WEDNESDAY MORNING, APRIL 21, 1999

7:00 a.m. GENERAL THORACIC SURGERY FORUM SESSION

Room 211-213, Ernest N. Morial Convention Center

Moderators: Mark K. Ferguson, M.D.

Valerie W. Rusch, M.D.

F1. A Rational Approach to Substitution of The NOcGMP Pathway in Lung Transplantation - Overview of a Series of Experiments

Ralph Alexander Schmid*, Sven Hillinger*, Gabriele Schoedon* and Walter Weder*, Zurich, Switzerland

Sponsored By: G. Alexander Patterson, St. Louis, Missouri

OBJECTIVE(s): Impairment of the NO pathway accelerates ischemia/reperfusion injury following lung transplantation (LTPL). Direct application of NO is not ideal because of its short half-life and toxic side effects. Tetrahydrobiopterin (BH4) is the essential coenzyme of the NO synthase (NOS). 8-Br-cGMP is a membrane permeable analogue of cGMP, second messenger of the NO pathway. We evaluated the effect of several treatment modalities with these compounds on posttransplant lung edema in a large animal model of LTPL.

METHODS: Unilateral left LTPL was performed in 25 weight matched out-bred pigs (all groups n=5). Donor lungs were flushed with 1.51 cold LPD solution and preserved for 20h at 1°C. All donors (except group III) received 250mg PGE1 into the pulmonary artery prior to flush. Treatment in experimental groups was as follows: I: BH4 (20mg/kg) to recipient over 30 min, starting before reperfusion; II: as in group I plus continuous dose BH4 (10mg/kg/h); III: 8-Br-cGMP (1mg/kg) to flush solution; IV: 8-Br-cGMP (0.2mg/kg/h) continuously to recipient; V: control. Extravascular lung water index (EVLWI) and hemodynamic parameters (PAP, PVR, CO) were assessed during a five hour observation period. Lipid peroxidation (TEARS) and neutrophil migration (MPO) in the allograft were measured at the end of the assessment.

RESULTS: EVLWI (ml/kg) in group I: 8.4±0.9; II: 7.0±.0.5; HI: 6.7±1.0; IV: 8.2±0.3; V: 10.1±0.6, MPO (DOD/mg/min) in group I: 1.1±0.2; II: 1.0±0.1; III: 0.9±0.1; IV: 1.0±0.2; V: 1.7±0.3, TEARS (pmol/g) in group I: 68.2±11.3; II: 65.7±7.9; III: 65.6±7.9; IV: 61.8±12.3; V: 120.8±7.2. No effect on pulmonary and systemic hemodynamic parameters could be detected with either treatment.

CONCLUSIONS: Our results show that substitution of the NO pathway by BH4 or 8-Br-cGMP reduces posttransplant pulmonary edema, neutrophil migration, and lipid peroxidation in the allograft. Addition of 8-Br-cGMP to the flush solution is superior to PGE1. Based on pharmacologic considerations of both substances and the presented results, administration of cGMP in the flush solution and BH4 during reperfusion seems to be very promising.

*By Invitation

F2. Cyclooxygenase-2 is Up-regulated in Lung Cancer

Fan Zhang*, Amit Kumar*, Yu-chang Wu*, Eun K. Yang*, Robert A. Soslow*, Jamie L. Masferrer*, Alane Koki-Goodson*, Andrew J. Dannenberg* and Nasser K. Altorki, New York, New York, St. Louis, Missouri

OBJECTIVE: Prostaglandin (PGs) are important in the pathogenesis of cancer because they affect mitogenesis, cellular adhesion, immune surveillance and apoptosis. Epidemiological studies have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs), known inhibitors of PG synthesis, protect against lung cancer. Two cyclooxygenase (COX) isoenzymes catalyze the conversion of arachidonic acid to PGs: constitutive COX-1 and inducible COX-2. The recent finding that a COX-2 null mutation protected against the formation of intestinal and skin tumors suggests that COX-2 represents an important target for anti-cancer therapy. The goal of this study was to determine whether COX-2 is a potential target for preventing or treating lung cancer.

METHODS: The expression and activity of COX-2 were compared in human lung cancer vs. adjacent nontumorous tissue. Levels of PGE₂ were determined by enzyme immunoassay. Amounts of COX-2 protein (immunoblotting, im-munohistochemistry) and mRNA (quantitative RT-PCR) were measured in surgical specimens.

RESULTS: Levels of PGE₂ were increased by about 4-fold in cancerous vs. adjacent non-rumorous tissue (n=19, p<0.001). A corresponding increase in amounts of COX-2 protein was detected in both adenocarcinoma and SCC of the lung. Amounts of COX-2 mRNA were also increased in the majority of cases of lung cancer. Immunohistocehmical evaluation indicated that COX-2 protein was expressed in 18/20 lung cancers and localized to malignant epithelial cells.

CONCLUSIONS: Levels of COX-2 and PGs are increased in adenocarcinoma and SCC of the lung. These results may explain the prior epidemiological findings that NSAIDs protect against lung cancer. Newly developed selective inhibitors of COX-2 may prove useful in preventing or treating lung cancer.

*By Invitation

F3. Proliferation of Airway Epithelial Cells Secondary to Anti-HLA Antibodies: A Potential Mechanism for Bronchiolitis Obliterans Syndrome.

Scott I. Reznik*, Andres Jaramillo*, Wan J. Zhang*, G. A. Patterson, Joel D. Cooper and T. Mohanakumar*, St. Louis, Missouri

OBJECTIVE: Development of antibodies to donor HLA after lung transplantation is associated with earlier onset of bronchiolitis obliterans syndrome (BOS) and decreased graft survival. We sought to determine the mechanism by which anti-HLA antibodies effect chronic chronic lung allograft rejection. We postulate that anti-HLA antibodies bind HLA class I molecules on the surface of donor lung epithelium and stimulate phosphorylation and proliferation.

METHODS: A549, a lung epithelial carcinoma cell line, was cultured in serum deficient media for 48 hours to produce static growth. Cells were then treated with complete media (15% FBS), media containing HLA-sensitized serum from lung transplant recipients, pooled HLA-sensitized sera, non-sensitized sera or deficient media containing either W6/32 anti-HLA monoclonal antibody or mouse IgG. [³H-]-thymidine was determined at 24,48 and 72 hours. In phosphorylation studies, cells treated as above were assayed for tyrosine phosphorylation at one minute by western blot analysis.

RESULTS: Cells treated with HLA-sensitized sera exhibited proliferation at 24,48 and 72 hours equivalent or greater than cells treated with complete media. Cells treated with non-sensitized serum showed significantly less proliferation. Treatment with W6/32 induced proliferation at levels equal to or greater than cells treated with complete media. Increased phosphorylation of cellular proteins was observed in cells treated with HLA-sensitized sera and W6/32.

CONCLUSIONS: These data indicate that anti-HLA antibodies stimulate lung epithelial cells and may play important role in the development of BOS. Immunosupression of the humoral immune response may be pivotal in the delaying the onset of BOS in patients with HLA reactive antibodies.

*By Invitation

F4. Transgenic Swine Lungs Expressing hCD59 in a Pig-to-Human Model of Xenotransplantation

David M. Kulick*, Christopher T. Salerno*, Agustin P. Dalmasso*, Soon J. Park*, Manuel Guzman-Paz*, ^§William Fodor*, ^§Steven Squinto* and R. M. Bolman, III, Minneapolis, Minnesota, New Haven, Connecticut

OBJECTIVE: Pulmonary Xenotransplantation is currently limited by hyper-acute rejection mediated in part by xenoreactive natural antibody and complement. Transgenic swine organs, expressing the human complement regulatory protein CD59 (hCD59), have demonstrated improved survival in models of pig-to-primate Xenotransplantation. Our objective was to evaluate transgenic swine lungs expressing hCD59 in an ex-vivo model of pig-to-human Xenotransplantation.

METHODS: Transgenic swine lungs (n=4, experimental group) and outbred swine lungs (n=6, control group) were perfused with fresh, whole, human blood via a centrifugal pump on an ex-vivo circuit. Functional data were collected throughout each perfusion. Immunoglobulin and comlpement studies were performed on perfusate samples, and both histologic and immunofluorescent analyses were performed on tissue sections.

RESULTS: Mean lung survival for the experimental group was significantly increased when compared to controls, 240±0 min vs. 35.3±5.9 min respectively, p<0.01. A decreased rise in pulmonary vascular resistance was observed in the experimental group 380±75 dynes-sec-cm^-5 in contrast to the control group 985±338 dynes-sec-cm^-5, p<0.01. Lung compliance was improved for the experimental group vs. the control group, 8.7±.97 ml-cm^-2 H₂O and 4.8±1.2 mi-cm² H₂O, respectively, p<0.05. SC5b9 levels measured by ELISA were significantly lower for the experimental group vs. controls 901±224 units and 4069±828 units repectively, p<0.01. Immunofluorescent examination of tissue sections demonstrated equivalent deposition of IgG, IgM, C1q, and C3 in both groups, with reduced deposition of C9 in the experimetnal group.

CONCLUSIONS: Transgenic swine pulmonary xenografts expressing hCD59, demonstrate improved function and survival in an ex-vivo model of pig-to-human Xenotransplantation.

^§Authors have a relationship with Alexion Pharmaceuticals

*By Invitation

F5. Prolonged Discordant Lung Orthotopic Xenograft Survival

Paolo Macchiarini*, Rafael Oriol* and Philippe Dartevelle, Robinson Le Plessis, France, Villejuif, France

OBJECTIVE(s): We used a pig-to-goat orthotopic lung xenograft model to test whether depletion of goat xenoreactive antibodies (XNA) against pig red blood cells would prolong pig lung xenograft survival and to study the late phase of discordant lung xenograft rejection.

METHODS: Adult goats with anti-pig XNA underwent left pneumonectomy followed by orthotopic transplantation of pig left lung (group 1) or immunodepletion of XNA by extra-corporeal right pig lung perfusion before transplantation without (group 2) or with (group 3) complete occlusion of the right pulmonary artery (RPA). In group 4, goat left lungs were orthotopically transplanted into pigs and served as negative controls (pig serum does not have anti-goat XNA). Each study group included 5 animals. Immunosuppression in surviving recipients included cyclosporin and azathioprine. Open chest xenograft biopsies were made on the 2nd postoperative day and at sacrifice.

RESULTS: Group 1 recipients died 7±3 hours following xenograft reimplantation due to severe pulmonary hypertension and right heart failure, with little evidence of histological xenograft injury. Group 2 xenografts had significantly (p<0.001) lower pulmonary vascular resistances and higher blood flow compared to group 1 animals, and recipients survived for 9±4 days. Group 3 animals tolerated also complete occlusion of the RPA, and the xenografts assured the total respiratory support for 4±1 days. After immunodepletion, goat serum showed no detectable titers of XNA which reappeared on the 2nd postoperative day. On the 2nd postoperative day, all immunodepleted xenografts showed features of delayed humoral (perivas-cular stromal cells, microvasculature thrombosis, interstitial hemmorrhages) and cellular (perivascular and interstitial lymphocyte infiltration) vascular rejection (DVR). At sacrifice, all xenografts were completely necrosed being the contralateral lungs intact. Group 4 xenografts showed scattered features of acute rejection.

CONCLUSIONS: Immunodepletion prolongs left lung xenograft survival, even after occlusion of the RPA. Despite immunosuppression, DVR and xenograft necrosis occurred and corresponded to the return of XNA titres.

*By Invitation

F6. Functional Interleukin-4 Receptor and Interleukin-2 Receptor Common Gamma Chain on Human Non-Small Cell Lung Cancers: Novel Targets for Directing Immune Therapy

Richard Essner*, Young Huynh*, Tung Nguyen*, Donald L. Morton and Dave S. B. Hoon*, Santa Monica, California

OBJECTIVE: IL-4R has been demonstrated on human non-small cell lung carcinoma cell lines (NSCLC) and rumor specimens. IL-4 causes G₁ phase cell-cycle arrest of lung cancer cells expressing IL-4R; the effect directly correlates with expression of IL-4R and is seen within 48 hr after treatment. We examined signal transduction pathways employed by IL-4R, which may account for growth arrest of established line LUst and but had no effect on a second cell line SK-MES-1.

METHODS: Western blot analysis was performed on both cell lines cultured up to 48 hr in the presence of IL-4 (500 U/ml). Cells were lysed, protein-extracted, and electroblotted; blots were then probed with murine monoclonal antibodies to specific intracellular proteins.

RESULTS: Western blotting of cell lines with anti-phosphotyrosine antibody (4G10) demonstrated multiple (140, 100-130, and 65 kD) phosphoproteins in IL-4 treated LUst but not in IL-4 treated SK-MES-1. Immunoprecipitation and blotting of LUst with specific secondary antibodies showed that the 140 kD phosphoprotein was IL-4R, the 100 kD phosphoprotein was nuclear transcription factor STAT 6, the 130 kD phosphoprotein was janus kinase (JAK-1), the 120 kD phosphoprotein was JAK-3, and the 65 kD phosphoprotein was IL-2R common gamma chain (IL-2Rgc). Specific binding was not observed in SK-MES-1, suggesting the absence of a functional IL-2Rgc. Southern blotting with cDNA probes to IL-2Rgc confirmed absence of this cytokine receptor on SK-MES-1.

CONCLUSIONS: These results suggest that human NSCLC may express functional cytokine receptors, including the IL-2Rgc commonly associated with lymphocyte IL-2R. These receptors may be novel targets for directing cytokine-based immune therapy for NSCLC.

*By Invitation

F7. Paclitaxel Induces Apoptosis in Lung Cancer Cells by Increasing Caspase-3 Activity But May Not Require Fas and Fas Ligand

Christine Odoux*, Michael Lotze*, Peter K. Kim*, Andy Amoscato*, James D. Luketich*, Robert J. Keenan and Tracey L Weigel*, Pittsburgh, Pennsylvania

OBJECTIVE: The Fas receptor when bound by its ligand (FasL) induces programmed cell death (apoptosis). Both Fas and FasL have been documented on the cell surface of lung cancer cell lines (LCCLs). Paclitaxel (PA), one of the most active chemotherapeautic agents for NSCLC, has been shown to induce apoptotic cell death.

METHODS: To elucidate the role of Fas/FasL in PA induced apoptosis of LCCLs, we cultured 2 squamous (A549, H226) and 1 bronchoalveolar (H358) patient-derived, NSCLC cell lines in 10 uM paclitaxel. Cell morphology was assessed @ 24 hrs for nuclear and cytoplasmic condensation by methylene blue/Azure A/eosin staining. Apoptosis was confirmed by DNA laddering. Caspase-3 (cytoplasmic protease in the apoptotic cascade) activity was measured by a Z-DVED cleavage assay. Expression of Fas and FasL was assessed immunohistochemically with human anti-Fas and anti-FasL antibodies.

RESULTS:Paclitaxel consistently induced apoptosis in LCCLs as determined by morphological analyses, DNA laddering and increased Caspase-3 activity. The effect of PA on Fas/FasL expression was variable (see table).

* Values are expressed as percentages of controls from untreated cells.

** Mann-Whitney U test, n = 4 experiments, NS = not significant

CONCLUSIONS: Caspase-3 activity was significantly increased in all LCCLs after culture in PA. Upregulation of Fas and FasL did not appear requisite for PA induced apoptotis of LCCLs.

*By Invitation

F8. Respiratory Failure after Thoracic Surgery: The Incidence of Aspiration can be Limited by GI Tract Management

John R. Roberts*, Karla R. Christian*, Richard R. Pierson*, Davis C. Drinkwater and Walter H. Merrill, Nashville, Tennessee

OBJECTIVE: Respiratory failure is the major cause of mortality after general thoracic surgery. However, respiratory failure may result from two very different causes: aspiration, which results from ileus and reflux, and pneumonia, which results from poor pain control and weak cough. Epidural catheters help control pain and prevent pneumonia, but contribute to ileus and may increase aspiration. We compared our results after thoracotomy before and after a change in management designed to decrease the risk of aspiration.

METHODS: All patients undergoing thoracotomy by a single surgeon over three years were evaluated for aspiration, pneumonia, morbidity, and mortality. For the first eighteen months, patients (N=129) did not receive an intraoperative NG tube and were started on "advance as tolerated" diet postoperatively. For the second 18 months (N=141) NG tubes were placed intraoperatively and diets advanced slowly. Patients were considered to have pneumonia if they developed infiltrates in a single or adjoining lobes and grew a dominant organism from their sputa. Patients were considered to have aspirated if they developed diffuse infiltrates and multiple organisms. Chi-square testing was used to test significance.

RESULTS: Two hundred seventy patients underwent thoracotomy at five different hospitals over a three-year period. Seven patients (5.43%) developed respiratory failure on ad lib diets in the first eighteen months, five due to aspiration and two due to pneumonia. Three (2.11%) developed respiratory failure in the second eighteen months, all due to pneumonia. The difference was highly significant (p=0.0183).

CONCLUSIONS: Careful GI tract management decreased the incidence of aspiration in this cohort of patients undergoing thoracotomy who received epidurals for pain control.

*By Invitation

F9. Sequence-dependent Enhancement of Taxol Sensitivity in Non-Small Cell Lung Cancer by the erbB-2 Tyrosine Kinase Inhibitor NSC 330507.

Dao Nguyen*, Aaron Chen*, Arnold Mixon* and David S Schrump*, Bethesda, Maryland

Sponsored By: Jack A. Roth, M.D., Houston, Texas

OBJECTIVE: Overexpression of the erbB-2 oncogene may contribute to chemoresistance in NSCLCs. The aim of this study was to determine if down regulation of this oncogene by NSC 330507 (NSC) could enhance taxol sensitivity in NSCLCs.

METHODS: Four NSCLC lines (H460, H1299: low erbB-2; H322, H358: high erbB-2) were treated with taxol(90 minutes exposure, 0.016 to 16 mM) and NSC (96 hrs continuous exposure, 20 to 80 nM) as follow: taxol + NSC (schedule 1) or 24 hrs NSC treatment prior to NSC (schedule 2). Taxol IC50 values of each groups were calculated. Apoptosis was evaluated by ApoBrdU technique.

RESULTS: Sensitivity to taxol was significantly enhanced (ØIC50) only in high erbB-2 cells treated with the schedule 1 combination compared to taxol alone controls: 0.12±0.08mM vs 0.45±0.13mM and 0.75±0.12mM vs 1.9±0.4mM in H322 and H358 cells respectively (n=4, p<0.01). Significant apoptosis was noted in H322 cells treated with the schedule 1 combination of taxol (0.5mM) and NSC (40nM): control:<1%, NSC:<2%, taxol:16.0±1.4%, taxol+NSC:48.5±8.0% (p<0.01, n=3). Moreover, all cells treated with the schedule 2 became less sensitive to cytotoxic effects of taxol, possibly related to G1 cell cycle arrest induced by NSC (as previously observed) prior to taxol treatment.

CONCLUSIONS: Downregulation of erbB-2 gene function by NSC results in sensitization of high erbB-2 NSCLC cells to taxol. This synergistic effect is sequence-dependent which has important implication in development of combination chemotherapy regimens for lung cancer. Mechanism(s) of this salutary effect of NSC 330507 is currently under investigation.

*By Invitation

F10. Decitabine-mediated Induction of NY-ESO-1 and MAGE-3 Cancer-Testis Antigen Expression in Lung Cancer Cells and EBV-transformed Lymphocytes

David S. Schrump*, Li Lee*, Dao Nguyen*, Rong F. Wang*, Xiang Wang* and Steven A. Rosenberg*, Bethesda, Maryland

Sponsored by: Jack A. Roth, M.D., Houston, Texas

OBJECTIVE: Previously we observed expression of NY-ESO-1 in 40% of NSCLC lines, and demonstrated that expression of this tumor antigen can be induced by the demethylating agent, decitabine (DAC). Recently we further defined the kinetics of NY-ESO-1 (and MAGE-3) induction in lung cancer cells.

METHODS: CALU-6 NSCLC cells, normal human bronchial epithelial (NHBE) cells, and EBV-transformed lymphocytes (EBV-L) were exposed to DAC at 4 mM x 8 h, 2 mM x 24h, 1 mM x 24h, and 1 mM x 96h. RT-PCR techniques were utilized to evaluate NY-ESO-1 and MAGE-3 expression 96h following initiation of DAC treatment. Cytokine release assays were utilized to evaluate recognition of EBV-L by an HLA restricted TIL clone specific for NY-ESO-1.

RESULTS: NY-ESO-1 expression was detected in CALU-6 cells following 8h DAC exposure, and induction following 24h treatment was comparable to that observed after continuous 96h exposure. MAGE-3 expression was similarly augmented under these conditions. No induction of NY-ESO-1 or MAGE-3 was observed in near confluent NHBE cells or EBV-L following similar treatment; however, expression of NY-ESO-1 could be detected in log phase NHBE cells and EBV-L following DAC exposure at 1 mM x 96h and 2 mM x 8d, respectively. EBV-L expressing NY-ESO-1 were recognized by an autologous melanoma TIL clone specific for this antigen.

CONCLUSIONS: NY-ESO-1 and MAGE-3 cancer-testais antigen expression can be induced under conditions which are known to facilitate p16 expression and have been achieved in leukemia patients. Demethylating agents may be useful for simultaneously enhancing immunogenicity and cell cycle arrest in lung cancer cells. Furthermore, decitabine treated EBV-L may prove efficacious for the immunotherapy of lung cancer patients whose tumors express NY-ESO-1.

*By Invitation

WEDNESDAY MORNING, APRIL 21,1999

7:00 a.m. ADULT CARDIAC SURGERY FORUM SESSION

Ballroom, Ernest N. Morial Convention Center

Moderators: Verdi J. DiSesa, M.D.

Edward D. Verrier, M.D.

F11. Cardiac Allograft Long-Term Functional Tolerance

Without Immunosuppressive Drugs in Mice Transgenic For a Suicide Gene

Eric Bratmberger*, Nathalie Raynal-raschilas*, David Klatzmann*, Olivier Boyer*, Patrick Bruneval*, Jean-Noel Fabiani*, Denis Glotz* and Alain Carpentier, Paris, France

OBJECTIVE: Life-long immunosuppression is a major cause of mortality and morbidity in transplant recipients. Gene therapy could provide new ways to obtain tolerance and avoid indefinite immunosuppression.EpTK mice are derived from the FVB/N strain (H2q) and express the Thymidine Kinase gene of the Herpes virus in all mature T cells. Thus, any mature dividing T cell can be killed in the presence of Ganciclovir (GCV). We investigated the survival of allo-incompatible C57B1 /6 (H2b) hearts heterotopically transplanted into EpTK mice given only GCV from day 0 to day 7 or 14.

METHODS: Abdominal cardiac transplantation was performed in 22 controls (untreated FVB n=15, GCV treated FVB n=5, untreated EpTK mice n=2), and in 28 EpTK mice given GCV from day 0 to day 7 (n=15) or 14 (n=13). Rejection was defined as complete cessation of cardiac beats. Histological examination of the grafts was performed at rejection or at day 100. Lymphocyte proliferation assays (Con A stimulation or Mixed Lymphocyte Reaction) were performed at day 100.

RESULTS: All control animals rejected in 7 days (5-9), whereas survival >100 days was observed in 89% of the GCV treated EpTK group, irrespective of the duration of GCV treatment. Graft histology showed extensive cellular infiltrates with myocyte necrosis and arteritis in the controls, but only a mild infiltrate without necrosis or arteritis in the GVC treated EpTK group. The proliferative responses of the tolerant mice at day 100 were identical to naive mice, including a preserved proliferation against the donor's lymphocytes in MLR.

CONCLUSIONS: Functional transplantation tolerance of a fully incompatible heart could be achieved without immunosuppressive drugs in this model of suicide gene therapy.

*By Invitation

F12. Development of an Autologous Bioengineered Cardiac Graft.

SRen-ke Li*, Zhi-qiang Jia*, ^§Richard D. Weisel, ^§Donald A. G. Mickle*, Eung-Joong Kim*, Shinji Tomita* and Tetsuro Sakai*, Toronto, Ontario, Canada

OBJECTIVE: Patients with congenital heart defect frequently require graft material for cardiac reconstruction. Currently available grafts lack growth potential, are not contractile and are thrombogenic. We developed a beating three dimensional mesh from fetal cardiomyocytes, which successfully repaired a cardiac defect but the cells were rejected. Then we developed a graft derived from autologous cardiomyocytes which could be employed to replace a cardiac defect.

METHODS: BEATING MESH: Fetal rat cardiomyocytes were seeded into a gelatin mesh and cultured. The cells formed a tissue in the mesh and after 7 days the mesh was beating regularly and spontaneously in culture. We transplanted the beating mesh into the subcutaneous tissue of an adult rat leg (N=14). After 21 days, the graft continued to beat with a fractional shortening of 35±9%. We attached the beating mesh to a cryoinjured rat heart scar (N=10). After 5 weeks, the mesh contracted synchronously with the heart and the gelatin matrix dissolved. Unfortunately, the fetal cardiomyocytes were rejected 24 weeks later.

RESULTS: AUTOLOGOUS GRAFT: We isolated and cultured juvenile rat cardiomyocytes which were seeded into a gelatin mesh and formed a tissue after 7 days. The tissue engineered mesh was then exposed to mechanical stretch and relaxation. After 7 days of contraction, the cardiomyocytes in the mesh joined together and oriented in the direction of the stress.

CONCLUSIONS: Fetal cardiomyocytes were grown into a three dimensional graft which contracted in vitro and in vivo. Juvenile cardiomyocytes were grown in a mesh which oriented to stretch. These studies suggest that an autologous cardiac graft could be employed to replace congenital cardiac defects.

^§Authors have a relationship with Genzyme, Corp.

*By Invitation

F13. Novacor LVAS versus HeartMate as a Long-term Mechanical Circulatory Support Device in Bridging Patients: a Prospective Randomized Study

Aly El-banayosy*, Latif Arusoglu*, Lukas Kizner*, Heinrich Koertke*, Michael Koerner*, Michel Morshuis*, Ullrich Schuett*, Peter Sarnowski*, Oliver Fey*, Kazutomo Minami* and Reiner Koerfer, Bad Oeynhausen, Germany

OBJECTIVE: The aim of our study was to investigate the reliability of both devices and to compare them with regard to morbidity following implantation.

METHODS: From October 1996 to March 1998, we randomized patients (pts) needing mechanical left ventricular support as a bridge-to-transplant procedure alternatingly between Novacor and HeartMate. Altogether, 40 pts were included in the study, 20 of whom were supported with Novacor and 20 with the VE HeartMate. Both groups were comparable as to age, preoperative laboratory parameters, hemodynamics and risk factors.

RESULTS: Mean duration of support in Novacor pts was 137.7 days, in HeartMate pts 134.6 days. 15 Novacor pts and 13 HeartMate pts could be discharged home while under support. A bleeding complication occurred in 15 pts (8 Novacor, 7 HeartMate), minor and major neurological disorders in 9 pts (7 Novacor, 2 HeartMate), device related infection complications (driveline infections, pocket infections, conduit endocarditis) in 16 pts (6 Novacor, 10 HeartMate), sepsis in 6 pts (2 Novacor, 4 HeartMate). 2 pts (1 Novacor, 1 HeartMate) suffered severe right heart failure requiring the implantaion of an additional right VAD (Novacor + Thoratec VAD, HeartMate + Medos HIA-VAD). Pump failure occurred in 1 HeartMate pt, a controller change was necessary 14 times in the HeartMate group and twice in the Novacor group. Driveline rupture was found in 2 HeartMate pts and in 1 Novacor pt.

CONCLUSIONS: Both systems have proved to be reliable devices for long-term support with slight advantage of the Novacor LVAS, which is also superior to the HeartMate device in terms of infection complications, whereas HeartMate patients suffer less neurological disorders.

*By Invitation

F14. Tissue Engineered Three Leaflet Heart Valves

Ulrich A. Stock*, Mitsugi Nagashima*, Philipe N. Khalil*, Georg D. Nollert*, Adrian M. Moran*, Tanja Herden*, Jason S. Sperling*, ^§David P Martin*, Jamie G. Lien*, Frederick J. Schoen*, Joseph P. Vacanti*,and John E. Mayer, M.D., Boston, Massachusetts and Cambridge, Massachusetts

OBJECTIVE: Bioprosthetic, mechanical valves or valved conduits have the disadvantage that they are not able to grow, repair or remodel. In an attempt to overcome these shortcomings, we have evaluated the feasibility of creating 3 leaflet pulmonary conduits from autologous ovine cells and biodegradable polymers.

METHODS: Endothelial cells(EC)were harvested from carotid artery segments using collagenase instillation and myofibroblasts(MF)by mincing of the remaining artery wall. Scaffolds of polyglycolic acid and polyhydroxyalkanoates (Metabolix Inc) were formed into a 3 leaflet valved conduit (18mm ∆,2cm length) and were seeded on 4 consecutive days with MF and finally once with EC. With the heart beating, 6 constructs were implanted 31 days(±3) after the vessel harvest using cadiopulmonary bypass. A segment of the native pulmonary artery and all 3 leaflets were removed and 5 seeded and 1 unseeded construct implanted. The animals received no postoperative anticoagulation. Valve function was evaluated by echocardiography before chest closure and before explantation after 1, 2, 4, 6, and 8 weeks. The conduits were evaluated histologically and biochemically for DNA, collagen(4-OHP) and elastin.

RESULTS: ALL animals survived the procedure. Postoperative echocardiography of the seeded constructs demonstrated no thrombus formation with mild non progressive valvular regurgitation up to 8 weeks after implantation. Histology showed progressive tissue formation. Biochemical assays revealed increasing DNA, 4-OHP and elastin content.The unseeded construct showed severe thrombus formation on all 3 leaflets after 1 week.

CONCLUSIONS: This preliminary experiment showed that 3 leaflet heart valves constructed from autologous cells and a biodegradble matrix can function in the pulmonary circulation. Tissue consisting of cellular and extracellular matrix increased over the observed time period.

^§Author has a relationship with Metabolix, Inc.

*By Invitation

F15. Transplantation of Fetal Cells Limits Postinfarct Ventricular Expansion: An Echocardiographic Study

Marcio Scorsin*, Alain A. Hagege*, Jean-Thomas Vilquin*, Francoise Marotte*, Marc Fiszman*, Jane-Lise Samuel*, Ketty Schwartz*, Lydie Rappaport* and Philippe Menasche, Paris, France

OBJECTIVE: Transplantation of fetal cardiomyocytes has been shown to improve function of the infarcted myocardium but the patterns of this improvement have not yet been documented by longitudinal studies.

METHODS: Myocardial infarction was created in 18 rats by ligation of the left coronary artery. One week later, the animals were reoperated on and randomly assigned to receive fetal cardiomyocytes (5 x 10⁶ cells harvested from 19-day fetuses and suspended in 250 mL) injected intramyocardially in the core and at the borders of the infarct or an equivalent volume of culture medium. Left ventricular function was assessed by echocardiography immediately before transplantation and 1 month later using a custom-made 13 MHz 2D transducer allowing to display 180 frames/sec. All rats were immunosuppressed by cyclosporin. Data were compared by Student's t tests and are reported as mean ± SD.

RESULTS: Left ventricular enddiastolic volume (LVEDV) significantly increased in controls (from 0.47±0.12 mL to 0.68±0.17 mL, p<0.003) while cell-treated hearts dilated to a lesser extent with LVEDV being 0.45±0.16 mL at baseline and 0.62±0.25 mL 1 month later, p=NS. Likewise, control hearts increased their endsystolic volumes to a much greater extent than cell-treated hearts (at 1 month: 0.44+0.17 mL and 0.36±0.19 mL, respectively). As a result, ejection fraction did not change in controls (from 37.7±14.4% to 34.2±9.4%) whereas it significantly increased after fetal cell grafting (from 33.9±12.7% to 44.9±13.7%, p<0.04 vs baseline).

CONCLUSIONS: Transplantation of fetal cardiomyocytes significantly improves function of the infarcted myocardium, primarily through a limitation of ventricular remodeling, and might represent a potential rescue therapy for the ischemically-damaged heart.

F16. Emboli Management with a Novel Aortic Filtration System: Histopathological Confirmation of Atheromatous Plaque Capture in Cardiac Surgery

Hermann H. Reichenspurner*, Jose Navia*, Gerald J. Berry*, Robert C. Robbins*, ^§Jefferey P. Gold and Bruno Reichart*, Munich, Germany, Buenos Aires, Argentina, New York, New York and Stanford, California

OBJECTIVE: Significant neurological morbidity following cardiac surgery is associated with aortic emboli released upon removal of the cross clamp. Initial clinical experience with an aortic filtration system was previously evaluated and shown to be safe and feasible for emboli capture in 58 patients. Further study was performed on 20 patients to evaluate the presence of atheromatous plaque.

METHODS: Elective cardiac surgery patients underwent cardiopulmonary bypass (CPB) with an aortic filtration system (average age; 63, female; 26%). Intra-aortic filters were inserted via a modified 24F arterial cannula (EMBOLΣX, Mt.View, CA) immediately prior to releasing the cross clamp and were deployed for an average of 23 minutes (range 5-30). Filters were subsequently examined by scanning electron microscopy (SEM) (11) and histology (20).

RESULTS: The insertion and removal of the filtration system was safe and uneventful in all patients. No morbidity due to stroke or neurological events was noted. Upon removal, visual inspection of the filters revealed particulate debris. SEM was performed and no thrombus formation was evident (few activated platelets 4/11 filters; and few fibrin strands 4/11 filters ). Histologic examination of 7/20 samples showed evidence of atheromatous material: mature collagen (4/20 filters); myofibroblasts compatible with fibroatheromatous caps (2/20 filters); aggregates of cholesterol grumous material and choleterol fragments indicative of atheromatous plaque (1/20 filters).

CONCLUSIONS: The intra-aortic filtration system can be safely deployed during CPB and captures atheromatous parriuclate material without generating thrombus. The impact of aortic fitlration on neurological outcomes is to be determined in prospective randomized studies.

^§Author has a relationship with Embolx

*By Invitation

F17. Sutureless Coronary Artery Bypass with Biologic Glued Anastomoses: Preliminary In Vivo and In Vitro Results

^§Steven R. Gundry, ^§Kirby S. Black* and ^§Hironori Izutani*, Atlanta, Georgia and Loma Linda, California

OBJECTIVE: As heart surgery becomes increasingly focused upon minimally invasive techniques, conventional anastomosis techniques will need to be severely altered. We developed and tested coronary artery bypass graft anastomoses using a biologic glue formulated from bovine albumin and glutaralde-hyde and using a double-balloon catheter temporary internal stent to create and seal the anastomosis during gluing.

METHODS: Anastomoses were made between cryopreserved human saphenous vein segments and coronary arteries in vitro on 12 intact bovine hearts. A total of 42 anastomoses were created using the catheter system introduced into the distal end of the graft exiting the back wall and entering the anterior wall of the coronary artery. Two balloons (one in the graft and one in the coronary) held the anastomosis stable while the biologic glue was applied externally and allowed to set for 2 min. The balloon catheter was then removed from the end of the graft. The open end of the graft was clipped, turning the anastomosis into an end to side graft.

RESULTS: A pressure transducer was then attached to the graft and saline forcefully infused. All grafts easily held 300 mmHg pressure. Distal and proximal coronary artery patency was checked by examining flow out of the coronary ostia and by cutting arteries distal to the grafts. All anastomoses were patent upon opening. Subsequently, three LIMA to LAD anastomoses have been constructed in goats, with ligation of the proximal LAD. Survival now exceeds 7 months, with normal echo function.

CONCLUSIONS: A biologic glue and catheter system has been developed which allows for a high bursting strength coronary anastomosis to be performed. When further developed and tested, truly minimally invasive heart surgery may be possible.

^§Authors have a relationship with Cryolife, Inc.

*By Invitation

F18. Hypothermic Retrograde Venous Perfusion Cools the Spinal Cord and Reduces Paraplegia During Thoracic Aortic Surgery

Scott D. Ross*, John A. Kern*, James J. Gangemi*, Char R. St. Laurent*, Kimberly S. Shockey*, Irving L. Kron and Curtis G. Tribble, Charlottesville, Virginia

OBJECTIVE: We evaluated the utility of retrograde venous perfusion to cool the spinal cord and protect neurological function during aortic clamping. We hypothesized that hypothermia-induced electrical silence would preserve the spinal cord during ischemia.

METHODS: Six swine(GroupI) underwent thoracic aortic occlusion for 30 minutes at normothermia. A second group(GroupII) underwent spinal cord cooling by retrograde perfusion of the paravertebral veins with a hypothermic(4°C) saline solution during 30 minutes of aortic occlusion. The spinal cords of a third group(GroupIII) were cooled with a hypothermic adenosine solution in a similar fashion. Intrathecal temperature was monitored and measurements of somatosensory evoked potentials(SSEP) assessed the functional status of the spinal somatosensory pathways during clamping, cooling, and recovery.

RESULTS: Spinal cooling without systemic hypothermia significantly improved neurological Tarlov scores in groupIII(4.7±0.2) and groupII(3.8±0.4) when compared to groupl(1.3±0.6)(p<0.001). Furthermore, 5 of the 6 animals in groupIII displayed normal neurological function, whereas only 1 animal in groupII and no animals in groupI did(p=0.005). SSEP were lost 10.6±1.4 minutes after ischemia in groupI. In contrast, spinal cooling resulted in rapid cessation of neural transmission with loss of SSEP at 6.9±1.2 minutes in groupll and 7.0±0.8 minutes in groupIII(p=0.06). SSEP amplitudes returned to 85% of baseline in groupIII and 90% of baseline in groupII compared to only 10% of baseline in groupI(p=0.01).

CONCLUSIONS: We conclude that retrograde cooling of the spinal cord is possible and protects against ischemic injury. Its efficacy may be attributed to induced electrical silence of the cord during ischemia, similar to cardiac electrical silence during cold cardioplegia.

*By Invitation

F19. Experimental Aortomyoplasty Improves Systolic and Diastolic Performance in Chronic Ischemic Cardiomyopathy

Thierry G. Mesana*, Olivier Y. Ghez*, Jeffrey S Martin*, Mark H. Danton*, Sergey D. Grachev*, Kathryn Q. Flores*, Rita G.. Laurence*, Lawrence H. Cohn and John G. Byrne*, Boston, Massachusetts

OBJECTIVE: Aortomyoplasty (AMP) may have a role in the treatment of chronic ischemic cardiomyopathy (CIC), but has not yet been evaluated for this purpose. Therefore we tested whether AMP improves global and regional myocardial performance and regional myocardial blood flow (RMBF), in an experimental model of CIC.

METHODS: Ameroid constrictors were placed on the proximal circumflex artery in goats. After six weeks, AMP (n=6) was performed by wrapping untrained latissimus dorsi muscle around the descending thoracic aorta and coun-terpulsation established at 1:2. Measurements were made with and without AMP-powered counterpulsation.

RESULTS:

*p<0.05 †p<0.01

CONCLUSIONS: Aortomyoplasty improves global systolic and diastolic performance and RMBF in this model of chronic ischemic cardiomyopathy.

*By Invitation

F20. Cardiac Performance After Deep Hypothermic Circulatory Arrest In Cyanotic Neonatal Lambs

Mitsugi Nagashima*, Georg Nollert*, Ulrich Stock*, Jason Sperling*, Dominique Shum-tim*, Koh Takeuchi*, Arther Nedder* and John E Mayer, Boston, Massachusetts

OBJECTIVE(s): Acute hypoxic stress before ischemia results in increased free radical production and depressed recovery of cardiac function. The relevance of the acute hypoxic stress model to chronic cyanosis in patients remains unclear. This neonatal lamb study assessed the effect of 1 week of cyanosis on recovery of cardiac function and free radical production after deep hypothermic circulatory arrest (DHCA).

METHODS: Cyanosis was created in 8 lambs (age 4.8 days) by an anastomosis between the pulmonary artery and the left atrium. Seven controls underwent thoracotomy and pericardiotomy. One week later, all animals underwent CPB with 90 min of DHCA at 18°C. Preload recruitable stroke work (Mw) was measured as LV contractility before CPB (FiO₂ =1.0 and FiO₂=0.21) and post CPB at 2 hr of reperfusion (FiO₂ =1.0). Malondialdehyde (MDA) levels in coronary sinus were measured. LV antioxidant reserve capacity (ARC) was assessed at 2 hr of reperfusion by measuring thiobarbituric acid reactive substance (TEARS) in LV biopsies incubated with t-butylhydroperoxide (t-BHP).

RESULTS: The cyanosis model produced a significantly (p < .01) lower arterial oxygen tension (35 ± 3 mmHg vs. 93 ± 9 in control, FiO2 = 0.21). Mw and MDA are shown in the Table (Data are mean ± SE. * = p < .05 vs. control, † = p <.05 vs FiO2 = 0.21 baseline). LV ARC was not significantly different between groups (TEARS, 1.5 ± 0.2 in cyanosis vs. 1.4 ± 0.1 mmol/g protein in control at 4mM t-BHP).

CONCLUSIONS: After one week of chronic cyanosis, acute increases in arterial oxygen before CPB result in improved LV function. Recovery of LV function after DHCA was worse in cyanotic animals than controls. The absence of a significant difference in coronary sinus MDA or LV antioxidant reserve capacity suggests that free radical injury may not responsible for worse LV functional recovery in chronically cyanotic hearts.

*By Invitation