. However, there is no information about its antitumor effects in vivo. We determined the effect of VES on the growth of the human breast cancer cell line, MDA-MB-231, in nude mice. Mice were inoculated in the right mammary fat pad with 4 3 10 6 cells. Mice growing with tumors were randomized to receive VES at 100 mg/kg/day by intraperitoneal injection (IP), 100 mg/kg/day by subcutaneous injection (SC), or vehicle injection. Tumor area was measured weekly, and animals were sacrificed 4 weeks after tumor inoculation. In vivo inhibition of tumor growth was profound with i.p. administration of VES decreasing tumor size 60% compared with controls at 26 days (p , 0.05). Stopping VES treatment resulted in resumption of tumor growth in the VES treated mice. Histological examination found that VES treated tumors were smaller; however, the rates of proliferation (determined by PCNA immunostaining) and apoptosis (determined by the TUNEL method) were not significantly different. In vitro data (by RNase protection assay) reveals that VES inhibits vascular endothelial growth factor (VEGF) expression suggesting that it may inhibit tumor angiogenesis. We conclude that VES promotes breast tumor dormancy. The mechanism of tumor growth inhibition is yet to be elucidated. This is the first report of VES antitumor growth in vivo. By promoting tumor dormancy, VES may be useful in preventing relapse after standard therapy of locoregional breast cancer. 56. Hypericin and Photodynamic Therapy Decreases Human Pancreatic Cancer in Vitro and in Vivo. C. D. Liu, M.D., R. E. Saxton, Ph.D., and D. W. McFadden, M.D. Department of Surgery, UCLA Medical Center, Los Angeles, California. A majority of patients who are diagnosed with pancreatic cancer are surgically unresectable. Recent preclinical data has revealed that hypericin, a photochemical dye, is activated by green light and generates toxic radical species in tumor. We hypothesized that interstitial hypericin and laser phototherapy would decrease pancreatic cancer growth. Methods. MiaPaCa-2 and PANC-1 cells were grown in tissue culture. In vitro experiments were performed with addition of 10 mg of hypericin/500,000 cancer cells. Cells were incubated with hypericin for 2 h. Cells were then exposed to KTP532 green laser light for 1 min at 0.6 W using a cylindrical diffuser tip. Cell growth was measured by MTT assay 24 h after laser treatment, N 5 12. MiaPaCa-2 cells were implanted subcutaneously and orthotopically in pancreas of nude mice. After 5 weeks, both tumors were injected with 100 mg of hypericin followed by insertion of a cylindrical diffuser tip into the tumor center. Mice received 200J KTP laser light

TABLE—ABSTRACT 56 In vitro MTT assay

In vivo SQ tumor

In vivo panc. tumor

66.1 6 0.2%* n 5 12, * P , 0.01

91.2 6 2.3%* n 5 12, * P , 0.001

42.2 6 8.1%* n 5 12, * P , 0.05

Note. Data are expressed as percentage of reduction vs paired controls in the MTT assay and vs prephotodynamic therapy in mice experiments. * Student’s t test performed.

at 1.0W in 2 sites. Tumors were measured before and 4 weeks after laser treatment. Results. Both in vitro and in vivo mice data showed a significant decrease in growth of pancreatic cancer (see table). Conclusion. Both in vitro and in vivo results revealed a significant decrease in pancreatic cancer cell growth. Laser or dye alone had no effect, indicating intra-tumor hypericin laser therapy may prove useful in unresectable pancreatic cancer. 57. Inhibition of Intestinal Tumors by Curcumin Is Associated with Changes in the Intestinal Immune Cell Profile. M. Churchill, A. Chadburn, M.D., R. T. Bilinski, and M. M. Bertagnolli. The New York Presbyterian Hospital and Weill Cornell Medical College, New York, New York. Curcumin is a phenolic antioxidant known for its anti-tumor and immune modulatory functions. Curcumin prevents tumors in the Min /1 mouse, an animal that develops spontaneous intestinal adenomas as a result of germline Apc mutation. Curcumin-associated tumor prevention may occur through an immunomodulatory mechanism. To study the relationship between intestinal immunity and anti-tumor response, we treated 10 Min /1 mice with a tumorpreventing dose of curcumin (AIN76A diet10.1% curcumin). 10 control Min /1 and 10 wild-type mice received AIN76A diet alone. After 10 weeks, tumors in the mice treated with curcumin decreased by 64% compared to untreated controls (p 5 .005). Intestinal immune cells in sections from treated and control animals were characterized by immunohistochemistry. The values are expressed as 0 – 41 staining (403) 6 SEM (see table). These results show that mucosal CD41 T cells and B cells increase in animals treated with curcumin, sug-

TABLE—ABSTRACT 57

T cells CD31 CD41 CD81 TCRab TCRgd B cells Monocytes

Wild-type

Min/1

Min/1 curcumin

1.5 6 0.2 1.4 6 0.2 1.9 6 0.4 1.1 6 0.2 2.9 6 0.5 0.9 6 0.2 2.1 6 0.1

2.3 6 0.3 2.3 6 0.6 2.8 6 0.3 1.2 6 0.2 2.8 6 0.3 1.5 6 0.2 3.3 6 0.4

3.4 6 0.3* 3.3 6 0.4* 2.3 6 0.4 1.2 6 0.3 3.7 6 0.5 2.5 6 0.1* 2.6 6 0.2

* P , 0.05 compared to Min/ 1; n 5 10 mice/group, 10 fields/ mouse. gesting that enhanced antigen presentation in the intestinal mucosa may be responsible for the anti-tumor effect. 58. Suramin Analogues Inhibit Human Angiogenesis in Vitro. M. O. Meyers, M.D., A. R. Gagliardia, M.D,. Ph.D.,* G. J. Flattmann, M.D., Y. Z. Wang, M.D., and E. A. Woltering, M.D. Louisisana State University Medical Center, Department of Surgery, New Orleans, Louisiana; and *Department of Internal Medicine, University of Kentucky Medical Center, Lexington, Kentucky. Suramin is a polysulfonated naphthylurea that inhibits tumor cell proliferation and angiogenesis. However, the use of this drug has been limited by significant toxicity. A series of suramin analogues that have less in vivo toxicity have been generated and we hypothesized that these would have equal to or greater anti-angiogenic properties as suramin in vitro. To test this hypothesis, we incorporated 2mm in diameter discs of human placental vein into a 0.3% fibrin matrix and supplemented them with medium 199 and 20% fetal bovine serum. Over time, these discs exhibit radial angiogenesis and comparison of initiation rates between control and test

261

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 58 n 5 72 per group n 5 141 for control

Initiation of angiogenesis (%), Day 12

Control Suramin (56 nM) Suramin (560 nM) NF 145 (56 nM) NF 145 (560 nM) NF 293 (56 nM) NF 293 (560 nM) NF 248 (56 nM)

48 39 3* 30* 1* 38 1* 36

* Denotes statistical significance (P , 0.05) by Cochran’s Q test. groups can be carried out. We tested three suramin analogues (NF 145, NF 248 and NF 293) in addition to suramin itself, at 56 and 560 nM concentrations (3 placentae tested). Only the 56 nM concentration of NF 248 was tested secondary to short supply. The percent of vein discs exhibiting angiogenesis in each group was determined on days 9 and 12 of culture. The three suramin analogues all inhibited angiogenesis in a dose-dependent fashion (see table) at day 12 with all analogs exhibiting near complete inhibition at 560nM concentration and the analog NF 145 demonstrating the greatest inhibitory effect, reaching significance at 56 nM concentration. Findings were similar at day 9. These findings show that suramin inhibits human angiogenesis at concentrations equivalent to those seen in vivo. It also demonstrates that suramin analogues with various structural alterations inhibit angiogenesis to an equal or greater degree than suramin, suggesting that these drugs may be as effective in vivo with potentially less toxicity.

PARALLEL SESSION II Cardiothoracic I 59. Adenosine Preconditioning Reduces Both Pre- and PostIschemic Arrhythmias in Human Myocardium. B. J. Pomerantz, M.D., K. Joo, Ph.D., B. D. Shames, M.D., J. C. Cleveland, Jr., M.D., A. Banerjee, Ph.D., and A. H. Harken, M.D. Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado. Supraventricular tachyarrhythmias occur in approximately 30% of patients following coronary artery bypass surgery (CABG). Ischemic preconditioning and adenosine preconditioning (Ado-PC) decrease post-ischemia/reperfusion (I/R) myocardial stunning, infarct size, and pharmacologically induced arrhythmias in both human and animal models. We hypothesized that adenosine preconditioning would decrease spontaneous pre- and post-ischemic atrial arrhythmias. Purpose. The purposes of this study were to determine the effect of in vivo and in vitro Ado-PC on atrial arrhythmias. Methods. Human atrial trabeculae were harvested from CABG patients, placed in organ baths and paced (1 Hz). Developed force (DF) was recorded during simulated I/R (30/45 min). Prior to I/R, trabeculae were treated with Ado (125 mM) for 5 min, or patients were treated with Ado (12 mg IV) 5 min prior to harvest of trabeculae. Contraction frequency (.4 Hz defined as atrial tachyarrhythmias) was calculated in all groups pre- and post-ischemia Results. Controls exhibited increased tachyarrhythmias post-ischemia. In vivo and in vitro Ado-PC suppressed both pre- and post-ischemic tachyarrhythmias. The effect of Ado-PC was evidenced by increased post I/R DF (in vitro 48 6 3%*, in vivo 46 6 2%*, and control 30 6 2% *P , 0.05 vs control) (see figure). Conclusions. Ado-PC suppresses the occur-

rence of pre- and post-ischemic arrhythmias in human myocardium. Ado-PC offers a potential means of reducing post-operative atrial arrhythmias.

60. Pinacidil Pretreatment Improves Ischemia Tolerance of Neonatal Hearts. J. Feng, M.D., Ph.D., H.-L. Li, M.D., and E. R. Rosenkranz, M.D. Division of CV Surgery, The Children’s Hospital of Buffalo, Buffalo, New York. Objectives. Blockade of the ATP-sensitive potassium (K ATP) channels with Glibenclamide diminishes ischemia tolerance of neonatal rabbit hearts. This study tests the hypothesis that exogenous K ATP channel activation with Pinacidil improves the neonatal heart’s tolerance to prolonged cardioplegic ischemia. Methods. 7-day-old rabbit hearts were perfused with Krebs-Henseleit buffer (KHB) on a Langendorff apparatus and underwent 90 min of normothermic ischemia with St Thomas’ cardioplegia (STCP) solution administered every 30 min. Six received no pretreatment. Six others were pretreated with a 10 min infusion of 1:M Pinacidil in KHB and then received STCP solution enriched with 1mM Pinacidil. Recovery of left ventricular developed pressure (LVDP) and coronary flow (CF) were measured after 60 min of KHB reperfusion. Results. As shown in the table, Pinacidil pretreatment significantly increased preischemia CF but did not effect LVDP. After reperfusion, Pinacidil treated hearts had significantly better recovery of LVDP and CF compared to untreated control hearts (see table: Preisch 5 Preischemia, Reperf 5 Reperfusion) Conclusions. Exogenous K ATP channel activation by Pinacidil pretreatment and cardioplegic enrichment significantly improved the neonatal rabbit heart’s tolerance to cardioplegic ischemia. This may be an important addition to myocardial protection during pediatric cardiac surgery.

TABLE—ABSTRACT 60 LVDP (mm Hg)

CF (ml/min)

Group

Preisch

Reperf

Preisch

Reperf

Control Pinacidil

78 6 4 76 6 2

16 6 3 27 6 3**

6.7 6 0.4 8.4 6 0.5*

3.9 6 0.6** 5.1 6 0.4* ,**

* P , 0.05 vs control; ** P , 0.01 vs preischemia. 61. A Technique to Protect Non-beating Donor Hearts from Ischemia–Reperfusion Injury. P. A. Fedalen, M.D., V. Piacentino III, B.A., D. A. Popowich, B.S., C. A. Fisher, B.A., J. P. Gaughan, Ph.D., V. Jeevanandam, M.D., B. I. Goldman, M.D., K. B. Margulies, M.D., S. Furukawa, M.D., and S. R. Houser, Ph.D. Division of Cardiothoracic Surgery and Cardiovascular Research Group, Temple University, Philadelphia, Pennsylvania.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

Ischemia–reperfusion injury renders non-beating donor hearts unsuitable for transplantation. We used pharmacological preconditioning followed by controlled reperfusion to protect non-beating hearts after 30 min of global hypoxic/ischemic (H/I) injury. Methods. Fifteen 100kg pigs were anesthetized, heparinized and maintained non-exsanguinated (chests closed) before heart explantation and 3 h of reperfusion on an ex-vivo working heart device. Functional recovery was assessed as LV systolic pressure (mmHg) vs preload (L/min). In Group I (N 5 6), non-ischemic control hearts were explanted and perfused with whole blood. In Group II (N 5 6), H/I and subsequent cardiac arrest were induced by discontinuation of ventilation, followed 30 min later by explantation and reperfusion with whole blood. In Group III (N 5 6), H/I hearts similar to Group II were precondi-

emia tolerance by initiating myocardial preconditioning pathways. This study tests the hypothesis that neonatal hearts are more tolerant to ischemia due to their greater expression and translocation of PKCe. Methods. Eighteen neonatal and 15 adult rabbit hearts were retrogradely perfused with Krebs-Henseleit buffer (KHB) on a Langendorff apparatus. Control hearts were perfused for 60 min without ischemia. Ischemic hearts (I) underwent 20 min of 37°C ischemia. Ischemia/reperfused hearts (I 1 R) underwent 20 min of 37°C ischemia and 10 min of KHB reperfusion. Recovery of LVDP (%preischemia) was measured. PKCe was measured in cytosol and membrane fractions by Western Immunoblotting (densitometer units). Results. Adult hearts showed significant depression of LVDP (54%)* compared to 97% recovery in neonates (see table). In the neonates, this was paralleled by an increase in PKCe expression in the cytosol and translocation of PKCe to the membrane fraction after ischemia and reperfusion. In contrast, PKCe remained unchanged in adult hearts. Conclusions. Better ischemia tolerance in neonates is due to their significantly greater expression of PKCe and its translocation to the membrane compared to adult hearts. 63. Reduction in the Level of Cardiac Cyclic GMP Worsens Myocardial Stunning. K. N. Patel, M.D., H. R. Weiss, Ph.D., and P. M. Scholz, M.D. Departments of Surgery and Physiology & Biophysics, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, New Brunswick, New Jersey.

tioned 10 min prior to discontinuation of ventilation with adenosine (40mg i.v.) and the Na 1/H 1 antiport inhibitor HOE 642 (400 mg i.v.) and reperfused with leukocyte-free blood containing free radical scavengers. Results. As shown in the figure, Groups I (F) & III (■) had similar functional parameters (I vs III at 1, 1.5, 2L/min preload, x 6 SD, ANOVA; p 5 ns) which were maintained for the entire 3hr reperfusion period. Also, a positive response to an increase in preload was demonstrated in these 2 groups (2 vs 1L/min preload for I & III; p , 0.05). Group II () hearts could not be resuscitated to perform work (II vs I & III; p , 0.05). Conclusions. Preconditioning and controlled reperfusion after 30 min of hypoxia/ischemia protect nonbeating hearts from ischemia-reperfusion injury. Protected hearts have functional capacities similar to non-ischemic controls. These results suggest that non-beating hearts can be protected and used for transplantation. 62. Protein Kinase Ce Upregulation Improves Ischemia Tolerance in Neonatal Hearts. H.-L. Li, M.D., J. Feng, M.D., Ph.D., and E. R. Rosenkranz, M.D. Division of CV Surgery, The Children’s Hospital of Buffalo, Buffalo, New York. Objective. Neonatal rabbit hearts are more tolerant to ischemia than adult hearts. Protein Kinase C (PKC) activation improves isch-

We tested the hypothesis that a reduction in the level of myocardial cyclic GMP would worsen the effects of myocardial stunning. Two groups of twelve anesthetized open-chest New Zealand white rabbits were utilized. Myocardial stunning was produced by two 15-min occlusions of the left anterior descending coronary artery followed by 15 min of reperfusion. Either control vehicle (saline11% DMSO) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 10 24 M, a guanylate cyclase inhibitor) was topically applied to the left ventricular surface of the rabbit hearts. Left ventricular and aortic pressures along with wall thickness parameters were determined. Coronary blood flow (microspheres) and O 2 extraction (microspectrophotometry) were used to determine myocardial O 2 consumption. Myocardial stunning was observed in the control group through an increased delay in onset of wall thickening (46.2 6 7.3 vs 76.6 6 17.5 ms). There was no significant effect of stunning on the rate of wall thickening (21.8 6 9.5 vs 18.1 6 3.4 mm/s) or O 2 consumption (stun 4.6 6 0.6, control 4.8 6 0.4 ml O 2/min/100g). After treatment with ODQ 10 24 M, both delay (43.9 6 9.6 vs 134.1 6 30.0 ms) and myocardial O 2 consumption (stun 5.9 6 0.6, control 5.9 6 0.7) increased significantly compared to no treatment. There was no significant change in the rate of wall thickening. We conclude that decreasing cyclic GMP worsens stunning by increasing delay in onset of wall thickening and increasing local O 2 costs in the stunned region. 64. Improved Biochemical Preservation of Lung Slices during Cold Storage. D. A. Bull, M.D., B. B. Reid, M.D., R. C. Connors, B.S., A. Albanil, B.A., J. C. Stringham, M.D., and S. V.

TABLE—ABSTRACT 62 Cytosol fraction

Membrane fraction

Group

Control

I

I1R

Control

I

I1R

Neo Adult

0.7 6 0.1 0.6 6 0.1

0.8 6 0.2 0.6 6 0.1

1.4 6 0.3* 0.3 6 0.1

0.6 6 0.1 0.8 6 0.1

1.2 6 0.2* 0.7 6 0.2

1.6 6 0.3** 0.4 6 0.1**

* P , 0.05 vs control; ** P , 0.01 vs control. Neo, neonatal.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Karwande, M.D. Division of Cardiothoracic Surgery, Department of Surgery, University of Utah Health Sciences Center, Salt Lake City, Utah. Development of lung preservation solutions typically requires whole organ models which are animal and labor intensive. These models rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual components difficult. We hypothesized that lung slices could be used to assess preservation of biochemical function during cold storage. Methods. Whole rat lungs were precision cut into slices with a thickness of 200 mm and preserved at 4°C in the following solutions: University of Wisconsin (UW), Euro-Collins (EC), low potassiumdextran (LPD), Kyoto (K), normal saline (NS), or a novel lung preservation solution (NPS) developed using this model. Lung biochemical function was assessed by ATP content (mmol ATP/mg wet wet) and capacity for protein synthesis (counts per minute (cpm)/mg protein) immediately following slicing (0 h), and at 6, 12, 18, and 24 h of cold storage. Six slices were assayed at each time point for each solution. The data were analyzed using analysis of variance and are presented as the mean 6 standard deviation. Results. For each solution, one pair of rat lungs provided sufficient slices to assess ATP content and protein synthesis over 24 h of cold storage. ATP content was significantly higher in the lung slices stored in the NPS com-

263

65. Increased Pulmonary Vascular Contraction to Serotonin after Cardiopulmonary Bypass: Role of Inducible Cyclooxygenase. K. Sato, M.D., J. Y. Li, M.S., C. Metais, M.D., C. Bianchi, M.D., Ph.D., and F. W. Sellke, M.D. Department of Surgery, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts. Pulmonary vascular resistance (PVR) is elevated after cardiopulmonary bypass (CPB). We examined if this altered pulmonary microvascular reactivity is due to altered expression of isoforms of nitric oxide synthase (NOS) or cyclooxygenase. Pigs (n 5 8) were heparinized and placed on total CPB for 90 min and then perfused off CPB for 90 min. Non-instrumented pigs (n 5 6) served as controls for vascular studies. Relaxation responses (% of precontraction) of microvessels (60 to 150 mm in diameter) were examined in vitro in a pressurized (20 mmHg) no-flow state with video-microscopic imaging. Expressions of constitutive (ec) and inducible (i) NOS and inducible (COX-2) and constitutive (COX-1) cyclooxygenases were examined with RT-PCR. PVR increased from 3166109 mmHg/L/min at baseline to 495612 at 60 min and 5656153 at 80 min after termination of CPB. In vitro, serotonin (5-HT) elicited a relaxation response (50 6 6%) in control microvessels precontracted by 20% of the baseline diameter. This response was not affected by the NOS inhibitor N Gnitro-L-arginine. After cardiopulmonary bypass, pulmonary microvessels contracted significantly to 5-HT (242 6 6%, p , 0.05 vs control, minus denotes additional contraction). This response was partially inhibited (210 6 12%) in the presence of the COX-2 inhibitor NS398, but was not significantly affected by the thromboxane synthase inhibitor U63557A (235 6 9%). Expressions of iNOS or COX-1 mRNA were not changed after CPB. Expression of COX-2 mRNA was increased slightly, but significantly by 30% (p , 0.02 vs control) while that of ecNOS was decreased by 50% (p , 0.05 vs control). In conclusion, PVR increases after CPB. This is associated with a hypercontractile response of isolated pulmonary microvessels to 5-HT that is mediated in part by the release of prostaglandins (but not thromboxane) due to increased expression of COX-2 and associated with decreased expression of ecNOS mRNA.

pared to all other solutions at each time point (p , 0.0001, Fig. 1). Protein synthesis was significantly higher in the lung slices stored in the NPS compared to all other solutions at 6, 12, and 18 h of

66. One Year Histologic Followup of Experimental Biologic Glued Coronary Artery Anastomoses. S. R. Gundry, M.D., C. W. Zuppan, M.A., K. S. Black, Ph.D.,* and L. L. Bailey, M.D. Division of Cardiothoracic Surgery, Loma Linda University School of Medicine, Loma Linda, California; and *Cryolife, Inc., Kennesaw, Georgia.

preservation (p , 0.05, Fig. 2). Conclusions. This rat lung slice model allows the rapid and efficient screening of lung preservation solutions and their components using quantifiable biochemical endpoints. Using this model, we have developed a novel solution which improves the biochemical preservation of lung slices during cold storage.

With the advent of minimally invasive coronary artery surgery, the construction of anastamoses (A) between the internal mammary artery (IMA) and coronary arteries becomes extremely difficult with conventional suturing techniques. Previous work in our laboratory has demonstrated the in-vitro effectiveness of biologic glue in coronary artery A and herein we report the one year followup of these A in an animal model. Goats weighing approximately 40kg were studied. Using a small left anterior thoracotomy, the IMA was taken down under direct vision. The pericardium was opened and the animal placed on cardiopulmonary bypass through traditional cannulation techniques. After cardioplegic arrest of the heart, the midportion of the left anterior descending coronary artery was opened. Two stay sutures were placed to orient the left IMA artery over the left anterior descending coronary artery. An incision was made on the back wall of the IMA, 1 cm from its distal end. Two modified angioplasty balloon catheters were placed through the distal IMA opening, one passing through the arteriotomy of the IMA and arteriotomy of the LAD and the other going up the IMA. These balloons were then inflated. The A was then covered with a thick layer of Bioglue, a proprietary combination of bovine albumin and gluteraldehyde and allowed to set for 2 min. The catheters were deflated and removed and the distal end of the IMA was sealed. Animals were weaned from coronary artery bypass and recovered. One animal was

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 67 % AST Leak with THBP (mM)** Group

% Weight change

Liver GSH mmol/g

0

1

2

4

Stv Chow

221 6 1* 8.1 6 1

5.0 6 0.3* 6.6 6 0.3

24 6 2 18 6 2

58 6 4 30 6 4

77 6 8 43 6 6

94 6 5 65 6 6

* P , 0.05 vs chow by t test; ** P , 0.001 Stv vs chow by ANOVA.

sacrificed at 24 h; the anastomosis was patent. The other two animals recovered for 10 months and one year respectively. At autopsy, both A were patent. Histology revealed no luminal irregularities at the anastomotic site and a persistence of Bioglue around the anastomosis with encapsulation of the Bioglue by a normal inflammatory reaction. This reaction did not extend into the luminal surface and no narrowing or aneurysm formation was identified. At one year followup in experimental animals, Bioglue has been shown to produce a patent and unobstructed IMA to coronary artery anastamosis without the use of sutures. This may represent the long sought for method of producing truly minimally invasive cardiac surgery.

PARALLEL SESSION II Metabolism, Endocrinology, & Nutrition 67. Starvation Increases Hepatocyte Susceptibility to Oxidant Injury. Y. P. Hsu, M.D., W. Wang, M.D., Ph.D., E. A. Chambers, M.S., J. D. Rounds, B.S., D. O. Jacobs, M.D., and M. K. Robinson, M.D. Department of Surgery, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts. Malnourished animals which become septic have enhanced hepatic oxygen free radical release. To determine if hepatocytes from starved animals have an altered susceptibility to oxidant stress, male Wistar rats (200 –211 g) were randomly assigned to receive water but no food (n 5 6, “Stv”) or rat chow and water (n 5 5, “Chow”) ad libitum. After 3 days, liver samples were taken to determine glutathione (GSH, a potent antioxidant) content. Hepatocytes were also isolated from each rat and incubated in wells containing RPMI 1640 at a density of 1310 5 cells/ml for 4 h. The cells were then plated for an additional 5 h in fresh RPMI with the oxidant t-butyl hydroperoxide (TBHP) and % leakage of aspartate aminotransferase (AST) was calculated. Results are shown as mean 6 SEM (see table). Starved rats had significant weight loss and a 25% reduction in liver GSH compared to chow animals. There was a significant, dosedependent increase in AST% leakage when hepatocytes from starved animals were incubated with TBHP compared to hepatocytes from chow rats. We conclude that hepatocytes from starved animals are more vulnerable to oxidant stress and that the altered hepatocyte susceptibility is associated with depleted liver oxidant stores. Thus, poor nutrition is associated with accelerated hepatic oxygen free radical release and impaired hepatocyte defense against oxidant stress in septic animals. This may partially explain why there is increased risk of organ failure and death in malnourished patients who become systemically infected. 68. Hyperventilation Increases Muscle Protein Synthesis in Critically Ill Trauma Patients. J. A. Vosswinkel, M.D., C. E. M. Brathwaite, M.D., T. R. Smith, M.D., J. M. Ferber, M.D., G. A. Casella, B.S., and P. J. Garlick, Ph.D. Department of Surgery, State University of New York Health Science Center at Stony Brook, Stony Brook, New York.

Background. Critically ill trauma patients are often in negative nitrogen balance and demonstrate advanced muscle protein wasting, which is in part due to a decrease in muscle protein synthesis. Animal studies suggest that alkalosis might enhance protein synthesis. The purpose of the present study is to determine whether protein synthesis is increased in trauma patients who have a respiratory alkalosis from hyperventilation. Methods. Trauma patients in the Intensive Care Unit (n 5 8) who were treated with hyperventilation for elevated intracranial pressures were enrolled. Muscle protein synthesis rates were determined in vivo using the flooding method with L-[ 2H 5]phenylalanine. Measurements were performed twice on each patient within a 36 h period, first during hyperventilation and then after hyperventilation was discontinued. Hemoglobin oxygen saturation was maintained above 95% for all measurements. Results. Protein synthesis in muscle was 1.38 6 0.11%/day during hyperventilation (pH 7.50 6 0.02, pCO 2 27.3 6 1.0) and 0.93 6 0.15%/day after respiratory parameters were normalized (pH 7.39 6 0.01, pCO 2 39.4 6 1.5). The synthesis rate was significantly higher (p , 0.01; paired t-test), 0.46 6 0.13%/day (32.6%), at the time of hyperventilation. Conclusion. Muscle protein synthesis is elevated during hyperventilation in critically ill trauma patients. These data suggest that this ventilatory therapeutic strategy may have a role in mitigating the negative nitrogen balance and muscle protein wasting that can impair the recovery of these patients. 69. Glutamine Starvation-Induced Cell Cycle Arrest Is Associated with the Induction of p21 Cip1 and Does Not Require Caspase Activation. H. T. Papaconstantinou, M.D., M. R. Hellmich, Ph.D., C. M. Townsend Jr., M.D., and T. C. Ko, M.D. Department of Surgery, UTMB, Galveston, Texas. Gln starvation induces both apoptosis and G1 cell cycle arrest in intestinal epithelial cells. We have shown that Gln starvationinduced apoptosis is mediated, in part, by caspase 3 activation and is blocked by ZVAD, a general caspase inhibitor. Caspase 3 can inactivate DNA replication factor C, and may be involved in Gln starvation-induced cell cycle arrest. p21 Cip1 is a cyclin-dependent kinase inhibitor, which is activated during DNA damage-associated G1-arrest. The purpose of this study was to determine whether G1 cell cycle arrest in Gln-deprived intestinal epithelial cells requires caspase activation and is associated with the induction of p21 Cip1. Methods. Rat intestinal epithelial cells (RIE-1) were incubated for 24 h 6 1 mM Gln and 6 80 mM ZVAD. p21 Cip1 levels were analyzed by Western blot. Caspase 3 activation was determined by fluorogenic substrate assay. Phase specific cell cycle distribution was determined by flow cytometric analysis. Results. Gln starvation resulted in an increase in p21 Cip1 expression (Fig. 1). Gln-starved cells had an 85% decrease in S phase population and a 30% increase in G1 phase population (Fig. 2). ZVAD treatment did not affect cell cycle distribution in Gln-starved cells (Fig. 2), but completely blocked activation of caspase 3 (not shown). Conclusions. We have demonstrated that Gln starvation results in the induction of p21 Cip1, which is associated with G1 cell cycle arrest. Furthermore, cell cycle arrest occurs independent of caspase activation suggesting that caspases are not required for Gln starvation-induced cell cycle arrest in intestinal epithelial cells.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

265

conclusion, the mSSTR5 KO model has been successfully achieved through gene ablation, and micro-manipulation techniques of ES cells and mouse embryos. The absence of mSSTR5 gene in these mice was confirmed by Southern blot and RT-PCR analysis. While no alterations of glucose homeostasis were seen in this model so far, further islet hormone measurements and hormone secretion studies are needed. 71. Parathyroid Induced Angiogenesis of Human Microvessels. W. B. Carter, M.D., and M. D. Ward, B.S. Department of Surgery, Eastern Virginia Medical School, Norfolk, Virginia.

70. Development of a Mouse Somatostatin Receptor 5 (mSSTR5) Gene Knockout Model. S. Moldovan, M.D., F. J. DeMayo, Ph.D., and F. C. Brunicardi, M.D. Departments of Surgery and Cell Biology, Baylor College of Medicine, Houston, Texas. We have recently cloned the mSSTR5 gene. The purpose of this study was to determine if genetic knockout (KO) of mSSTR5, using gene targeting techniques in embryonic stem cells (ES cells), results in alterations of glucose homeostasis. Gene KO. A targeting construction was developed using DNA fragments from the SSTR5 and the Neomycin resistance genes. The DNA was introduced into AB 2.2 ES cells by electroporation and heterozygous (1/2) cells for mSSTR5 gene were generated through homologous recombination. 1/2 cells were injected into C57 mouse blastocysts, which were then transferred into the uterus of recipient females to generate chimeric mice. Chimeras were crossed with wild type C57 mice to generate heterozygous mice (1/2 SSTR5), and these were then bred to each other to generate knockout mice (2/2 SSTR5). 1/2 and 2/2 SSTR5 mice were genotyped by Southern blot analysis and the absence of SSTR5 mRNA in the knockout mice was demonstrated by RT-PCR. Phenotype analysis. Body weight measurement (n 5 8), basal insulin levels (n 5 10), IPGTT and OGTT (1g/kg bw glucose, n 5 6) were performed in 4-6 month old wild type and KO mice (see table). In

TABLE—ABSTRACT 70

Weight Basal insulin Mean glucose at 10 min (IPGTT) Mean glucose at 10 min (OGTT)

Wild type mice

KO mice

24.14 6 1.6 g 81.7 6 16.9 pM 175.4 6 8.3 mg%

26.94 6 0.5 g 69.1 6 13.5 pM 166 6 13.7 mg%

152.25 6 7.1 mg%

140 6 20.1 mg%

Angiogenesis is the development of new blood vessels from preexisting vessels, a complex process involving endothelial cell proliferation and migration, with microvessel elongation and branching. The mechanism of angiogenesis is poorly understood, but is influenced by matrix, vascular pericytes and numerous growth factors, including VEGF. In contrast to rat microvessels, intact human microvessels will not spontaneously undergo angiogenesis, nor have they been induced to undergo angiogenesis in vitro. We have identified VEGF mRNA production in human parathyroid tissue. Using a 3 dimensional assay, we tested the ability of human parathyroid cells to induce angiogenesis of intact human microvessels, and further tested to determine if the mechanism is parathyroid production of VEGF. Telomerase-transformed, normal human parathyroid cells were cocultured with freshly isolated human fat microvessels in a 3 dimensional collagen I matrix. After 14 days in culture (DMEM 1 10% FBS), the microvessels were evaluated for elongation and branching by phase contrast and fluorescent microscopy, and microvessel density was determined by image analysis software. In some cocultures, parathyroid cells were treated with flt-1 soluble receptor fusion protein to bind VEGF. Other microvessels were treated with VEGF 165 alone. Parathyroid cells induced angiogenesis in human microvessels, demonstrated by elongation and branching. Control microvessels were completely static, as were microvessels treated with VEGF 165 alone. Cocultures treated with flt-1 soluble receptor (0.01 and 0.1 mg/ml) did not significantly reduce microvessel density. We conclude that human parathyroid cells can induce angiogenesis of human microvessels in vitro. The mechanism may not require VEGF participation, but clearly involves other parathyroid produced factors. 72. A Single Intramuscular Injection of Erythropoietin Gene Using Adeno-Associated Viral Vector Provides Sustained Elevation of Cynomolgus Monkey Hematocrit. S. Rudich, M.D., Ph.D., S. Zhou, Ph.D., J. Escobedo, Ph.D., R. Perez, M.D., and W. Manning, Ph.D. Department of Surgery, UC Davis Medical Center, Sacramento, California; and Chiron Corporation, Emeryville, California. Chronic anemia is a significant problem in many disease states. This study investigates the dose response profiles of a single intramuscular (im) injection of a recombinant adeno-associated virus vector (rAAV) containing the erythropoietin (Epo) gene with the goal to achieve a sustained elevation of hematocrit (hct). At each specified viral load (total particles injected), a single im injection of rAAV-cmEpo was placed into the tibialis muscle of cm. The biological effect of Epo gene expression was monitored by determining the hct levels and circulating hormone level by ELISA. Antibody to the AAV capsid protein was also measured (see figure). Differences between hct levels during weeks 3–10 were significantly different (p , 0.001). At 6 weeks, Epo levels were nearly 50 fold greater than baseline in the group of animals injected with the highest load of virus (p , 0.001). We conclude that dose responses to rAAV-Epo are achievable via a one-time single im injection. Significant increases in Epo are noted within one week of injection and are sustained for a prolonged period of time.

266

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS activity is a post-translational effect. PMA decreased GLN transport by 30 –50% in all cell formats tested. Conclusions. 3-D tumor spheroid formats offer a clinically relevant model for studying size-dependent nutrient uptake in the growing tumor. Although transporter regulation remained unchanged, the increase in GLN uptake in large MTS suggests that alterations in the tumor microenvironment affect nutrient uptake. Further studies in this spheroid model will define the relationship between transport and progressive tumor growth.

73. Alterations in Amino Acid Transport in a ThreeDimensional Tumor Model. T. Pawlik, M.D., T. Sweeney, M.D., W. Souba, M.D., and B. Bode, Ph.D. Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts. Isolated cultured cancer cells are frequently used to study tumor metabolism, but there is a concern that they may not reflect the growing tumor in vivo. Because tumor cells grown in a 3-D configuration more closely resemble the in vivo environment, we examined, for the first time, amino acid uptake and regulation in a multicellular 3-D tumor spheroid (MTS) format. Methods. After culturing SK-Hep hepatoma cells as single cells and spheroids, the transport of radiolabeled glutamine (GLN), arginine (Arg), leucine (Leu), and MeAIB was measured in single cell suspensions and MTS (0.50-0.60 mm) for 30 s by rapid filtration. To determine the effect of MTS size on amino acid uptake, L- 3H-GLN was also measured in larger spheroids (0.85-1.5mm). Transporter regulation was examined after 1/2 exposure to the phorbol ester PMA (10 27M), a protein kinase C activator. ATB° GLN transporter mRNA was assessed by Northern blotting in isolated cells, and in small and large MTS. Results. Small MTS formed by one week, while large MTS developed at 2-3 weeks. Histologic examination of the MTS revealed concentric arrangements of cell populations with proliferative cells at the periphery and a necrotic core in large MTS. In the smaller MTS population, arginine transport rates were 35% higher than those in single cell suspensions (Fig. 1). While there was no difference in GLN transport in small MTS, uptake in MTS $ 1mm exhibited a 40-53% increase as compared to single cells. No difference in ATB° mRNA levels was observed (Fig. 2), suggesting that the increase in GLN transporter

74. Integrin-Dependent Signaling Is Enabled during Keratinocyte Activation. L. T. Kim, M.D., J. Wu, M.D., Ph.D., C. Bier-Laning, M.D., B. T. Dollar, B.S., and R. H. Turnage, M.D. Surgical Service, Veterans Affairs North Texas Health Care System, and University of Texas Southwestern Medical Center, Dallas, Texas. During wound healing keratinocytes undergo a process called “activation” that enables the cells to spread and migrate on wound matrix molecules. Resting keratinocytes exhibit integrin a 2b 1 collagen receptors but are unable to use them to spread or migrate while activated cells can. Focal adhesion kinase (FAK) is a key component of integrinmediated intracellular signaling. We hypothesize that induction of FAK accompanies keratinocyte activation. Keratinocytes were harvested from normal human skin. Freshly isolated, un-activated (Un-act) cells were compared to activated (Act) cells from culture. In other experiments growing colonies of keratinocytes (Col) were compared to activated cells replated for 1 hour on collagen (Repl). Indirect immunofluorescence was performed using a polyclonal anti-FAK antibody. FAK content was also assessed by Western blotting. Tyrosine phosphorylation of FAK was assessed by Western blotting with a monoclonal antiphosphotyrosine (P-T) antibody. FAK was not detectable by immunostaining or Western blotting in freshly isolated cells. In contrast FAK was detected in growing colonies. Immunostaining showed a diffuse, cytoplasmic pattern. In activated cells re-plated on collagen FAK became concentrated in focal adhesions. Lysates from re-plated cells showed increased tyrosine phosphorylation of FAK (see figure). In summary FAK is induced with KC activation and undergoes redistribution to focal adhesions when cells are plated on b 1 integrin ligands. This

redistribution is accompanied by tyrosine phosphorylation of FAK. These data suggest that induction of FAK and subsequent FAK-induced signaling, rather than changes in integrins themselves, may be responsible for changes in integrin function in activated KC during reepithelialization.

PARALLEL SESSION III Pediatrics 75. Mesenchymal–Epithelial Conversion in Embryonic Mouse Pancreas: Implications for Lineage Selection. A. S. Kadison, M.D., T. S. Maldonado, M.D., C. A. Crisera, M.D., J. B. Grau, M.D., S. L. Alkasab, B.A., M. Li, M.S., M. T. Longaker, M.D., and G. K. Gittes, M.D. Department of Surgery, NYU Medical Center, New York, New York. The pancreas begins to form as an evagination of the foregut at embryonic (E) day 9.5 in the mouse. The epithelium subsequently branches and differentiates into exocrine and endocrine structures.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS We now provide indirect evidence suggesting that the embryonic pancreatic mesenchyme may contribute cells to the epithelium. Such a mesenchyme-to-epithelial conversion has previously been shown to occur in the kidney and testes. Pancreases were microdissected from mouse embryos at E9.5-16.5. RT-PCR was performed for the duct markers carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR) on pooled whole pancreases at E9.5, and pooled isolated epithelia and mesenchyme at E10.5 and E11.5. Immunohistochemistry (IHC) for CAII, glucagon and vimentin and in situ hybridization for CAII and CFTR were performed on whole pancreases at E11.5-16.5. RT PCR revealed CAII and CFTR expression in the mesenchyme at E10.5 and E11.5, with sparing of the epithelium. In situ hybridization showed expression of CAII and CFTR only in the mesenchyme at E12.5, but in the newly forming ductal epithelium at E16.5. IHC on whole pancreas at E11.5 revealed co-expression of CAII, glucagon and vimentin in clusters of cells at the interface between the epithelium and mesenchyme. At E16.5, CAII is present in the ducts by IHC and is co-expressed with glucagon in cells of the peri-ductal region. These data represent the first evidence of cells that co-express exocrine, endocrine and mesenchymal markers. We suspect that these cells may represent pluripotential cells that are in transition from mesenchymal cells to epithelial cells. Cells at E16.5 that co-express CAII and glucagon may represent more differentiated pluripotential cells that can further differentiate into exocrine or endocrine cells. In addition, evidence showing duct marker gene expression present only in the mesenchyme early on, and then in the ductal epithelium by E16.5, further supports the hypothesis that there is a mesenchymal– epithelial conversion in the developing pancreas. These data raise the possibility that the long sought-after pancreatic stem cell may actually reside in the mesenchyme.

76. AAV Mediated Gene Transfer to the Developing Murine Fetus: Organ Tropism and Expression of a Reporter Gene. G. S. Lipshutz, M.D., and K. M. L. Gaensler, M.D. Departments of Surgery and Medicine, University of California, San Francisco, California. Recombinant adeno-associated viruses (rAAV) are promising gene delivery vectors as they direct long-term gene expression with little toxicity or immunogenicity. Our goal was to assess the efficacy and safety of these vectors following delivery in the developing fetus. We have determined the survival rates of pups, and the level of luciferase gene expression in neonatal tissues, following in utero intraperitoneal, delivery of a rAAV-luciferase (rAAV-luc) vector. Methods. rAAV-luc was constructed by deleting the rep/cap genes from plasmid SSV9 and inserting an EcoRI fragment containing the EF1a promoter, a multi-cloning site, and the bovine growth hormone polyadenylation signal. Subsequently, an NcoI-XbaI fragment containing the luciferase reporter gene was cloned into the SmaI site of the multicloning region. The activity of the EF1a-luciferase cassette was verified by transfection. Then the rAAV was produced by triple transfection in 293 cells with adenoviral and AAV helper plasmids. rAAV-luc was isolated, concentrated, and titered. Three pregnant female CD 1 mice were anesthetized at day 15 of gestation (E15) and 1310 10 rAAV-luc genomes delivered intraperitoneally (IP) to all fetuses (n 5 30). At birth, pups were sacrificed and their tissues were analyzed for luciferase activity (normalized and expressed as relative light units/mg tissue protein). Results. All females delivered at term with 90% of pups liveborn. Highest levels of luciferase were observed in the liver (72548.1 RLU/mg) although significant luciferase activity was observed in multiple other tissues: brain (168.7 RLU/mg), heart (4044.8 RLU/mg), intestine (907.7 RLU/mg), lung (441.4 RLU/mg), kidney (380.6 RLU/mg), and spleen (444.0 RLU/mg) (control level 0.01 RLU/mg). Conclusions. Transuterine, IP injection of rAAV vectors at E15 is tolerated well and produces high level gene expression in many tissues. Future studies will establish the

267

duration of gene expression following in utero delivery and the feasibility of correcting congenital disorders due to single gene defects. 77. Ontogeny of IGF-1 in a Rabbit Model of Growth Retardation. A. Thakur, M.D., M. Sase, M.D., J. J. Lee, M.D., V. Thakur, M.D., and T. L. Buchmiller, M.D. UCLA School of Medicine/ Harbor UCLA Medical Center, Los Angeles, California. Many cases of intrauterine growth retardation (IUGR) result from placental insufficiency, but the molecular signals controlling IUGR are unknown. Insulin-like growth factor-1 (IGF-1) is a potent mitogen for fetal tissues and is lowered in the serum of human IUGR infants. To study IGF-1 expression before birth, a fetal rabbit model of naturally occurring IUGR was used. Four fetal rabbit pairs (normal vs growth-retarded) were harvested on days 23, 25, 27, 29, and 31 of a 31-day gestation. Fetal weights were recorded; fetal serum, amniotic fluid, liver, kidney, proximal, middle, and distal small intestine (SI) were collected. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine IGF-1/beta-actin mRNA densitometric band ratio in all tissues. Radioimmunoassay (RIA) was done to determine IGF-1 protein levels in serum and amniotic fluid. Statistical analysis was performed using ANOVA and the paired Student’s t-test. Weights were decreased in IUGR fetuses at all time points (p , 0.05). Liver, proximal, and distal SI IGF-1 mRNA decreased during late gestation (p , 0.01). Kidney IGF-1 RNA increased throughout late gestation (p , 0.01). Compared to normals, IUGR fetuses had a trend to decreased IGF-1mRNA in the kidney, liver and SI at all time points, reaching significance in the liver at day 27 (p 5 0.002). Serum IGF-1 decreased throughout gestation in all fetuses (p , 0.05). Compared to normals, IUGR fetuses had lower serum IGF-1 at all time points, reaching significance at day 27 (p 5 0.02). Amniotic fluid IGF-1 was lowered in IUGR fetuses compared to normals, though not quite reaching significance. Serum IGF-1 correlates with differential somatic growth in the normal and IUGR fetus (p 5 0.03 and 0.17). Varied tissues express IGF-1 mRNA during late gestation in a rabbit model of IUGR. The kidney and liver continue to express IGF-1 mRNA at birth. Compared to normals, growth-retarded fetal rabbits have depressed liver, kidney, and intestinal expression of IGF-1 mRNA and lower serum and amniotic fluid IGF-1 protein. Serum IGF-1 levels correlate with fetal somatic growth. This fetal rabbit model is further supported as a correlate to study human IUGR. Investigations into the prenatal manipulation of IGF-1 expression are supported as a potential prenatal treatment of IUGR. 78. Regional Differentiation of Dura Mater Determines Fate of Cranial Sutures. J. A. Greenwald, M.D., B. J. Mehrara, M.D., J. A. Spector, M.D., D. S. Steinbrech, M.D., P. B. Saadeh, M.D., G. K. Gittes, M.D., and M. T. Longaker, M.D. Department of Surgery, NYU Medical Center, New York, New York. Introduction. Craniosynostosis, the premature fusion of cranial sutures, occurs with a frequency of 1 in 2,000 and presents a substantial biomedical burden. Studies of murine cranial suture fusion have implicated the dura mater (DM) in the regulation of sutural fate with regional differentiation of the underlying DM guiding sutural fusion (posterior frontal; PF) or patency (sagittal; SAG). The purpose of this study was to further assess the regional differentiation of cranial suture-derived dural cells both in vivo and in vitro. Methods. DM underlying the PF and SAG sutures were harvested from 6 day old rats (prior to evidence of suture fusion) and plated in 100 mm dishes. First passage cells were analyzed. TGF-b1 and FGF-2 immunoassays were used to assess the expression of these cytokines by isolated dural cell cultures. To assess mRNA expression of osteogenic growth factors (GFs) and extracellular matrix (ECM) molecules, Northern blot analysis was performed on snap-frozen dural tissues isolated from the PF and SAG sutures of 6-day old rats

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using radioactive probes against TGF-b1, collagen I, alkaline phosphatase (AP) and osteocalcin (OC). Statistical analysis was performed using Student’s t-test with p#0.05 considered significant. Results. Analysis of FGF-2 and TGF-b1 protein expression by isolated dural cell cultures revealed a greater than 43 increase in both proteins in PF-derived dural cells vs SAG-derived dural cells (p , 0.01). Similarly, northern analysis for TGF-b1, collagen I, AP and OC revealed an approximately two-fold increase in mRNA expression for all genes in the PF vs SAG dural tissues. Conclusions. We have demonstrated differential protein and mRNA expression of growth factors and ECM molecules that are critical for successful osteogenesis from PF and SAG dural tissues. These data support the hypothesis that the DM underlying fusing (PF) and patent (SAG) sutures is regionally differentiated and that this differentiation precedes active sutural fusion. The high level of expression of osteogenic GFs as well as bone ECM molecules by the PF dura mater is critical for the programmed osseous obliteration of the overlying PF suture. 79. Mediators of Oxygen Induced Vasodilation in Isolated Third Generation Pulmonary Arterioles from Fetal Rats. J. R. Gosche, M.D., Ph.D., Z. Vukcevic, M.D., and C. P. Coppola, M.D. Yale University School of Medicine, New Haven, Connecticut Purpose. We have observed that third generation pulmonary arterioles from rats with nitrofen induced CDH are poorly responsive to oxygen. Our goal was to identify mediators of oxygen induced vasodilation in isolated third generation pulmonary arterioles from normal (control) rats. Methods. Third generation arterioles were isolated from the right lung of term fetal rats. Arterioles were cannulated and pressurized at a pressure that caused maximal constriction to K 1. Arterioles were initially suffused with “hypoxic” K 1 solution (PaO 2 20-40mmHg), and then with “normoxic” K 1 solution (PaO 2 100-140mmHg) for 60 min. Responses were measured in control vessels, or in the presence of 0.1 mM L v-nitro-L-arginine, or 28 mM indomethacin, or following endothelial removal. Results are expressed as % changes at 60 min. from the preconstricted baseline diameter (mean 6 SEM). Differences were detected by ANOVA and SNK tests. Results. Control arterioles gradually dilated over the period of observation to 73 6 10% at 60 min. Responses were not significantly changed (p . 0.05) by NOS blockade (66 6 10%) or endothelial removal (64 6 4%). In contrast, indomethacin significantly (p , 0.05) blunted the vasodilation that was observed at 60 min (7 6 18%). Conclusion. Our results suggest that cyclooxygenase products contribute significantly to oxygen induced vasodilation in third generation pulmonary arterioles from term fetal rats. A defect in this mechanism may contribute to blunted vasodilation in the pulmonary arterioles of rats with nitrofen induced CDH. 80. Kupffer Cell Inactivation Delays Repair in Rat Model of Reversible Biliary Obstruction. K. K. Roggin, M.D., E. Papa, B.S., A. G. Kurkchubasche, M.D., and T. F. Tracy, Jr., M.D. Department of Surgery, Brown University School of Medicine, Providence, Rhode Island. During cholestatic liver injury, Kupffer cells (KC) modulate cell proliferation and matrix deposition. The role of KC in the restoration of cell architecture and matrix metabolism during repair following chronic cholestatic liver injury is unknown. To determine the effect of KC inactivation, adult male Sprague-Dawley rats underwent bile duct obstruction (BDO) for 5 days followed by reversal of the obstruction. Saline (control) and gadolinium chloride (10mg/kg) were administered one day prior to BDO and one day prior to reversal, to inactivate KC both during injury and repair. Serum bilirubin and quantitative cell morphometry were compared to verify the reversibility of the model. Collagen content was measured in trichrome stained liver sections using NIH imaging software. Results. Reversibility of the obstruction was verified (see table). H&E sections of livers from gadolinium treated animals, at 4 and 7 days post-

TABLE—ABSTRACT 80 Group

Bilirubin (mg/dL)

Collagen/mm 2, saline

Collagen/mm 2, Gado

Sham Reversal Repair d7

0.15 6 0.2 8.86 6 4.1* 0.42 6 0.5

1.23 6 0.10 7.03 6 0.81 2.80 6 0.04

0.94 6 0.34 7.21 6 5.62 5.17 6 0.03**

Note. Results expressed as means 6 SD, analyzed by Student paired t test. * P , 0.05 compared to sham; ** P , 0.05 compared to saline group.

reversal, exhibited persistent bile duct proliferation, matrix deposition, and inflammation. Conclusion. These results demonstrate that inactivation of resident hepatic macrophages during liver repair impairs collagen metabolism, inhibits the resolution of fibrosis and allows the persistence of inflammatory cell infiltrates in the portal areas. This is the first evidence of positive cellular and matrix control by KCs in liver repair. 81. Is Admission for Pediatric Trauma Patients with Isolated Head Injury and Normal Head CT Scan Necessary? B. Benedetto, M.D., I. A. Munshi, M.D., D. E. Biggs, R.N., M.S., J. L. McCall, M.S., K. Smith, R.N., E. Vitorino, P. Letourneau, B.S.N., R. Courtney, M.D., and R. Wait, M.D., Ph.D. Department of Surgery, Division of Trauma, Tufts University School of Medicine, Baystate Medical Center, Springfield, Massachusetts. The purpose of the study was to assess the need for hospitalization of children with Glasgow Coma Score (GCS) of 14-15 after mild head injury with brief loss of consciousness (LOC), normal neurological exam, and normal head CT scan. Methods. Retrospective chart review of children aged 4 to 12 years with documented LOC after isolated head injury at a regional Level 1 Trauma Center between 1992-1998. All patients had a GCS of 14-15 on initial ED evaluation and underwent physical and neurological examinations. Subsequently, patients either had a head CT scan and were admitted for observation, had a head CT scan and were discharged home, or were admitted for observation without a head CT scan. All patients received follow-up by a pediatric surgeon. Results: 60 patients with a mean age of 7.1 years were evaluated. 3 patients had abnormalities on initial head CT scan. These were frontal contusion, subarachnoid hemorrhage, and occipital skull fracture (see table). None of the patients had any neurological problems requiring surgical intervention on follow-up (95% CI 5 0-6%). For the 42 patients without neurologic findings and a normal head CT scan, a cost savings of $83,197 would be realized if they were discharged home with outpatient follow-up. Conclusion. Hospitalization in pediatric trauma

TABLE—ABSTRACT 81 Abnormal head CT, n 5 3 Mechanism MVC Fall Hit by car Hit by object Disposition Admit Discharge Avg LOS (days)

Normal head CT, n 5 50

Observation only, n 5 7

2 1 0 0

6 25 12 7

2 4 1 0

3 0 3.0

42 8 2.2

7 0 2.9

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TABLE—ABSTRACT 83

patients with isolated head injury, no neurologic findings, and a normal head CT scan may not be necessary. Group 82. The Use of Extracellular Matrix for Esophageal Repair. M. K. Chen, M.D.,* S. Meurling, M.D.,† and S. F. Badylak, D.V.M., Ph.D., M.D., ‡ *Department of Surgery, UMDNJ, Camden, New Jersey; †Department of Pediatric Surgery, University Hospital, Uppsala, Sweden; and ‡Purdue University, West Lafayette, Indiana. Introduction. Techniques for esophageal repair or replacement are imperfect. Advances in biomaterial science may provide a better solution. Small intestinal submucosa (SIS) is a resorbable, naturally occurring matrix scaffold derived from pigs. It has been used for regeneration of various other organs. We propose to use this extracellular matrix material as a scaffold for esophageal regeneration in a dog model. Methods. Thirteen healthy adult dogs were used. Two had a 6-cm segment removed and replaced with pre-constructed tube of SIS and 11 had a portion (approximately 40%) of the wall removed and patched with SIS devices. The animals were sacrificed at times ranging from 1 week to 1 1/2 year. Measured endpoints included macroscopic and microscopic morphology. Results. The 2 dogs with full circumference defects survived for 15 and 45 days. Both had significant morbidity due to stricture at the graft site. One of 11 dogs with a patch repair was euthanized due to leakage. The other 10 dogs were clinically normal and tolerated a regular diet up to the time of sacrifice. The perimeter of the patch was marked with silk sutures and this area was markedly smaller at necropsy. However, the diameter of the esophagus was not narrowed and was isodiametric with adjacent normal esophagus. The graft site was slightly firmer. Histologically, there was complete epithelialization and replacement of the submucosal tissues by connective tissue and smooth muscle. The further out from placement of the SIS scaffold, the better the organization of the regenerated esophageal tissue Conclusions. Extracellular matrix such as small intestinal submucosa may be used as a scaffold to repair defects in the esophagus. The regenerated tissue is similar to native esophagus on histologic examination and the functionality is excellent.

PARALLEL SESSION III Gastrointestinal II 83. Preoperative Radiation, Colonic Healing, and Transforming Growth Factor-b 1 (TGF-b 1) Expression. S. G. Fukuchi, M.D., J. L. Seeburger, Ph.D., G. Parquet, M.D., A. K. Devabhaktuni, Ph.D., C. T. Miyamoto, M.D., and R. H. Rolandelli, M.D. Department of Surgery and Radiation Oncology, MCP Hahnemann and Temple University, Philadelphia, Pennsylvania. Adjuvant treatment modalities, including radiation therapy (XRT), have a central role in the treatment of colorectal cancer. Since TGF-b 1 is an important mediator of colonic healing, this study investigates the effects of preoperative XRT on mechanical parameters of colonic healing and the expression of TGF-b 1 . Methods. Forty-eight male Sprague-Dawley rats (250 –275 g) were randomized to receive either XRT or sham treatment (NX). XRT consisted of four fractions of 7.7 Gy to the abdomen using 6 MeV photons with 1.5 cm bolus on the anterior fields. Dose fractionation and total dose was based on a biological equivalent standard fractionation dose of 46 Gy (2 Gy/fraction), which is comparable to preoperative conventional human therapy for colonic carcinoma. XRT or NX sessions were performed 14, 10, 7, and 3 days prior to transection of the descending colon with primary anastomosis. On postoperative days (PODs) 3, 5, and 7, in-situ bursting pressure (BP) and bursting energy (BE) determinations

POD 3

POD 5

POD 7

NX 66.6 6 10.4 114.8 6 4.6 137.1 6 8.0 XRT 73.6 6 11.9 108.0 6 7.5 122.6 6 20.7 BE b NX 958.5 6 443.9 2626.9 6 362.4 2568.4 6 543.7 XRT 1233.9 6 486.3 2305.9 6 443.9 3002.0 6 411.0 NX-AC 0.51 6 0.13 0.36 6 0.20 0.39 6 0.13 TGF-b1 XRT-AC 0.47 6 0.09 0.50 6 0.17 0.43 6 0.08 exp. NX-NC 0.48 6 0.09 0.34 6 0.09 0.38 6 0.10 XRT-NC 0.80 6 0.18 0.66 6 0.16 0.43 6 0.08 BP a

Note. Data: mean 6 SEM.

a

mm Hg.

b

mm Hg 3 s.

were obtained. Expression of TGF-b 1 normalized, to a constitutive gene, was determined by semi-quantitative RT-PCR in anastomotic (AC) and non-operated colon (NC). Data were analyzed by two-way ANOVA; students t-test used for post-hoc comparisons. Results. (See table.) Conclusions. Both groups, XRT and NX, exhibited a trend towards increases in BP and BE through the postoperative period. However, these differences were not statistically significant. No differences in BP and BE were detected between XRT and NX. The increased expression of TGF-b 1 in XRT animals suggest an effective compensatory mechanism that nullifies the effect of XRT on colonic healing. 84. Lysophosphatidic Acid (LPA) Stimulates Intestinal Restitution via Cytoskeletal Activation and Remodeling. O. J. Hines, M.D., J. Chu, and D. W. McFadden. Department of Surgery, UCLA School of Medicine, Los Angeles, California. Superficial injury to the gastrointestinal tract is followed by rapid repair and closure of the mucosal defect through restitution. The application of LPA, a product of activated platelets, to an epithelial wound in vitro increases migration 3 fold. The purpose of this study was to evaluate the activity of LPA on intestinal cell cytoskeletal structure. Methods. IEC-6 cells were grown in serum-free media and exposed to 100 nM LPA or 100 nM LPA and 100 ng/mL pertussis toxin (PTX) for varying time intervals. Cells were stained for actin with FITC-phalloidin 15 min, 2 and 6 h following exposure. Protein levels for actin and focal adhesion kinase (FAK) were determined by Western analysis. Comparison between groups was made by Student’s t-test (P , 0.05) Results. Cells exposed to LPA showed initial actin stress fiber formation at 15 min following exposure and substantial cytoskeletal rearrangement by 2 and 6 h following exposure. This effect was completely blocked with PTX. Western blot analysis showed a 54% increase in actin levels (p , 0.001), and a 31% increase in FAK levels (p , 0.01) with LPA treatment. Conclusions. Exposure of IEC-6 cells with LPA induces significant cytoskeletal remodeling within minutes which is blocked by the G-protein inhibitor PTX. This is accompanied by increased actin and FAK levels. The ability of LPA to potentiate intestinal cell restitution appears, in part, to be mediated by effects on intestinal cell cytoskeletal structure. 85. Systemic Effects of Acute Terminal Ileitis on Uninflamed Gut Aggravate Bile Acid Malabsorption. M. Stelzner, M.D., S. Somasundaram, Ph.D., and T. Khakberdiev, M.D., Ph.D. Department of Surgery, University of Washington, Seattle, Washington. Introduction. Patients with terminal ileitis suffer from significant bile acid malabsorption even if the inflammation is locally limited. We hypothesized that inflammation in the terminal ileum may lead to changes in mucosal absorption in more proximal intes-

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tinal segments and aggravate bile acid absorption. Methods. Six hamsters underwent laparotomy and localized instillation of 2,4,6trinitrobenzenesulfonic acid in 10 % ethanol into the last 4 cm of ileum to create terminal ileitis. A control group (n 5 6) underwent instillation of saline. Animals were sacrificed after 24 h. Active and passive bile acid transport was measured in the proximal and terminal ileum and glucose absorption in the jejunum using an everted sleeve technique. Myeloperoxidase (MPO) activity and histomorphology were determined by standard methods. Results. In animals with ileitis, active bile acid uptake decreased by 84 % in the terminal ileum (t-test; p , 0.001), and in the proximal ileum by 58 % (p , 0.05) compared to saline-treated controls. Jejunal glucose absorption decreased by 59 % (p , 0.01). Passive bile acid and glucose absorption rates were not significantly changed in treated animals vs controls. Histologic examination of the treatment group revealed signs of acute terminal ileitis, however, no changes in the proximal ileum and jejunum. All control tissues were uninflamed. MPO activity was 13-fold increased in the inflamed ileal samples compared to controls (p , 0.001). No significant changes were seen in the proximal ileum and jejunum. Nominal mucosal surface area values were showed no significant changes between groups. Conclusion. These data suggest that acute ileitis inhibits active bile acid uptake in the terminal ileum. It also diminishes bile acid and glucose absorption in more proximal segments of the small intestine. This systemic effect will further aggravate bile acid malabsorption.

87. Withdrawn per author request. 88. P21 (WAF1/CIP1) Is Required for the Mitogenic Adaptive Response to Intestinal Resection. L. E. Stern, M.D., R. A. Falcone Jr., M.D., C. R. Erwin, Ph.D., and B. W. Warner, M.D. Division of Pediatric Surgery, Children’s Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio. Introduction. Increased rates of enterocyte proliferation and apoptosis characterize the intestinal adaptive response to massive small bowel resection (SBR). P21 (WAF1/CIP1), a G1 cyclindependent kinase inhibitor, has been implicated in playing a role in cellular differentiation and apoptosis. This study tested the hypothesis that p21 is obligatory for adaptation. Methods. P21-null (n 5 36) and wild-type mice (C57Bl/6; n 5 19) underwent a 50% SBR or sham operation. After 3 days, an enterocyte proliferation index (PI; % BrdU 1 cells per crypt) and apoptosis index (AI; # apoptotic bodies per crypt) were determined in the residual ileum. Results. The expected increase in AI and PI occurred after SBR in the wild-type mice. In the p21-null mice, SBR augmented AI, but did not affect the PI. After SBR, other adaptive parameters (villus/crypt morphology, ileal DNA and protein content) increased in the wild-type, but failed to increase in the p21-null mice (data not shown) (see figures).

86. Signal Transduction Pathways in Glucagon-like Peptide 2 (GLP-2) Induced Intestinal Epithelial Cell Proliferation. J. Jasleen, M.D., R. Shen, M.D., A. Tavakkolizadeh, MBBS, E. E. Whang, M.D., D. O. Jacobs, M.D., M. J. Zinner, M.D., and S. W. Ashley, M.D. Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. GLP-2 stimulates intestinal epithelial growth with high potency and specificity. The purpose of this study was to characterize the signal transduction pathways through which GLP-2 exerts its growth-stimulatory actions. Methods. Caco-2 cells were sub-cultured under serum-deprived conditions in the presence or absence of GLP-2 (10 mM). Proliferation was assessed using [ 3 H]thymidine incorporation. Relative abundance of the activated (phosphorylated) forms of two specific mitogen-activated protein kinases (MAPKs: ERK1 and ERK2) was assessed by Western blotting. In separate experiments, cells were treated with the tyrosine kinase inhibitor genistein, the MAPK kinase (MEK) inhibitor PD98059, or the PI-3 kinase inhibitor LY294001. Results. GLP-2-treated cells demonstrated a greater than 10-fold increase in proliferation (p , 0.05). This response was inhibited by each of genistein, PD98059, and LY294002 in a dose-dependent fashion. A significantly greater abundance of the phosphorylated forms of both ERK1 and ERK2 were present in cells within 5 min of treatment with GLP-2 (see figure). Conclusions. GLP-2 stimulates the proliferation of Caco-2 cells in vitro. The initial events in this stimulation appear to involve activation of tyrosine kinases. The downstream signaling appears to involve both MAPK and PI-3 kinase activation. These observations suggest possible targets in designing therapeutic strategies to optimize intestinal adaptation in patients with intestinal insufficiency.

Conclusion. p21 is critical for increasing enterocyte proliferation and other adaptive parameters; however, this factor does not appear necessary to elevate enterocyte apoptosis during adaptation. In the absence of p21, the proliferative and apoptotic responses to SBR are uncoupled. These results suggest a differential mechanism for the regulation of proliferation and apoptosis in the adapting intestine. 89. VIP Is a Neuropeptide Mediator of the Chloride Secretory Response to Serotonin in Rat Colon. J. Arcuni, M.D., M. Stoner, M.D., and J. M. Kellum, M.D. Department of Surgery, Virginia Commonwealth University, Richmond, Virginia. The chloride secretory response to serotonin (5-HT) has both nonneural and neural mechanisms, the latter mediated through a 5-HT 3 receptor. We hypothesized that 5-HT 3-induced Cl 2 secretion is partially mediated by VIP and should be inhibited by a VIP receptor antagonist, VIP 6-28. Furthermore, exogenous VIP should induce secretion in the presence of tetrodotoxin (TTX). Methods. Unstripped sheets of rat colon (n 5 6) were mounted in Ussing chambers. The 5-HT 3 receptor agonist, 2-methyl-5-HT (10 mM), was added in the absence and presence of VIP 6-28 (30 mM). In companion studies VIP (10 mM) was added to tissue incubated with or without TTX. Short circuit current was recorded and repeat-measure ANOVA used to analyze data. Results. Addition of 2-methyl-5-HT induced a rise in short circuit current (DI SC) seen in controls at 1 to 5 min (p , 0.02, see figure). VIP 6-28 blunted the DI sc (p , 0.01 as compared to controls, see figure.). In companion studies, VIP caused a DI sc over baseline in 15 min (4.7 6 2.6 mA/cm 2 to 10.4 6 3.0 mA/cm 2, p , 0.01). The addition of TTX prior to VIP did not alter the DI sc.

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consistent with its putative role in stimulating goblet cell differentiation; and 3) diversion alters the profile of MUC isoform expression by ileal and colonic mucosa.

PARALLEL SESSION III Shock II 91. Neutrophils Regulate Their Own Apoptosis through an Autocrine Mechanism via Preservation of CXC Receptors. A. Dunican, M.D., P. Grutkoski, Ph.D., S. Leuenroth, M.S., A. Ayala, Ph.D., and H. H. Simms, M.D. Department of Surgery, Rhode Island Hospital, Providence, Rhode Island.

Conclusion. The activation of the neural 5-HT 3 receptor by 2-methyl-5-HT induces a secretory response in rat colon which is inhibited by a VIP receptor antagonist. This response is mimicked by exogenous VIP, which is unaffected by TTX. VIP is a likely nonadrenergic and noncholinergic neurotransmitter in this pathway. 90. Mucin Expression in Distal Intestine Mucosa after Luminal Diversion. R. R. Cima, M.D., V. B. Tola, M.D., M. A. Doble, B.A., M. A. Klein, M.D., H. Mashimo, M.D., M. J. Zinner, M.D., and D. I. Soybel, M.D. Departments of Surgery, Medicine, and Pathology, West Roxbury VAMC and Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. The distal intestine secretes mucins as lubricants to minimize mechanical injury and to promote mucosal integrity. MUC-2 is the dominant mucin isoform secreted by distal intestine and colon. ITF (intestinal trefoil factor) is present in the mucus gel, and is thought to promote goblet cell differentiation and mucosal repair. To evaluate whether diversion influences expression of mucin-secreting cells and mucin genes in distal intestine, adult male Sprague-Dawley rats underwent sham operation or loop ileostomies constructed proximal to the cecum, leaving 5 cm of ileum diverted. Rats (6 in each group) were fed ad libitum and sacrificed 8 or 21 days after procedures. Mucosa was harvested from the 5cm of ileum proximal to the stoma (Pre-Ileo), the 5 cm segment distal to the stoma (Post-Ileo), and the diverted colon. Samples were taken from each segment for histology and mucosal scrapings for northern analysis of mRNA levels for Muc-2 and ITF (see table: Densitometry units means 6 SE, p , 0.05 compared to sham by ANOVA). Results. Histologically, at both 8 and 21 days, the crypts and surface epithelium in diverted ileum and colon were markedly enriched with mucin-secreting goblet cells. As shown in the table, Levels of ITF mRNA were higher in diverted segments at 8 days. Levels for MUC-2, thought to be a predominant colonic MUC isoform, were significantly decreased at 8 and 21 days. Conclusions. (1) Luminal diversion causes proliferation of mucincell lineages in distal intestine mucosa; (2) ITF levels increase,

Dysregulated neutrophil (PMN) apoptosis (A o) is thought to contribute to an exaggerated inflammatory response in diseases such as ARDS or MOF. The CXC chemokines, interleukin-8 (IL-8) and growth related oncogene-a (Gro-a), contribute to the inflammatory response and suppress PMN A o. We hypothesized PMN generation of CXC chemokines is an autocrine/paracrine mechanism for amplification of the PMN inflammatory response via suppression of A o. Methods. IL-8 or Gro-a (100ng/ml) was incubated with freshly isolated human PMN from healthy donors for 0-12 h and A o was analyzed at 24 h. De novo synthesis of IL-8 or Gro-a was measured using an ELISA assay. To determine if the newly synthesized ligands had available receptors mAbs specific for each receptor (CXCR I, II) were radiolabeled with I 125, incubated with PMN and CPM specificly bound were measured. Comparison was by one-way ANOVA. Results. Significant suppression of apoptosis was seen after 4h incubation with IL-8 or Gro-a (n 5 5,*p , 0.05). PMN cultured with IL-8 for 4 h produced 31 6 4.3 ng/ml of IL-8 by 24h; PMN cultured with Gro-a produced 19.7 6 4.0 ng/ml (n 5 6, p , 0.05). Neither chemokine induced significant production of the other chemokine. The addition of either ligand promoted upregulation of CXCR1 (*p , 0.05) at 24 h. However, the CXCR2 receptor was down regulated by Gro-a or IL-8 to 71 6 7.5 and 79 6 6.3 percent of control, respectively (#p , 0.05) (see figures). Conclusion. IL-8 and Gro-a which suppress A o stimulate their own production after short term incubation

with PMN. PMN maintain the ability to respond to these chemokines through expression of the CXC receptors which shows that PMN are active participants in the suppression of apoptosis at inflammatory sites.

TABLE—ABSTRACT 90 Pre-ileo

Muc-2 (8 day) Muc-2 (21 day) ITF (8 day) ITF (21 day)

Post-ileo

Colon

Sham

Ileo

Sham

Ileo

Sham

Ileo

100 6 5 100 6 1 100 6 2 100 6 6

100 6 2 100 6 4 106 6 9 113 6 4

100 6 5 100 6 4 100 6 5 100 6 1

63 6 8 64 6 8 117 6 6 95 6 2

100 6 7 100 6 7 100 6 6 100 6 10

72 6 13 62 6 6 157 6 9 124 6 18

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

92. Trauma Plasma Suppresses Bone Marrow Progenitor Proliferation via Bone Marrow Stroma. J. Wu, M.D.,* P. Rameshwar, Ph.D.,† X. Song, M.D.,* J. Qian, B.S.,† and D. Livingston, M.D.* *Department of Surgery and †Department of Medicine, UMDNJ-New Jersey Medical School, Newark, New Jersey. Severely injured trauma patients often exhibit depressed bone marrow (BM) functions evidenced by the predisposition for infections and the persistent anemia long after acute injuries. Prior work in our laboratory demonstrated that trauma plasma inhibits the BM progenitors in vitro. As BM stroma is important in regulating hematopoiesis, we postulate that BM stroma is involved in the suppression of BM progenitors by trauma plasma. Method. BM was obtained from the posterior iliac crest of normal volunteers and BM mononuclear cells (BMNC) separated using Ficoll-Hypaque density gradient. Stroma was depleted by passage through nylon wool column twice. Trauma plasma obtained from patients admitted to Surgical Intensive Care Unit (N 5 26, ISS 5 21 6 5), at 2% v/v basis, were added to both the stroma-depleted and non-stroma-depleted groups while the control group received plasma from normal volunteers. BMNCs (10 5/mL cells) were then plated in duplicate for both myeloid (CFUGM) and erythroid progenitors (BFU-E) in short term methylcellulose culture. CFU-GM cultures were supplemented with GM-CSF and BFU-E cultures were supplemented with erythropoietin and IL-3. The cultures were incubated at 37°C for 10 days (CFU-GM) and 15 days (BFU-E) respectively. Colonies were enumerated at the end of incubation period by a single, blinded observer. Results. Colonies with normal plasma were normalized to 100 %. Colonies with trauma plasma were represented as a percentage of colonies with normal plasma. CFU-GM and BFU-E colonies in the non-stroma depleted group were significantly depressed compared to normal controls while the stroma-depleted group showed no suppression when compared to normal controls. The observed difference between stromadepleted group and non-stroma-depleted group is highly significant statistically (p , 0.001 for both CFU-GM and BFU-E; see table). Conclusion. Trauma plasma is inhibitory to the BM progenitors in

TABLE—ABSTRACT 92

W/stroma W/o stroma

CFU-GM

BFU-E

57 6 23% 100 6 31%

45 6 14% 85 6 15%

an in vitro setting. This inhibition on progenitor cultures is abrogated when stromal element is removed. This suggests that BM stroma is intimately involved in the suppressive effect of trauma plasma on BM progenitors.

93. Inhibition of Alveolar Neutrophil Immigration in Endotoxemia Is MIP-2 Independent. A. J. Duffy, M.D., B. Nolan, M.D., H. Collette, R.N., M. De, and P. E. Bankey, M.D., Ph.D. Department of Surgery, UMass Medical School, Worcester, Massachusetts. Altered transendothelial migration and delayed apoptosis of PMN have been implicated as contributing to lung injury in patients with gram negative sepsis. Macrophage inflammatory protein 2 (MIP-2) signals PMN immigration and may alter other PMN functions. We tested the hypothesis that sequential endotoxin challenge in vivo alters PMN function and MIP-2 responses. Endotoxemia was induced in male Wistar rats (250g) via intraperitoneal (IP) administration of LPS (E.coli 0111:B4, 4 mg/kg). After 18 h, intratracheal (IT) injection of LPS (400 mg/kg) was performed. Control animals

received saline injections. After 4 h, circulating and bronchialalveolar lavage (BAL) PMN were isolated. PMN yields were calculated, and apoptosis was quantified after 18 h of culture by annexin V-FITC FACS analysis. BAL MIP-2 concentrations were determined by ELISA. Endotoxemia (IP/IT-LPS) significantly decreases BAL PMN yield in response to an in vivo IT-LPS challenge (Fig. 1).

Alveolar MIP-2 concentrations are equal in the two groups (Fig. 2). IT-LPS inhibits BAL PMN apoptosis to the same extent as sequential IP/IT-LPS, 25.562.1 and 21.764.4%, respectively (Control 60.466.5%). These data demonstrate that endotoxemia inhibits PMN migration despite similar MIP-2 concentrations in the alveolus. The inhibition of apoptosis is unaffected by sequential insults. Impaired lung PMN migration in the presence of chemokines suggests an induced dysfunction in septic neutrophils. This may result in an inadequate host defense that contributes to increased ICU acquired pneumonia. 94. Immune Suppression in Polymicrobial Sepsis: Differential Regulation of Th1 and Th2 Lymphocyte Response by P38 MAPK. G. Y. Song, B.A., C. S. Chung, Ph.D., I. H. Chaudry, Ph.D., and A. Ayala, Ph.D. Center for Surgical Research, Brown University School of Medicine Rhode Island Hospital, Providence, Rhode Island. Studies have shown that following the induction of sepsis there is a shift from a Th1 to a Th2 response that contributes to immune suppression. It is suggested that the family of mitogen activated protein kinases (MAPK) plays a role in transducing signals from septic stimuli which in turn alter lymphocyte activity; however, the roles of the various MAPK isoforms (p38 MAPK isoforms, JNK and ERK) remain unclear. Prior studies have shown that there is a significant change in p38 MAPK (1) activity and expression in splenic lymphocytes (SPL) following the induction of sepsis by cecal ligation and puncture (CLP). Here we further demonstrate that not only are there changes in p38, but also that ERK expression markedly decreases after CLP, while no signal for JNK is detectable (see figure and table). Subsequently we assessed the contribution of these MAPK to the development of the

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 94 Treatment

IFN-g

IL-2

IL-10

SH CLP SH PD9 CLP PD9 SH PD1 CLP PD1 SH SB CLP SB

6.8 6 1.0 3.6 6 0.61* 5.2 6 0.6 3.2 6 0.6* 6.1 6 0.49 1.5 6 0.35* 5.2 6 0.69 3.06 6 0.42*

3.2 6 0.16 1.7 6 0.13* 2.7 6 0.5 1.3 6 0.17* 2.3 6 0.16 1.2 6 0.27* 2.9 6 0.3 2.55 6 0.4

0.2 6 0.04 0.51 6 0.07* 0.16 6 0.04 0.43 6 0.07* 0.15 6 0.03 0.12 6 0.06** 0.01 6 0.004 0.09 6 0.02**

* P , 0.05 vs SH; ** P , 0.05 vs CLP alone; Mann-Whitney U; mean 6 SE, n 5 5/group.

Th1-Th2 response in splenocytes from male C3H/HeN mice who were subjected to CLP or Sham-CLP (SH) 24h prior to isolation. SPL were pretreated with 10mM of either the p38 inhibitor SB203580 (SB) or PD169316 (PD1), or the MEK inhibitor PD98059 (PD9), and IL-2, IFN-g, and IL-10 release was determined by ELISA (ng/mL). The results indicate that only pretreatment with SB, an inhibitor of the a/b isoforms of p38 MAPK, restored the release of IL-2 from septic SPL, while treatment with either SB or PD1 suppressed IL-10 release. Alternatively, the ERK inhibitor PD9 had no effect. These data document not only the overall role of p38 MAPK as opposed to ERK in splenic immune suppression seen in sepsis, but also the contribution of specific p38 MAPK isoforms to Th1 as opposed to Th2 responses.

273

M.D., C. Murphy, M.D., V. Wong, M.S., A. A. Kramer, M.D., K. F. Salhab, B.S., L. C. Carey, M.D., K. J. Tracey, M.D., and J. G. Norman, M.D. Department of Surgery, University of South Florida, Tampa, Florida. Exposure to sublethal hemorrhage (SLH) makes rats tolerant to subsequent hemorrhagic or septic shock. We have shown that this tolerance leads to alterations in macrophage (Mf) NF-kB activation. The purpose of this study was to explore whether changes in p38 MAP-kinase activity occur in the induction of tolerance by SLH. Rats were made tolerant by SLH (mean arterial pressure 5 30 mmHg for 15 min with shed blood returned). CNI-1493, a p38 inhibitor, was given prior to SLH. Shams had anesthesia and instrumentation only. Lung and peritoneal Mf were harvested 1 and 24 h after SLH and Mf were stimulated with LPS (10mg/ml 3 1hr). Protein was isolated for determination of p38 dual phosphorylation by Western blot. TNF was measured in cellular supernatants by ELISA 18 h after LPS. SLH activated p38 in the lung and this was inhibited by CNI-1493 (Fig. 1). All groups showed increased Mf p38 activity when exposed to LPS (Fig 2). TNF production by tolerant Mf in response to LPS

95. A Lethal Systemic Inflammatory Response Is Induced by Administration of Two Macrophage Derived Cytokines. J. Dierksheide, H. Yu, M. Caligiuri, and W. Carson, III, M.D. Department of Surgery, Ohio State University, Columbus, Ohio. Activation of macrophages by microbes or their products results in the rapid production of monokines (TNF-a, IL-1b, IL-12, IL-15, and IL-18) which induce natural killer (NK) cell production of IFN-g and TNF-a. The ability to obtain synergistic NK cell cytokine production with IL-15 and IL-12 suggests that NK cells and macrophages may interact during the course of severe systemic infections. In this study, we examined the effects of administering IL-15 in combination with IL-12 in a murine toxicity model. The daily, simultaneous administration of recombinant (r) IL-15 (3 3 10 5 U) and rIL-12 (1 mg) to normal mice resulted in shock and 100% mortality within 4-10 days depending on the strain employed, whereas minimal toxicity and no deaths followed administration of IL-15 or IL-12 alone. Mice treated with IL-15 plus IL-12 exhibited pulmonary edema, hepatocellular injury, degenerative lesions of the gastrointestinal tract, elevated serum levels of acute phase reactants, and NK cell apoptosis. Serum levels of IFN-g, TNF-a, and IL-1b rose rapidly in mice treated with IL-15 plus IL-12, and remained elevated until the death of the animal. Neutralization of IFN-g, TNF-a, and IL-1 was not protective to mice, nor was pretreatment with dexamethasone, TGF-b1, or ibuprofen. However, IL-15 plus IL-12 induced toxicity and death could be completely abrogated by antibody depletion of NK cells or partially arbrogated by deactivation of the macrophage compartment. Thus, treatment of mice with IL-15 plus IL-12 results in a fatal systemic inflammatory response that is critically dependent upon the NK cell compartment but does not require the actions of traditional proinflammatory mediators such as TNF-a or IL-1b. These results suggest a role for NK cells in the pathogenesis of other macrophage-mediated processes such as septic shock. 96. Involvement of p38 MAP-Kinase in the Induction of Tolerance to Hemorrhagic and Endotoxic Shock. C. Mendez,

was significantly (p , 0.05, t-test) decreased and p38 inhibition with CNI-1493 at the time of SLH reversed the inhibitory effects of tolerance on TNF production (Fig 3). TNF production by tolerant Mf

following a second insult (LPS) is attenuated despite preservation of normal p38 MAP-kinase activity. However, activation of this intracellular second messenger is a necessary step in the “cellular reprogramming” that occurs during the induction of tolerance by SLH.

97. LBP Accelerates and Augments E. coli Phagocytosis by Alveolar Macrophages. R. D. Klein, M.D., M.P.H., C. Schmidt, M.D., A. Aminlari, B.S., W. H. Alarcon, M.D., G. L. Su, M.D., and

274

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

S. C. Wang, M.D., Ph.D. Departments of Surgery and Medicine, University of Michigan Medical School, Ann Arbor, Michigan. Nosocomial pneumonia is an important cause of mortality in critically ill patients. LPS binding protein (LBP) is best known for potentiating LPS-induced cytokine production. However, recent studies suggest that LBP plays a critical role in clearance of Gram negative bacteria (GNB) and is essential for survival after bacterial challenge. The first step in GNB clearance by leukocytes is attachment and phagocytosis. We therefore sought to determine LBP’s effect on phagocytosis of GNB by alveolar macrophages. SpragueDawley rat alveolar macrophages (AM) were incubated in the presence of increasing doses of either recombinant LBP (rLBP), negative control protein (CAT), normal rat serum or saline. They were then challenged with heat inactivated FITC labeled E. coli. Phagocytosed bacteria were assayed after 120 min by measuring fluorescence. A time course study was also performed. Statistical analysis was performed using unpaired t-test (see figure, *p,0.05). The left graph

Regulation of the phagocyte apoptotic (A o ) response appears to play a significant role in the pathophysiology of sepsis. In this regard, prior studies have shown that the onset of phagocyte A o , as well as those agents which regulate it at the nidus of infection, differ significantly from those seen in circulation. The aim of this study therefore was to determine if the increase in inducible phagocyte A o and caspase activities seen in the peritoneum during sepsis are due to endotoxin (ETX) or FasL. To study this, male C3H/HeN (ETX-sensitive, HeN) C3H/HeJ (ETX-tolerant, HeJ) and C3H/HeJ-FasL gld (ETX-tolerant/FasL deficient, FasL 2 ) mice were subjected to cecal ligation and puncture (CLP) or sham operation. 24h later, phagocytes were collected and cultured with lipopolysaccharide (LPS), then harvested for A o (PI-cell cycle analysis) and caspase activity (fluorogenic assay) determination (see table). The data indicate that there was a marked increase in A o in LPS stimulated phagocytes which was associated with a significant increase in caspase 3, 8 and 9 activities from HeN and FasL 2 septic mice and an increase in caspase 3 and 8 activities in phagocytes from HeJ septic mice. The inability to suppress these changes suggests that neither ETX nor FasL regulate the peritoneal phagocyte apoptotic responses seen during the late phase of polymicrobial sepsis/peritonitis.

PARALLEL SESSION III Oncology II

demonstrates that rLBP markedly potentiates phagocytosis of E. coli by AM in a dose dependent fashion as compared to both serum and CAT. RLBP also accelerates phagocytosis of E. coli with significant augmentation seen as early as 30 min after initial challenge. These results clearly demonstrate for the first time that LBP plays an important role in both acceleration and augmentation on GNB phagocytosis by alveolar macrophages. These findings indicate a potential rose for LBP in the treatment of pneumonia. 98. Neither Fas Ligand nor Endotoxin Is Responsible for Inducible Peritoneal Phagocyte Apoptosis during Sepsis/ Peritonitis. C. S. Chung, Ph.D., G. Y. Song, B.A., I. H. Chaudry, Ph.D., and A. Ayala, Ph.D. Department of Surgery, Brown University School of Medicine, and Rhode Island Hospital, Providence, Rhode Island.

99. Inactivation of p16 by Promotor Hypermethylation or Deletion in Esophageal Adenocarcinomas. K. A. Skinner, C. A. Eads, K. Danenberg, M. E. McGarvey, M. L. Skinner, J. H. Peters, T. R. DeMeester, P. V. Danenberg, and P. W. Laird. Department of Surgery, University of Southern California, Los Angeles, California. Loss of function of the p16/CDKN2A (p16) tumor suppressor gene has been implicated in the progression of esophageal adenocarcinoma (EAC). The aim of this study was to investigate the mechanisms underlying loss of function of p16 in patients with Barrett’s adenocarcinoma. Methods. Resection specimens from 7 patients with EAC were studied. p16 expression was determined by quantitative real-time RT-PCR normalized to b-actin. p14ARF(p14) expression was also determined, and when both levels were undetectable, homozygous deletion was suspected. Promoter methylation was determined using a quantitative real-time PCR method to analyze DNA methylation. Absence of p16 amplification in the methylation assay was considered confirmation p16 of a homozygous deletion.

TABLE—ABSTRACT 98 Caspase 3, pmol/mg/min HeN Sham CLP HeJ Sham CLP FasL 2 Sham CLP

Caspase 8, pmol/mg/min

Caspase 9, pmol/mg/min

A 0 1 (%)

0.06 6 0.003 0.28 6 0.04*

0.09 6 0.006 0.11 6 0.001*

0.03 6 0.002 0.05 6 0.003*

6.1 6 0.8 27.6 6 4.9*

0.10 6 0.007 0.28 6 0.03*

0.16 6 0.008 0.24 6 0.06

0.04 6 0.007 0.05 6 0.004

5.3 6 1.0 17.5 6 3.7*

0.04 6 0.003 0.06 6 0.004*

0.04 6 0.005 0.08 6 0.01*

0.001 6 0.0001 0.01 6 0.003*

4.0 6 0.3 9.2 6 1.5*

* P , 0.05 vs sham, Mann-Whitney U, mean 1 SE, n 5 5–8/group.

275

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 99 Esophagus

Barrett’s

Tumor

Pt

Expression

DNA

Expression

DNA

Expression

DNA

1 2 3 4 5 6 7

1 1 1

2 2 2 2 2 2 2

1111

2

11111

0 111111 1

Del 2 Meth 2 Del

0 1 1 11 1

2 Meth Del 2 Meth Meth Meth

1 1 0

0

Results. (See table.) Conclusions. In normal esophagus, expression of p16 is uniformly low and its promoter is uniformly unmethylated, suggesting an alternative regulatory mechanism. Inactivating alterations in p16 occur with high frequency in EAC, with promoter hypermethylation occurring in 57% of cancers and deletions in 14%. Such changes also appear to be an early event in the progression to cancer, seen in up to 50 % of patients with Barrett’s metaplasia in the setting of esophageal adenocarcinoma. 100. Overexpression of TIMP-1 in Human Pancreatic Cancer Cells Reduces in Vitro Invasion and Reduces in Vivo Tumor Growth. M. Haq, M.D., A. Shafii, B.S., E. E. Zervos, M.D., and A. S. Rosemurgy, M.D. Department of Surgery, University of South Florida, Tampa, Florida. Background. Tissue inhibitor of matrix metalloproteinases (TIMP) are critical in regulating matrix metalloproteinases (MMP). Using pancreatic cancer cells, we sought to determine the effects of TIMP-1 expression on invasive ability in vitro and tumor growth in vivo, and MMP inhibition on cancer cells overexpressing TIMP-1. Methods. In vitro, poorly differentiated human pancreatic adenocarcinoma cells (PANC-1) were transfected to overexpress TIMP-1, with confirmation by repeated western blot and reverse zymography. Matrigel invasion assay compared the invasive potential of cells overexpressing TIMP-1 (CD-1 cells) with or without MMP inhibitor BB-94 to PANC-1 cells. In vivo, ten million CD-1 or PANC-1 cells

were injected into the back of 10 Balb-c nu/nu mice. Tumor growth was recorded every week until sacrifice at 16 weeks. Results. In vitro, CD-1 cells showed significantly less invasion than PANC-1 cells; this was further reduced by BB-94 (400 ng/ml) (Fig. 1). In vivo, CD-1 tumors grew to smaller size (0.08 cc6 0.02 vs 3.5 cc 6 0.5 for PANC-1, p , 0.05) (Fig. 2) with delayed appearance of tumor (45 days 6 5 vs 25 days 6 4 with PANC-1, p , 0.05) (Student’s t-test). Conclusions. Overexpression of TIMP-1 by human pancreatic cancer cells reduces in vitro invasion, which is further reduced by MMP inhibition. As well, TIMP-1 overexpression reduces in vivo tumor growth, providing further evidence of the importance of TIMP-1 in regulating human pancreatic cancer. 101. Rendering Immune Effector Cells Resistant to Fas Ligand-Induced Apoptosis. J. S. Goydos, M.D., M. P. Kadakia, Ph.D., M. V. Tirabassi, M.D., M. Doshi, B.A., D. Perez, Ph.D., and E. White, Ph.D. Department of Surgery, UMDNJRobert Wood Johnson Medical School, Piscataway, New Jersey. Tumor cells have developed ways to avoid immune surveillance. The recent finding that many tumor types produce Fas ligand (FasL) may mean that tumors mimic immune privileged sites as one mechanism. Immune effector cells may be induced to undergo apoptosis prior to the initiation of an immune response. If this is true, then producing immune effectors that are resistant to FasL-induced apoptosis may lead to a more effective immunotherapy. Methods. Peripheral blood lymphocytes were cultured in IL-2 to produce lymphokine activated killer (LAK) cells in vitro and then exposed to antibodies (CH11) that mimic the effects of FasL. We then produced a retroviral vector that codes for a fusion protein between E1B 19K, a viral protein that blocks FasL-induced apoptosis, and the enhanced green fluorescent protein (causes infected cells to glow green under the UV microscope). We infected cells with this retrovirus and tested to see if it would render them resistant to FasL-induced apoptosis. Results were compared using the Wilcoxon Rank-Sum Test. Results. CH11 antibodies were able to induce apoptosis in 75% of LAK cells in 48 h compared to a relative steady-state in the control cultures that lacked CH11 (p , 0.01). This apoptosis could be blocked with antagonistic anti-Fas antibodies (ZB4) that block CH11 binding. Infection of cells with our retroviral vector protected them from apoptosis and caused the protected cells to glow green. There was a two-fold increase in infected cells as compared to control cells (noninfected) in our apoptosis assay (p , 0.01). Conclusions. We have demonstrated that LAK cells are sensitive to FasL-induced apoptosis and that we can protect these cells from this form of apoptosis by infection with our retroviral vector. This may lead to a more effective adoptive immunotherapy of cancer. 102. Phorbol Esters Attenuate Glutamine Uptake in Colon Carcinoma Cells. T. Pawlik, M.D., T. Sweeney, M.D.,W.

276

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Souba, M.D., and B. Bode, Ph.D. Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts.

Cancer cells are known to extract glutamine (GLN) at rates several-fold greater than normal cells, but the regulation of this response is unclear. The present studies were undertaken to determine whether GLN transport in colon carcinoma cells is regulated by a phorbol ester-dependent protein kinase C (PKC) pathway. Methods. The WiDr colon carcinoma cell line was cultured in the presence and absence of the PKC activator phorbol 12-myristate 13-acetate (PMA) at10 27 M for 10 and 30 min and 1, 3, and 24 h. Transport of L- 3 H-GLN was assessed after cell exposure to vehicle or PMA 1/2 cycloheximide (CHX), 1/2 staurosporine (a PKC inhibitor), and 1/2 4-aPMA (a biologically inactive form of PMA). PMA’s effect on GLN transport was also studied in the HT29 colon carcinoma cell line. In addition, to determine if PMA’s effect was specific to GLN transport, the uptake of L- 3 H-arginine (Arg) and leucine (Leu) was assessed. Statistical analysis by t-test. Results. PMA treatment induced a rapid time- and concentration-dependent inhibition of

103. Contact Inhibition of the Menin Tumor Suppressor Gene. A. Y. Wu, M.D., E. Srivatsan, Ph.D., C. J. Arora, A. T. Shimode, and M. P. Sawicki, M.D. Department of Surgery, West Los Angeles VAMC and the UCLA School of Medicine, Los Angeles, California. Introduction. MENIN is a tumor suppressor gene that is inactivated in the multiple endocrine neoplasia type 1 (MEN1) syndrome and associated sporadic forms of these tumors. MENIN is a nuclear transcription factor that binds JunD and acts, in part, by inhibiting AP-1 transcriptional activation. We hypothesized that MENIN function is regulated by nuclear transport similar to other AP-1 proteins. Methods. The MENIN gene was isolated from a leukocyte cDNA library and cloned into a mammalian expression vector as an enhanced green fluorescent protein fusion product (MENIN-EGFP). This gene construct was stably transfected into mammalian fibroblasts that were grown under standard conditions. MENIN-EGFP expression was confirmed by western blot and visualized in live cells by UV microscopy. Results. Western blot analysis of the stable cell line protein demonstrated appropriate expression of the MENINEGFP fusion product with both anti-MENIN and anti-EGFP. While the fibroblasts were actively proliferating MENIN was primarily expressed within the nucleus. When the cells were allowed to grow to 100 % confluence, however, the gene expression shifted to the cytoplasm. When the same confluent cells were split and replated MENIN expression shifted back to the nucleus. Conclusions. The MENIN tumor suppressor gene plays an important role in endocrine cell growth. It functions primarily as a nuclear transcription factor and its nuclear localization is inhibited by cell contact.

104. Production of the Proto-oncogene blk Is Directed by Internal Ribosomal Entry. C. H. Cha, M.D., T. Eisenbraun, Ph.D., G. D. Kennedy, M.D., and J. E. Niederhuber, M.D. Departments of Surgery and Oncology, University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin

GLN uptake rates in WiDr (Fig 1) and HT29 cells by 25-30% and 50%, respectively. CHX did not block this response, indicating that the mechanism by which PMA exerts its effects are posttranslational. The inhibition of GLN uptake by PMA was PKCdependent as staurosporine abrogated the effects of PMA on GLN uptake (Fig 2). The effect of PMA was specific, as the analog 4-aPMA had no effect on transport activity. PMA was also found to inhibit arginine uptake by 50%, and to a lesser degree leucine uptake (27%). Conclusions. GLN uptake by colon carcinoma cells is regulated through a PKC-dependent post-translational pathway. Similarly, PMA inhibits the activity of the arginine and leucine transporters. This PKC pathway functions to regulate nutrient uptake in colon carcinomas and may constitute a potential therapeutic target.

Blk is a member of the src family tyrosine kinases, many of which are proto-oncogenes. Overexpression of activated blk has been shown to induce malignant transformation in both NIH 3T3 cells and transgenic mice. Translational regulation of oncogene expression is becoming recognized as an important target of malignant conversion. Internal Ribosomal Entry Sites (IRES) enable ribosomes to bind directly to the interior of an mRNA 59 leader region and initiate translation of genes that would otherwise be translationally repressed due to the size and structure of their 59 leader sequence. Only a select few eukaryotic genes demonstrate IRES activity, including the 59 leaders of FGF-2 and the proto-oncogene c-myc. In both cases, IRES activity is implicated in malignant transformation. Blk mRNA has an unusually large 588 nt leader sequence upstream of the protein coding region that forms a stable secondary structure which ordinarily would inhibit any translation of the blk protein. To determine whether blk synthesis is directed by an internal ribosomal entry site, a reporter vector was constructed to produce a bicistronic mRNA in which the blk 59 leader sequence was placed between a renilla luciferase coding region and a firefly luciferase coding region (see Fig. 1). A stop codon and stable hairpin loop were engineered between the renilla luciferase and the blk 59 leader sequence, preventing ribosomes from scanning into the second luciferase cistron. Plasmids were then transfected into a Daudi cell line via electroporation and IRES activity was measured as the ratio of firefly luciferase light activity relative to renilla. Translation of the downstream Firefly luciferase gene only occurred in constructs that contained the

277

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

106. Angiostatin Binds a 33 Kilodalton Protein in Endothelial Cells. M. C. Sharma, Ph.D., M. Sharma, Ph.D., and D. H. Berger, M.D. Department of Surgery, MCP Hahnemann University, Philadelphia, Pennsylvania.

blk 59 leader sequence (28.3 6 4.6) and in a positive IRES control (6.9 6 1.7) whereas removal of the 59 leader resulted in complete loss of IRES activity (1.0 6 0.1, p 5 .001) (see Fig. 2). As expected, renilla light output was detected in all three constructs. Deletional analysis and S1 nuclease assays mapped essential IRES sequences to the first 80 nt of the blk 59 leader sequence. These data prove that an IRES site exists within the 59 leader of the blk gene and provides a mechanism for the upregulation of blk synthesis. 105. Optimization of Antisense Oligonucleotides Targeting HER2/neu Expression. C. B. Hirose, M.D., H. Roh, Ph.D., C. B. Boswell, M.D., J. Pippin, B.A., and J. A. Drebin, M.D., Ph.D. Department of Surgery, Washington University School of Medicine, St. Louis, Missouri. Introduction. The HER2/neu oncogene encodes a cell surface glycoprotein, p185, which is involved in mitogenic signaling. Overexpression of this protein in human breast carcinomas is associated with a poor prognosis. A number of laboratories including our own have demonstrated that oligonucleotides (ODNs) targeting the 59 region of the HER2/neu mRNAs down-regulates the expression of HER2/neu and inhibits the growth of cancer cells which overexpress this gene. We sought to optimize this effect by identifying HER2/neu mRNA sequences particularly sensitive to antisense targeting. Methods. HER2/neu overexpressing human breast carcinoma cells, BT474, were exposed to HER2/neu specific antisense (AS) or scrambled antisense (SC) phosphorothioate ODNs at varying concentrations in the presence of 10 mg/ml Lipofectin. Overlapping 20mers from position 215 to 117 (around the transcription initiation codon AUG) were studied. Cell growth was determined by MTT assay. 50% inhibitory concentrations were compared. Down-regulation of p185 was demonstrated by Western blotting. For the in vivo studies, 17 b-estradiol pellets were implanted in 4 week old female nude mice prior to 1 3 10 7 s.c. tumor cell implantation. The mice were injected with ODNs at 10 mg/kg i.p. for 14 days. Results. Treatment of BT474 breast carcinoma cells with HER2/neu AS ODNs result in growth inhibition as well as p185 protein down-regulation compared with control ODNs. There is a significant difference in potency of distinct ODNs differing by only a few bases. The sequence with optimal in vitro activity also displays enhanced in vivo tumor growth inhibition when compared with other sequences (see table). Conclusions. AS ODNs targeting a number of oncogene proteins, including HER2/neu, are undergoing evaluation as anticancer agents. Small differences in sequence targeting can result in significant alterations in AS activity and resulting tumor growth inhibition. Sequence optimization is a critical step in developing compounds for potential therapeutic application.

Angiostatin (AS), an internal fragment of plasminogen, is one of the most potent of the known angiogenesis inhibitors. Currently the mechanism by which AS exerts its specific action on endothelial cells is unknown. It was our hypothesis AS binds to a protein present in mammalian endothelial cells. Total cell lysates were prepared from bovine aortic endothelial cells (BAE), human umbilical vein endothelial cells (HUVEC), human fibroblasts (HF), and human lung cancer cells (A5439). Lysates were run on SDS-PAGE, incubated with AS, washed and reprobed with anti-AS antibody. Immunoblots were developed by enhanced chemiluminescence (ECL). Subsequently BAE cells were lysed and subject to ultracentrifugation in order to facilitate fractionation. Specific cellular fractions were run on SDSPAGE and probed as above. Immunoblotting revealed a 33-kD band expressed in endothelial cells which bound AS (Fig. 1). Cell fractionation experiments demonstrate that this 33kD protein is localized to the cytosol of BAE (Fig. 2). We conclude that there is a 33kD protein localized to the cytosol of endothelial cells which is capable of binding AS. Experiments are in progress to identify this protein and its role in AS action.

PARALLEL SESSION IV Transplant/Immunology 107. Kupffer Cells Regulate Th1 and Th2 Cytokine Profiles: A Chemokine-Dependent Mechanism of Immunosuppression? M. A Beneke, M.D., J. R. Johnson, B.A., T. Paglieroni,

TABLE—ABSTRACT 105 MTT assay BT 474 lipofected with HER2 AS

C 50 (mM)

A

B

C

D

Control

0.35 6 0.003

0.20 6 0.006

0.20 6 0.01

0.10 6 0.013

.10

278

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 107

IFN-g (%) IL-4 (%)

PVS

PVT

PVT 1 anti-MCP

PVS sup

PVT sup

PVT sup 1 anti-MCP1

46.7 0.5

6.2 6.3

25.5 2.8

47.2 1.5

18.3 3.6

29.2 1.7

Ph.D.,* and R. V. Perez, M.D. Department of Surgery, UC Davis, Sacramento, California; and *Sacramento Medical Foundation, Sacramento, California. Introduction. Alloantigen from portal venous transfusion (PVT) leads to an allospecific immunotolerance. Lymphocyte-kupffer cell (KC) interactions are hypothesized to suppress immunostimulatory Th1 cytokines (eg, IFN-g) or enhance immunosuppressive Th2 cytokines (eg, IL-4). KC elaboration of MCP-1 may mediate this effect on helper T-lymphocytes. Methods. Lewis rats underwent heart transplant from donor Wistar-Firth rats after either PVT or portal venous saline infusion (PVS). Graft survival was monitored by palpation. In parallel studies, Lewis rat KC were isolated after PVT or PVS. MCP-1 was measured in KC supernatants by ELISA. These KC or their supernatants were also co-cultured with naı¨ve lymphocytes in the presence or absence of anti-MCP-1. Intracellular cytokine expression was then assessed by flow cytometry for Th1 or Th2 cytokine profile predominance. Results. Graft survival was longer with PVT when compared to PVS (9.6 v. 5.9 days, p , 0.01). PVT also led to a higher KC production of MCP-1 than did PVS (1208 v. 31.7 pg/ml, p , 0.01). PVT resulted in a smaller percentage of lymphocytes producing IFN-g and greater percentage producing IL-4 when KC or their supernatants were in contact with the lymphocytes. Incubation with anti-MCP-1 partially abrogated this response (see table). Conclusions. KC regulation of Th1/Th2 cytokine profiles may be an important immunosuppressive mechanism mediated in part by MCP-1. 108. Kupffer Cell Role in Chemokine Production in Hepatic Ischemia/Reperfusion Injury in Mice. B. D. Mosher, M.D., J. Palma, M.D., C. Remelius, B.S., J. Kisala, M.D., R. E. Dean, M.D., and E. Crockett, Ph.D. Michigan State University Integrated General Surgery Residency, College of Human Medicine, East Lansing, Michigan. Kupffer cells play a critical role in chemokine production, which are important mediators in neutrophil recruitment and subsequent hepatocellular damage. Methods. Adult male C57BL/6 mice were injected with gadolinium chloride (GC) or normal saline (NS), 48 and 24 h before operation. GC is an inhibitor of Kupffer cell functional activity. Twenty-four hours following the second injection the mice underwent 90 min of hepatic lobar ischemia followed by reperfusion. Plasma levels of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC were measured by enzyme linked immunosorbent assay (ELISA). Neutrophil infiltration was determined by myeloperoxidase (MPO) assay. Liver injury was determined by plasma levels of alanine transaminase (ALT). ANOVA was applied to compare the results between NS vs GC-treated animals. Differences were assessed at p , 0.05. Results. Plasma levels of KC after 6 h of reperfusion were significantly higher in the NS vs GC-treated mice, 5660 6 1375 pg/ml vs 1359 6 134 pg/ml (p , 0.05). MIP-2 plasma levels following the same amount of reperfusion were 988 6 195 pg/ml vs ,50 6 5 pg/ml (NS vs GC-treated mice), p , 0.05. The tissue MPO levels were higher in the NS vs the GC-treated animals, 3.70 6 1.54 U/ml vs 1.17 6 0.10 U/ml (p 5 0.13) after 6 h of reperfusion indicating moderate inhibition of neutrophil recruitment upon GC treatment. ALT levels were significantly higher in the NS

vs GC-treated mice, 5616 6 875 IU/L vs 791 6 114 IU/L, p , 0.05, indicating less hepatocellular damage in the GC-treated animals. Error was reported as 6the standard error of the mean (SEM). The number of animals in each group was five. Conclusions. Kupffer cells appear to be major contributors of chemokine production in hepatic ischemia/reperfusion. Modulation of chemokine production or their action may serve as a potential target for therapeutic intervention. 109. NFkB Nuclear Expression during Cold Ischemia Correlates with Post Reperfusion Function. R. Ricciardi, M.D., R. D. Kim, M.D., T. P. McDade, M.D., R. A. Perugini, M.D., S. H. Quarfordt, M.D., R. S. Chari, M.D., and W. C. Meyers, M.D. Department of Surgery, UMASS Medical School, Worcester, Massachusetts. In liver transplantation, activation of transcription factors (TF) occurs upon reperfusion (R), yet no data exists regarding TF activation during cold ischemia (CI). We hypothesized that activation of TF may initially occur during CI, prior to reperfusion, and serve as an important determinant of post reperfusion function. Methods. Serial biopsies during porcine liver harvest were obtained immediately upon laparotomy (LAP), upon completion of dissection (ED), after 45 min (m) and 120 m of CI, and 60 m and 180 m after R. Nuclear extracts were isolated for Western Blot analysis of NFkB. Hepatic function was assessed through bile output and sorbitol dehydrogenase (SDH) activity. Statistical significance was determined with correlation coefficients. Results. NFkB expression was maximal at 45 m of CI and decreased by 120 m (Fig 1). Increased NFkB expression at 120 m of CI correlated with poor graft function: decreased bile flow (n 5 6, r 5 0.85, p , 0.05) and increased SDH activity (n 5 5, r 5 0.99, p , 0.05) (Fig 2). During R a second distinct peak occurred at

180 m. Increased expression of NFkB at 180 m of R correlated directly with prior expression at 120 m of CI (n 5 6, r 5 0.83, p , 0.05). The magnitude of the 180 m R peak also correlated directly with SDH activity (n 5 5, r 5 0.91, p , 0.05). Conclusions. Nuclear expression of NFkB demonstrates distinct peaks of activity, one during CI and one after R. These data suggest that prolonged activation of NFkB during CI not only correlates directly with NFkB expression during R, but also correlates inversely with post reperfusion graft function.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS 110. Mixed Hematopoietic Chimerism Induces DonorSpecific Tolerance to Cardiac Allografts in Rats. Y. Huang, X. Que, and S. T. Ildstad. The Institute for Cellular Therapeutics, University of Louisville, Glenolden, Pennsylvania. We previously reported that depletion of ab-TCR 1 and gd-TCR 1 T-cells (TCD) from bone marrow (BM) while retaining facilitating cells (CD8 1/CD3 1/TCR 2) allows reliable engraftment yet avoids GVHD completely. We have now investigated whether the mixed hematopoietic chimerism that results from the transplantation of this TCD donor BM induces donor-specific tolerance to cardiac allografts. TCD of donor ACI rat BM was performed using anti-ab TCR and gd-TCR mAbs and immunomagnetic beads. Wistar Furth (WF) rat Recipients were conditioned with 950 cGy of total body irradiation and reconstituted with 100 3 10 6 TCD BM cells. All recipient rats exhibited stable mixed hematopoietic stem cell chimerism with 8 to 89% of total peripheral lymphoid cells of donor derivation 4 months after BMT. Mixed allogeneic chimeras received cardiac transplantation with donor-specific (ACI) or third party (F344) allografts. No immunosuppressive agents were administered after cardiac transplantation. All donor-specific cardiac allografts were accepted by mixed chimeras, while all third party grafts were rejected within 10-20 days as rapidly as naı¨ve WF rats (see figure). Our data indicate that depletion of ab-TCR and gd-TCR T-cells produces stable mixed chimerism and tolerance for cardiac allografts. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.

279

18-22, and 26-30 (n 5 3-5 mice per time point). eNOS 2/2 grafts showed significantly increased intima/media ratios at days 26-30 compared to iNOS 2/2 and wild-type (WT) grafts. There was no difference in intimal thickening between iNOS 2/2 and WT grafts. Immunostaining was performed to determine the expression and localization of eNOS and iNOS. In eNOS 2/2 grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In iNOS 2/2 and WT grafts, eNOS expression was preserved in the endothelium even by day 26, and associated with a decrease in intimal thickening. In conclusion, we found that eNOS-deficient allografts developed significantly worse vasculopathy, and preserved expression of eNOS seems to protect allografts from vasculopathy (see figure).

112. Removal of Host T-Cells Enhances Bone Marrow Engraftment in NOD Mice. M. A. Domenick, M.D., L. A. Mills, B.S., and S. T. Ildstad, M.D. Department of Surgery, MCP/ Hahnemann University, Philadelphia, Pennsylvania; and Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky.

111. Endothelial Nitric Oxide Synthase Protects Allografts from Transplant Vasculopathy. P. C. Lee, M.D., Z. L. Wang, M.D., S. C. Watkins, Ph.D., S. Qian, M.D., T. R. Billiar, M.D., L. L. Shears, M.D. Department of Surgery and Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. Inducible nitric oxide synthase (iNOS) has been shown to be upregulated and plays a protective role in grafts with transplant vasculopathy; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) remains unclear. We examined the effects of selective eNOS and iNOS deficiency in aortic allografts in a murine chronic rejection model using eNOS and iNOS 2/2 (C57BL/6 background; H2 b ) donors and C3H (H2 K ) recipients. Aortic grafts were harvested and analyzed morphometrically at days 10-14,

Bone marrow transplantation (BMT) impacts on Type 1 diabetes in two ways: (1) through the induction of donor-specific tolerance with hematopoietic stem cell (HSC) chimerism for islet/pancreas transplantation; (2) to halt the autoimmune process itself. However, BMT is limited by the morbidity and mortality associated with lethal conditioning. The development of minimal conditioning strategies may allow the use of HSC chimerism for the induction of tolerance. The nonobese diabetic (NOD) mouse is a model for Type I diabetes. While 600cGyTBI (total body irradiation) conditioning allows HSC chimerism in normal mice, a minimum of 750cGyTBI is required in NOD mice. The aim of this study was to decrease the irradiation dose in NOD mice by targeting specific cells in the host allowing donor cells to engraft. Methods. Monoclonal antibody (mAb) treatment was utilized to remove cells in the recipient. NOD mice were pretreated on Day –2 with anti-CD8 and/or anti-CD4 mAb, or saline via a single injection. On Day 0 the mice were irradiated with 700, 600, or 500 cGy TBI and reconstituted with 30310 6 bone marrow cells from B10.BR donors. Chimerism was evaluated by flow cytometry of peripheral blood one month after BMT. Results: Removal of host CD4 1 allows only 14% of animals to engraftment. However, targeting of the CD8 1 cells allows 100% of the animals to engraft with 700cGy TBI. When anti-CD4 and anti-CD8 are combined, TBI can be further reduced to 600cGy. When TBI is reduced to 500cGy there is no engraftment with this combined mAb therapy. These data show that CD8 1 together with CD4 1 T-cells in the recipient play a role in engraftment of donor HSC (see table). Minimal conditioning strategies may allow HSC chimerism to be applied clinically in tolerance induction.

280

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 112 Antibody

TBI (cGy)

Engraftment (%)

Chimerism (%)

Saline CD4 CD8 CD8 1 CD4 CD4 CD8 CD8 1 CD4

700 700 700 700 600 600 600

0 14 100 100 0 33 100

0 97.4 90.7 73.5 6 24 0 98 60.2 6 35

113. A Murine Model for Coronary Vasculitis by Immunization with Artery-Specific Antigenic Proteins (ASAPs). W. M. Park, M.D., J. R. Borromeo, M.D., N. Koshy, M.D., K. Hardy, B.S., S. Xia, M.D., and M. D. Tilson, M.D. Department of Surgery, Columbia University and St. Luke’s/Roosevelt Hospital Center, New York, New York.

through the pump was monitored continuously while PV, IVC, and HA pressures were maintained within the physiologic range. Bile output (cc) was recorded every hour. Results. Comparative results are summarized in the table. Conclusions. Dual vessel hepatic inflow provides better liver function and longer recipient survival, compared with single vessel ECLP.

TABLE—ABSTRACT 114

12-h recipient survival Blood flow (ml/min) b VO 2 (ml/min) c NH 3 Cl. (mmol/ml) c Lactate Cl. (mmol/ml min) c Factor V (%) b Bile output (ml/h) a

Background. Syphlitic coronary ostial stenosis and Kawasaki’s Disease have features of autoimmunity, including aggregates of Ig and infiltration with inflammatory cells. We have described a family of artery-specific antigenic proteins (ASAPs), among which AAAP-40 was the first to be discovered and is the best characterized. We have also described sequence similarities between AAAP-40 and a protein of T pallidum. The present studies were performed to evaluate the occurence of vasculitis in an autoimmune mouse model. Methods. Murine MHC Class II genes were searched for homologs to human HLA DR 1501, and E-b-b and E-b-k were highly similar (p , 10 231). Homozygous inbred mice bearing these alleles were immunized with intradermal injection of 100 mg of a 12-mer synthetic polypeptide from the AAAP-40 sequence. Results. A mouse sacrificed 8 weeks post-injection had intense coronary vasculitis, with inflammatory infiltrates of lymphocytes, plasma cells, and rective histiocytes (Anitschkow cells). Additional mice sacrificed at 5 months showed persistent vasculitis, but to a lesser degree. Conclusion. The microscopic findings are typical of the coronary vasculitis of rheumatic fever, but the coronary aneurysms of Kawasaki’s disease and the ostial lesions of syphilis may also be on the basis of autoimmunity against this or other ASAP’s. 114. 24-hr Extracorporeal Pig Liver Perfusion (ECLP): A Comparative Study about Single versus Dual Hepatic Inflow. N. P. Mora, M.D., L. Kaptanoglu, M.D., V. J. Siddall, M.S., J. Zhang, M.D., M. Abecassis, M.D., D. Kaufman, M.D., J. Leventhal, M.D., F. Stuart, M.D., A. Blei, M.D., and J. P. Fryer, M.D. Division of Organ Transplantation, Northwestern University. Chicago, Illinois. The shortage of human donors has led to renewed interest in ECLP as a bridge to transplantation. In recent applications of ECLP, the nature of hepatic inflow has been inconsistent. While some advocate the portal vein (PV) only (single vessel), others have argued that hepatic artery inflow (HA) is also required (dual vessel). The aim of our study is to evaluate the utility of single vs dual vessel perfusion. Methods. Female White Landrace pigs were used for the study. 10 as liver donors, 10 as recipients for ECLP, Group 1: Single vessel perfusion (n 5 5). Group 2: Dual vessel perfusion (n 5 5). Livers were procured in standard fashion. Veno-venous bypass was established in the recipient using a Biomedicus pump and a membrane oxygenator to oxygenate all blood flow into the liver. Liver function was monitored by taking simultaneous pre- and post-hepatic blood samples. These were taken at: Baseline, 1, 3, 6, 12, 24 h. The following parameters were determined at each step: CBC, platelets, Factor V, AST, ALT, Bilirubin total, Ammonia, Lactate, and VO 2 . Total blood flow

b c

Single vessel

Dual vessel

P value a

1/5 55.1 6 2.2 92.9 6 7.3 31.8 6 8.3 30.5 6 14.3 21.6 6 0.5 0.34 6 0.04

4/5 62.1 6 1.1 89.9 6 11.0 50.7 6 8.7 58.9 6 22.5 0.3 6 0.2 0.66 6 0.11

0.031 0.006 ns ns ns 0.015 0.0002

Mann-Whitney U. Per 100 g/liver. Per g/liver.

PARALLEL SESSION IV Shock III/Gastrointestinal III 115. Mechanism of the Beneficial Effects of Pentoxifylline during Sepsis: Maintenance of Adrenomedullin Responsiveness and Downregulation of Proinflammatory Cytokines. P. Yoo, B.A., D. J. Koo, B.A., W. G. Cioffi, M.D., K. I. Bland, M.D., I. H. Chaudry, Ph.D., and P. Wang, M.D. Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, Rhode Island. Although it is known that pentoxifylline (PTX) produces various beneficial effects during sepsis, it remains unknown whether this agent has any salutary effects on the depressed vascular responsiveness to adrenomedullin (ADM, a novel potent vasodilatory peptide). To study this, male rats (275-325g) were subjected to sepsis by cecal ligation and puncture (CLP). At 1 h after CLP, PTX (50 mg/kg BW) or vehicle (normal saline) was infused IV over 90 min. At 20 h after CLP (i.e., the late, hypodynamic stage of sepsis), the thoracic aorta and small intestine were isolated and preconstricted by norepinephrine. Rat ADM (10 27 M) was applied and the percentage of ADMinduced relaxation in the aortic rings and resistance vessels in the small intestine was determined. Plasma ADM was determined by RIA and TNF-a, IL-1b, and IL-6 levels were measured by ELISA. The data (mean 6 SE, n 5 6 – 8/group) are shown in the table. The results indicate that ADM-induced vascular relaxation in the aortic

TABLE—ABSTRACT 115 Sham

CLP-vehicle

Aortic rings (%) 76.4 6 2.6 56.8 6 4.2* Intestinal vessels (%) 94.5 6 2.4 78.4 6 2.9* Plasma ADM (pg/ml) 164 6 12.7 673 6 142* Plasma TNF-a (pg/ml) 2.5 6 1.5 31.1 6 4.8* Plasma IL-1b (pg/ml) 9.7 6 5.3 89.5 6 12.2* Plasma IL-6 (pg/ml) 1.7 6 0.9 2000 6 614*

CLP-PTX 73.2 6 5.3** 87.8 6 1.9** 661 6 95* 14.0 6 4.5** 16.1 6 5.9** 591.6 6 43.2**

Note. ANOVA and Tukey’s; * P , 0.05 vs sham; ** P , 0.05 vs. CLP-vehicle.

281

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS rings and arterioles of the isolated gut was significantly reduced at 20 h after CLP. Administration of PTX early after the onset of sepsis, however, prevented the decrease in vascular ADM responsiveness at the macro- and microcirculatory levels. Plasma ADM levels increased after CLP, irrespective of PTX infusion, indicating that the effect of PTX was not mediated by altering plasma ADM. The upregulated TNF-a, IL-1b, and IL-6 were, however, reduced by PTX treatment, suggesting that maintenance of ADM responsiveness by this agent appears to be mediated by downregulation of these cytokines. Since early administration of PTX maintains vascular ADM responsiveness even during the late stages of sepsis, this agent appears to be a useful adjunct in preventing the deterioration in hemodynamics during polymicrobial sepsis. 116. Maintenance of Endothelial NOS Activity during Intestinal I/R Limits Capillary Leak. D. T. Ward, M.D., S. A. Lawson, M.D., W. C. Connor, M.D., and T. Shea-Donohue, Ph.D. Department of General Surgery, Walter Reed Army Medical Center, and Departmens of Medicine and Surgery, USUHS, Bethesda, Maryland. Nitric oxide synthase (NOS) activity diminishes with intestinal ischemia-reperfusion (IR), concurrent with onset of capillary leak in the lung and gut. Aim. To examine the effect of the nitric oxide (NO) substrate, l-arginine, on PMN activation and infiltration, capillary leak (gut, lung), and mucosal injury following IR. Methods. Male rats received saline (IR) or l-arginine 4mg/kg/min (LARG) for 1 h, followed by 30 min of superior mesenteric artery occlusion and 4 h of reperfusion. Evan’s Blue dye was given IV 1 h before harvest. Mucosal injury [0 (normal)-5 (severe)] and PMN infiltration (#PMN/hpf) were assessed in sections of intestine. Dye concentration was assayed in serum, lung and small bowel lavage (BAL, SBL) via spectrophotometer. The neutrophil “priming state” (PS) was defined as the product of oxidative burst activity (%ACT), as determined by flow cytometry, and the PMN number from CBC (PMN/CBC). Results. L-arginine reduced IR-induced intestinal PMN infiltration and mucosal injury (3.9 6 0.4 vs 2.2 6 0.5; p , 0.05). L-arginine had no effect on elevated systemic PMN number after IR, but reduced PMN oxidative activity and thus “priming state”. The gut and lung leaks induced by IR were reduced significantly by l-arginine (see table). These data show that l-arginine reduces the inflammatory response by sustaining NO levels and limiting capillary leak. Secondly, both the PMN number and their activity (Priming State) determine leak. Thus, nitric oxide plays an anti-inflammatory role during IR by reducing intestinal PMN infiltration, systemic activation and PMN priming state. 117. Taurine Attenuates Lipopolysaccharide-Induced Rolling and Adhesion in the Rat Mesenteric Microcirculation. B. Egan, FRCSI, G. Chen, M.D., C. Kelly, FRCSI, and D. J. Bouchier-Hayes, FRCSI. Department of Surgery, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland. Adhesion of polymorphonuclear leucocytes (PMN) to endothelial cells and subsequent transendothelial migration is an early event in the inflammatory response and plays an important part in the patho-

genesis of septic shock, contributing to vascular and tissue injury. We hypothesised that taurine (2-aminoethanesulfonic acid) a known anti-oxidant and endothelial protector would attenuate leucocyte endothelial interactions and microvascular permability observed during endotoxaemia. Sprague-Dawley rats (300-350g) were randomised into Control, LPS and LPS1taurine groups. Taurine was administered orally as a 4% solution. Endotoxamia was induced using 15mg/kg E. coli endotoxin (Serotype 0.55B5) via a slow intravenous infusion. Using mesenteric post-capillary venules (28-32mm diameter) the number of adherent and migrated leucocytes and their rolling velocity were measured by intravital microscopy at baseline and subsequently at 10, 30, 60 and 90 min post administration of LPS (see table). These results demonstrate that taurine attenuates endotoxin induced alterations in leucocyte-endothelial cell adhesion and migration. It also prevents the endotoxin associated decrease in leucocyte rolling velocity,suggesting that Taurine may be therapeutic in the prevention of endothelial damage in sepsis.

TABLE—ABSTRACT 117

Na (/100 mm), 30 s Nm (/field), 90 s RV (mm/s), 30 s

Control

LPS

LPS 1 taurine

3.4 6 0.4 1.6 6 0.2 48.0 6 1.4

11.8 6 0.9* 8.2 6 2.1* 35.8 6 1.9*

4.3 6 0.9** 2.8 6 0.9** 44.4 6 1.1**

Note. Results, means 6 SEM; statistics Student’s t test; * P , 0.05 LPS vs control; ** P , 0.05 LPS 1 taurine vs LPS. 118. Matrix Metalloproteinase (MMP)-2 and -9 Contribute to Lung and Liver Injury Following Hepatic Ischemia/ Reperfusion (I/R). S. Patel, M.D., H. L. Pachter, M.D., J. Mitchell, M.D., H. Yee, M.D., P. Mignatti, M.D., and P. Shamamian, M.D. Departments of Surgery and Pathology, S. A. Localio Laboratory for Surgical Research, NYU School of Medicine, New York, New York. MMPs play a key role in extracellular matrix degradation during tissue remodeling and neutrophil (PMN)/monocyte diapedesis. Previously we have shown that TNF-a-stimulated PMNs released MMP-9 in a dose-dependent manner, and that endothelial cellderived MMP-2 is activated by PMN serine proteases. PMN activation, with release of proteinases has been implicated as a central mechanism in the pathogenesis of hepatic and pulmonary injury following hepatic I/R. The role of MMP-2 and -9 in hepatic and distant organ injury following hepatic I/R is unknown. We hypothesize that elevated levels of MMP-2 and -9 contribute to acute liver and lung injury following hepatic I/R. Twelve SpragueDawley rats were randomized to control (laparotomy only) or partial hepatic I/R (90 min/12 h reperfusion). Animals were sacrificed after 12 h. Hepatic injury was assessed by histologic scoring of necrosis (0 5 no necrosis to 3 5 .60% necrosis) and PMN infiltration (PMN/HPF). Pulmonary PMN infiltration was assessed by myeloperoxidase (MPO) activity. Pulmonary vascular permeability index (PI) was calculated as the ratio of Evans Blue

TABLE—ABSTRACT 116

Sham IR L-arginine

PMN/hpf

%ACT

PMN/CBC

PS

SBL

BAL

4 6 0.1 22 6 1.6* 16 6 1.1*

36 6 3.5 60 6 10 46 6 2.1

7 6 1.9 40 6 3.5* 41 6 2.5*

2.7 6 1.0 24 6 2.1* 19 6 0.2*

6.2 6 2.1 27 6 7.0* 10.1 6 2.1

9.4 6 2.4 28.3 6 6.3* 13 6 1.9**

* P , 0.05 vs Sham; ** P , 0.05 vs IR; n 5 6–8; PMN/CBC 5 No. of PMNs 3 10 2.

282

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

(EB) uptake in the lung: EB in serum. Total MMP-2 and -9 were isolated from tissue homogenate by heparin sepharose affinity chromatography and activity assayed by scanning densitometry of gelatin zymograms and reported as arbitrary units (AU) of density. Data is reported as mean 6 SEM. Statistical analysis was performed using Students t-test. P values ,0.05 were considered statistically significant. Liver necrosis (2.5 6 0.3 vs 0.007; p , 0.05) and PMN infiltration (6.5 6 1.2 vs 1.5 6 0.4; p , 0.05) were significantly increased in hepatic I/R animals compared to controls. Lung injury as measured by MPO activity (4407 6 206 vs 384 6 22; p , 0.05) and PI (0.2 6 .005 vs 0.05 6 .002; p , 0.05) were similarly increased in hepatic I/R animals (see table). Hepatic I/R animals had increased (p , 0.05) expression of total

TABLE—ABSTRACT 118 Lung

Control Hepatic I/R

Liver

MMP-2

MMP-9

MMP-2

MMP-9

361 6 61 635 6 69

109 6 50 334 6 35*

728 6 22 1114 6 109*

19 6 10 831.2 6 257*

* P , 0.05. MMP-2 and -9 in lungs and liver compared to controls. Our results suggest that MMP-2 and -9 may mediate PMN migration into the lung and liver following hepatic I/R as well as subsequent alveolocapillary membrane destruction and hepatocellular injury. 119. Effects of Aging on Gut Responses to Severe Burn. S. E. Wolf, M.D., M. A. DebRoy, M.D., M. G. Jeschke, M.D., and J. C. Thompson, M.D. Department of Surgery, University of Texas Medical Branch, Galveston, Texas. Introduction. Aging increases complications after severe burn. Cutaneous burns are associated with gut mucosal atrophy and increased gut epithelial cell death by apoptosis. We wondered whether aging affects the balance between gut epithelial cell growth and cell death after injury. Methods. Thirty-six pair-fed 2 month old (young) and 24 month old (aged) C57BL-6 mice underwent 30% TBSA burn (B) or sham burn (SB). Mice were sacrificed 12, 24 and 48 h later. Small bowel mucosal weights and gut epithelial apoptosis and proliferation were analyzed. Apoptosis was measured by counting gut epithelial cells with fragmented DNA (TUNEL), and proliferation for cells expressing proliferative cell nuclear antigen (PCNA). Four young and aged mice were sacrificed without intervention as controls. x 2 and one-way ANOVA was used for statistical analysis. Results. Mortality was higher in both B and SB groups of aged mice compared to young mice (p , 0.05). Mortality rates were not different between B and SB in either the aged or young. Mucosal weights indexed to total body weights increased 12 h after burn in both aged and young B groups compared to controls (p , 0.05). Gut epithelial apoptosis was increased at 12 h in both young and aged mice after burn compared to controls (p , 0.05), while gut epithelial proliferation was not different. Conclusions. Mortality increased in the aged mice compared to the young, however, the response of increased gut epithelial apoptosis was maintained. We conclude that gut responses to injury are preserved with aging. We speculate that effects outside the gut are responsible for higher morbidity and mortality in the aged after severe burn. 120. Cyclooxygenase-2 (COX-2) Inhibitors Attenuate Serum Stimulated Proliferation of Gut Derived Smooth Muscle Cells. E. M. Grossmann, M.D., D. L. Kaminski, M.D., J. E. Mazuski, M.D., Ph. D., and W. E. Longo, M.D. Department of

Surgery, St. Louis University School of Medicine, St. Louis, Missouri. COX-2 inhibitors have been shown to decrease intestinal epithelial cell proliferation. The effect of these compounds on intestinal smooth muscle remains poorly defined. We have previously demonstrated that the selective COX-2 inhibitors decrease prostaglandin E 2 release from a human intestinal smooth muscle cell line (CRL-1692). The present study was undertaken to determine the effect two selective COX-2 inhibitors (SC-58125 and NS-398) on proliferation of CRL-1692 cells. Methods. 70% confluent cells were exposed for 24 h to media that was serum-free, serum supplemented, or serum supplemented with either SC-58125 or NS398. Proliferation was determined by 3 H-thymidine incorporation (CPM 6 SEM, n 5 5). ANOVA was employed for statistical analysis. To evaluate the effect of SC-58125 on apoptosis, 5x10 6 cells/ well were incubated for 96 h with or without SC-58125 and the ratio of apoptotic floating cells was quantified (apoptotic cells 4 total cells 3 100) (see table). Results. SC-58125 and NS-398 decreased 3 H-thymidine incorporation in a dose dependent fashion. SC-58125 was demonstrated to increase the proportion of floating apoptotic cells. Conclusions. Proliferation of gut derived smooth muscle is decreased by COX-2 inhibitors. This effect may be mediated by the induction of apoptosis.

TABLE—ABSTRACT 120 SC-58125 Txt CPM % Apoptosis

Serum free

Serum

10 mg/ml

50 mg/ml

669 6 33* 3171 6 318 2402 6 276* 1519 6 251* 34.4 11.1 21.0 55.5

* P , 0.05 vs serum.

121. Role of Stasis and Oxidative Stress in Ileal Pouch Inflammation. K. O. Shebani, M.D., A. F. Stucchi, Ph.D., J. McClung, M.S., E. Beer, B.S., W. LaMorte, M.D., Ph.D., M.P.H., and J. M. Becker, M.D., FACS. Department of Surgery, Boston University School of Medicine, Boston, Masschusetts. Pouchitis remains a significant post-operative complication following ileal pouch-anal anastomosis and it has been suggested that stasis and/or oxidative stress may be important etiologic factors. The goal of this study was to test these hypotheses in a rat model. Methods. Lewis rats underwent the construction of an ileal U-pouch proximal to the ileocecal junction without colectomy. Treated animals were fed vitamin E (Vit E; 1000IU/kg), allopurinol (Allo; 15mg/kg/d), or a combination of both (Allo 1 E). After 30 days, mucosa was obtained from the ileal U-pouch and myeloperoxidase (MPO) (units/gm) activity was assessed as a marker of inflammation. Results. Serial radiographic studies over a 48 h period and the presence of semi-solid intestinal contents in all pouches indicated chronic stasis. Mucosa from operated (Op) ileal U-pouches was grossly normal, but had significantly increased MPO activities compared with non-operated ileum, allopurinol, vitamin E or both (see table). Conclusions. 1) Ileal pouch stasis was associated with biochemical evidence of inflammation; 2) Antioxidants significantly reduced MPO activity suggesting a potential role for oxidative stress in the etiology of pouchitis; and 3) These data suggest a potential role for antioxidants in the treatment of pouchitis. 122. Intestinal Fatty Acid Binding Protein: A Marker of Intestinal Ischemia in Peritoneal Fluid. J. Arcuni, M.D.,* G. Ereso, B.A., M.S.,* R. Franson, Ph.D.,* and R. E. Sonnino,

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 121 Operated ileal U-pouch

MPO

Non-op ileum, n 5 3

No Rx, n 5 12

Allo, n 5 7

Vit E, n 5 7

Allo 1 E, n 5 7

0.17 6 0.05

19.1 6 4.1*

4.4 6 1.0

7.2 6 0.7

5.1 6 1.5

Note. Mean 6 SEM. * P , 0.05 compared with all other groups. M.D.† †The University of Kansas School of Medicine, Kansas City, Kansas; and *The Medical College of Virginia, Richmond, Virgina. There is no reliable biochemical marker for intestinal ischemia. Experimental studies by radioimmunoassay have shown elevated serum levels of human intestinal fatty acid binding protein (hIFABP) during intestinal ischemia. The purpose of this study was to test a newly developed ELISA, determine whether hI-FABP could be measured in human peritoneal fluid and whether it could reliably predict the presence of intestinal pathology. Methods. Sample collection: With IRB approval, random samples of peritoneal fluid were collected from pediatric and adult patients with either no bowel pathology (n 5 11) or intestinal ischemia of variable degrees (n 5 5), including incarcerated bowel (1), appendicitis (1), ischemic bowel (2) and frankly necrotic bowel (1). ELISA: The assay was developed using specific, affinity-purified antibodies raised against purified recombinant hI-FABP in male New Zealand White rabbits. Microtiter plates were coated overnight at 4°C with purified hI-FABP, and blocked with 5% milk protein in isotonic saline. The samples or hI-FABP standards were pre-incubated for 30 min with an equal volume of antibody. The plates were decanted and reaction mixture (sample 1 antibody) was added to the wells and incubated for 90 min. The wells were washed, the secondary antibody was added and allowed to incubate for 90 min. After washing again, substrate (TMB) was added and incubated for 20 min. The reaction was stopped and the absorbance was read at 450 nm. The ELISA showed no cross-reactivity with other serum or peritoneal fluid components. The mean analytical recoveries were 99% and 95%, respectively. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The standard curve was linear between 2.7 ng/ml and 1,000 ng/ml (r 5 0.97). Results. hI-FABP levels in in patients without intestinal pathology were , 2.71 ng/ml (the limit of detection of our assay), while in patients with intestinal pathology they averaged 155.17 6 94.43 ng/ml (range 20.42 to 527.74 ng/ml). The difference was highly significant (p , 0.0025). Conclusions. These findings demonstrate the presence of hI-FABP as a marker of injured bowel in peritoneal fluid, and strongly suggest a positive correlation between hI-FABP levels and degree of intestinal ischemia. No correlation with patient outcome has been made in this study and warrants further investigation. Since the assay may be shortened considerably to meet clinical needs, this ELISA may offer a method of early detection of intestinal ischemia, leading to treatment of the underlying pathology before the onset of irreversibile injury.

PARALLEL SESSION IV Cardiothoracic II/Peripheral Vascular II 123. Extracorporeal Circulation Exacerbates Microvascular Permeability after Endotoxemia. C. S. Cox, M.D., S. J. Allen, M.D., D. A. Butler, M.D., and J. Frederick, B.A. Extracorporeal life support without prior inflammatory stimuli results in a modest increase in microvascular permeability. Clinically, initiating ECLS after shock, sepsis or hypoxia results in

marked increases in interstitial fluid and total body water. We sought to determine if an inflammatory stimulus prior to initiating ECLS exacerbates microvascular permeability to protein. Methods. An anesthetized canine model was used for this study (n 5 12). One group received LPS (1 mg/kg) 1 hour before ECLS, and the control group did not. To determine mesenteric microvascular permeability, a mesenteric lymphatic was cannulated and mesenteric venous pressure was elevated to 3261 mm Hg to reach a minimal lymph protein concentration (C L). With simultaneous measurement of plasma protein concentrations (C P), the reflection coefficient, s, was calculated using the formula: s 5 1 2 C L/C P. Transvascular protein clearance, and intestinal tissue water were all measured using standard techniques. Normothermic, atrial to aortic ECLS (flow 5 75-80 ml/kg/ min) was initiated after a steady state was achieved, and ECLS was continued for 2 h. and then discontinued. Measurements were repeated 30 min after ECLS was discontinued. In another group, LPS was given without ECLS to determine if LPS alone altered microvascular permeability. Comparisons were made using ANOVA and t-tests where appropriate. Results. LPS alone did not alter s. s decreased significantly from .77 6 .02 to .53 6 .07 with LPS/ECLS, and was lower compared to ECLS alone (.77 6 .02 to .65 6 .03). Transvascular protein clearance increased significantly from 266646 to 8196125ml/min in LPS/ECLS compared to ECLS alone (from 284649 to 568688ml/min). Ileal tissue water increased in LPS/ECLS (84.86.5%) compared to ECLS (83.16.8%), (NS). Conclusions. Initiation of ECLS after endotoxemia exacerbates the modest increase in microvascular permeability associated with ECLS alone. 124. Dexamethasone Inhibits Phosphorylation of Retinoblastoma Protein in the Suppression of Vascular Smooth Muscle Cell Proliferation. T. Reil, M.D., R. Sarkar, M.D., Ph.D., V. Kashyap, M.D., and H. Gelabert, M.D. Division of Vascular Surgery, UCLA School of Medicine, Los Angeles, California. Dexamethasone (DEX) suppresses neointimal hyperplasia and proliferation smooth muscle cells (SMC) by inducing a late G1-phase cell cycle arrest. Phosphorylation of retinoblastoma protein (Rb) regulates cell proliferation by controlling progression from G1 to S-phase. We hypothesized that DEX inhibits human vascular SMC proliferation and causes cell cycle arrest through inhibition of Rb phosphorylation. Methods. Human aortic SMC were cultured and treated with incremental doses of DEX. Cell counts and [ 3H]thymidine uptake were determined after 72 h. To examine the effects of DEX on cell cycle, cells were synchronized by serum deprivation, re-stimulated to enter G1-phase, treated with 10 25 M DEX, and protein extracted at sequential time points. Western blots were performed to examine Rb phosphorylation. Flow cytometry was performed to track cell cycle progression. Results. DEX inhibited smooth muscle cell proliferation (see figure A) and DNA synthesis in a concentration-dependent fashion. Flow cytometry indicated DEX causes cell cyle arrest in G1-phase. DEX inhibited the phosphorylation of Rb protein compared to control (see figure B). Conclusions. DEX inhibits the proliferation of human vascular SMC by inducing G1-phase cell cycle arrest. DEX inhibited the phosphorylation of Rb,

284

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

a key step in the progression of the cell from G1 to S-phase. Elucidation of the mechanism of DEX may be helpful in treatment strategies for preventing neointimal hyperplasia as well as other disorders of cell proliferation. 125. Opening of Potassium Channels Protects Mitochondria from Calcium Overload. J. A. Crestanello, M.D., N. M. Doliba, Ph.D., A. M. Babsky, Ph.D., K. Niborii, M.D., M. Osbakken, M.D., Ph.D., and G. J. Whitman, M.D. Division of Cardiothoracic Surgery University of Maryland, Baltimore Maryland; and the Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania. One of the mechanisms involved in ischemic preconditioning (IPC) is opening of mitochondrial (mito) potassium channels. This activation would theoretically decrease mito Ca 21 overload during reperfusion (RP). The purpose of this experiment was to evaluate the effects of channel openers (KCO) on mito function and to determine whether it protects mito from Ca 21 overload in an isolated mito preparation. Mito was isolated from rat hearts by differential centrifugation (n 5 5/group). Mito respiratory function was measured by polarography without (Control) or with a KCO (pinacidil,10mM) in the presence of physiologically high Ca 21 concentration (5310 27M) to simulate Ca 21 overload during RP. State 2: oxygen consumption with substrate only; State 3: oxygen consumption stimulated by ADP; State 4: oxygen consumption after cessation of ADP phosphorylation, and respiratory control index (RCI: ratio of state 3 to 4) were measured. Paired and non paired t test were used (see table). KCO depolarizes mito membrane as evidenced by significantly decreased RCI at baseline (Ca 21: 0M) and by decreased State 3 (NS). This depolarization prevents Ca 21 influx through voltage sensitive channels and protects mito from Ca 21overload as evidenced by improved State 3 and RCI compared to control (Ca 21: 5310M). We conclude that opening of potassium channels protects mito from Ca 21overload and that this mechanism may play an important protective role in IPC. 126. The Incorporation of Fibronectin into Fibrin Matrices Modulates Intracellular Signaling Events in Cells That Express the Integrin a5b1. S. A. Corbett, M.D. UMDNJRobert Wood Johnson Medical School, New Brunswick,New Jersey. Background. Retraction of the blood clot by nucleated cells

contributes both to hemostasis and to tissue remodeling. While plasma fibronectin (FN) is a key component of the clot, its role in clot retraction is not defined. Our previous work has demonstrated that the incorporation of FN into fibrin matrices improves clot retraction by nucleated cells. We have hypothesized that FNfibrin clots may support distinct intracellular signaling events. Methods. Chinese hamster ovary cells expressing the integrin a5b1 (CHO-a5) were cultured in either fibrin or FN-fibrin clots for 24 h at 37°C. Clot retraction was measured by detaching the clots from the dish and calculating the surface area at individual time points. Alternately, the cell lysates were analyzed by SDS-PAGE and immunoblotting with an anti-phosphotyrosine antibody or with an antibody against focal adhesion kinase to confirm equal loading. Results. CHO-a5 cells in FN-fibrin matrices demonstrated significantly increased clot retraction when compared to cells cultured in fibrin matrices (see figure, panel A; ‡ indicates p , 0.002, Student’s unpaired t-test). As demonstrated in panel B, FN-fibrin clots (1) support increased levels of protein tyrosine phosphorylation when compared to fibrin clots (2). Conclusion. The incorporation of FN into a fibrin matrix promotes clot retraction by cells that express the integrin a5b1. The ability of cells to generate tractional forces in FN-fibrin clots may be associated with increased intracellular protein tyrosine phosphorylation. This provides strong evidence that a5b1-FN interactions may contribute to early wound remodeling.

127. Characterization of Dopamine Responses in Experimental Vein Grafts. M. G. Davies, M.D., T. T. T. Huynh, M.D., and P-O. Hagen, Ph.D. Department of Surgery, University of Rochester, New York; and Duke University Medical Center, Durham, North Carolina. Background. Dopamine is a commonly used inotropic agent during coronary artery surgery and in the medical therapy of a previously revascularized patient. The responses of intimal hyperplastic vein grafts to dopamine are examined in this study. Methods. The in vitro isometric tension responses to dopamine (DA) of common carotid jugular vein bypass grafts in New Zealand White rabbits were determined and compared to those obtained in the jugular vein and in the common carotid artery. Both endothelialized and denuded vessels were precontracted with prostaglandin F2a. The contributions of nitric oxide and prostanoid were also

TABLE—ABSTRACT 125 Ca 21, 5 3 10 27 M

Ca 21, 0 M

Control KCO

State 2

State 3

RCI

State 2

State 3

RCI

6.1 6 0.7 8.2 6 1

44 6 4 39 6 5

5.8 6 0.5 4.1 6 0.1*

18 6 2** 27 6 2* ,**

24 6 2** 52 6 8* ,**

1.2 6 0.03** 1.7 6 0.2* ,**

Note. Values are means 6 SEM. * P , 0.05 vs control; ** P , 0.05 vs Ca 21, 0 M.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT 127

Control Denuded

Jugular vein

Vein graft

Carotid artery

7.86 6 0.08 7.82 6 0.15

6.62 6 0.22* ,*** 8.91 6 0.09** ,†

7.89 6 0.47 7.13 6 0.15*

Note. Mean 6 SEM (2log 10[IC 50]); * P , 0.05, ** P , 0.001 vs. jugular vein; *** P , 0.05.† P , 0.01 vs carotid artery by ANOVA with post hoc Tukey-Kramer testing.

assessed. Results. Each vessel had a biphasic response to DA with relaxation followed by contraction. DA relaxation in the jugular vein and carotid artery was equal and was significantly greater than the vein graft (see table). After endothelial denudation, DA mediated relaxation of the vein graft was significantly enhanced and was greater than either the jugular vein or the carotid artery. Denudation had no effect on DA relaxation in the jugular vein, while that in the carotid artery was significantly decreased. Preincubation with L-NMMA (to block NO synthesis) or with indomethacin (to block cyclo-oxygenase activity) had no effect. Addition of phenoxybenzamine, an a-adrenergic antagonist, enhanced DA relaxation in the jugular vein and depressed the relaxation in the carotid artery. There was no effect on the DA response in the vein graft. Jugular vein, carotid artery and vein graft responded to DA with pertussis toxin insensitive responses. In the presence of cholera toxin, DA relaxation was inhibited in the vein and artery but in contrast it was enhanced in the vein graft Conclusion. DA relaxation in vein grafts is diminished compared to native vessels due to an endothelium dependent, a-adrenergic, cholera toxin-sensitive G-protein activated pathway. 128. The Effect of RAS N17 Transfection on ECM Stimulated VSMC Migration. A. I. Willis, M.D., X. J. Wang, M.D., H. Kito, M.D., N. Azuma, M.D., E. Chen, B.A., G. P. Tuszynski, Ph.D.,* B. E. Sumpio, M.D., Ph.D., and V. Gahtan, M.D. Yale University School of Medicine, Department of Surgery, New Haven, Connecticut; and *MCP/Hahnemann University, Philadelphia, Pennsylvania.

285

t-test, n 5 3. Results. TSP, Fn, and Vn stimulation significantly increased VSMC migration. Ras N17 transfection inhibited VSMC migration induced by TSP by 57.3% (p , 0.05), Fn by 38.1%, and Vn by 35.1% versus their respective pcDNA positive controls (see figure). Conclusions. These results show that Ras N17 transfection more strongly inhibits TSP induced VSMC migration. This suggests that Ras plays a greater role in TSP induced VSMC migration than in that of Fn and Vn. 129. Cytoskeletal Changes in Inflammation Are Prevented by Adenosine A1 Receptor Pathway. B. A. Laureano, M.D., T. E. Dahms, Ph.D., and T. A. Miller, M.D. Department of Surgery and Anesthesiology, St. Louis University Health Science Center, St. Louis, Missouri. Cytoskeletal changes directly influence endothelial permeability. Adenosine is known to have anti-inflammatory effects which act through a G-protein coupled mechanism. Using an endothelial monolayer, this study determined the influence of adenosine and related molecules on cytoskeletal rearrangements. Endothelial cells confluent on fibronectin coated surfaces were fixed, permeabilized and stained for actin. Confocal micrographic images assessed actin morphology; actin was quantified optically using phalloidin labeling. The role of G-protein was investigated with cholera toxin and pertussis toxin, and that of Adenylate Cyclase (AC) with forskolin. Adenosine analogs for A1 (CHA) and A2 (DPMA) receptors were used to study pathway-specific signaling. Cytoskeletal actin rearranged in the presence of the inflammatory agent, thrombin, with an increase in filamentous (F)-actin, and enhanced actin density at the cell periphery with formation of gaps between cells. Adding adenosine, CHA, cholera toxin and forskolin prevented these changes in quantity and morphology. DPMA and pertussis toxin had no such preventative effect. We conclude that adenosine has a protective effect on the cytoskeletal changes that take place during inflammation. This protective action appears to involve an A 1 receptor pathway requiring AC activation and G I protein inhibition (n 5 14 ea, statistics by ANOVA) (see figure).

Purpose. To assess the effect of p21 ras (Ras) upon vascular smooth muscle cell (VSMC) migration induced by the extracellular matrix (ECM) proteins thrombospondin-1 (TSP), fibronectin (Fn), and vitronectin (Vn). Methods. Bovine aortic VSMC were transfected with Ras N17 (dominant negative mutant, 1.8 mg/mL) or pcDNA (vector control, 1.8 mg/mL), and b-Gal (transfection efficiency marker, 0.6 mg/mL) plasmids in lipofectamine (15 mg/mL, ;33% transfection rate). Migration studies were performed using a modified Boyden chamber. TSP, Fn, or Vn (20 mg/mL) or serum free medium (SFM) was placed in the bottom chamber and quiescent Ras N17 or pcDNA transfected VSMC were placed in the top chamber. Cells were stained with hematoxylin for total cell counts. Results were recorded as VSMC per 5 fields (4003), and analyzed by paired 130. Development of a Novel Murine Model of Arterial Thrombosis: Experiments Using the b3 Integrin Knockout Mouse. E. D. Reis, M.D.,* S. S. Smyth, M.D.,† H. Va¨a¨na¨nen, M.Sc.,‡ W. X. Zhang, M.D.,* L. Scudder,† and B. S. Coller, M.D.† Departments of *Surgery, †Medicine, and ‡Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York. Models of arterial thrombosis applied in genetically modified mice provide important information on hematologic and vascular dis-

286

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

eases, including Glanzmann thrombasthenia, coronary events, and cerebrovascular and peripheral vascular disease. Mice deficient in b3 integrins (GPIIb/IIIa and avb3) have been generated to study these processes. A model of carotid thrombosis in mice was developed that accurately assessed thrombosis time in response to external application of FeCl 3 in wild-type (1/1) mice, mice heterozygous (1/2) for the b3 integrin, and mice lacking the b3 integrin completely (2/2). Twelve mice, 4 of each genotype, were tested in a blinded fashion. The left common carotid artery was exposed. A silicone elastomer periarterial container was placed on the carotid and filled with 20% FeCl 3 gel solution to induce thrombosis. The time of thrombotic occlusion was determined by assessment of blood flow using a thermistor placed distal to the FeCl 3 solution. Representative carotid specimens underwent histology, immunohistochemistry, and electron microscopy. The time to occlusion in b3 1/1 mice was 8 6 3 min; in b3 1/2 mice, 11 6 1 min; and in b3 2/2 mice, .30 min (p , 0.001 versus 1/1 combined with 1/–). In b3 1/1 and b3 1/2 mice, the carotids showed acute platelet-rich thrombus; in b3 2/2 mice, virtually no thrombus formation was present. The complete lack of b 3 integrins is associated with severe impairment of arterial thrombus formation in this model. This model will be valuable to further investigate the mechanisms of thrombosis in genetically modified mice and to test therapeutic interventions.

in the embryonic ducts is particularly interesting as it may relate to the formation of pancreatic ductal carcinoma. These carcinomas may arise from such “stem” cells in the adult duct. Furthermore, we have, for the first time, identified a possible endogenous mechanism for regulating a previously undefined “stem” cell in the pancreas. 132. Mediators in Neuroblastoma and Hepatocyte Coculture Conditioned Media Alter Apoptosis. E. A. Beierle, M.D., L. F. Strande, M.S., B. D. Geldziler, B.S., and M. K. Chen, M.D. Department of Surgery, UMDNJ, Camden, New Jersey.

PARALLEL SESSION IV

Introduction. Alterations in apoptosis may be responsible for neuroblastoma’s unique properties of spontaneous regression and site specific metastasis. We hypothesize that neuroblastoma cells interact with host tissues to release mediators that affect apoptosis. Methods. Human neuroblastoma cells (NB) and human Chang hepatocytes (CH) are grown in a noncontact, co-culture system, and the media are collected [conditioned media (CM)]. NB and CH are then plated separately with CM. The cells are harvested and cytospins made for immunostaining to measure Bcl-2, FasL, and TNF-a. Apoptosis is identified using the TUNEL method. Data are interpreted with computer image analysis and reported as a mean intensity stain index. The Student’s t-test is used for statistical analysis with significance determined at p , 0.05. Results. Results are reported in the table as means 6 SEM. Conclusions. Mediators in

Pediatrics II/Oncology III

TABLE—ABSTRACT 132

131. TGF-b Regulation of Pancreatic Differentiation: Insights into Stem Cell Regulation and Pancreatic Ductal Carcinoma Formation. C. A. Crisera, M.D., A. S. Kadison, M.D., T. S. Maldonado, M.D., J. B. Grau, M.D., M. Li, M.S., S. L. Alkasab, B.A., M. T. Longaker, M.D., and G. K. Gittes, M.D. Laboratory of Developmental Biology and Repair, New York University Medical Center, New York, New York. TGF-b superfamily signaling has been implicated in both pancreatic cancer and pancreatic organogenesis. To better understand the role of TGF-b in pancreatic duct, acinar, and endocrine development, and to better understand how defects in TGF-b signaling may lead to pancreatic cancer, we studied a new transgenic line of mice. In these mice, TGF-b signaling is blocked by the overexpression of a dominant-negative type II TGF-b receptor (DNTbRII). Transgenic mice expressing DNTbRII were bred. Pancreases from the embryos were isolated at serial ages and processed for histology. Embryos were genotyped via Southern blot hybridization using a radiolabeled probe for the transgene. The pancreases were analyzed with H&E and immunofluorescent staining for insulin, glucagon, carbonic anhydrase II (CAII), and amylase. The most striking finding in the homozygotic transgenic E16.5 pancreas was the presence of a hyperproliferative region around the ductules. In this region, there were a large number of unusual cells that stained simultaneously positively for endocrine markers (insulin or glucagon) and a duct marker (CAII). This co-expression of endocrine and duct markers in normal pancreas has been thought to represent stem cells in the duct that could give rise to more pancreatic tissue after injury, or in the event of b-cell destruction in diabetes mellitus. The proliferation of these “stem” cells in the transgenic pancreas implies a normal regulatory role of TGF-b signaling in maintaining these “stem” cells in the quiescent, non-proliferative state in the lining of the embryonic ducts. Further evidence for TGF-b signaling controlling pancreatic proliferation and differentiation was found in the acini. The acini were dysmorphic. They still stained positively for amylase, but they were generally smaller and had less round cells. These results demonstrate interesting potential roles for TGF-b in controlling the normal state of the pancreas during development. The need for TGF-b signaling to prevent proliferation of the potential “stem” cells

TNF-a FasL Bcl-2 TUNEL

Control NB

CM-NB

Control CH

CM-CH

5.0 6 1.4 17.6 6 4.6 0.3 6 0.1 0.0

0.7 6 1.4 0.02 6 0.01* 7.7 6 2.4* 0.0

15.0 6 3.0 21.4 6 2.3 0.0 0.0

22.5 6 2.8* 33.2 6 7.1* 0.0 1.4 6 0.5*

* P , 0.05, control vs CM. conditioned media resulted in a significant increase in Bcl-2 and a marked decrease in FasL in NB, both of which protect the tumor cells from apoptosis. In contrast, the conditioned media resulted in an increase in FasL, TNF-a, and apoptosis in CH. The CM promoted the ability of NB to avoid apoptosis and induce apoptosis in the host tissue (hepatocytes), thereby providing the tumor with a survival or metastatic advantage. 133. Inhibition of IL-12 Production by Dendritic Cells in Neuroblastoma Bearing Mice. E. M. Barksdale Jr., G. S. Shurin, Ph.D., and M. T. Lotze, M.D. University of Pittsburgh, Pittsburgh, Pennsylvania. Introduction. Interleukin 12 (IL-12), a potent antitumor cytokine released by dendritic cells, induces natural killer (NK) cell activity and cytotoxic T cell (CTL) proliferation. In our previous in vitro studies we demonstrated that human CD 341 stem cells (DC progenitors) cocultured with NB are dysfunctional, while IL-12 transduced DC are potent immunoadjuvants. The purpose of this study was to determine if NB inhibits IL-12 production by DC in tumor bearing mice as a mechanism of immune dysfunction. Methods. A/J mice underwent subcutaneous inoculation with either the highly aggressive TBJ (n 5 8) or the less aggressive N2a (n 5 8) murine NB cell lines or media control (n 5 8). Bone marrow (BM) and tumors were harvested at 14 and 21 days. Morphometric and histologic studies of the tumors were performed. BM derived CD 341 cells were isolated from all groups and cultured with GMCSF and IL-4 for 7 days to stimulate DC differentiation. Phenotypic DC maturation markers were analyzed by FACS,

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS mixed lymphocyte reaction (MLR) was performed to assess DC function, and fibroblasts were transfected with CD40L (IL-12 production assay). Data were analyzed by Student’s t test. Findings. Both TBJ and N2a NB subclones had a significant decrease in CD341 cells in the BM relative to control at 14 and 21 days (p , 0.01). DC markers CD 80 (B7.1) and CD40 were significantly downregulated (p , 0.01) relative to control, while no significant differences were seen in CD 54, Class II, and CD 86, indicative of maturational delay. These APC from the tumor bearing mice were significantly poorer stimulators in MLR relative to control TBJ (p , 0.005) and N2a (p , 0.01). Striking functional changes were also noted in the 7-day cultured DC ability to induce IL-12 production in the fibroblasts assay below measuring IL-12 (pg/ml) (see table). Conclusions. Murine NB inhibits both DC maturation assessed by downregulation of differentiation markers and function as assessed by MLR and IL-12 production. Immunoadjuvant strategies directed at inducing IL-12 may improve prognosis in this tumor.

287

disrupt gut glutathione (GSH) metabolism and this disruption would correlate with mammary cell carcinogenesis. Methods. 64 Sprague-Dawley rats were randomized to the DMBA vs CONTROL group. At age 50 days, rats were gavaged with a one-time dose of 20mg DMBA or sesame oil. Rats from each group were sacrificed at 1 week (n 5 16), 2 weeks (n 5 16), 4 weeks (n 5 16) and 11 weeks (n 5 16). Tumor appearance, arterial and gut GSH concentration and gut GSH extraction were measured overtime. Results are shown in the figure (*p , 0.05, DMBA vs CONTROL, 6 SEM, unpaired t-test. Open: DMBA; Closed: CONTROL). Gut

TABLE—ABSTRACT 133

24 h 48 h

Control

N2a

TBJ

15.14 6 0.5 83.10 6 3.7

10.37 6 0.1 9.18 6 1.0

0.8 6 0.01 0.8 6 0.01

134. Comparison of Telomerase Levels before and after Differentiation of Two Cell Lines of Human Neuroblastoma Cells. C. K. Sanborn, M.D., A. O’Connor, M.D., R. S. Sawin, M.D., K. Moore, Ph.D., M. J. Dehart, B.S., and K. S. Azarow, M.D. Departments of Surgery and Clinical Investigation, Madigan Army Medical Center, Tacoma, Washington. Introduction. This study was performed to determine if the level of telomerase activity in human neuroblastoma cell lines correlated with their level of differentiation. Telomerase is the enzyme that is responsible for maintaining telomere length in human germs cells, tumor cells, and immortalized cell lines. It is undetectable in or very low in most mature somatic cells. We proposed that as neuroblastoma cells differentiated into more mature or benign cells, the levels of telomerase expression would decrease. Methods. Two human neuroblastoma cell lines, SKN-AS and SK-N-DZ, were differentiated using retinoic acid. These cells were assayed for telomerase activity by the telomere repeat amplification protocol (TRAP) before, during, and after treatment with retinoic acid for eight days. Differentiation of the cell lines was confirmed by assaying expression of the RET proto-oncogene using reverse transciptase polymerase chain reaction (RT-PCR) and Northern blot analysis. Results. No statistical difference in telomerase activity was noted between control and treated groups. Conclusion. While telomerase activity has been shown by others to correlate with tumor aggressiveness in human neuroblastoma cells, the mechanism that is involved in this process appears to be separate from cellular differentiation. 135. Effect of DMBA on Gut GSH Metabolism. Y. Cao, M.S., J. Kornbluth, Ph.D., J. Wang, M.D., R. Henry-Tillman, M.D., and V. S. Klimberg, M.D. Surgery, University of AR and VA, Little Rock, Arkansas. One mechanism of the mammary carcinogenesis of 7,12dimethybenz[a]anthracene (DMBA) is thought to be the generation of reactive oxygen species known to play an important role in initiation and progression. We hypothesized that DMBA would

GSH extraction (normally negative; production) was significantly depressed over the time points, even showing uptake (positive extraction) at week 1 and 2. Tumors developed in all animals in the DMBA group by 11 weeks. Conclusions. A one time oral administration of DMBA has a significant and prolonged depressive effect on gut GSH production that has not previously been described. This data supports the hypothesis that the carcinogenic effect of DMBA is mediated, at least in part, by oxidative damage and that the disruption of gut GSH metabolism may play a greater role in carcinogenesis than previously realized.

136. b Cell Specific Cytotoxicity Using a Rat Insulin Promoter Thymidine Kinase Construct. T. A. Tirone, M.D., S. P. Fagan, M.D., M. K. Ray, Ph.D., and F. C. Brunicardi, M.D. Department of Surgery, Baylor College of Medicine, Houston, Texas. The ability to express selected genes in a tissue specific manner is an important goal in gene therapy. The purpose of this study was to 1) investigate if use of the rat insulin promoter (RIP) would result in the b-cell specific expression of a transfected gene and 2) induce b-cell specific cytotoxicity using RIP with the thymidine kinase gene (tk). 1) 0.448 kb of RIP was ligated to the reporter gene lac-Z and transfected into several cell lines: insulinoma (NIT-1), embryonic carcinoma (F9), fibroblast (3T3), and lung (H441) cells in vitro. X-gal staining analyzed the lac-Z gene where blue nuclei represented cellular expression. 2) RIP was ligated to tk and transfected into NIT-1 and F9 cells. A hollow vector (v) and tk under the control of a ubiquitous promoter (MC-1tk) were used as negative and positive controls, respectively. The cells were treated daily with ganciclovir (GCV). Cell viability was ascertained on day six using an MTS assay (see figure). The data suggests that the RIP is a b-cell specific promoter. A b cell specific and GCV dose dependent decrease in NIT-1 cell survival was demonstrated with the RIPtk gene. F9 cell death was shown using the MC-1tk gene at similar doses of GCV, but not with the RIPtk gene suggesting that b-cell targeted cell death can be accomplished by the tissue specific expression of tk by RIP.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS and Molecular Medicine, Mount Sinai School of Medicine, The Mount Sinai Medical Center, New York, New York.

137. Activation of Tumor Cell-Derived MMP-2 Is Mediated by PMN Elastase: Implication for Inflammatory Cells in Tumor Progression. D. Whiting, M.D., M. Schursky, B.S., J. D. Schwartz, M.D., P. Mignatti, M.D., and P. Shamamian, M.D. Department of Surgery, S. A. Localio Laboratory for Surgical Research, New York University School of Medicine, New York, New York. MMP-2 degrades many constituents of the basement membrane and extracellular matrix (ECM). Degradation of the ECM is vital for a variety of tissue remodeling processes, including tumor invasion and angiogenesis. MMP-2 is secreted as an inactive proenzyme and activated extracellularly; its activation is an important step in the regulation of MMP-2 activity. In HT-1080 cells, MMP-2 can be activated by plasmin (PL) and PMN-derived elastase. We hypothesized that the phenomenon of PMN-derived serine proteinase activation of MMP-2 is not unique to HT-1080 cells but is a common mechanism for tumor cells. Confluent cultures of MDAMB-231 (human ductal breast cancer) or SK-HEP-1 (human hepatoma) cells were incubated in serum-free media with or without addition of either purified PMN elastase or PL. HT-1080 (human fibrosarcoma) cells were used as positive controls. MMP-2 activity was assessed by gelatin zymography of the conditioned media. HT-1080, MDA-MB-231, and SK-HEP-1 cells express MMP-2 in its inactive (72 kDa) form. Addition of 0.5 mg/ml of elastase resulted in the generation of fully active MMP-2 (62 kDa) in all three cell lines. Addition of PL resulted in the generation of fully active MMP-2 (62kDa) in the HT-1080, MDA-MB-231, and SKHEP-1 cells. These findings indicate that MMP-2 activation can be mediated by PMN elastase in a variety of tumor cells. MMP-2 activation by PMN-derived proteinases indicates the potential involvement of inflammatory cells in the tissue remodeling that occurs during tumor invasion and angiogenesis (see figure).

138. A Novel Immunocytolytic Factor Secreted by Pancreatic Adenocarcinoma. L. P. Angel, M.D., C. M. Divino, M.D., S. T. Brower, M.D., and S. Chen, Ph.D. Division of Surgical Oncology, Department of Surgery, and the Institute of Gene Therapy

Introduction. We have observed a putative immunocytolytic factor secreted by pancreatic adenocarcinoma cell lines that mediates a potent cytolytic effect on lymphocytes and sought to investigate the mechanism of this action and characterize the factor. Methods. Isolated murine splenocytes were incubated with the supernatants of various pancreatic adenocarcinoma cell lines (human, hamster, murine). Cell viability after 48 h was assessed by Trypan blue exclusion and mechanism of cell death evaluated by the TUNEL assay. Results. A marked reduction was present in the viability (%/control) of the target splenocytes after incubation with the conditioned media from hamster PAN-1 (15%), PC 1.0 (22%), Taka-1 p70 (12%), Taka-1 p79 (8%), murine PANCO2 (16%), and human Capan-1 (14%) pancreatic adenocarcinoma cell lines. Significant lymphoid apoptosis was evident at 16 h. The immunocytolytic effect was specific for lymphocytes and not evident with the supernatants of other nonpancreatic tumor cells. Biologic activity of the supernatants was heat sensitive and resides in a 30-50 KD range. Immunoassays excluded the presence of known immunosuppressive cytokines such as TGF-b, IL-10, and TNF-a. Coincubation of Fas-sensitive Jurkat cells with the pancreatic adenocarcinoma supernatant resulted in no killing, and immunoblotting confirmed absence of the 27 KD soluble FasL. Conclusions. These findings suggest pancreatic adenocarcinoma cells secrete a potent cytolytic factor that induces apoptosis in lymphocytes. Potential clinical applications of this protein include its use as a tumor marker for early detection of pancreatic cancer and its implementation in immunomodulatory gene therapy.

SCIENTIFIC POSTERS P1. In Vitro and in Vivo Analysis of the Mouse Clara Cell Secretory Protein Promoter. A. S. Y. Chang, M.D., P. L. Ramsay, M.D., M. J. Reardon, M.D., and F. J. DeMayo, Ph.D. Departments of Surgery, Pediatrics, and Cell Biology, Baylor College of Medicine, Houston, Texas. The Clara cell is a major cell type comprising the epithelial lining of the respiratory and terminal bronchioles in the lung. It is a non-ciliated secretory cell which secretes a 10 kDa protein, the Clara Cell Secretory Protein (CCSP), that has been shown in vitro to have anti-inflammatory properties by the inhibition of phospholipase A 2 and in vivo to have a protective effect on the lung under hyperoxic conditions. Promoter analysis of the CCSP gene has been hampered by the lack of an authentic Clara cell line in which to do the analysis. Studies in H441 cells, a Clara cell-like cell line, have previously been utilized which recognize the proximal promoter elements, while a new cell line generated in our lab, mouse transformed Clara cell (mtCC) cells, recognizes both the proximal and distal promoter elements found to be critical for full CCSP expression in a transgenic mouse model. Transient transfection studies in the mtCC cells have demonstrated that CCSP gene regulation by the cytokine interferon gamma (INFg) and by hyperoxia is mediated, at least partially, at the level of transcription. To further characterize CCSP gene regulation we sought to develop an in vivo mouse model for analysis of Clara cells and CCSP production. Therefore, we generated a transgenic mouse line that expresses green fluorescent protein (GFP) under the transcriptional control of the mouse CCSP promoter. Enhanced GFP (Clontech, Inc.) was fused downstream of the combined distal and proximal mouse CCSP promoter regions and microinjected into pseudopregnant mice. Four lines of mice were generated that incorporated the transgene. These transgenic mice will provide a valuable tool for the isolation Clara cells and further analysis of the regulatory elements involved in CCSP expression. P2. Saphenous Vein Endothelial Cell Viability: A Comparative Study of Endoscopic and Open Incision Saphenoc-

289

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS tomy for Coronary Artery Bypass Grafting. F. Balaya, M.D., S. Alrawi, M.D., G. Alshkaki, M.D., R. Raju, M.D., A. J. Acinapura, M.D., and J. N. Cunningham, Jr., M.D. Departments of Surgery, Maimonides Medical Center and Lutheran Medical Center, Brooklyn, NewYork. Background. Persuasive evidence supports that care be taken not to compromise the integrity of harvested saphenous veins by excessive handling during dissection for coronary artery bypass grafting (CABG). Although endoscopic saphenous vein harvesting (EndoSVH) ostensibly violates this so-called “no touch” axiom, its use as an alternative to standard open incision saphenectomy (StdSVH) is growing. The extent of EndoSVH’s injurious effects (if any) on vein endothelium was the central focus of this study. Methods. Forty-five consecutive patients underwent saphenectomy for CABG. Saphenous vein samples (approximately 1 inch each) were endoscopically removed via a small incision from the knee joint to the inguinal ligament. Through the same incision a small sample of vein was retrieved by no touch technique representing the StdSVH for CABG. Vein samples from each group were divided into 8 subgroups of 5 each, incubated in plasma-lyte solution in combination with or without papaverine, at distending pressures of 100 or 300 mmHg, and at either 4°C or 28°C respectively. A ninth subgroup was preserved with 3% glutaraldehyde as a control. The viability of cultured saphenous vein endothelial cells was assessed following incubation in Dulbecco’s culture medium for 72 h. Results. The z-statistic comparing the proportion of viable endothelial cells maintained in papaverine solution (70.2%) verses those without papaverine (63.7%) was significant (z 5 3.80, p 5 0.001), as were those at 4°C (71.1%) to those maintained at 28° C (62.5%), (z 5 5.03, p , 0.001). Likewise, cells subjected to greater distending pressure (300 mmHg) were generally found to be of greater viability. In contras, the absolute ratio of viable tissue of EndoSVH (66.9%) to StdSVH (68.1%) did not demonstrate significance (z 5 0.80, p 5 0.788). Conclusion. The absence of statistical differences between StdSVH and EndoSVH suggests that both techniques are comparable for CABG, in conduit endothelial tissue viability, and thus would presuppose parity in long-term outcome. P3. Influence of IBS Symptoms on the Outcome of Laparoscopic Anti-Reflux Procedures. D. A. Axelrod, M.D., M. M. Ajluni, F. E. Eckhauser, M.D., and L. M. Colletti, M. D. Department of Surgery, University of Michigan, Ann Arbor, Michigan. Patients with symptomatic gastro-esophageal reflux disease have a co-existing diagnosis of irritable bowel syndrome in approximately 30% of cases which may impact the success of laparoscopic anti-reflux surgery. Methods. A retrospective chart review of all patients undergoing laparoscopic anti-reflux procedures during 1997-98 was performed to identify preoperative symptoms of irritable bowel syndrome and poor outcomes post-operatively. Patients reporting diarrhea, constipation, irregular bowel habits, or a history of IBS were included in the IBS symptoms group. Poor outcomes included a delay in eating requiring tube feeds, dysphagia requiring dilation or re-operation, recurrent cough, recurrent heartburn, or gasbloat symptoms. Results. This study included 80 patients, 56% were women, with an average age of 45.1 years. The average follow-up was 17.1 months. This group included 26 patients (33%) who were identified as suffering from IBS symptoms. The IBS patients were 50% male, and an average age of 43. Overall, 36% of patients reported a post-operative bad outcome. This included delayed eating in 3%, dysphagia 6%, recurrent asthma 2%, recurrent cough 9%, recurrent heartburn 16%, and gasbloat 11%. However, patients with IBS symptoms experienced a bad outcome 47% more often than in cases without IBS symptoms (46% vs 31% p 5 .22). Furthermore, recurrent symptoms (heartburn, cough, asthma) occurred in 30% with IBS symptoms vs 17% in normals (p 5 .16). Conclusions.

IBS symptomatology may be a risk factor for poor outcome after laparoscopic anti-reflux surgery and should be carefully evaluated in a prospective trial.

P4. The Economic Impact of Postoperative Nosocomial Infections: A Prospective Study of Outcomes, Health Care Utilization and Costs. S. Bailey, B.S., J. J. Cullen, M.D., D. Scholz, M.B.A., B. Zimmerman, Ph.D., L. Herwaldt, M.D., and T. M. Perl, M.D. Departments of Surgery, Internal Medicine, and Preventive Medicine, University of Iowa, Iowa City, Iowa; and Johns Hopkins University, Baltimore, Maryland. As cost-effectiveness of hospital care becomes a more important issue, accurate estimates of health care utilization and costs attributable to nosocomial infections (NI) are of increasing importance. The aim of our study was to determine prospectively the outcomes, utilization of health care resources, and hospital costs for patients who acquire postoperative nosocomial infections (NI). Patients who underwent general, cardiothoracic, and neurosurgical operations (n 5 3864) at the University of Iowa Hospitals and Clinics (UIHC) or the VA Medical Center were followed postoperatively for 1 month. Patients were followed twice weekly while hospitalized and were contacted weekly, either in clinic or by phone, for the duration of the follow-up period. NIs were defined using modified CDC criteria. The NI rate was 11.3%. NI increased postoperative length of stay (for inpatients), outpatient antibiotic use, readmissions and inpatient costs at the UIHC (see table). Postoperative NIs increase the cost of medical care and should be taken into account when determining accurate estimates of health care utilization and costs.

TABLE—ABSTRACT P4

NI

Postoperative stay a

Outpatient antibiotic use

Hospital readmission

Total inpatient costs a

1

8.0 (5–14)*

64.8%*

24.2%*

2

5.0 (3–7)

22.2%

8.0%

$8,545* (4,345–17,191) $3,343 (992–6,742)

a

Median (25 th–75 th percentile). * P , 0.001 vs no NI.

P5. Patterns of Care in Patients with Blunt Splenic Injuries at Trauma Centers (TC) Compared To Non-Trauma Centers (NTC). B. Putnam, M.D., D. Abele, R.N., C. Rosati, M.D., D. Kuehler, M.D., and P. Kispert, M.D. Department of Surgery, Albany Medical Center, Albany, New York. Trauma center care should reduce morbidity and mortality associated with severe injuries. We analyzed New York State (NYS) Trauma Registry Data to evaluate patterns of care of 161 patients with splenic injuries treated at TC and NTC from 1996 to 1997. The NYS Trauma Registry is unique in that data is collected for patients cared for at TC and NTC. Transferred patients and DOA were excluded. The rates of splenectomy were found to be slightly higher at NTCs, and non-operative management (NOM) of splenic injuries more often failed in NTC patients (23.5 vs 13.3%). Neither finding was statistically significant. Mortality rates at TC (3.8%) and 4.8% (NTC) were not significantly different. The AIS severity of splenic injuries was similar at TC and NTC, but overall injury severity scores were higher at trauma centers. The time from admission to the emergency department to laparotomy for splenic injuries was longer at NTC (2.9 h) vs TC (1.7 h) [p 5 0.0002, Students paired

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

t-test]. At NTC, patients with splenic injuries who were hemodynamically unstable (BP , 90 S or pulse . 120) were more likely to undergo CT scan than patients at TCs (85% vs 33%, p 5 0.025, Chi-squared analysis). No clear correlation existed between hemodynamic instability and the need for laparotomy in patients with blunt splenic injuries. Variation existed in operation rates for splenic trauma at NTC. Two of 14 NTC included had significantly increased rates of splenectomy compared to TC. No patient in this study died of a spleen-related complication. Although TC care resulted in more rapid access to the OR and a trend toward decreased failure of non-operative management, no differences were noted in mortality. Dissemination of this information using a regional quality assurance process may lead to improvement in the care of patients with splenic injuries. P6. The Effect of Ketorolac Tromethamine (Toradol) on Whole Blood Coagulation. J. I. Kamelgard, M.D., and C. R. Spillert, Ph.D. Department of Surgery, UMDNJ/NJMS, Newark, New Jersey. Postmarketing reports have indicated an apparent increase in the incidence of wound hematomas following preoperative use of Ketorolac Tromethamine (Toradol), a non-steroidal anti-inflammatory drug (NSAID) with opioid analgesic strength. However, Toradol likely remains an effective preemptive analgesic. The purpose of this study was to determine whether a single dose of Toradol would adversely effect the coagulation properties of whole blood from healthy volunteers. Blood samples were drawn from 12 fasting subjects. A single 60mg oral dose of Toradol was administered, thereby achieving blood levels equivalent to those following a single 30mg preoperative I.V. bolus. One hour later, a second blood sample was drawn from each subject. Prothrombin time (PT), partial thromboplastin time (PTT),

troenterology, Kaplan Cancer Center, Bellevue Hospital Center, NYU School of Medicine, New York, New York. An increasing proportion of East Asian patients are being diagnosed with gastric adenocarcinoma. H. pylori (HP) and intestinal metaplasia (IM) are both associated with progression to gastric adenocarcinoma. The purpose of this study is to examine whether a local population of East Asian immigrants is at high risk for premalignant lesions of the stomach and the development of gastric adenocarcinoma. All patients with gastrointestinal tract adenocarcinomas diagnosed January 1996 –December 1998 at Bellevue Hospital Center (BHC), a large urban municipal hospital, were identified. Presentation characteristics were compared between patients with gastric adenocarcinoma and other pathologies using Chi square analysis. Prospective endoscopic evaluation of a cohort of dyspeptic East Asian patients was then performed. Of 40 patients diagnosed with gastric adenocarcinoma, 21 (53%) were East Asian, 10 (25%) Hispanic, 5 (13%) African-American, and 4 (10%) Caucasian. In comparison, during the same time period, East Asians comprised 24% of 192 other patients diagnosed with gastrointestinal adenocarcinomas at BHC (p , 0.05): 3 of 14 (21%) esophageal adenocarcinomas, 24 of 118 (20%) colorectal adenocarcinomas, 5 of 21 (24%) periampullary/biliary adenocarcinomas, and 9 of 13 (69%) hepatocellular carcinomas. In this same time period, approximately 14% of all Dept of Surgery discharges and 9% of all BHC discharges were East Asian (p , 0.05). 67% and 37% of 30 patients with resected gastric adenocarcinoma had IM and HP, respectively. Of 56 East Asian patients undergoing endoscopy for dyspepsia, 43% had evidence of IM and 55% had HP. These data reveal that our local population of East Asians is at increased risk for both gastric adenocarcinoma and pre-malignant changes. This population appears ideal to study preventative and therapeutic measures that may impact on the progression of pre-malignant gastric lesions to adenocarcinoma as well as the biology of this process.

P8. Risk Factors for Mortality and Associated Injuries Following Blunt Splenic Trauma. W. C. Fang, M.D., M.H.Sc., H. L. Collette, R.N., C. Hartigan, B.S.N., and P. E. Bankey, M.D., Ph.D. Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts.

viscosity, and whole blood clotting time were determined. Results were compared for similarity of variance, and the appropriate paired t-test was applied for calculation of p values. P , 0.05 was used as the threshold for statistical significance. All baseline values measured fell within accepted normal ranges for their respective tests. Following administration of Toradol, no significant change was noted for PT, PTT, viscosity, or whole blood clotting time. In conclusion, the results of this study suggest that a single preoperative dose of Toradol may be administered without any resultant adverse effect on whole blood coagulation properties.

P7. Increased Risk of Gastric Adenocarcinoma in East Asian Immigrants. S. G. Marcus, M.D., G. Villanueva, M.D., P. Shamamian, M.D., E. Newman, M.D., J. Ortega, B.A., Y. Lam, B.A., and S. Garbers, M.P.A. Departments of Surgery and Gas-

Splenic laceration is the most common injury following blunt abdominal trauma. This study was designed to explore the risk factors for mortality for patients with blunt splenic trauma. Methods. From 1989 to 1998, 530 consecutive patients admitted to a level I trauma center with the diagnosis of splenic laceration were included in this study. Thirty-six risk factors for each patient were obtained from the Trauma Registry for statistical analysis. Results. The overall mortality rate was 11.9% (63/530). Operative treatment was performed in 31% (164/530) of the patients with total or partial splenectomy. Univariate analysis revealed eleven variables which were statistically significant risk factors for mortality: age, injury severity score (ISS), Glasgow coma score (GCS), initial hematocrit count (HCT), Revised Trauma Score (RTS), grade of laceration, liver laceration, hemothorax, pneumothorax, subdural hematoma, and subarachnoid hemorrhage. Multiple logistic regression confirms that age, ISS, GCS, RTS, and HCT were statistically significant independent predictors for mortality (p , 0.05). Splenic laceration was associated with a pelvic fracture 21.9% (116/530), liver laceration 19.4% (103/ 530), gastrointestinal tract injury 14.7% (78/530), renal injury 12.8% (68/530), and hemopneumothorax 30% (161/530) of the time. Trauma/Injury Severity Score (TRISS) was found to be an accurate predictor for mortality with the area under Receiver Operating Characteristic curve of 0.94. Conclusion. Multiple risk factors for mortality and associated injuries following blunt splenic trauma were identified. The recognition of risk factors should improve trauma system efficiency and patient outcomes.

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P11

P9. Withdrawn per author request.

P10. TIPS in Alcoholic and Non-Alcoholic Cirrhosis. J. Ayerdi, M.D., S. Leon, M.D., S. K.Gupta, M.D., and C. Joy, M.D. Department of Surgery, Robert Packer Hospital, Sayre, Pennsylvania. Transjugular intrahepatic portosystemic shunts (TIPS) are being used more frequently in the treatment of patients with both alcoholic and non-alcoholic cirrhosis. Traditionally, however, the etiology of cirrhosis has played an important role in dictating the appropriate type of portosystemic shunt procedure. In the present study, we evaluated the importance of the etiology of cirrhosis on the clinical outcome after non-selective portosystemic shunting with TIPS. Twenty patients with alcoholic cirrhosis (Group I), were compared to 17 patients with non-alcoholic cirrhosis (Group II). TIPS were placed for variceal hemorrhage and ascites. ChildPugh class was similar in both groups. Both groups had similar reductions in portal vein pressures and portosystemic gradients. Portocaval gradients were 9.9 6 1.9 mm Hg (Group I) and 10.1 6 4.5 mm Hg (Group II) (p 5 0.89) after the procedure. TIPS were successful in controlling hemorrhage in all cases. One patient in each group developed recurrent variceal hemorrhage. Ascites improved in 50% of patients in Group I and 66% in Group II. Two patients in Group I and four patients in Group II developed encephalopathy requiring hospitalization. Overall shunt patency was 90% in Group I and 100% in Group II at one year. Three patients in Group I and one patient in Group II required shunt revision. The survival rates at one year were 80% and 100% for Groups I and II, respectively. We found no statistically significant differences with respect to portal pressure decompression, shunt patency, complication, and survival rates in alcoholic and nonalcoholic cirrhotic patients. We conclude that TIPS is safe and effective in treating patients with both alcoholic and non-alcoholic cirrhosis.

P11. Laparoscopic Knot Tying Techniques: A Comparision of Performance and Preference among Surgical Residents. N. T. Nguyen, M.D., H. S. Ho, M.D., K. L. Mayer, M.D., R. J. Bold, M.D., L. S. Palmer, B.S., and B. M. Wolfe, M.D. Department of Surgery, University of California Davis Medical Center, Sacramento, California. Laparoscopic suturing and knot tying are an integral part of advance laparoscopic surgery training. The Endostitch (USSC) was introduced to facilitate the performance of laparoscopic suturing and knot tying. The aim of this study was to evaluate the performance and preference of surgical residents with regards to four different types of laparoscopic knot tying techniques. Thirtynine surgical residents were evaluated as they performed laparoscopic knot tying using either conventional laparoscopic instruments (Conv) or the Endostitch (Endo) in a laparoscopic simulator. There were 24 junior (PGY 1-2) and 15 senior residents (PGY 3-6). Time to completion of four knots and preference of knot tying technique were recorded, comparing the instruments used as well as intracorporeal (Intr) versus extracorporeal (Extr) knot tying (see table). Surgical residents performed laparoscopic knot tying faster using the Endostitch compared to conventional laparoscopic instruments. Senior residents performed knot tying faster than junior residents did on 3 of 4 different types of laparoscopic knot tying techniques. The overwhelming preferred method of knot tying among both senior and junior residents is intracorporeal technique using the Endostitch.

Time (s) Overall Senior Junior

Conv/Intr

Conv/Extr

Endo/Intr

Endo/Extr

185 6 84 153 6 67† 205 6 88

102 6 35* 100 6 37 104 6 35

107 6 49* 82 6 33† 121 6 52

76 6 22** ,† 67 6 17† 81 6 23

First choice preference (number of residents) Overall Senior Junior

4 2 2

2 0 2

28 12 16

2 0 2

* P , 0.05 vs Conv/Intr; ** P , 0.05 vs Conv/Extr; *** P , 0.05 vs Endo/Intr; † P , 0.05 senior vs junior group; determined by two-tail t tests.

P12. Comparison of Edited Videotape to Direct Observation to Assess Operative Performance after Laparoscopic Skills Training. D. J. Scott, M.D., P. C. Bergen, M.D., D. M. Euhus, M.D., W. A. Guo, M.D., Ph.D., D. R. Jeyarajah, M.D., R. Laycock, M.D., R. V. Rege, M.D., S. T. Tesfay, R.N., W. M. Thompson, M.D., R. J. Valentine, M.D., and D. B. Jones, M.D. Southwestern Center for Minimally Invasive Surgery, UT Southwestern Medical Center, Dallas, Texas. Global assessment by direct observation has been validated for evaluating operative performance of surgery residents after formal skills training, but is time consuming. The purpose of this study was to compare global assessment performed from edited videotape to scores from direct observation. Junior surgery residents (n 5 22) were randomized to 2 weeks of formal video-trainer skills training or control. Laparoscopic cholecystectomy was performed at the beginning and end of rotation and global assessment scores were compared for training and control groups. Laparoscopic videotapes were edited: initial (2 min.), cystic duct/artery (6 min.), and fossa dissection (2 min.). Two raters performed both direct observation and videotape assessments and scores were compared for each rater and for inter-rater reliability using a Spearman correlation (perfect correlation 5 1). Correlation coefficients for videotape versus direct observation for 5 global assessment criteria were less than 0.33 for both raters (NS for all values). The correlation coefficient for interrater reliability for the overall score was 0.57 (p 5 0.01) for direct observation versus 0.28 (NS) for videotape. The trained group had significantly better overall performance than the control group per assessment by direct observation (p 5 0.02) but not by videotape assessment (NS). Direct observation demonstrated improved overall performance of junior residents after formal skills training on a video-trainer. Global assessment from an edited 10-min videotape did not correlate with direct observation and had poor inter-rater reliability. Efficient and valid methods of evaluating operative performance await development. P13. Increasing Advance Directive Use among Patients Undergoing High-Risk Operations. P. Angelos, M.D., Ph.D., and C. Johnston, B.A. Department of Surgery, Northwestern University, Chicago, Illinois. Since 1991, all patients entering Medicare-reimbursed institutions are required by law to be asked if they have an advance directive (AD) and be given information about ADs if they do not have one. ADs (e.g., a living will or durable power of attorney for health care) are documents that allow patients to extend autonomy over their health care decisions into those periods when they are no longer competent to make decisions. Little attention has

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

been paid to AD use among patients undergoing elective high-risk operations. Methods. Patients being explored for possible pancreatico-duodenectomy or esophagectomy, which are both “high-risk” operations, between 1996 and 1998 at a university teaching hospital were identified. Review of these patients’ medical records was undertaken to determine whether they had an AD and what impact the presence of an AD had on their care. Results. Between 1996 and 1998, the number of patients undergoing whipples or esophagectomies who have ADs has significantly increased (see table). As shown in the table, even though AD use by

TABLE—ABSTRACT P13 Advance Directive Use in High Risk Procedures

1996 1997 1998

Whipple or esophagectomy

AD declared by the patient

AD present in chart

43 38 39

3 2 14

2 1 1

patients has increased, few patients actually bring a copy of the AD to the hospital when having surgery. Despite the increasing use of ADs, in only one medical record was the presence of an AD noted by the attending surgeons or the housestaff caring for the patient. Conclusions. AD use among patients who might be best able to benefit from them is increasing. In order to be prepared to care for such patients, surgeons must become more familiar with the implications and limitations of ADs on the care of their patients. P14. Student Reaction to the Inclusion of a Surgery Resident in a Medical Anatomy Course. E. S. Lee, M.D., I. B. Harris, Ph.D., S. M. Santilli, M.D., Ph.D., and R. L. Shew, Ph.D. University of Minnesota, Minneapolis, Minnesota. Although gross anatomy has always been a core basic science discipline, lecture and dissection time available for students has decreased over recent years. The purpose of this study was to evaluate student reaction to a surgery resident introducing anatomic detail important in clinical practice. A general surgery resident served as a co-teacher in an accelerated summer anatomy course for 27 medical students who were chosen based on their superior qualifications. The resident attended all student lectures and laboratory dissections. At the conclusion of the course, surveys were distributed to gauge student reaction to a surgical resident offering clinical relevance in the medical anatomy course. Student reaction was excellent with a mean rating of 1.37 (1-Excellent and 5-Poor). Students were equally positive about each aspect of the resident’s teaching: participation in the anatomist’s lectures, co-teaching in the laboratory dissection groups, and the resident’s weekly clinical perspectives lecture. The initial impact appears to be that students are more motivated to study anatomic structures. The surgery resident also reported benefits derived from reviewing anatomy and preparing the lectures. This approach, pairing anatomist and surgical resident for teaching, provides mutual benefits. Continuing to offer this team teaching opportunity enhances medical education to the students while challenging surgery residents to review their anatomy. P15. Outcome and Quality of Life after Severe Acute Pancreatitis. A. Soran, M.D., L. Chelluri, M.D., K. K. W. Lee, M.D., and S. A. Tisherman, M.D. Departments of Surgery and Anesthesiology/CCM, University of Pittsburgh, Pittsburgh, Pennsylvania.

Patients with severe acute pancreatitis (AP) often require intensive care unit (ICU) admission, have multiple complications, spend months in the hospital, and consume a large amount of resources. The aim of this study was to evaluate the ICU course, costs, mortality, and quality of life of patients who require ICU admission for AP. Methods. Patients with AP requiring ICU admission were identified retrospectively. Data regarding in-hospital morbidity, mortality, and hospital costs was obtained. Long-term quality of life was assessed using the Short Form-36 Health Survey (SF-36). Results. 52 patients were identified: 31 men, 21 women; mean age 53 years (range, 22– 89). The most common causes of AP were gallstones (44%) and alcoholism (17%). Pulmonary failure (52% required mechanical ventilation) and renal failure (21% required dialysis) were common. There were 39 (75%) hospital survivors and 13 (25%) non-survivors. In the first 24 h, the mean Acute Physiology and Chronic Health Evaluation (APACHE) II scores were 1066 in survivors and 1664 in the non-survivors (,0.01). Length of ICU (15618 and 28631 days) and hospital (40634 and 38634 days) stays were similar between survivors and non-survivors, respectively (NS). The mean hospital cost for survivors was $83,611688,434 and for non-survivors was $136,730695,045 (p 5 0.09). The estimated cost to obtain one hospital survivor was $129,188. Of the 39 hospital survivors, 5 died later, 21 completed the SF-36, and 13 were lost to follow-up. Longterm quality of life (SF-36) was similar to that of an age-matched population. Twenty of 21 felt their general health was at least as good as it had been one year previously. Conclusions. Patients with severe AP need prolonged ICU and hospital stays. APACHE II may be a good predictor of outcome; prospective evaluation is needed. Although resource utilization is high, most patients survive and have good long-term quality of life. P16. Nitrous Oxide May Not Be a Safe Alternative to Carbon Dioxide for Prolonged, Complex Laparoscopic Surgery. A. E. Roja, M.D., H. S. Ho, M.D., L. S. Palmer, B.S., and B. M. Wolfe, M.D. Department of Surgery, University of California Davis School of Medicine, Davis, California. We investigated the hemodynamic and renal effects of prolonged nitrous oxide (N 2 O) pneumoperitoneum with extensive dissection in a swine model. Eighteen pigs underwent laparotomy, dissection of the mesenteric and renal vessels and had transonic flow probes placed around the SMA and left renal ar-

TABLE—ABSTRACT P16 CI Baseline

100 6 2

SVR

%RBF

UO

1501 6 198 100

0.99 6 0.2

1964 6 348 1449 6 194 1215 6 134

38* ,** 79 78

0.83 6 0.8* 1.20 6 0.7 0.25 6 0.2

1488 6 359 1424 6 169 1234 6 94

6* ,** 69 71

0 (Anuric)* ,** 0.50 6 0.5 0.26 6 0.2

Pneumoperitoneum (at 2 h) Euvol N 2O Hyper N 2O Euvol CO 2

60 6 1* 100 6 1 90 6 1

Pneumoperitoneum (at 4 h) Euvol N 2O Hyper N 2O Euvol CO 2

40 6 1* ,** 90 6 1 90 6 12

Note. CI, cardiac index (ml/kg/min); SVR, systemic vascular resistance (mm Hg/L/min); %RBF, percentage of baseline renal blood flow; UO, urine output (ml/kg/h). * P , 0.05 vs baseline; ** P , 0.05 vs. Hyper N 2O and Euvol CO 2, by ANOVA for repeated measurements. Data are means 6 SEM.

293

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS tery. The abdomen was closed and the pigs allowed to recover before the three groups of pigs (n 5 6 each) underwent either N 2 O or CO 2 pneumoperitoneum. The euvolemic pigs (Euvol) received 3 ml/kg/hr of LR and the hypervolemic pigs (Hyper) had 15 ml/kg/hr of LR. Minute ventilation was adjusted to maintain normal PaCO 2 (35-45 mmHg) and arterial pH (7.35-7.45). Hemodynamics and renal function were monitored for 5 h. Half of the euvolemic N 2 O pigs (3/6 pigs) died at the end of the third hour (see table). The data suggest that extensive abdominal dissection results in a relative hypovolemic state that, in the setting of prolonged N 2 O pneumoperitoneum, leads to severe hemodynamic instability and renal dysfunction. Although intravascular volume expansion may offer some protection from these adverse effects, N 2 O may not be a safe alternative gas to CO 2 for providing pneumoperitoneum during prolonged or complex laparoscopic procedures. P17. Bile Acids Induce Activator Protein-1 (AP-1) in Human Pancreatic Cancer Cell Lines. O. N. Tucker, M.D., J. Mestre, M.D., P. J. Mackrell, M.D., and T. J. Fahey III, M.D. The New York Presbyterian Hospital, New York, New York. Bile acids are known promoters of gastrointestinal cancer. Recent evidence suggests that cyclooxygenase-2 (COX-2) is important in carcinogenesis. We have demonstrated upregulation of COX-2 in human pancreatic adenocarcinoma biopsies, and bile acid induced dose and time dependent increase in COX-2 protein and PGE 2 in vitro in the human pancreatic cancer cell lines, BxPC-3 and Su86.86. This suggests a potential role for bile acids in the pathogenesis of pancreatic cancer. To investigate the mechanisms of upregulation of COX-2 by bile acids, we examined the effects of a tumor promoting phorbol ester, PMA (phorbol 12myristate 13-acetate), and bile acids on COX-2 expression and activity of the transcription factor AP-1. AP-1 has been shown to play a crucial role in tumor promotion in vivo. BxPC-3 and Su86.86 cell lines were treated with deoxycholate (DC), tauro(TCDC) and glycochenodeoxycholate (GCDC), chenodeoxycholate (CD), PMA or vehicle for 2 h in serum free media. 5mg of nuclear protein was incubated with a 32 P-labeled oligonucleotide containing an AP-1 consensus site. The protein-DNA complexes were separated on 4% nondenaturing polyacrylamide gels (see figure).

PMA induced increased binding of AP-1 to DNA. Both unconjugated and conjugated bile acids induced AP-1 DNA-binding activity, which was greater than with PMA treatment. Bile acids and PMA induced a dose and time dependent increase in COX-2. These data suggest that COX-2 induction by bile acids may be mediated via AP-1. Future work will concentrate on identifying other potential transcription factors in bile acid mediated induction of COX-2.

P18. Hyperlipidemia in Acute Pancreatitis: Risk Factors and Outcomes. B. Ngo-Nonga, M.D., and B. Donaldson, M.D. Department of Surgery, Woodhull Medical and Mental Health center, New York, New York. Objectives. To determine the risk factors and outcomes of patients admitted with acute pancreatitis (AP) associated with hyperlipidemia (HL). Setting. Community Teaching Hospital. Patients and methods. We performed a retrospective chart review on 180 patients admitted for AP from Jan. 1995 to Feb. 1998; 35 patients (Group 1) were found on admission to have HL (Group 1). Forty patients were selected randomly from the remaining 145 patients without HL to serve as controls (Group 2). Both groups were compared for risk factors and complications of AP. Ranson’s criteria were used to assess the severity of patients condition on admission. Chisquare and Fisher Exact tests (2-sided) were used for statistical analysis. Results. Both groups were comparable as to age and Ranson’s criteria. Males predominated in Group 1 (94%) over Group 2 (55%). Twenty six patients (74%) in the hyperlipidemic group had alcohol related AP and no patient with gallstone pancreatitis had hyperlipidemia. There were 11 complications (31.4%) in the hyperlipidemic group including: 3 deaths (8.5%), 2 pancreatic abscesses, 3 pseudocysts, 2 severe sepsis and one pneumonia. There were 4 complications (10%) in the control group: one subphrenic abscess, 2 severe sepsis and one delirium tremens. The difference was significant (p 5 0.02, 95% confidence interval). Conclusions. AP associated with HL on admission is most common in men with history of alcohol abuse. Gallstone pancreatitis is not usually associated with hyperlipidemia. This is the first study to show that significant hyperlipidemia in acute pancreatitis is associated with more complications and therefore should be considered a poor prognosis sign. P19. Synthetic Biotinylated Peptide YY(22-36) Binds to Human Pancreatic Cancer Cells. C. D. Liu, M.D., N. Simon, B.S., and D. W. McFadden, M.D. Department of Surgery, UCLA Medical Center, Los Angeles, California. Peptide YY (PYY) is a naturally occurring gut hormone. We have previously shown that PYY(22-36) inhibits pancreatic cancer growth. We tested three biotinylated analogs of PYY: PYY(9-36), PYY(14-36), and PYY(22-36). We hypothesized that biotinylated analogs of PYY would allow binding of fluorescent conjugates to the cell surface of human pancreatic cancer cells. Methods. MiaPaCa-2 and PANC-1 human ductal adenocarcinoma cell lines were incubated in serum free media with PYY analogs for one hour. Cells were washed and incubated with streptavidin-phycoerythrin (PE), a fluorescent conjugate. Quantitative flow cytometry was performed. Paired controls received streptavidin-PE without PYY analogs. Results. Significant binding is observed in both tissue culture lines with 10 nmol of PYY(22-36)/500,000 cells by quantitative flow cytometry. PYY(9-36) and PYY(14-36) did not have cell binding. Data in the table are percentages of specific binding to cancer cells above background PE binding (N 5 6, *p , 0.05, Student’s t test). Conclusions. Biotinylated PYY(22-36) binds to MiaPaCa-2 and PANC-1 cell lines suggesting that a receptor exists on the cell surface. Longer isoforms, including native PYY, do not bind to pancreatic cancer cells. Biotinylated PYY(22-36) may be useful for fluorescent imaging of pancreatic tumors and it may be useful as a ligand to deliver therapeutic agents.

TABLE—ABSTRACT P19 MiaPaCa-2, 10 nmol

MiaPaCa-2 100 pmol

PANC-1, 10 nmol

PANC-1 100 pmol

47 6 3%*

5 1 1%

44 1 2%*

27% 1 3%*

294

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P20

Jejunal weight (mg) Villus height (mm) Crypt depth (mm)

Control

Chemo

G-CH-G

CH-G

0.21 6 0.004 491.9 6 9.9 169.8 6 4.1

0.17 6 0.01* 384.1 6 8.8* 134.3 6 5.0*

0.20 6 0.02 379.9 6 10.8* 155.3 6 6.6

0.23 6 0.03** 453.4 6 11.7* ,** 168.6 6 10.0**

Note. Data are expressed as means 6 SEM, n 5 8/group. * P , 0.05 vs. control; ** P , 0.05 vs. Chemo.

P20. Glucagon-like Peptide-2 (GLP-2): A New Treatment for Chemotherapy-Induced Enteritis. A. Tavakkolizadeh, M.B., B.S., R. Shen, M.D., P. Ibrahim, B.Sc., N. Kormi, A.B., D. O. Jacobs, M.D., E. Edelman, M.D., Ph.D., P. Seifort, Ph.D., M. J. Zinner, M.D., S. W. Ashley, M.D., and E. E. Whang, M.D. Departments of Surgery and Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; and MIT, Cambridge, Massachusetts. GLP-2 is a recently identified intestinal epithelium-specific growth factor. We hypothesized that GLP-2 administration would be beneficial in chemotherapy-induced enteritis by either preventing injury or promoting recovery. Methods. Rats received no drug (control), chemotherapy alone (5-FU, 190 mg/kg, i.p.) (Chemo), 5-FU followed by 3 days of GLP-2 analog (ALX-0600, 0.1 mg, s.c. bid) (CH-G), or GLP-2 analog for three days prior to 5-FU and for three days afterward (G-CH-G). Rats received BrdU (50mg/kg, 2.5 h prior to harvest on day 3 post-chemotherapy) for immunochemical identification of cellular proliferation. Results. (See table.) BrdU labeling was confined to the crypts of control tissue; however, it was present in both villi and crypts of treated groups. Conclusions. These results suggest, for the first time, that GLP-2 treatment begun after administration of chemotherapy enhances intestinal recovery. In contrast, pre-treatment to prevent injury had no beneficial effect. GLP-2 administration may be beneficial to patients suffering from chemotherapy-induced enteritis. P21. Opiate Receptor Antagonist Attenuated Atrophy of the Gut Mucosa in Fasting Rats. T. Tajiri, M.D., S. Yoshida, M.D., T. Yoshizumi, M.D., K. Ozaki, M.D., T. Yahara, M.D., K. Yamasaki, M.D., H. Kamei, M.D., and K. Shirouzu, M.D. Department of Surgery, Kurume University, Kurume, Japan. The objective of this study was to determine whether the gut mucosal atrophy in fasting rats was inhibited by m-receptor antagonist of opioid (Naloxone). Methods. Male SD rats (n 5 24, BW:200220g) were catheterized into the jugular vein on day 0. The animals were fed standard rat chow and water ad libitum for 4 days. On day 4, the rats were allowed free access to standard liquid diet. On day 7, the animals were randomized into 4 groups:1) free fed plus saline infusion (FF), 2) free fed plus Naloxone (FFN), 3) fasting plus saline (FA), 4) fasting plus Naloxone (FAN). Naloxone was continuously infused via i.v. catheter at the rate of 0.16mg/kg/hr. On day 8, the animals were decapitated, and blood, brain, small intestine and large

TABLE—ABSTRACT P21 FF Morphine brain Morphine plasma Jejunum Large bowel

434 6 60 a

FFN 924 6 86 b

FA 814 6 97 b

FAN 994 6 163 b

368 6 34 a 3374 6 552 b

329 6 28 a 4637 6 1030 b

733 6 67 a 217 6 35

513 6 49 c 198 6 42

710 6 71 ab 220 6 20

690 6 27 b 207 6 47

bowel harvested. Morphine levels in the brain (fmol/g) and plasma (fmol/ml) were determined by coulometric analytical system and mucosal thickness of jejunum and colon was measured in micrometers (mm) (see table: data are mean 6 SEM, Stat. Calc. by ANOVA; different superscripts indicate significant differences, p , 0.05, and N 5 6 in each group). In conclusion, the atrophy of the jejunal mucosa induced by 24 h of fasting was attenuated by naloxone probably through blocking m-receptors in the brain. P22. Effects of Intestinal Ischemia/Reperfusion (I/R) Injury on Gastric Function. A. Castaneda, M.D., L. Chang, B.S., and D. Mercer, M.D. Department of Surgery, University of Texas Medical School, Houston, Texas. The mechanism responsible for gastric colonization in critically injured ICU patients remains to be fully elucidated. Moreover, the effects of traumatic stress on gastric function are unclear. It was our hypothesis that small intestinal (I/R) injury, a model that mimics organ dysfunction in ICU patients also causes gastric dysfunction. Thus, rats were anesthetized and via laparotomy, the superior mesenteric artery (SMA) was clamped at its aortic origin for 45 min followed by clamp removal. Control animals underwent laparotomy with isolation of the SMA. Rats were allowed to awaken and then killed after 6 h of reperfusion. Stomachs were removed, gastric fluid aspirated and the volume, pH, protein, and bicarbonate content determined. In addition, serum was prepared for gastrin radioimmunoassay (see table: data are reported as mean 6 SEM (ANOVA; n$6). SMA I/R causes gastric luminal alkalinization and shedding of protein consistent with gastric injury. SMA I/R also caused gastric

TABLE—ABSTRACT P22 Group

Vol (ml)

pH

Protein (mg)

HCO 3 (mEq)

Sham I/R

0.2 6 0.1 5.4 6 0.6*

4.1 6 1.2 7.4 6 0.3*

0.2 6 0.01 5.7 6 0.6*

763 82 6 20*

* P , 0.05 vs control. surface epithelial cell injury (light microscopy) and a gastric ileus as evident by the significant increase in gastric residual volume. Furthermore, SMA I/R significantly increased serum gastrin levels (35.6 6 4 vs 16.7 6 3 pg/ml, P 5 0.002), an expected response in the non-acid secreting stomach. These findings suggest that SMA I/R causes gastric injury and a gastric ileus that may predispose patients to gastric colonization due to stasis and loss of the natural bactericidal effect of gastric acid. P23. TGF-b Isoforms 1, 2 and 3 Are Elevated in Early Gestational Age Rat Amniotic Fluid. H. Levinson, M.D., G. S. Chin, M.D., W. Liu, M.D., Ph.D., M. Hsu, B.S., S. Lee, M.D., G. K. Gittes, M.D., M. T. Longaker, M.D., and H. P. Ehrlich, Ph.D. Departments of Surgery, New York University, New

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS York, New York; and Penn State University, Hershey, Pennsylvania. The effects of amniotic fluid (AF) on fibroblast populated collagen lattice (FPCL) contraction are species dependent, suggesting alternative roles in fetal wound healing (J. Cell Physiol, 149; 1991). Although stimulation and inhibition are species specific, scarless fetal healing is not. We first investigated the role of rat AF on collagen lattice contraction and then sought to identify potential underlying mechanisms. Our results suggest that early gestational age AF is rich in pro-fibrotic growth factors. Three sets of gestational age rats 14, 16, 18 and 21 were anesthetized and a 21 gauge needle was inserted into the fetal sacs to aspirate AF under sterile conditions. Adult dermal fibroblasts seeded in collagen lattices were exposed to AF. Areas of FPCL were measured daily for three days. AF was analyzed under reducing conditions by SDS polyacrylamide gel electrophesis and Western Blotting, using antibodies specific for TGF-b isoforms 1, 2, and 3. A bioassay, using mink lung epithelial cells stably transfected with a construct containing the PAI-1 promoter and luciferase reporter genes, was used to determine total and active TGF-b in AF. AF stimulated lattice contraction maximally within 24 h (area of FPCL in mm 2 at): Control 5 74 6 7, Day 14 5 47 6 6, Day 16 5 133 6 17, Day 18 5 124 6 25, and Day 21 5 118 6 18. A trend toward decreasing amounts of TGF-b in older age AF was detected by Western Blotting. There was 2-3 fold more TGF-b 1, 2, and 3 activity in day 14 relative to other age AF. Day 14 gestational age rat AF contained 2-3 fold more total and active TGF-b 1, 2, and 3 than AF from gestational ages 16, 18 and 21. Despite higher levels of TGF-b in AF, early gestational age rat fetal skin heals without scar. This finding is interesting, since the fetal skin is bathed in AF and would thus appear to have a role in wound healing. We hypothesize this phenomenon may be related to altered growth factor signal transduction in early gestational age fetal dermal fibroblasts. P24. EGF Enhances Pro-Survival Gene Expression Following Massive Small Bowel Resection. L. E. Stern, M.D., R. A. Falcone Jr., M.D., C. R. Erwin, Ph.D., and B. W. Warner, M.D. Division of Pediatric Surgery, Children’s Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio. Introduction. Following massive small bowel resection (SBR), the expression of various pro- and anti-apoptotic Bcl-2 gene members is associated with increased enterocyte apoptosis. Epidermal growth factor (EGF) has been shown to enhance enterocyte proliferation and retard enterocyte apoptosis within the adapting bowel. This study sought to determine the effect of EGF on the expression of several pro- and anti-apoptotic genes during adaptation. Methods. Mice (C57Bl/6; n 5 16) underwent a 50% SBR or sham operation and were randomized to twice daily orogastric EGF (50mg/kg/day) or saline. After 3 days, the remnant ileum was removed, apoptotic index (AI; # apoptotic bodies/crypt) calculated, and expression of mRNA for Bclx L, Bfl-1, Bcl-w, Bax, Bad, and Bak determined by ribonuclease protection assay. Results. EGF prevented the increased AI after SBR and increased expression of all pro-survival genes. Proapoptotic gene expression was minimally affected (see figure). Conclusion. Following massive SBR, EGF retards rates of enterocyte apoptosis and increases the expression of pro-survival members of

295

the Bcl-2 family. By increasing pro-survival expression, the balance between pro- and anti-apoptotic genes is shifted in favor of cell survival. Alteration of Bcl-2 gene expression may be an important mechanism by which EGF reduces the increased enterocyte apoptosis that occurs following massive SBR. P25. The Angiogenesis Inhibitor Endostatin Does Not Affect Murine Cutaneous Wound Healing. A. C. Berger, M.D., A. L. Feldman, M.D., E. A. Kruger, B.A., B. K. L. Sim, Ph.D., W. D. Figg, Pharm.D., H. R. Alexander, M.D., and S. K. Libutti, M.D. The Surgery and Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland; and EntreMed Inc., Rockville, Maryland. The purpose of this study was to determine whether the potent antiangiogenic agent endostatin has an effect on wound healing in a murine model. Methods. Full-thickness incisions were made on the dorsum of athymic nude mice and closed primarily with skin staples. Subsequently, 15 mice per group were treated BID with recombinant human endostatin (Pichia pastoris, ENDO) at 50 mg/kg/dose or PBS via subcutaneous injection away from the site of the wound for a total of 14 days. On days 2, 4, 8, 12 and 14, three mice per group had serum samples drawn and were sacrificed. Perpendicular breaking strength (N) of 3 strips per wound was determined using an Instron 5540 tensometer. The wound length was kept constant and the product of the width and thickness of the wound determined the wound area (cm 2). Wound strength was determined by dividing breaking strength by wound area (N/cm 2). Data were compared using Student’s t-test. Serum endostatin concentrations were determined using a commercially available ELISA kit. The function of ENDO was confirmed using a human microvascular endothelial cell (EC) proliferation assay in which cells are treated for four days with growth media plus or minus ENDO and viable cells are counted by trypan blue exclusion. Results. ENDO caused a significant reduction of EC proliferation after four days compared to media alone (72%, p 5 0.031). At all time points tested, there was no statistical difference in the wound breaking strength between ENDO and PBS treated mice. Serum endostatin levels were consistently 10-fold higher in ENDO- treated mice than in PBS controls. Conclusions. Therapy with human endostatin does not induce a significant decrease of breaking strength in cutaneous wounds in mice. P26. Construction of a Recombinant Adenovirus Expressing Constitutively Active Rat Bone Morphogenetic Protein Receptor Ib. D. C. Hitt, M.D., S. Harada, M.D., Ph.D., G. M. Pighetti, Ph.D., R. A. Squires, M.D., L. R. Pennington, M.D., and J. M. Gimble, M.D., Ph.D. Department of General Surgery, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; and Department of Bone Biology and Osteoporosis Research Laboratories, West Point, Pennsylvania. We have used the AdEasy system, a novel prokaryotic based method to construct a recombinant adenoviral vector coding for a hemagglutin tagged, constitutively active rat bone morphogenetic protein receptor Ib (caBMPRIb). The vector also codes for green fluorescent protein (GFP) to track cellular infection. Bone morphogenetic protein (BMP) and its receptor are known to control bone formation. In vitro studies with the constitutively active form of the receptor have reported induction of an osteoblastic phenotype in the absence of BMP. The long-term objective of this research is to create an adenoviral expression vector which can be used to force cellular differentiation along the osteoblast pathway. The vector could have gene therapeutic applications in the healing of clinically problematic fractures such as those in diabetic and elderly patients. The cDNA encoding caBMPRIb was excised and subcloned into the linker region of the shuttle vector (pAdTrack-cmv) under the control of the cytomegalovirus promoter. The resulting plasmid (pAdTrack-cmv-caBMPRIb) was linearized and cotransformed with the pAdEasy-1 viral backbone by electroporation into BJ5183 bacteria.

296

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

Correct clones were confirmed by BamHI restriction enzyme digestion and purified. The linearized plasmid was transfected with lipofectamine into 293 renal epithelial cells (viral packaging cell line) and initial virus isolated. High titer virus was isolated after repeated infection and purified. Expression of the viral encoded caBMPRIb and GFP was confirmed by Western blot. To assess function, GFP expression was visualized by fluorescence microscopy, while caBMPRIb function was demonstrated by the presence of alkaline phosphatase, an early marker of the osteoblast phenotype. In conclusion, we have successfully constructed a functional recombinant adenovirus expressing caBMPRIb and GFP. The constitutively active BMP receptor will potentially promote bone formation without requiring exogenous BMP ligand and may have applications to help heal problematic fractures.

received systemic cyclosporin preoperatively at days 228 to 25 and BN allografts at day 0. All grafts were explanted at various time points between days 1-101 and divided into proximal, medial, and distal segments. Histopathology and morphometry were performed for all samples and quantified using computerized digital image analysis. Pearson correlation and analysis of variance were performed for statistics. SC isografts showed significantly decreasing proximal intimal thickness, increasing distal media area and thickness, and adventitial nucleus number (NN) over time. Results of AC allografts are shown in the table (NS, not significant). CsA allografts demonstrated significantly progressive intimal thickening in medial

TABLE—ABSTRACT P28 P27. The Effects of the Microenvironment on Osteoblast VEGF Expression. J. A. Spector, M.D., J. A. Greenwald, M.D., B. J. Mehrara, M.D., P. B. Saadeh, M.D., G. K. Gittes, M.D., and M. T. Longaker, M.D. New York University Medical Center, New York, New York. Introduction. Vascular disruption after osseous injury is thought to result in an acidic, hypoxic wound environment. We have previously shown that osteoblasts can produce Vascular Endothelial Growth Factor (VEGF), one the most potent endothelial cell mitogens, in response to a variety of stimuli. In this study we examined pH and lactate concentration, two variables of the microenvironment of an early fracture, and determined their relative contribution to regulation of osteoblast VEGF protein production, under both normoxic and hypoxic conditions. Methods. Confluent plates of first passage osteoblasts derived from neonatal rat calvaria, and 24 day old mouse hemi-mandibles, which had been cleaned free of all loose connective tissue except the periosteum, were placed in serum-free media of varying pH (7.0, 7.2, 7.4, 7.6) under either normoxic (pO2 5 21%) or hypoxic (pO2 5 5%) conditions. The media were conditioned for 24 h and then examined for the production of VEGF protein by immunoassay. The immunoassays were also performed on media conditioned by cells exposed to an elevated lactate concentration [23 mmol/l], at a pH of either 7.0 or 7.4. Statistical analyses were performed using Student’s t-test with significance set at *p , 0.05. Results. Immunoassay of media conditioned by cells grown in normoxic conditions demonstrated significant increases in VEGF expression with increasing pH (p , 0.05). Cells cultured in a hypoxic atmosphere produced significantly more VEGF than those cells cultured under normoxic conditions (p , 0.05). This trend was also seen in the media conditioned by the mouse hemi-mandibles. However, when cells were cultured in a hypoxic atmosphere in media with a low pH or increased lactate concentration, the increase in VEGF production was significantly blunted as compared with the cells cultured in media of neutral pH and no lactate (p , 0.05). Conclusions. Our results demonstrate that decreased pH alone is an insufficient stimulus for increased expression of VEGF by osteoblasts. Furthermore, acidic pH or elevated lactate levels, as would be expected in the local microenvironment of a fracture, do not appear to be important independent stimuli of VEGF protein production in osteoblasts but rather inhibit its production. P28. Cyclosporin Pretreatment Inhibits Distal Vasculopathy in Femoral Arterial Allograft. H. S. Tran, M.D., M. M. Puc, M.D., N. Patel, M.D., B. Geldziler, B.S., J-L. Tran, B.S., C. Brimer, B.S., M. S. Matthews, M.D., A. J. DelRossi, M.D., and C. W. Hewitt, Ph.D. Department of Surgery, Cooper Hospital/ UMC, Camden, New Jersey. To quantify the pattern of vasculopathy in femoral arterial allografts. Lewis rats (RT1 l) underwent interpositioned femoro-femoral artery transplantation. Syngeneic controls (SC, n 5 7) received Lewis isografts. Allogeneic controls (AC, n 5 15) received BrownNorway (RT1 n) allografts, and the cyclosporin group (CsA, n 5 5)

Proximal Lumen Intima

NS 1 thickness

Media Adventitia

NS NS

Medial

Distal

1 % narrowing 1 % intimal thickness 1 NA, NN 2 elastin content NS

1 % narrowing 1 % intimal thickness NS NS

segments with decreasing adventitial thickness in proximal and medial segments. AC allografts had greater media NN in medial segments and proximal adventitial NN and nucleus area (NA) compared with SC (p , 0.05) and CsA (p , 0.05). There was a unique pattern of progressive arterial vasculopathy characterized by increasing intimal hyperplasia and luminal narrowing with decreasing medial elastin content proceeding in the medial to distal segments of peripheral femoral allografts. Preoperative CsA immunosuppression prevented medial elastin resorption in the medial and intimal hyperplasia and luminal narrowing in the distal peripheral allograft segments. P29. Carotid Endarterectomy in Patients with Renal Insufficiency. J. Ayerdi, M.D., L. N. Sampson, M.D., N. Deshmukh, M.D., and S. K. Gupta, M.D. Department of Surgery, Robert Packer Hospital, Sayre, Pennsylvania. Objective. The purpose of this study was to elucidate the relationship between outcomes from CEA in patients with renal insufficiency. We hypothesized that the outcomes from CEA are negatively influenced by renal dysfunction. Methods. Retrospective review of all consecutive CEAs performed at our institution between 1990 and 1995. Patients were grouped into two different categories according to their renal function. Group A: 448 patients (90.1%) with creatinine level less than 1.8 mg/dl. Group B: 49 patients (9.9%) with creatinine levels more than 1.8 mg/dl. Information regarding patients on dial-

TABLE—ABSTRACT P29 Groups Perioperative Cardiac events TIAs Stroke Mortality 2-year follow-up TIAs Stroke Mortality

A(%)

B(%)

P

(5.5) (2.0) (2.9) (0.9)

13 (26.5) 0 1 (2.0) 4 (8.2)

,0.001 NS NS 0.004

25 (5.6) 21 (4.7) 98 (21.9)

3 (6.1) 2 (4.1) 25 (51.0)

NS NS ,0.001

24 9 13 4

297

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS ysis was available but it was excluded for the purpose of analysis. Results. 497 patients were included in the study with a mean age of 70 6 8.9 and 74 6 8.9 for groups A and B, respectively. Preoperative mean creatinine was 1.1(6 0.25) mg/dl for group A and 2.5(6 0.81) mg/dl for group B. Outcomes are shown in the table. Conclusion. While patients with chronic renal insufficiency have no increased risk of perioperative or long term neurologic events, their perioperative and long term mortality are significantly increased. This significant reduction in survival should prompt a more cautious application of CEA in patients with increased creatinine. P30. Videoendoscopic Thoracic Aorta to Femoral Artery Bypass in the Pig. A. A. Noel, M.D., P. Gloviczki, M.D., M. Young, M.D., K. Karnicki, M.D., C. Deschamps, M.D., and C. Moir, M.D. Division of Vascular Surgery, Mayo Clinic, Rochester, Minnesota. Thoracoscopic approach to the aorta has advantages of easy aortic dissection, excellent inflow, improved exposure in the thorax without insufflation, and the ability to employ both laparoscopic and traditional instruments. Our aim was to develop a thoracoscopic technique for descending thoracic aorta to femoral artery bypass (TAFB) in the pig that results in acceptable shortterm survival and graft patency. Thoracoscopic TAFB was performed in eleven pigs. Using two-lung ventilation, the animals were placed in a 45-degree left semi-decubitus position. A fan lung retractor, 2 dissecting ports, intercostal artery loops, and camera were placed through five 10-20 mm thoracoscopic incisions. After aortic dissection, an 8mm graft was passed through a retroperitoneal tunnel. Rumel tourniquets were used for aortic occlusion after placement of a shunt. End-to-side endoscopic anastomosis was completed with knots tied extracorporeally. The left femoral anastomosis was completed under direct vision. Duplex ultrasound of the graft was done at postoperative days one, three and seven. Thoracoscopic TAFB was completed in all animals. Mean aortic anastomosis time: 57 min (range: 34 –145); mean crossclamp time: 74 min (range 53–155). Mean operative time was 310 min; the first six operations lasted longer than the last five (338 vs 276 min, p , 0.04). Average blood loss was 611 ml (range: 250 – 1300 ml). Two animals died due to anesthetic complications. One (11%) of the nine surviving pigs died on day two due to bleeding. Complications were paraplegia in one (11%) and graft thrombosis in another (11%). Videoendoscopic TAFB can be completed in pigs with acceptable short-term patency and survival. Further experience in thoracoscopic techniques can make TAFB a feasible and low-risk option for selected patients with aorto-iliac occlusive disease. P31. Thrombolysis Stimulates TGF-b 1 Release in a Canine Vein Graft Model. D. S. Feuer, M.D., R. G. Ciocca, M.D., A. M. Graham, M.D., T. Stefan, M.D., D. Thompson, B.A., and G. B. Nackman, M.D. Division of Vascular Surgery, Robert Wood Johnson Medical School, New Brunswick, New Jersey. Thrombolysis of vein bypass grafts has resulted in poor long term patency. A possible explanation is that thrombolysis results

in an alteration in the vascular microenvironment which predisposes for intimal hyperplasia. TGF-b 1 , activated by plasmin, is associated with intimal hyperplasia. We hypothesized that thrombolysis causes an increase in TGF-b 1 which could result in late graft failures. Femoral vein grafts were placed bilaterally in 4 dogs, and 1 week later, the inflow vessels were ligated. 1 day after thrombosis, balloon catheter thrombectomy (BT) of one graft was performed followed by thrombolysis with urokinase (UK) of the contra-lateral graft. Blood was taken from the graft (G), outflow artery (Art), and vena cava (VC) preceding, during and following thrombus elimination. Active and latent TGF-b 1 were determined by ELISA (see table: mean ng/ml 6 SE, *p , 0.05 ANOVA with post-hoc comparisons). Thrombolysis resulted in significantly increased TGF-b 1 in the graft, outflow artery and vena cava that was not noted after thrombectomy. The active fraction of TGF-b 1 in serum was barely detectable and significant differences were not noted. The increased availability of latent TGF-b 1 for activation on a cellular level may stimulate the development of intimal hyperplasia or progression of atherosclerotic disease which could explain the poor patency after thrombolysis of infra-inguinal vein grafts.

P32. Formation of Arteriovenous Fistulas Enhances Venous Ecnos Gene Expression. H. M. Choi, M.D., M. B. Silva, Jr., M.D., P. J. Pappas, M.D., M. Kissin, M.D., R. You, M.D., R. W. Hobson II, M.D., and W. N. Dura´n, Ph.D. Departments of Surgery and of Pharmacology and Physiology, UMDNJ-NJMS, Newark, New Jersey. Purpose. Shear stress induces endothelial cell nitric oxide synthase (ecNOS) expression in arteries. We hypothesized that NO signaling is involved in the patency and maturation of arteriovenous fistulas (AVF). As a first approach to test this hypothesis, we investigated the changes in gene expression of ecNOS and iNOS in the arterial and venous limbs of AVF in a rat model. Methods. Eighteen male Sprague Dawley rats were separated into two groups: The AVF group (n 5 12) received a femoral vein to femoral artery fistula in an end to side fashion. The control group (C, n 5 6) had their femoral vessels harvested at day zero without external manipulation. The AVF group had the femoral vessels harvested at post-operative day (POD) 1 and 3. All specimens were rinsed in PBS and snap frozen in liquid nitrogen. Competitive RT-PCR was used to analyze ecNOS and iNOS RNA gene expression in the femoral vessels. Results. Arterial and venous gene expression of ecNOS was increased at POD 3, compared to control vessels (p , 0.05). Venous ecNOS expression was increased at POD 1 compared to control veins (p , 0.05). Arterial ecNOS expression at POD 1 showed a similar trend relative to control arteries. Arterial and venous expression of iNOS was decreased at POD 3 relative to control vessels (p , 0.05). Femoral arteries showed a similar trend at POD1 regarding iNOS expression compared to control arteries but the differences were not significant (see figures). Conclusions. Increased shear stress, associated with AVF, enhances the gene expression of ecNOS in the arterial and venous limbs of AVFs. These molecular events

TABLE—ABSTRACT P31 Pretreatment

2 min post-thrombus elimination

30 min post-thrombus elimination

Site

Art

VC

G

Art

VC

G

Art

VC

UK BT

14.1 6 1.2 14.0 6 3.7

16.3 6 3.2 17.5 6 1.7

*24.1 6 3.5 13.7 6 2.9

*22.2 6 5.0 12.0 6 4.9

*22.9 6 3.8 10.4 6 0.4

17.4 6 3.2 12.8 6 1.8

24.4 6 7.5 15.8 6 3.0

*31.9 6 5.8 14.0 6 1.8

298

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P34

support a role for NO in the patency and maturation of arteriovenous fistulas.

P33. Calcium Chloride Induces Murine Abdominal Aortic Aneurysm Formation. A. C. Chiou, M.D., M.P.H., B. Chiu, B.S., W. F. Oppat, M.D., R. L. Chisholm, Ph.D., and W. H. Pearce, M.D. Division of Vascular Surgery, Northwestern University Medical School, Chicago, Illinois. Introduction. Recent research into aortic aneurysm pathogenesis has increasingly utilized small animal models. We hypothesize that aortic dilatation in the murine model may be achieved via an inflammatory pathway initiated by peri-arterial calcium chloride application. Methods. Nine C57BL/6 mice were anesthetized, infrarenal aortas were exposed and skeletonized, and calcium chloride was applied topically in the peri-arterial region for 15 min. Nine littermates of similar age received peri-arterial normal saline topically and served as controls. Three mice and their controls were each examined at 7 days, 14 days and 21 days. At laparotomy, the infrarenal aortic diameter was measured. Images were acquired with NIH Image software and aortic measurements made. Results. (See figure.) Control mice revealed only a 3%-14% increase in aortic diameter. A marked inflammatory infiltrate was seen in all calcium chloride treated mice. Conclusion. Topical calcium chloride induces aneurysmal dilatation in the murine aorta.

P34. Neuroprotective Effect of Endorphin Antagonist in a Rabbit Model of Spinal Ischemia. Krish Soundararajan, M.D., Larry H. Hollier, M.D., Azhar Hossain, M.D., Mohammed Siddiqui, M.D., Ernane D. Reis, M.D., and Morris D. Kerstein, M.D. Departments of Surgery and Pathology, Mount Sinai Medical Center, New York, New York. The neuroprotective effect of naloxone, an endorphin antagonist was evaluated in a rabbit model of spinal ischemia. Method. Three groups of ten New Zealand rabbits had a circum-aortic snare placed through a retroperitoneal incision. Forty-eight hours later, in a fully awake state, the infrarenal aorta was occluded for 18 min by tightening the snare. Two groups received naloxone before occlusion as a bolus followed by six hour infusion of 1mg/kg/hr and 1mg/kg/hr. The control group received saline. Animals were observed 72 h for neurological deficit and graded with modified Tarlov scale. Results. Neurological outcome is shown in the table. The incidence of com-

Group

Early

Delayed

Grade 0

No deficit

Control 1 mg/kg naloxone 1 mg/kg naloxone

6 6 4

4 3 6

7 (70%) 3 (33%) 3 (30%)

0 1 0

plete paraplegia in the naloxone group was 30 and 33%. This was significantly lesser than the 70%in the control group (p , 0.04). Histology confirmed spinal tissue necrosis in the paralyzed animals. Conclusion. Endorphin antagonist did not prevent complete paraplegia but diminished the degree of neurological injury in this model. In combination with other preventive measures this could reduce the incidence of paraplegia following TAA repair. P35. Vascular Invasion May Predict Lymph Node Metastasis in Early Rectal Cancer. S. Bayar, M.D., R. Saxena, M.D., B. Emir, Ph.D., and R. R. Salem, M.D. Departments of Surgery and Pathology, Yale University School of Medicine, New Haven, Connecticut. Introduction. Early carcinoma of the rectum is defined as lesions limited to mucosa or the submucosal layer. The aim of this study was to evaluate the value of histopathologic examination in predicting lymph node metastasis in early rectal cancer. Methods. Between 1970 and 1999 fifty-eight patients with early rectal cancer underwent resection of the rectum including lymph nodes and 5 showed lymph node metastasis (8.6%). Pathology slides of these patients were reexamined by a single pathologist. We compared the demographic and clinical characteristics of these 58 patients with the existence of lymph node metastasis. Formal tests of comparability were carried out by using Fisher’s exact test. Logistic regression models were fitted to data to examine possible relationships with 12 covariates measured from each patient and to obtain corresponding odds ratios (as well as a 95% confidence interval for the odds ratios). These covariates included: Age at surgery, gender, morphology of polyp (pedunculated or sessile), histologic type of polyp (nonpolypoid, tubular, tubulovillous, and villous), degree of differentiation (well to poor), Haggitt’s classification for polyps according to the level of invasion (level 0-4), presence or absence of lymphatic and vascular invasion, presence or absence of desmoplastic reaction, levels of lymphocytic infiltration (none to severe), presence or absence of lymphoid follicles and presence of infiltrating or pushing margins. Results. Statistical analysis showed a significantly higher lymph node metastasis rate in the presence of vascular invasion (p 5 0.01). For all other variables, the characteristics of the covariates were similar between the metastasized and non-metastasized patients. Conclusion. From this analysis, only the existence of vascular invasion was found to be highly significant. The odds ratio of lymph node metastasis increased 18 fold for a patient who had vascular invasion versus a patient who did not. These results suggest that vascular invasion in early rectal cancer may provide valuable information to determine which patients would benefit from radical surgery or adjuvant radiation therapy after sphincter sparing surgery as a result of increased risk of lymph node metastasis. P36. Endothelial Monocyte Activating Polypeptide-II (EMAPII) Selectively Induces Apoptosis in Endothelial Cells (EC). A. C. Berger, M.D., H. R. Alexander, M.D., G. Tang, Ph.D., and S. K. Libutti, M.D. Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Recombinant human EMAP-II (rhEII) is a cytokine that induces E-selectin, TNF-R1, and tissue factor on EC. The purpose of this study was to determine the effects rhEII would have on EC with

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS respect to proliferation, apoptosis, and expression of the apoptosisrelated proteins, Bcl-2 and FADD. Methods. Endothelial cells (HUVECs), fibroblasts (NIH 3T3), and colon cancer (HT29) cells were treated for 24 h with media alone, rhEII (100 and 500 mg 6 EII blocking Ab), or TNF (100 ng/ml). Percent apoptotic cells was determined by FACS analysis using fluorescent antibody labeling of DNA strand breaks with Br-dUTP. Cell proliferation was assessed using EC treated for 4 days with the same agents and counting viable cells by trypan blue exclusion. Western blotting for FADD and Bcl-2 was performed on lysates from cells treated in a similar fashion. Results. The effects of rhEII on EC proliferation, apoptosis, Bcl-2 and FADDexpression are illustrated in the table. These effects were specific for EC and not seen with fibroblasts or colon cancer cells. Conclusions. Our data suggest that EMAP-II inhibits EC proliferation and

TABLE—ABSTRACT P36 Relative expression % Inhibition of proliferation % Apoptosis bcl-2 Media EII (500 mg) EII 1 blocking Ab TNF (100 ng/ml)

0 82 50 79

0 18 1 12

11 1 11 1

FADD 0 11 Not done 11

selectively induces apoptosis in EC. This is associated with downregulation of Bcl-2 and increased expression of FADD. Thus, EMAP-II has potent and specific regulatory effects on EC indicating a potential role in regulating angiogenesis. P37. Patient Follow-Up after Resection of Melanoma. C. A. Paul, M.D., K. S. Virgo, Ph.D., T. P. Wade, M.D., R. A. Audisio, M.D., F. E. Johnson, M.D., and contributors to the International Registry of Adrenal Metastases. Department of Surgery, St. Louis University School of Medicine, Department of Veterans Affairs Medical Center, St. Louis, Missouri; and European Institute of Oncology, Milan, Italy. Purpose. Follow-up care for patients after potentially curative resection of cutaneous melanoma varies widely among physicians. To determine current practice patterns, a custom-designed questionnaire was mailed to U.S. and non-U.S. surgeons, all of whom were members of the American Society of Plastic and Reconstructive Surgeons (ASPRS). Methods. Surveys were mailed to 3,032 ASPRS members, chosen randomly from the 4,320 total. 1,142 were returned, of which 395 were evaluable. 415 of the 1,142 respondents do not provide post-operative follow-up. The data were analyzed using ANOVA. Results. Follow-up of patients after resection of Stage I (T2NOMO) melanoma relies most heavily on office visit, chest x-ray, CBC and liver function tests. There was little influence of elective node dissection and more advanced initial TNM stage on follow-up practices. Imaging tests such as CT, MRI and PET scan are rarely employed. Conclusions. This survey provides the first direct, empirical information regarding current follow-up strategies recommended by ASPRS surgeons after potentially curative resection of Stage I (T2NOMO) cutaneous melanoma with or without elective node dissection. Office visit, chest x-ray, CBC, and liver function tests are the main tools employed. There is considerable variation among practitioners, representing a lack of consensus. P38. Implementing Computer-Based Breast Cancer Risk Assessment in the Clinic. D. M. Euhus, M.D., A. M. Leitch, M.D., J. F. Huth, M.D., and G. N. Peters, M.D. Division of

299

Surgical Oncology, U.T. Southwestern Medical Center, Dallas, Texas. Many women who have witnessed a breast cancer death in a close relative experience significant anxiety over their own prospects of developing breast cancer and welcome the opportunity to measure and discuss breast cancer risk. In addition, with a recent report demonstrating that tamoxifen reduces breast cancer risk in increased risk women by 50%, there appears to be value in implementing community-based risk screening and counseling services. We have recently developed an interactive computer program for comprehensive breast cancer risk analysis which collects a detailed risk factor history; generates age-specific breast cancer probabilities based on the models of Gail, Claus, Bodian, and Berry; calculates the probabilities of harboring a BRCA1 or BRCA2 gene mutation; and generates personalized reports for the client and referring physician. Of the first 103 women evaluated with this program, thirty-eight (37%) perceived themselves to be at slightly increased risk and 40 (39%) perceived themselves to be at very increased risk. Based on the Gail model alone, 66% of these women were at slightly increased risk (lifetime risk 20 –29%) and 24% at very increased risk (lifetime risk $30%). Thirty-nine women were identified as fully eligible for the upcoming NSABP P2 trial of tamoxifen vs raloxifene. Twenty-one women were referred for formal genetic counseling and six of these underwent BRCA gene mutation testing. Three pathologic mutations were detected. Community-based breast cancer risk screening has the potential to positively impact public health by identifying large numbers of increased risk women who would benefit from chemoprevention or BRCA gene mutation testing. P39. Peptide YY and Vitamin E Inhibit Hormone Sensitive and Insensitive Breast Cancer Cell Growth. T. Heisler, M.D., S. Towfigh, M.D., N. Simon, B.S., and D. W. McFadden, M.D. Department of Surgery, UCLA Medical Center, Los Angeles, California. We have shown peptide YY, an endogenous gut hormone, and vitamin E succinate (VES) inhibit cancer cell growth in vitro. We hypothesized that PYY with VES would inhibit breast cancer cell viability regardless of the hormone receptor status. Human breast ZR-75 ductal carcinoma (estrogen receptor negative) and MCF-7 adenocarcinoma (estrogen receptor positive) cells were cultured and exposed to VES (10 pg/ml), PYY (500 pmols), or both agents together. MTT assay was performed for cell viability at 24, 48 and 72 h. Student’s t-test was used to analyze the data. At every time interval, PYY and VES significantly inhibited cell growth compared to control. The effects of PYY were similar in magnitude to those of VES. Combining the agents resulted in a significant additive inhibition of growth with the greatest effect seen at 72 h. We have shown for the first time that PYY and vitamin E inhibit growth of breast cancer cells in vitro regardless of hormone receptor status. When used in combination, the agents have a significant increase in effect. P40. Bcl-2 and Bax Apoptotic Proteins Are Not Involved in the Development of Doxorubicin Resistance by Breast Metastatic Cells. P. L. Mojica, M.D., and E. M. Mora, M.D., M.S. Departments of Surgery and Pathology, UPR School of Medicine, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico. Resistance to chemotherapeutic agents, especially doxorubicin, is one of the most common problems in breast cancer patients. The apoptosis cascade participates in the development of tumor resistance to chemotherapuetic agents. The relationship of Bcl-2 (antiapoptotic protein) and bax (pro-apoptotic protein) had been shown to correlate with breast carcinoma progression and resistance of tumor cells to cytotoxic drugs and irradiation. Manipulation of the expression of the apoptotic proteins could be a target to: 1) develop new

300

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

methods to determine doxorubicin resistance prior to treatment, and 2) development of new therapeutic strategies to decrease the development resistance of breast cancer cells to doxorubicin We hypothesized that Bcl-2 and bax are involved in the mechanism of resistance to doxorubicin by breast cancer cells. To test this hypothesis we exposed pleural-metastatic breast carcinoma cells (MCF-7) to doxorubicin (0.2 mg/cc) for 48 h. Cellular proteins were extracted from exposed and non-exposed cells. Western blots and Immunoblotting were performed using 80mg/well. Polyclonal antibodies to Bcl-2 and bax were used to identify the proteins by peroxidase system. Positive and negative controls were run concurrently. The results showed that the qualitative expression of both Bcl-2 and Bax in pleuralmetastatic breast cancer cells and doxorubicin-resistant pleural metastatic breast carcinoma cells were the same. These results suggest that other proteins, besides Bcl-2 and Bax, could be involved in the resistance of metastatic breast carcinoma cells to doxorubicin. In conclusion, manipulation of the Bcl-2 and Bax apoptotic proteins in metastatic breast cancer cells is not an alternative to decreased resistance of metastatic breast cancer cells to doxorubicin or the development of new methods to evaluate doxorubicin resistance. P41. Radiation Inhibits Basic Fibroblast Growth Factor (bFGF) Induced Angiogenesis. N. Chuang, M.D., R. Shetty, M.S., J. S. Cooper, M.D., R. L. Shapiro, M.D., P. Mignatti, M.D., and P. Shamamian, M.D. S A Localio Laboratory for Surgical Research, Department of Surgery, NYU School of Medicine, New York, New York. Angiogenesis is required for tumor growth and metastasis. Tumor cells secrete a variety of factors, including bFGF, to induce the formation of new blood vessels. Agents that inhibit angiogenesis have been shown to inhibit tumor growth and induce tumor regression. We hypothesized that radiotherapy inhibits angiogenesis and thus may potentiate the efficacy of other anti-angiogenic agents. The mouse corneal pocket assay was used to study the effect of radiation on angiogenesis. Slow-release pellets containing 50 ng of bFGF were implanted into the corneas of ten 8-week old Swiss Webster mice. On the second post-operative day, the mice underwent selective irradiation of one of their eyes using a specially designed applicator containing 90 Sr in equilibrium with 90 Y. The dosage of radiation, 1500 reps, was based on a preliminary dose-response curve. Six days after pellet implantation, the amount of corneal neovascularization was quantified using slitlamp microscopy. The length of the newly formed vessels (VL) and their angular extent (CH) were used to calculate a vascular area (VA) according to the formula: 0.2p*VL*CH. Vascular area and vessel length were decreased by 51% and 36% (p , 0.05), respectively, in the ten irradiated eyes (VA 5 3.13 6 0.49 SEM mm 2 ,

VL 5 1.19 6 0.06 SEM mm) compared to the ten non-irradiated control eyes (VA 5 1.52 6 0.33 SEM mm 2 , VL 5 0.76 6 0.09 SEM mm) (see figure). It has been proposed that radiation induced tumor regression is mediated by inhibition of tumor angiogenesis.

These data show that radiation does have a direct effect on angiogenesis. Furthermore, the combination of radiation with other anti-angiogenic agents may have additive or synergistic effects and be of use clinically. This model can be used to investigate this hypothesis. P42. Generation of Long-Term Murine Dendritic Cell Lines. D. S. Park, B.S.,* H. Ho¨rig, Ph.D.,* and H. L. Kaufman, M.D.* ,† †Department of Surgery and *Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, New York. Dendritic cells (DC) are potent antigen presenting cells that sample antigen in the local environment and then migrate to lymph nodes where they regulate T-cell responses. DC are attractive targets for immunotherapy but are difficult to obtain in large numbers due to low yields from DC harvesting and limited growth in culture. Long-term DC lines would provide large quantity, high purity dendritic cells for an array of applications. Current protocols using GM-CSF alone can only propagate DC in culture for approximately 3 months. Long-term DC cultures can be achieved by immortalization; however this may alter DC maturation and antigen presentation while also precluding their use for autologous administration. We have developed a long-term DC line (DC1) derived from bone marrow progenitor cells in C57Bl/6 mice. The DC1 line was grown in GMCSF (10ng/ml) and murine fibroblast-conditioned media producing CSF-1, and has been in continuous culture for over 5 months. The growth and morphology of DC1 cells are consistent with immature dendritic cells as demonstrated by their expansion in loosely adherent clusters and their fine cytoplasmic extensions. The cell line is being characterized by LPS induced maturation evidenced by changes in cell surface expression of CD11c, CD11b, CD14, MHC class I, MHC class II, and CD80. Furthermore, redistribution of MHC class II molecules and the rate of apoptosis after maturation are being evaluated. To determine antigen processing function in these cells, antigen uptake is being followed by cytofluorometry, which detects endocytosis of FITC-labeled OVA protein. Antigen presentation is being analyzed by coculture of DC1 cells with OVA protein or a K b-restricted OVA peptide and monitored for specificity with an OVA/K b- restricted T-cell hybridoma. This technique of generating stable long-term cultures of immature DC may have a wide variety of applications, including immune monitoring, elucidation of novel epitopes and direct use in therapeutic adoptive transfer or vaccine administration. P43. A Simple Tissue Implant Model to Study Xenogeneic and Allogeneic Rejection. H. S. Tran, M.D., M. M. Puc, M.D., N. G. Patel, M.D., S. Goldstein, Ph.D., J. Ollerenshaw, Ph.D., K. S. Black, Ph.D., A. J. DelRossi, M.D., and C. W. Hewitt, Ph.D. Department of Surgery, Cooper Hospital/UMC, Camden, New Jersey; and CryoLife, Inc., Kennesaw, Georgia. In order to facilitate the study of allogeneic and xenogeneic rejection mechanisms, a simple in vivo tissue implant model was developed. Cryopreserved tissues from bovine (n 5 6), porcine (n 5 3), and Lewis rats (n 5 5), which included aorta, aortic valves, peripheral arteries, fascia lata, tendon, and ureters were implanted into the subcutaneous tissues of Sprague-Dawley rats without immunosuppressive therapy. At day 7, the tissues were explanted and stained with H&E. Intrinsic cell numbers and intensity of tissue stain were quantitated. A subjective grading system was devised for preimplant and explant histology (1 to 4 scores). Tissues were graded for morphology/architecture, cellular infiltration, xenogeneic rejection, allogeneic rejection, or non-specific inflammation associated with a foreign body reaction. Statistical analysis was performed using twoway ANOVA with Student-Newman-Keuls test for multiple pairwise comparisons (statistical significance when p#0.05). There were significant differences in cell numbers (p , 0.05), morphology (p 5

301

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P45 B6

B6/GFP

Stain

MHC-I

GFP

MHC 1 GFP

MHC-I

GFP

MHC 1 GFP

Fresh cells CK 1 IFN-g CK 2 IFN-g

0.05 23.43 0.10

0.04 0.53 1.69

0.00 0.35 0.12

0.35 22.69 0.17

3.85 3.37 5.37

0.52 37.11 0.47

0.02), and intensity of tissue stain (p 5 0.002) between rejection responses of xenografts and allografts. Additionally, the rejection reactions significantly differed in all tissue types with respect to cell numbers (p , 0.0001) and morphology (p , 0.001). This tissue implant model provided evidence that the immune/inflammatory response was related to both tissue and species specificity. Our model establishes a simplistic, relatively inexpensive approach that can be used to study cellular as well as humoral mechanisms of allogeneic and xenogeneic rejections. P44. Improved Organ Yield Using High-Dose Steroids during Donor Management. R. C. Kincade, M.D.,* W. D. Babcock, R.N.,† and D. M. Follette, M.D.‡ *Department of Surgery, UC Davis Medical Center, Sacramento, California; †California Transplant Donor Network, San Francisco, California; and ‡Cardiothoracic Surgery, UC Davis Medical Center, Sacramento, California. Solid organ transplantation remains limited due to a shortage of available organs; this study was done to ascertain the effect of high dose steroids during organ donor management on donor yield of transplantable organs and specific organ recovery rates. A retrospective study was conducted on 704 organ donors occurring between January 1, 1995, and June 30, 1998, from our local organ procurement organization (OPO). The donors were divided into two groups. The steroid donors (SD, n 5 270) were treated with single dose of 15mg/kg methylprednisolone as soon as possible following consent and brain death declaration. The non-steroid donors (NSD, n 5 434) were not. Both groups were clinically managed by a standardized protocol of the OPO. Statistical analysis was done using the students t-test for numerical data and chi-square analysis for categorical data. Significance was for p#0.05. Both groups were similar in regards to age, gender, race, cause of death, and ABO blood type. The mean organs transplanted from the SD group was significantly higher (3.84, SEM 6 0.10), than from the NSD group (3.40 SEM 6 0.6) [p,0.001]. There was no difference in the rate of transplanted kidneys or livers between the two groups. Although not significant, there was a slight improvement in the rate of hearts transplanted from the SD group (49.6%) vs the NSD group (43.1%) [p 5 0.102]. The donors producing a transplanted pancreas was significantly higher in the SD group (23.3%), as compared to the NSD group (16.8%) [p 5 0.039]. Donors producing at least one transplanted lung was also greater in the SD group (26.7%) as compared to the NSD group (12.9%) [p,0.001]. High dose steroids, when administered early on during donor management has a positive impact on the total number of transplanted organs produced per donor. This increase is largely due to an enhanced rate of transplanted lungs and pancreata from organ donors. This study strongly suggests that the routine administration of high dose steroids may help to lessen the shortage of transplantable organs. P45. Induction of Green Fluorescent Protein in Cultured Keratinocytes from Transgenic Mice. W. G. Schooler, M.D., S. deSerres, B.A., J. A. Frelinger, Ph.D., A. A. Meyer, M.D., Ph.D., FACS. Departments of Surgery and Microbiology/

Immunology, University of North Carolina, Chapel Hill, North Carolina. Cultured keratinocyte (CK) grafts have been used as temporary and permanent skin replacements after massive burn injury, but the prolonged survival of these grafts has not been well established. A murine model for long-term assessment of CK autografts and allografts using transgenic mice containing the green fluorescent protein (GFP) gene from the jellyfish Aequorea victoria would permit the in vivo tracking of grafts without wound biopsy or sacrifice of the animal. In order to determine if this gene, attached to an MHC Class I (MHC-I) promotor, would express high levels of GFP, keratinocytes were exposed to IFN-g and assayed for MHC-I expression and GFP expression independently and simultaneously. CK were grown from tail skin harvested from either C57BL/6 (B6) mice or B6 transgenic mice expressing GFP (B6/GFP). Cultures were stimulated with IFN-g and compared to freshly harvested B6 and B6/GFP tail keratinocytes. Cells were stained with phycoerythrin (PE) labeled antiMHC-I antibody and were assessed for PE and GFP using FACScan analysis. The data are summarized in the table. The cells exposed to IFN-g express high levels of MHC-I. The transfected CK only express elevated levels of GFP if MHC-I is also increased, demonstrating that GFP expression is linked to MHC-I expression in this model. Since GFP expression is linked to MHC-I expression in this model, assays for CK graft persistence may also quantitate dynamic immunogenicity of the graft. P46. Liver Transplant Waiting List Morbidity: A Preliminary Report. T. Cowling, B.A., G. J. Jung, M.D., E. P. Molmenti, M.D., R. M. Goldstein, M.D., T. A. Gonwa, M.D., G. B. Klintmalm, Ph.D., and M. F. Levy, M.D. Transplant Services, Baylor University Medical Center, Dallas, Texas. We initiated a prospective study to assess the impact of the growing liver transplant waiting times on the intended recipients. Methods. All consecutive non-comatose adult patients (pts) placed on our liver transplant waiting list (W/L) (primary transplant, liver only) from 5-98 to 10-98 were asked to complete diaries describing days not working, outpatient medical procedures undergone, doctor visits, and lab tests obtained. Inpatient hospital days, ICU days, procedure/events, and diagnostic tests

TABLE—ABSTRACT P46 Parameter

Total

Patient days on W/L Outpatient doctor visits Hospital days (9 pts) ICU days (2 pts) Procedures (inpatient and outpatient) Outpatient lab draws

4566 217 98 13 45 132

302

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P47

Bld DO 2 BF LR DO 2 BF None DO 2 BF

Brain

Lung

Heart

Intestine

364 6 37 27.5 6 0.8

121 6 22 8.9 6 1.6

946 6 81 69.6 6 6.0

323 6 50* 23.8 6 3.7*

3263 6 158** 240 6 11.6**

354 6 48 47.3 6 1.7* ,**

263 6 57** 37 6 8.0* ,**

1658 6 116* ,** 234 6 16.4* ,**

575 6 65 81 6 9.2* ,**

1868 6 103* ,** 263 6 14.5**

417 6 139 31.6 6 2.4

26.5 6 12* 1.8 6 0.8

929 6 77 62.8 6 5.2

were also tracked. Fifty of 82 pts placed on the W/L (22 pts refused enrollment, 10 others were encephalopathic) enrolled in the study with a mean follow-up of 91 days (10 to 177 days). Follow-up ceases at patient death or transplantation. Results. 28 men (56%) and 22 women (44%) are now enrolled, ages 27 to 69 (median 49). Data are summarized in the table. Three patients were transplanted, one of whom died 45 days later. Two other patients with a combined W/L time of 141 days died before reaching transplant. One third (17 of 50) of the pts required five or more outpatient doctor visits. Most (34 of 47, 72%) of the pts were medically unable to work at time of W/L placement. Thirteen (28%) were working when placed on the W/L, 3 having to stop working during their wait. Conclusions. These data document, in concrete terms, the difficulty pts face while awaiting liver transplantation. Though these results are preliminary, they point to the large personal and societal burden of the liver transplant W/L and may help shape discussions on organ allocation and alternate sources of organs.

P47. Safety of Delayed Resuscitation as Shown by OrganSpecific Oxygen Delivery. R. Miraliakbari, M.D., J. M. Philpott, M.D., R. M. Lust, Ph.D., P. C. Ng, M.D., V. Kim, M.D., M. S. Swanson, M.D., Y. Sun, M.D., and W. R. Chitwood, Jr., M.D. Departments of Physiology and Surgery, East Carolina University School of Medicine, Greenville, North Carolina. Different methods of resuscitation have been evaluated using either blood flow (BF) or oxygen delivery (DO 2) but rarely both. The success of various resuscitation approaches has varied considerably depending on the chosen end point. The purpose of the present study was to simultaneously document regional BF and DO 2 in assessing basic forms of resuscitation and to evaluate the sequelae of delayed resuscitation (DR). Seventeen dogs were acutely bled to and maintained at a mean arterial pressure (MAP) of 45 mmHg for 1 h and then assigned to three treatment groups: 1) Blood (Bld, n 5 6, transfused shed blood), 2) lactated ringers (LR, n 5 6, received 33 shed blood volume), 3) None (n 5 5, no resuscitation). The experiment was completed 2.5 h after the initial hemorrhage. Final MAP was less than 50% of baseline in None group and final Hct was 50% of baseline in LR group (both p , 0.05). Final values are summarized in the table (mean 6 SEM, *p , 0.05 vs baseline, **p , 0.05 vs none, BF was measured using radioactive microspheres). By using only BF, crystalloid would have been thought to be adequate in resuscitation of most organs. In contrast, the DO 2 observed with DR was comparable to either blood or LR resuscitation for almost all the organs surveyed (except lungs). These results are consistent with the growing understanding that a judicious resuscitation delay will be adequate until the definitive treatment of the hemorrhage is addressed.

Kidney

*306 6 54 *20.6 6 3.6

1231 6 129* 83 6 8.7*

P48. Reasons for Systemic Hypertension (HTN) during Acute Elevation of Intra-abdominal Pressure (IAP) with CO 2 Pneumoperitoneum (PNP). M. Ben-Haim, M.D.,* R. J. Rosenthal, M.D.,* J. Mandeli, Ph.D,† and R. L. Friedman, M.D.‡ Departments of *Surgery and †Biomathematical Sciences, The Mount Sinai Medical Center, New York, New York; and ‡Department of Surgery, Beth Israel Medical Center, New York, New York. Introduction. In previous studies we described mechanisms in which acute elevation of the IAP induces intracranial hypertension (ICHTN). Here we sought to define the role of the ICHTN in mediating systemic HTN during CO 2 PNP. Materials and Methods. Six large animals (swine) were hyperventilated to buffer hypercarbia. Intracranial pressure (ICP) was monitored with a subdural bolt. A Foley catheter was introduced intracranially via a separate Burr hole. At phase 1, changes in ICP, central venous pressure (CVP) and mean arterial pressure (MAP) were recorded during periods of CO 2 PNP at IAP levels of 15, 20, 25 and 30 mm Hg. At phase 2 ICHTN was produced directly by inflating the intracranial balloon to the same ICP levels, which had been measured in phase 1 for each degree of IAP. CVP and MAP were recorded. Repeated Measures Analysis of Variance was applied. Results. At phase 1 the mean DCVP, DICP and DMAP increased relatively to the degree of IAP (p 5 0.0001, 0.0004 and 0.024, respectively; Table 1). At phase 2, the increments in DMAP were significant (p 5 0.024) and in the same direction and amplitude as at phase 1 (Table 2). Conclusions. In this study we could produce a comparable systemic HTN by increasing the IAP with CO 2 PNP and via direct manipulation of the ICP. We believe that it further supports our hypothesis: Elevated IAP produces immediate increase in the CVP, which impairs the venous drainage from the central nervous system (CNS), increases the ICP and initiates a CNS-mediated (Cushing’s type) response and systemic HTN.

TABLE 1—ABSTRACT P48 Phase 1: Mean Changes (D) in CVP, ICP, and MAP for Each Level of IAP (mm Hg) IAP

DCVPB

DICP

DMAP

15 20 25 30 p

12.16 6 3.9 16.16 6 1.9 19.33 6 1.8 24.6 6 2.6 0.0001

2.3 6 1 3.8 6 1.2 4.7 6 1 6.6 6 0.9 0.0004

8.5 6 6.3 7.2 6 3.4 15.3 6 7 13.2 6 7.6 0.024

303

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE 2—ABSTRACT P48 Phase 2: Mean DMAP for Each Level of Balloon Produced ICP DICP

DMAP 3 6 2.8 7.5 6 7.4 12.3 6 7.4 0.024

2–3 3–5 6–8 P

P49. Nitric Oxide (NOz) Inhibition and Dobutamine Augmentation of Cardiac Output in Shock. M. Golshan, M.D., R. Goldfarb, Ph.D., and R. Kilbourn, M.D. Department of Surgery and Medicine, Rush University, Chicago, Illinois. Introduction. NOz overproduction has been implicated in the pathological vasodilation of hemorrhagic shock. Nitric oxide synthase inhibitors such as N G -methyl-L-arginine (NMA) reverse this vasodilatation, but also cause a decreased cardiac output (C.O.). The effect of dobutamine on the NMA induced decrease in C.O. in hemorrhagic shock was studied. Methods. Ten endotracheally anesthetized pigs had pulmonary and femoral artery catheters placed and were divided into two groups of five. Hemorrhage was induced until mean arterial pressure (MAP) dropped below 40 mm Hg. Two volumes (800cc) of normal saline were rapidly infused; the animals were monitored until hypotension recurred (decompensatory phase of shock). NMA (20 mg/kg) was given and hypotension reversed, but the C.O. decreased. Upon a .20% drop in C.O. five pigs in the experimental group received dobutamine at 3mg/kg/min. Saline was infused in the controls. Means 6 SEM and ANOVA were used for analysis. Results. (See table.) Basal, hemorrhage, saline bolus and NMA C.O. in both groups were equivalent. C.O. was significantly elevated with dobutamine treatment after NMA. C.O. continued to decrease with

TABLE—ABSTRACT P49 Cardiac output, L/min Event

Control

Dobutamine

Basal readings Hemorrhage Saline bolus NMA Placebo/dobutamine

4.78 6 0.36 4.08 6 0.47 4.27 6 0.47 3.43 6 0.52 2.67 6 0.20

4.88 6 0.31 4.20 6 0.37 4.39 6 0.256 3.44 6 0.37 4.76 6 0.37* ,**

* P , 0.01 dobutamine vs NMA; ** P , 0.01 dobutamine vs placebo.

placebo after NMA. Conclusion. Dobutamine significantly ameliorates the decreased cardiac output seen after the infusion of NMA in NOz mediated shock. NMA in combination with dobutamine may lead to a decreased morbidity and mortality in patients in hemorrhagic shock.

P50. Rule of 9’s Is Not Valid for Obese Patients. S. Lee, B.S., H. Chan, and E. Livingston, M.D. UCLA Bariatric Surgery Program and the GLA-VA Health Care System, Los Angeles, California.

The rule of 9’s is often used to estimate the percentage of the body surface area involved with a burn. There have been no systematic studies of the body surface area in obese patients. Clinically this is important being that 1/3 the American population is now obese. The purpose of this study was to measure the body surface area of obese individuals to determine if the rule of 9’s accurately estimates the surface area of their major body segments. Methods. Body surface area was measured by the linear technique of Du Bois (Arch. Int. Med. 17: 863, 1916) in 47 healthy volunteers. Patients were classified as normal size if their BMI was less than 30, overweight if it was 30 –39, and obese if the BMI . 40. The % of body surface area attributable to a body part was calculated by dividing the actual measured surface area for that part divided by the total measured body surface area. Results. The % of the total body surface area represented by each major body part is presented in the table.

TABLE—ABSTRACT P50

BMI Head Arms Trunk Legs

Rule of 9’s (%)

Normal (%), n 5 18

Overweight (%), n 5 6

Obese (%), n 5 23

9 18 36 36

25.0 6 0.8 3 17 38 42

32.6 6 1.1 3 16 41 41

56.3 6 2.6 2 13 48 38

Conclusion. Body surface area of the head is far less than the often quoted figure of 9% and ranges from 2-3%. The rule of 9’s seriously underestimates the surface area of the trunk and overestimates the arm surface area in obese individuals. When estimating the contribution of a body location to the percentage of total body burn surface are we propose a rule of 5’s for obese individuals: 5% for each arm, 50% for the trunk and 20% for each lower extremity.

P51. eNOS Does Not Regulate Hepatic Perfusion in Hemorrhagic Shock. V. Schuchert, M.D., J. Baust, B.S., A. Peitzman, M.D., T. Billiar, M.D., B. Harbrecht. Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania. The constitutive endothelial isoform of nitric oxide synthase (eNOS) has been implicated in the development of hepatic dysfunction in hemorrhagic shock (HS). We and others have shown that eNOS contributes to hepatic dysfunction in shock partly by regulating sinusoidal blood flow. Previous studies utilizing pharmacologic inhibitors of eNOS are limited due to cross effects of these agents on the inducible NOS (iNOS). Mice genetically deficient in the eNOS gene have not been previously studied in models of shock or sepsis. We therefore utilized this powerful resource to investigate the specific effects of eNOS on hepatic perfusion in shock. Our hypothesis was that eNOS plays a significant role in the regulation of hepatic perfusion in HS. To test our hypothesis, eNOS deficient [eNOS(2/2)] (n 5 4) and wild type (WT) (n 5 6) mice were subjected to hemorrhage and resuscitation. Laser Doppler flowmetry was used to assess hepatic perfusion. Mean arterial pressure did not differ between groups (Fig. 1). Hepatic perfusion also did not differ significantly between eNOS(2/2) and WT mice at the end of shock, or at 1 and 2 h after resuscitation (Fig. 2). We conclude that eNOS does not play a significant role in the regulation of hepatic perfusion in early resuscitated HS.

304

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS ratio of dye in serum, pulmonary and intestinal lavages determined by spectrophotometry. PMN oxidative burst (activation) was determined by flow cytometry and number by CBC. “Priming state” (PS) was defined as PMN activation 3 number. Results. Euglycemic (EU) rats had significant pulmonary and intestinal capillary leak with 30/240 but not with shorter periods of ischemia or reperfusion. HG rats required 30 min ischemia but leaked after 1 h of reperfusion. 8 wk diabetics exhibited leak after sham and IR did not increase leak further. PMN number (#) was elevated in all sham-operated groups. PMN activation (act) was elevated in diabetic control (no laparotomy) compared to EU control. PS was positively correlated (r 5 0.9) with capillary leak (see table). The corrrelation between PS and capillary leak indicates that PS can predict those at risk for ARDS. Due to the baseline PMN activation, diabetics are predisposed to systemic injury with minimal injury. P53. Hepatic Integrity Dependent on Matrix Metalloproteinase Inhibition Not TNF-a or Different Bleeding Rates. A. S. Santibanez-Gallerani, M.D., A. E. Barber, M.D., S. J. Williams, M.S., Y. Zhao, B.S., and G. T. Shires, M.D. Department of Surgery. University of Nevada School of Medicine, Las Vegas, Nevada. Discrepancies in the levels of tumor necrosis factor-a (TNF-a) following hemorrhagic shock (HS) may be due to the inconsistent rate of bleeding. The purpose of this study was to investigate the effects of rapid vs slow bleeding rates on TNF-a levels, and if inhibition of TNF-a convertase by a Matrix Metalloproteinase Inhibitor (MMPI) affects hepatic integrity when exposed to 35% HS. SpragueDawley male rats (n 5 24) (300-350 grams) were divided into 4 groups: HS 15 (produced over 15 min), HS 30 (produced over 30 min) and with MMPI (2.5 mg/kg – British Biotech 1101). Mean arterial blood pressure (MAP), serum TNF-a, and hepatic resting membrane

TABLE—ABSTRACT P53 P52. Diabetes Predisposes to Systemic Capillary Leak through an Elevated Priming State. S. Lawson, M.D., D. Ward, M.D., W. Conner, M.D., and T. Shea-Donohue, Ph.D. Department of Surgery, Walter Reed Army Medical Center and Departments of Surgery and Medicine, USUHS, Bethesda, Maryland. Diabetics exhibit increased complications compared to normal subjects following trauma. Aim. To investigate the effects of diabetes on ischemia-reperfusion (IR) injury in rat small intestine. Methods. Rats were studied 8 wks post streptozotocin (65mg/kg ip) to induce diabetes or during an intravenous glucose infusion causing acute hyperglycemia (HG). Rats underwent sham operation (midline laparotomy) or 30 min of SMA occlusion and 1(30/60) or 4(30/240) h of reperfusion. 1 h prior to sacrifice, rats received Evan’s blue dye IV to assess intestinal and pulmonary capillary permeability based on the

HS HS HS HS

15 30 15 1 MMPI 30 1 MMPI

MAP (mm Hg) pre/post

TNF-a (pg/ml)

Em (mV)

115/32* 117/30* 115/34* 118/32*

600* 75 170* 70

226* 223* 230 231

* P , 0.05. potentials (Em) were obtained. Student t-test was performed (see table). In conclusion, rate of bleeding affects TNF-a levels. However, despite the differences in the magnitude of TNF-a in untreated animals, hepatic integrity was compromised. Interestingly, MMPI, an inhibitor of TNF-a convertase, stabilizes the membrane potential in both types of hemorrhagic shock.

TABLE—ABSTRACT P52 Pulmonary leak

EU HG diab

PMN data

Sham

30/60

30/240

362 661 16 6 2*

462 13 6 2*

23 6 1* 16 6 2* 12 6 2*

* P , 0.05; Dbc, diabetic control; Dbs, diabetic sham.

PS # Act

Cont

Eu/ir

Dbc

Dbs

2.7 7.1 0.4

25* 40* 0.6

3.4 7.1 0.5

16.3* 29.7* 0.6

305

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

TABLE—ABSTRACT P55

P54. The Effects of Interleukin-10 in Hemorrhagic Shock. S. Karakozis, M.D., M. Hinds, M.D., D. Kim, M.D., H. Provido, B.S., and J. R. Kirkpatrick, M.D., FACS. Department of Surgery, Washington Hospital Center, Washington, DC. Interleukin-10 (IL-10) counteracts the effects of the proinflammatory cytokines: interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor (TNF). Experimental data suggest that inhibition of proinflammatory cytokines may improve the outcome in hemorrhagic shock. We hypothesize that the use of IL-10 in hemorrhagic shock will attenuate the detrimental effects of the proinflammatory cytokines and improve survival. Methods. To test this hypothesis, twenty rats were randomized into two groups: control and experimental. All rats were bled to maintain the mean arterial pressure below 50mmHg for 120 min. The rats were resuscitated with the shed blood and an equal volume of normal saline. The experimental group received 5 mg of IL-10 prior to the initiation of shock. Blood samples were obtained prior to shock, at the end of shock and one hour after resuscitation. We measured the serum levels of lactate, IL-1, IL-6 and TNF at the above time intervals. The rats were followed for 72 h to calculate the survival rates. The Kruskal-Wallis test was used for comparisons. Results. 1. Similar levels of ischemia were obtained as evidenced by the levels of lactate and the amount of shed blood. 2. Survival rates were equal (70%). 3. Serum TNF levels (pg/ml) were significantly lower at the end of shock in the experimental group, (p , 0.05) (see figure). 4. Serum levels of IL1 and 6

were not significantly different between the groups. Conclusion. IL-10 suppresses the level of TNF but does not improve survival in hemorrhagic shock. P55. Thermotolerance Protects against Endotoxin-Mediated Microvascular Injury. G. Chen, M.D., C. J. Kelly, FRCSI, H. Chen, M.D., A. Leahy, FRCSI, and D. J. Bouchier-Hayes, FRCSI, Department of Surgery, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland. Thermotolerance has been shown to protect against acute lung injury in sepsis and endotoxemia. However, the effect of thermotolerance on lipopolysaccharide (LPS)-mediated leukocyte-endothelial interactions in vivo is not known. The aim of this study was to investigate the effect of thermotolerance on leukocyte adherence and migration mediated by LPS. Intravital video microscopy was used to examine hemodynamic parameters, leukocyte rolling, adhesion and migration in rat mesenteric postcapillary venules. Sprague-Dawley rats were randomized into saline, LPS and thermotolerance (T)1LPS groups. Thermotolerance was induced 18 h prior to administration of LPS by elevating core body temperature to 4110.5°C for 15 min. LPS (W055:B5 15mg/kg) was administrated via the jugular vein after baseline record. Leukocyte rolling velocity (RV), the number of adherent (Na) and migrated (Nm) leukocytes were measured by intravital microscopy at baseline (0 min), 2, 10, 30, 60 and 90 min after LPS administration. HSP72 expression in tissues was determined by Western immunoblotting (see table). LPS significantly increased the Na at 30, 60, and 90 min and Nm at 60 and 90 min and decreased the RV of leukocytes at 10, 30, 60 and 90 min after administration. Thermotolerance significantly reduced the increase in Na and NM and the decrease in RV induced by LPS. Expression of HSP72 was induced in mesentery, gut and lung by T. Thermotoler-

RV (mm/sec) Na (/100 mm) Nm (/field)

Saline

LPS

T 1 LPS

49.3 6 1.96 1.8 6 0.60 2.7 6 0.42

38.0 6 2.52** 10.0 6 1.57* ,*** 8.3 6 1.76* ,***

41.3 6 2.38 4.0 6 0.68 3.0 6 0.68

Note. Results, mean 6 SEM; statistics, ANOVA with Scheffe posthoc correction, at 90 min after LPS. * P , 0.01 vs saline; ** P , 0.05; *** P , 0.01 vs T 1 LPS.

ance attenuated LPS-induced microvascular injury by decreasing leukocyte-endothelial adhesion and migration. P56. Shifts in IL-4 Are Significantly Associated with Adverse Events in Burn Patients. R. D. Robertson, M.D., J. B. Cone, M.D., B. H. Wallace, MHSA, C. D. Bridges, B.S., and P. J. Bond, R.N. Department of Surgery, University of Arkansas, Little Rock, Arkansas. Cytokines in response to sufficient stimulus may “spillover” into the circulation serving as a marker for the magnitude of the stimulus. This study attempts to determine whether serum markers of dysregulation of the inflammatory response were associated with adverse events. Sixteen seriously burned patients (47 6 23% BSA; 31 6 21% FT BSA; Age 43 6 17 years) had daily determinations of IL-4, IL-10 and IFN-g during the first two weeks postburn and twice weekly thereafter. Invasive wound infection, bacteremia, organ failure requiring ventilation, dialysis or pressors and death were scored as adverse events. Each week post burn, plasma was analyzed for IL-4/INF-g ratio and whether an adverse event occurred that week. Mortality was 31% with one-half of the patients experiencing an adverse event. Initial values of IL-4, IL-10 and INF-g were elevated (22 6 47, 72 6 73, 25 6 17pg/ml). Overall, males had significantly higher levels of IL-4 (9 6 25 vs 3 6 6; p 5 0.002) and IL-10 (31 6 48 vs 13 6 27; p 5 0.0005) when compared to female patients. Patients experiencing adverse events had higher levels of IL-4 (10 6 26 vs 1.3 6 6.2; p 5 0.0001) and IL-10 (34 6 50 vs 8 6 16; p 5 0.0001) and lower levels of IFN-g (20 6 5 vs 24 6 14; p 5 0.04). When the occurrence of adverse events was compared on a weekly basis with the highest ratio of IL-4/INF-g for the week, a strong association was noted between adverse events and ratios greater than 0.05 (p , 0.0001). Of 27 adverse events, 24 occurred when the ratio exceeded 0.05. Adverse events are strongly associated with an increase in circulating IL-4 and a smaller but significant decrease in IFN-g. These shifts parallel those seen in T-cell populations shifting from Th1 to Th2 that are associated with decreased cell mediated immunity. This shift was associated with an increased incidence of both infectious and non-infectious complications. P57. Lisofylline Protects IEC-6 Cells against CytokineInduced Apoptosis Independent of Nitric Oxide (NO) Production. E. P. Nadler, M.D., J. S. Upperman, M.D., and H. R. Ford, M.D. Department of Surgery, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania. Introduction. We have previously shown that endotoxemia promotes gut barrier failure and bacterial translocation (BT) by upregulating intestinal NO production. Lisofylline (LSF), a novel anti-inflammatory compound, inhibits endotoxin (LPS)-induced gut barrier failure and BT in vivo. The mechanism may involve decreased enterocyte apoptosis in the villus tips. This experiment was designed to study the mechanism by which LSF prevents enterocyte apoptosis in vitro. Methods. Rat embryonal intestinal cells (IEC-6) were pre-incubated with various doses of LSF for 1 h.

306

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

The cells were then stimulated with a combination of LPS, interleukin-1, tumor necrosis factor-a, and interferon-g (cytomix or CM). At 24 h, apoptosis was measured using annexin V and propidium iodide labeling and FACS analysis; supernatant nitrite/nitrate (NO 22 /NO 32 ) was measured using the Greiss reaction. Results. CM significantly increased the percentage of apoptotic cells and NO 22 /NO 32 production (see table). LSF treatment

TABLE—ABSTRACT P57

% Apop. NO 2/NO 3

Media

CM

CM 1 LSF (10 mM)

CM 1 LSF (100 mM)

19.7% 0

28.5% 15.2 6 1.9*

17.9%** 18.6 6 6.5*

11.7%** 14.5 6 0.6*

* P , 0.05 vs media, Student’s t test; ** P , 0.001 vs CM, x 2 analysis.

during murine P. aeruginosa bacteremia. This protein warrants further testing as a therapeutic reagent that may have clinical utility for the treatment of serious gram-negative bacterial infection and sepsis.

reduced apoptotic rates below baseline, but had no effect on NO 22 / NO 32 production. Conclusions. LSF abrogates CM-stimulated apoptosis in IEC-6 cells. The reduction in apoptosis is independent of NO production. These findings corroborate our previous in vivo data and support the conclusion that LSF may be a useful adjunct in preventing the gut barrier dysfunction associated with sepsis through NO-dependent mechanisms.

P59. Sublytic Complement Activation Increases Intracellular Sodium in Isolated Rat Skeletal Muscle. K. Okamoto, M.D., W. Wang, M.D., J. Rounds, B.S., E. Chambers, M.S., and D. O. Jacobs, M.D. Laboratory for Surgical Metabolism & Nutrition, Department of Surgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts.

P58. Limulus Anti-LPS Factor (LALF) Is Bactericidal and Improves Survival during Murine P. aeruginosa Bacteremia. C. A. Weiss III, M.D., K. R. Wasiluk, Ph.D., T. A. Kellogg, M.D., and D. L. Dunn, M.D., Ph.D. Department of Surgery, University of Minnesota, Minneapolis, Minnesota. LALF is a 12 kDa protein from the American horseshoe crab that binds the toxic lipid A moiety of gram-negative lipopolysaccharide (endotoxin, LPS) in vitro. However, its activity in vivo has not been well characterized. Purpose. Characterize LALF: 1) bactericidal activity and 2) protective capacity during P. aeruginosa bacteremia. Methods. Mice received intraperitoneal (ip) 1) LALF (4 mg/kg), 2) Polymyxin B (PMB, an antibiotic that also neutralizes lipid A, but which is toxic in vivo to humans, 1 mg/kg), or 3) BG16 (an irrelevant control peptide, 12 mg/kg) immediately before and 30 min after receiving ip P. aeruginosa. Blood was collected 60 min after bacterial challenge, diluted and plated on MacConkey agar (see figure). Survival data were analyzed by x 2 or ANOVA and Student’s t test. Results. LALF and PMB treatment improved survival (9/10, 10/10, respectively) and reduced bacteremia (figure) when compared to BG16 (1/10, p , 0.01). Conclusions. LALF is bactericidal and improves survival

Previous in vivo studies have demonstrated that complement activation during sepsis is associated with alterations in transmyocellular sodium gradients. To determine if activated complement proteins can directly mediate changes in sodium permeability, fast-twitch extensor digitorum longus (EDL) muscles were isolated from infant rats weighing 40-60g. EDL muscles were incubated under normoxic conditions at 30°C for 60 min in: 1) Krebs-Henseleit buffer (KHB); 2) KHB containing 10% normal human serum (NHS); 3) 10% zymosan activated NHS (ZAS); 4) 10% heat-inactivated NHS (HIS); or 5) 10% C7 deficient human serum (C7D), which is unable to form the terminal complement complex (C5b-9). Intracellular sodium ([Na 1 ] i ) and potassium ([K 1 ] i ) contents of the muscles were determined after washing in ice-cold Na 1 and K 1 free Tris-sucrose buffer. LDH release (%LDH) from the muscles was determined during incubation (see table). Average LDH release was identical in all groups and less than 5%, which indicates that sublytic complement activation had occurred (,10% LDH release in the presence of excess complement). Two to 2.5 fold increases in [Na 1 ] i and the [Na 1 ] i /[K 1 ] i ratio were detected in muscles incubated with NHS as well as ZAS, which suggests that the effects of activation by heterogeneity and zymosan were physiologically equivalent. Changes in [Na 1 ] i and [K 1 ] i did not occur in muscles treated with HIS or C7D. We conclude that sublytic complement activation and C5b-9 formation can directly alter intracellular sodium content in fast-twitch skeletal muscle.

TABLE—ABSTRACT P59

[Na 1] i (mmol/g) [Na 1] i/[K 1] i %LDH (%)

KHB (n 5 14)

NHS (n 5 6)

ZAS (n 5 6)

HIS (n 5 4)

C7D (n 5 4)

10.1 6 1.1 0.14 6 0.01 3.1 6 0.6

25.2 6 1.9* 0.29 6 0.02* 4.9 6 0.7

24.6 6 2.9* 0.33 6 0.03* 4.8 6 1.2

11.1 6 4.0 0.13 6 0.04 4.4 6 0.9

14.9 6 0.8 0.17 6 0.01 3.9 6 1.4

Note. Mean 6 SEM, one-way ANOVA, LSD, * P , 0.01 vs KHB, HIS, C7D.

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

307

P60. Hypertonic Saline Primes the PMN Respiratory Burst via Activation of p38 MAPK. D. J. Ciesla, M.D., E. E. Moore, M.D., G. Zallen, M.D., W. L. Biffl, M.D., and C. C. Silliman, M.D., Ph.D. Department of Surgery, Denver Health Medical Center and University of Colorado HSC, Denver, Colorado. Priming increases PMN O 2- production and posttraumatic acute lung injury. p38 MAPK activation is a key intracellular signaling event in PMN priming and activation. HTS activates p38 MAPK at high concentrations ([Na 1] . 300mM) and would be expected to exacerbate acute lung injury. However, HTS resuscitation reduces lung injury in rat hemorrhagic shock models and decreases PMN O 2production in vitro. This apparent paradox is explained by the finding that HTS in the clinical range ([Na 1] , 200mM) does not activate p38 MAPK. Since the HTS effects on PMN cytotoxicity are reversed upon return to normotomicity, we hypothesized HTS 300 activates p38 MAPK and primes PMN O 2- production upon return to normotonicity. Methods. Isolated PMNs in KRPD were made hypertonic (HTS 300) by addition of 4M NaCl and incubated for 5min at 37°C then returned to normotonicity by addition of Na 1 free buffer for 5min at 37°C. p38 MAPK activation was assessed by western analysis. PMN O 2- production was assessed by reduction of cytochrome c after PMA

activation. Data are means 6 SEM. Results. HTS 300 activated p38 MAPK and attenuated PMA mediated O 2- production. Upon return to normotonicity, p38 MAPK activation returned to baseline but PMA mediated O 2- production was increased (see figure, *p , 0.05 vs PMA control). Conclusions. HTS 300 activates p38 MAPK and primes PMN O 22 production upon return to normotonicity.