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Wednesday Morning, April 26, 1995

Back to Annual Meeting Program


WEDNESDAY MORNING, APRIL 26, 1995

7:00 am FORUM SESSION II

Grand Ballroom, Sheraton Boston Hotel

Moderators: Robert A. Guyton, M.D.

Shukri F. Khuri, M.D.

F9. THE EXPRESSION OF TNFα AND CHEMOKINES (ENA-78 AND GROα) DURING ISCHEMIA/ REPERFUSION INJURY ASSOCIATED WITH LUNG TRANSPLANTATION

K..E. Magliato, M.D.*, D.L. Swiderski, Ph.D.*,

S.L. Kunkel, Ph.D.*, C.A. Wilke, B.S.*, M.D. Burdick, B.S.*, G.M. Deeb, M.D. and R.M. Stricter, M.D.*

Akron, Ohio and Ann Arbor, Michigan

The reimplantation response or ischemia/reperfusion injury is potentially an important cause of morbidity during lung transplantation. While a number of cellular events are involved in this response, the neutrophil appears to play a pivotal role in mediating lung injury during reimplantation. The mechanisms that lead to neutrophil sequestration and activation are complex and have not been fully elucidated. We postulated that the generation of the early response cytokine, tumor necrosis factor alpha (TNFα), and the chemokines, epithelial cell neutrophil activating protein (ENA-78), and growth related oncogene alpha (GROα ), would be significantly elevated during the reimplantation response and be associated with maximal neutrophil sequestration and subsequent lung injury. To test this hypothesis, we evaluated pulmonary microvascular permeability as index of injury, neutrophil influx , and expression of TNFα, ENA-78, and GROα during the reimplantation response using a rat orthotopic single-lung syngeneic transplant model. Cytokine expression in plasma, lung tissue homogenates, and bronchoalveolar lavage (BAL) at 4, 12, 24, 36, and 48 hours post-reimplantation was measured by specific ELISAs (n=30). Cytokine levels from the transplant recipient animals were similar to sham operated control animals until 24 hrs post-reimplantation. At 24 hrs post-reimplantation, TNFa, ENA-78, and GROα were all significantly elevated (2-fold) from the lung tissue homogenates of the transplant recipients, as compared to sham operated controls. The concentrations of all cytokines returned to levels similar to the sham operated animals by 48 hours. Pulmonary neutrophil sequestration, as evaluated by BAL cell counts, paralleled the changes in lung tissue cytokine levels. At 24 hours, neutrophil absolute and relative levels were higher in transplant recipients (6.5± 4.4 x 105, 69.7±5.8% of the total) than in sham operated controls (2.4±1.1 x 105, 12.3±5.7% of the total) (p=.002). The findings of BAL neutrophil levels were corroborated by elevated levels of myeloperoxidase activity from transplanted lung tissue homogenates (100.8±22.8) as compared with sham controls (35.1±8.1) (p=.02). Using Evan's blue dye as marker of pulmonary microvascular permeability, transplant recipients at 24 hours post-reimplantation demonstrated a 2.5-fold greater index of lung permeability, as compared to sham operated control animals (p=.01). In addition, transplanted animals pretreated with neutralizing TNF antisera showed a 50% decrease in lung injury by Evan's blue dye lung permeability assessment. These findings suggest that the temporal expression of TNFcc, ENA-78, GROa correlates with maximal neutrophil sequestration and lung injury associated with the reimplantation response and neutralizing TNF antisera has a protective role in ischemia/reperfusion injury.

*By invitation


F10. THERAPEUTIC ANGIOGENESIS: ACIDIC FIBROBLAST GROWTH FACTOR ACCELERATES COLLATERAL VESSEL FORMATION AND REVASCULARIZATION OF ISCHEMIC TISSUES

Todd K. Rosengart, M.D.*, Martin A. Duenas, B.S.*, Qing-Xue Zhang, Ph.D.*, Karl H. Krieger, M.D. and O. Wayne Isom, M.D.

New York, New York

Angiogenesis, the growth of new blood vessels, is thought to be an important biological mechanism allowing for the revascularization of ischemic tissues. Acidic fibroblast growth factor (aFGF) is a potent stimulant of angio-genesis that may prove useful in accelerating collateral vessel development following vascular occlusion. We therefore assessed the ability of aFGF to enhance angiogenesis and improve perfusion in the setting of acute ischemia. Ligation of the left common femoral artery was performed in the rat followed by the daily transcutaneous injections into the ischemic hind limb region of saline (1ml) or recombinant human aFGF (1 mcgm in 1 ml saline) for 10 days following ligation (n=4). Following the treatment period, 2 x 106 color micro-spheres were injected via the infrarenal abdominal aorta and the relative perfusion of the ligated limb versus the unligated, contralateral control limb was determined from the microsphere counts per gram of tissue harvested from each limb. Relative perfusion of the ligated limb in the aFGF-treated animals was 62 ± 16% (mean ± SEM) of that to the unligated, contralateral control limb compared with 10 ± 3% relative perfusion in the saline-treated animals (p=0.004). These results were confirmed by histologic analysis of hind limb tissue specimens, which demonstrated 27 ± 2 vs 4 ± 1 vessels per high power field in the aFGF and saline-treated animals, respectively (p=0.001). Proliferating cell nuclear antigen (PCNA) analysis was utilized to assess mitogenic activity and demonstrated that the percentage of PCNA-labelled cells per total cell nuclei was 68 ± 4% vs 5 ± 1% in the two respective groups (p<0.001). The six-fold increase in perfusion noted in these studies, confirmed by analysis of vascularization and cell replication, suggest that exogenous aFGF administration may represent a useful means of enhancing collateral vessel growth and relieving acute ischemia. The administration of aFGF may potentially allow the revascularization of myocardial or other tissues not salvageable by bypass procedures or other conventional means.

*By invitation


F11. MYOBLAST TRANSPLANTATION IN THE PORCINE MODEL. A POTENTIAL TECHNIQUE FOR MYOCARDIAL REPAIR

Clifford H. Van Meter, Jr., M.D.*, Frank Smart, M.D.*, William Claycomb, Ph.D.*, Joseph DelCarpio, Ph.D.* and John L. Ochsner, M.D.

New Orleans, Louisiana

The increasing shortage of organ donors continues to fuel the search for other methods to manage heart failure. The use of transgenic cells transplanted in syngeneic rodents has shown modest success, but allogeneic and xenogeneic transplants have not been uniformly successful. To assess the feasibility of xenogeneic and allogeneic myoblast transplantation, six adult swine underwent transplantation of murine atrial tumor cells (xenogeneic) and neonatal porcine (allogeneic) into the left ventricular wall. A subsequent seventh animal underwent transplantation of human neonatal myoblasts (16 weeks of age). Following general anesthesia, isolated cells were injected along the anterior and posterior wall of the porcine left ventricle (6 to 8 sites per animal). All the animals were immunosuppressed with cyclosporine and prednisone and were followed for one month post-injection at which time they were sacrificed.

In all injected sites, the transplanted cells proliferated within the host myocardium with no significant rejection. CPK MB isoenzymes did not increase after the procedure indicating that there was no damage to the host myocardium from the injection of cells. Moreover, transplant cells formed close associations with host myocytes that resembled intercalated discs on electron mycroscopy, and were composed of PAN cadherin on immuno-fluorescent staining. These cells also contained myofibrils and other cell architecture that resembled normal AT-1 or neonatal myocytes. Additionally, these cells produced angiogenic factors resulting in a proliferation of the surrounding microvasculature. In conclusion, these findings indicate successful xenogeneic and allogeneic myocyte cell transplantation in a large animal model. These experiments set the stage for future studies to test the ability of these cells to form a syncytium, to contract, and potentially repair failed myocardium.

*By invitation


F12. EFFICIENT CARDIAC GENE TRANSFER BY INTRA-CORONARY INFUSION OF ADENOVIRUS VECTOR MEDIATED REPORTER GENE IN TRANSPLANTED MOUSE HEART

Jeongryul Lee, M.D.*, Alistair I. Fyfe, M.D.*, Hillel Laks, M.D., Davis C. Drinkwater, M.D., Yuji Shiraishi, M.D.* and Paul Chang, B.S.*

Los Angeles, California

Gene transfer into the coronary arteries may offer an opportunity to modulate alloreactivity after cardiac transplantation. In the present study, we perfused the coronary arteries of donor mouse hearts with 107 pfu (200 |aL) of a recombinant adenovirus containing P-galactosidase encoding gene (AdCMV P gal) just prior to harvesting to demonstrate the feasibility, efficacy, and safety of adenovirus mediated in vivo transfer. The donor hearts were transplanted into the abdomen of the recipient mice of the same strain (BIO. BR). Both the grafts and recipient hearts were harvested at 3, 7, 15 and 30 days (n=6 in each group) after transplantation and P-galactosidase activity was assessed by immunohistochemistry. All grafts were functioning adequately at the time of harvest and P-galactosidase expression was seen throughout the myocardium, epicardium, and the surrounding coronary arteries. There were no significant differences in expression at least up to 30 days after transplantation. Staining was not seen in recipient hearts or sham control isografts (n=6) which were sacrificed 7 days after infusion of saline into the coronary arteries. This study revealed that intracoronary adenovirus mediated gene transfer is an efficient and feasible method for transfecting genes, with widespread expression, and there does not appear to be significant early cardiac graft dysfunction associated with the use of adenovirus. This approach may allow modification of graft immunogenicity in the future by allowing the production of therapeutic proteins that inhibit or delay the development of cardiac allograft vasculopathy.

*By invitation


F13. INTRATHYMIC STEM CELL TRANSPLANTS PROLONGSKIN GRAFT SURVIVAL IN UNRELATED PRIMATES

†Margaret D. Allen, M.D., Hiroji Akimoto, M.D.*, John T. Weyhrich, D.V.M.*, Karen A. Nelson, Ph.D.*, Robert Thomas, B.S.* and Robert G. Andrews, M.D.

Seattle, Washington

The intrathymic implantation of stem cells in transplant recipients may resolve several problems limiting the attainment of tolerance to transplanted organs. 1) Peripheral blood microchimerism has been associated with reduced rejection in clinical liver and renal transplantation but peripheral infusion of donor marrow to induce chimerism carries a risk of graft-versus-host disease (GVHD). Recent bone marrow transplants in primates suggest that stem cell fractions may result in less GVHD. 2) Intrathymic implantation of lymphocytes, splenocytes, class II peptides, and even islets in rodents pre-treated with myeloablation or anti-lymphocyte globulin can extend subsequent donor organ or skin graft survival, but hematopoietic microchimerism has not been reported. Stem cells might provide a self-perpetuating source of donor antigens. 3) If suppressor mechanisms are operative, less, rather than more, immunosuppression may be beneficial. This study examines whether the intrathymic implantation of donor CD34+ enriched marrow (97% CD34+) might produce hematopoietic chimerism and induce tolerance to donor-derived grafts in unrelated primates with or without immunosuppression.

Four juvenile female baboons underwent intrathymic transplantation of CD34+ enriched marrow (1 x 106 cells) from unrelated male baboon donors through a left thoracotomy approach. Peripheral blood was sampled at biweekly intervals for the presence and persistence of donor cells using PCR for Y chromosome determinants. Two recipient animals were treated with standard triple drug immunosuppression consisting of cyclosporine, azathioprine, and steroids, in doses equivalent to the clinical cardiac transplant protocol. No myeloablative therapy or anti-lymphocyte serum induction was used. Two other recipients received no immunosuppression. All 4 recipients, both with and without immunosuppression, developed peripheral blood microchimerism within 2 weeks which is still persistent at 9-16 months. No clinical evidence of GVHD was observed.

To test whether the implantation of intrathymic stem cells resulted in tolerance, two further juvenile female baboons were implanted with male donor CD34+ marrow without recipient immunosuppression. Chimerism was detected in both animals. Three months following the intrathymic implants, 9 skin grafts were placed on each recipient: 3 from autologous skin, 3 from the stem cell donor, and 3 from a third party baboon. Autologous grafts were incorporated without rejection. Third party grafts sloughed at 24 days post-transplant and, in both recipients, the grafts from the stem cell donor persisted DNA. Repetition of the skin grafts again demonstrated prolongation of the stem cell-donor over the third party-donor grafts.

These results demonstrate that: l)the intrathymic implantation of donor CD34+ enriched marrow can induce peripheral microchimerism in non-human primates with or without immunosuppression; 2) 1 x 106 CD34+ cells is sufficient to induce peripheral chimerism; 3) donor-specific skin graft, prolongation was achieved; and 4) graft versus host disease was not observed. These findings have current applications in the field of thoracic transplantation tolerance as well as future potential in the treatment of genetic deficiency diseases of the newborn.

†Nina S. Braunwald Career Development Award Recipient

*By invitation


F14. INHIBITION OF INDUCIBLE NITRIC OXIDE

AMELIORATES RAT LUNG ALLOGRAFT REJECTION

Takeshi Shiraishi, M.D.*, Neil K. Worrall, M.D.*, Steven R. DeMeester, M.D.*, Jon H. Ritter, M.D.*, Thomas P. Misko, Ph.D.*, T. Bruce Ferguson, M.D., Joel D. Cooper, M.D. and G. Alexander Patterson, M.D.

St. Louis, Missouri

Inhibition of the production of nitric oxide (NO) by inhibition of inducible nitric oxide synthase (iNOS) has recently been demonstrated to attenuate the pathogenesis of acute cardiac allograft rejection. This study examined the effect of inhibition of NO production on rejection in a rat lung transplant model. Syngeneic (F344 to F344) (n=7) and allogeneic (Brown Norway to F344 recipient) (n=8) left lung transplants were performed. In this latter strain combination complete rejection occurs by postoperative day 8. Using the same allogeneic combination, a third group of animals (n=9) underwent allotransplantation and received aminoguanidine (a specific iNOS inhibitor) 200 mg/kg IP q6h from transplantation until sacrifice on postoperative day 7 (POD-7). NO production was demonstrated by measuring serum nitrite/nitrate levels (stable end products of NO metabolism). Allograft serum nitrite/nitrate levels [16.5 ± 1.7mM, n=4 (p<.01 by ANOVA)] were elevated above normal control (8.0 ± 1.0 mM, n=3) and isograft levels (9.0 ± 1.4 mM, n=4) on postoperative day 4 (POD-4), indicating that NO is produced during lung allograft rejection. Aminoguanidine treatment prevented the increased serum nitrite/nitrate levels in the allografts on POD-4 (9.8 ± 1.0 mM; p<0.01 vs untreated allografts). The effect of iNOS inhibition on acute rejection was determined by using three scoring systems. Severity of rejection was evaluated using a radiographic score (Aeration Score; 0=opaque to 6=normal appearing lung). The histologic rejection grade was determined at sacrifice on POD-7 using the International Working Formulation (I.W.F. score: 0=normal to 4=diffuse infiltration of mononuclear cells) and Prop's scoring system (0=latent phase to 3=destruction). Aminoguanidine treatment significantly attenuated acute rejection as determined radiographically [AS= 4.1 ± 1.4 vs 1.8 ± 0.9 for untreated allografts on POD-6 (p<.05) and 3.9 ± 1.5 vs 0.6 ± 0.5 on POD-7 (p<.01)]. Aminoguanidine treatment also significantly ameliorated the histologic degree of rejection as determined at sacrifice on POD-7 [2.6 ± 0.2 vs 3.8 ± 0.5 for untreated allografts by IWF score (p<.005) and 1.3 ± 0.4 vs 2.6 ± 0.7 by Prop's score (p<.005)]. These results demonstrate that NO is produced during acute lung allograft rejection and that selective inhibition of the inducible isoform of nitric oxide synthase significantly attenuated acute rejection. NO production may serve as a marker of early rejection and selective inhibition of iNOS may be a novel therapeutic modality in the management of acute lung allograft rejection.

*By invitation


F15. THE EFFECT OF N-co-NITRO-L-ARGININE ON PULMONARY VASCULAR TONE IN THE MATURING NEWBORN PIG

Patrick W. Domkowski, Ph.D.*, John T. Cockerham, M.D.*, Peter A. Kot, M.D.*, Jeffrey L. Myers, M.D.*, Yi-Ning Wang, M.D.*, Robert B. Wallace, M.D. and Richard A. Hopkins, M.D.

Washington, DC

Current therapeutic modalities for treatment of newborn pulmonary hypertensive crisis include the administration of nitric oxide (endothelium derived relaxing factor, EDRF). However, few data are available on the role of endogenously produced EDRF in the modulation of newborn pulmonary arterial tone. We investigated the acute effects of a 10-minute intravenous infusion of N-oo-nitro-L-arginine (L-NA), a potent competitive inhibitor of EDRF synthase on the pulmonary vascular response, in 48-hour and 2-week-old anesthetized open-chest Yorkshire pigs. Fourier analysis of high fidelity main pulmonary artery pressure (PAP) and flow (PAF) waveforms was employed to distinguish between proximal and distal pulmonary arterial responses to L-NA. Input mean impedance (Zm, reflective of distal vasocon-striction) and characteristic impedance (Z0, reflecting proximal arterial disten-sibility) were calculated. Pulmonary vascular resistance (PVR) was also calculated. Data were collected at baseline and during intravenous infusion of L-NA.

PAP

PAF

PVR†

Zm†

Z0†

48-hour-old pigs (n=8)

17.0 ± 1.1

4.0 ± 0.6

5171 ± 805

6343 ± 806

2073 ± 418

L-NA (100mg/kg/min) infusion

17.6 ± 1.2

4.0 ± 0.5

5189 ± 1058

6529 ± 1043

2033 ± 466

2-week-old pigs (n=7)

13.7 ± 1.1

8.4 ± 1.0

810 ± 137

2302 ± 251

282 ± 40

L-NA (100mg/kg/min) infusion

18.0 ± 1.4*

8.7 ± 0.7

1519 ± 239*

2900 ± 255*

234 ± 21

mean ± S.E.M. *p<0.05 v. corresponding baseline; PAP = mmHg, PAF = ml/sec, †units = dyne sec cm-5

Baseline hemodynamic parameters were significantly diminished in 2-week-old pigs, reflecting the expected decrease in both proximal and distal pulmonary arterial vasoconstriction with maturation. L-NA produced no significant pulmonary hemodynamic changes in 48-hour-old pigs. However, in 2-week-old pigs, L-NA caused a significant pulmonary arteriolar vasoconstriction. L-NA infusion did not cause any significant alterations in zq in either group. These data indicate that endogenously produced EDRF modulates distal pulmonary arterial tone in 2-week-old pigs, but not in 48-hour-old pigs. Pulmonary hypertensive crisis of the newborn may reflect an inability of the pulmonary endothelium to synthesize and release nitric oxide in quantities sufficient enough to modulate pulmonary vascular tone.

*By invitation


F16. ELUCIDATION OF A TRIPARTITE MECHANISM UNDERLYING THE IMPROVEMENT IN CARDIAC TOLERANCE TO ISCHEMIA BY COENZYME Q10 PRETREATMENT

Juan A. Crestanello, M.D.*, Joseph Kamelgard, M.D.*, David M. Lingle, M.D.*, Svend A. Mortensen, M.D.* and Glenn J.R. Whitman, M.D.

Philadelphia, Pennsylvania and Copenhagen, Denmark

CoQ10, involved with mitochondrial ATP production, is also a powerful antioxidant. We hypothesize that CoQ10 pretreatment protects myocardium from ischemia reperfusion injury by both its ability to increase aerobic energy production and by its ability to protect creatine kinase (CK) from oxidative inactivation during reperfusion.

Isolated hearts (n=6/group) from rats pretreated with either CoQ10 20 mg/kg i.m. and 10 mg/kg i.p. (CoQ) or vehicle only (CTRL) 24 and 2 hours prior to the experiment were subjected to 15 minutes of equilibration (EQ), 25 minutes of ischemia, and 40 minutes of reperfusion (RP). Developed pressure (DP), ±dP/dt, myocardial oxygen consumption (MVO2), and myocardial aerobic efficiency (DP/MVC^) were measured. In parallel groups of hearts, 31P NMR spectroscopy was used to determine P-ATP and PCr concentrations. Hearts were assayed for myocardial CK activity at end RP. Values are expressed as mean ± SEM with t-test used for statistical evaluation.

Reperfusion 40

PCr

ATP

DP

(% EQ)

+dP/dt

(% EQ)

-dP/dt

(% EQ)

CK

IU/g V

DP/MVOZ

mmHg/(m O2/min/g V

EQ

(%MDP)

RP40

(%MDP)

EQ

(%MDP)

RP40

(%MDP)

CTRL

37 ± 4

37 ± 5

36 ± 5

391 ± 10

0.621 ± 0.069

35 ± 3

23 ± 4

33 ± 3

11 ± 2

CoQ

62 ± 2**

64 ± 2**

61 ± 2**

435 ± 17*

0 835 ± 0 076*

49 ± 3**

45 ± 4**

49 ± 2**

18 ± 3*

(MDP: Methylenediphosphonic acid standard) *p<0.05 vs Control **p<0 005 vs Control

CoQ10 pretreatment improves myocardial function after ischemia reperfusion. This results from a tripartite effect: 1) higher ATP and PCr concentrations initially and during reperfusion, 2) improved myocardial aerobic efficiency during reperfusion, and 3) protection of CK from oxidative inactivation during reperfusion with an improved bioenergetic profile.

*By invitation


F17. INDUCED IMMUNOLOGIC UNRESPONSIVENESS INHIBITS CARDIAC GRAFT ARTERIOSCLEROSIS

Yong T. Shin, M.D.*, Mohamed H. Sayegh, M.D.*, Lauri R. Wyner, B.A.*, Morris J. Karnovsky, M.B., B.Ch.* and †David H. Adams, M.D.*

Boston, Massachusetts

Sponsored by: Lawrence H. Cohn, M.D., Boston, Massachusetts

Chronic rejection, manifested by the development of graft arteriosclerosis, remains the leading cause of late death in cardiac transplant recipients. Immunologic mechanisms have been implicated in both experimental and clinical studies, yet the pathogenesis and therapy of chronic allograft rejection remains poorly defined. Recent studies have shown that intrathymic (i.t.) injection of donor cells or processed allo-MHC antigens with or without transient immunosuppression induces specific systemic T cell tolerance and prevents acute allograft rejection in several experimental trasnplantation models. In this study we examined the effects of i.t injection of donor cells with or without systemic anti-lymphocyte serum (ALS) on the development of chronic rejection in the Lewis-to-F344 rat cardiac allograft model. Recipients were divided into 4 groups: control group A was pre-treated with saline i.t., group B received donor (Lewis) splenocytes i.t. (2x106), group C was pre-treated with a one-time dose of ALS (1 ml) via intraperitoneal injection (i.p.), and group D received both the splenocytes i.t. and a one-time dose of ALS i.p. two weeks prior to transplantation. Allografts were followed by daily palpation and graded from 0-4 (grade 0=non-palpable, grade 4=normal heartbeat). Graft survival on post-op day 90 for all three treated groups was 100% vs. 33% for the control (n=6/group), and mean heartbeat grade for the three treated groups (B=2.0±0.41, C=2.83±0.17, D=2.67±0.21) was significantly higher (p<0.01) vs. the control group (A=0.4±0.24). Histologic and immunocytochemical analysis of 90-day old grafts revealed the following results (*(p<0.02, **p<0.003vs Control):

Groups

Inflammation

% Vessels Diseased

% Luminal Occlusion

Intima:Media Ratio

Control (A)

severe

89 ± 1

64 ± 5

0.97 ± 0.33

Splenocyte only (B)

moderate-severe

79 ± 8

57 ± 7

0.68 ± 0.16

ALS only (C)

minimal

25 ± 3**

8 ± 3**

0.09 ± 0.04*

Splenocyte+ALS (D)

minimal

27 ± 3**

8 ± 3**

0.07 ± 0.03*

Conclusions: (1) Treatment with donor splenocytes i.t. alone partially suppresses the immune response, prolonging graft survival and preserving function without inhibiting graft inflammation or arteriosclerosis. (2) The induction of systemic unresponsiveness with ALS i.p. or splenocytes i.t. plus ALS i.p. markedly reduces inflammation and inhibits graft arteriosclerosis, suggesting the development of therapies resulting in better control of the immune response remains the key to ameliorating this disease process.

†Alton Ochsner Research Scholar .

*By invitation


F18. MOLECULAR GENETIC DIFFERENTIATION BETWEEN PRIMARY LUNG CANCER AND LUNG METASTASES OF OTHER TUMORS

Daniela Kandioler, M.D.*, Gerhard Dekan, M.D.*, Adelheid End, M.D.*, Michael Mttller, M.D.*, Michael Gnant, M.D.*, Eva Pasching*, Christine Mannhalter, Ph.D.*, Franz Eckersberger, M.D.*, and Ernst Wolner, M.D.

Vienna, Austria

When single pulmonary tumors are observed in patients with history of other cancer, differentiation between metastasis and second primary lung cancer is crucial for appropriate therapy. Particularly in low differentiated tumors this determination by means of histology alone may be difficult or impossible even with the help of immunochemistry. Implicating that p53 mutations are conserved in metastases, we hypothesized that a genetic fingerprinting using the p53 gene could be a valuable tool in differentiating between metastases and second primary carcinomas since the p53 gene is frequently affected by various mutations in many different types of human carcinomas.

Out of 267 resected lung tumors we analysed 9 cases of solid lung tumors whose origin could not exactly be defined histologically. As a control we used tumor material of a patient with adenocarcinoma of the lung and histologically defined brain and lymphnode metastases.

Cases

History of
Lung Tumor

5

colon carcinoma

adenocarcinoma

1

breast cancer

adenocarcinoma

1

soft tissue sarcoma

undifferentiated blastoma

1

renal & thyroid cancer

clear cell carcinoma

1

head and neck

squamous cell carcinoma

We extracted DNA from the shock frozen tissue of the unclassified lung tumors, from the paraffin embedded tissue of the "past primary cancers" and from peripheral blood of the patients. Exons 2 to 11 of the p53 gene were amplified separately by polymerase chain reaction (PCR) and directly sequenced. In all cases the presence of germline mutations could be excluded by analysis of the peripheral blood. The p53 mutation detected in the control tumor proved to be conserved in the lymph node as well as in the brain metastasis.

In two cases the lung tumor was identified as (second) primary lung cancer since the genetically compared tumors exhibited different mutations. In five cases the lung tumors proved to be metastases exhibiting the identical p53 mutation as their "primary." In one case the lung tumor could be identified as metastases from renal cancer whereas a thyroid cancer metastasis could be excluded. One case remained inconclusive since no p53 mutation could be found in any tissue. Thus, due to genetic analysis using the p53 gene, correct surgical treatment could be approved in six cases regarded as metastatic disease of the lung, whereas further pulmonary surgery was mandatory in two cases identified as second primary lung cancer.

We conclude that analysis of p53 mutations is a valuable tool in the exact diagnosis of metastases and recurrent tumors which are difficult to classify by standard methods. Since the information about tumor origin is essential in assessing an individual patient's treatment strategy, these results are clinically relevant.

*By invitation


9:30 am SIMULTANEOUS SCIENTIFIC SESSION D ADULT CARDIAC SURGERY

Grand Ballroom, Sheraton Boston Hotel

Moderators: D. Glenn Pennington, M.D.

Robert L. Hardesty, M.D.

41. REFERRAL SOURCE AS A RISK FACTOR FOR

CORONARY BYPASS SURGERY: HMO VS. PRIVATE PRACTICE

Albert Starr, M.D., Aftab Ahmad, M.D.* Anthony Furnary, M.D.*, Guo Wei He, M.D.*, Jeffrey Swanson, M.D.* and Gary Grunkemeier, Ph.D.*

Portland, Oregon

We began performing coronary bypass surgery (CBS) for a large HMO in 1974, as the sole provider of their cardiac surgery. The HMO system entails pre-intervention, multi-disciplinary screening conferences and is absent of self-referral and personal financial incentives. Since 1985, the operative mortality for HMO patients has consistently been lower than for private patients. There were 8, 483 surgeries during this study period: 3, 168 (37%) HMO, with an overall operative mortality of 2.7%, and 5, 315 (63%) non-HMO, with an operative mortality of 4.6% (P=.00002). All patients were operated on in the same hospital by the same surgical group.

We investigated this difference with univariate and multivariable analyses. 19 risk factors were found to univariately affect the risk of operative mortality (p<.05). For 11 of these risk factors there was a significant maldistribution between HMO and non-HMO patients; HMO patients had worse average values for 3 factors and better values for 8 (the latter included age, previous CBS, abnormal wall motion, chronic angina, and emergency surgery).

We used logistic regression to explore the influence of this imbalance in risk factors. The model found 7 factors to significantly affect operative mortality, after which the variable indicating HMO membership was no longer significant (p=.006). Because of missing data, only 1, 279 (40%) HMO patients and 2, 407 (45%) non-HMO patients, with operative mortalities of 2.6% and 4.1%, respectively, could be used.

A reasonable conclusion is that the difference in operative mortality between a coherent and chaotic system of cardiologic management results in a distribution of risk factors that enhance the operative results in the coherent group. The question of whether this selection process would be beneficial to a large population at risk has not been addressed.

*By invitation


42. REDUCING THE RISK OF INFECTIVE ENDOCARDITIS AFTER AORTIC VALVE REPLACEMENT

Arvind K. Agnihotri, M.D.*, David C. McGiffin, M.D.*, Andrew J. Galbraith, M.D.* and Mark F. O'Brien, M.D.

Birmingham, Alabama and Brisbane, Australia

Replacement valve endocarditis (RVE) occurred on 3.9% of 2, 686 aortic valve replacement devices inserted between December 31, 1969 and January 1, 1992, based on a cross-sectional follow-up in 1992 which was 98.8% complete. As shown by others, the risk (hazard function) for RVE was highest early after valve replacement, peaking 3 months after operation, then decreasing to reach a constant hazard by about 6 months after operation. By multivariable analysis, patients with active preoperative endocarditis (p<.0001) or those receiving a synthetic aortic root replacement (p=.0006) were at an increased early risk for RVE, but this increased risk was neutralized when an allograft valve was used (p<.0001; figure A [figures A and B are nomograms of the multivariable equation]). Late risk (later than 6 months after operation) was increased in younger patients (p<.0001), those with renal failure (p=.01), and in patients with active or remote preoperation endocarditis (p=.04). Late risk was particularly high in patients with preoperative Staphylococcus aureus endocarditis (p=.05), which tended to recur as another staphylococcal endocarditis. Use of some mechanical valves resulted in a reduction in late risk (p=.05; figure B, while use of some xenograft valves increased late risk (p=.02).

In patients without risk factors, valve replacement with any modern device results in a similar infectious risk (figure C). In the presence of early phase risk factors use of an allograft valve reduces the risk of RVE. In patients with only late phase risk factors the use of some mechanical valves may be preferable.

*By invitation


43. SURGICAL TREATMENT OF PROSTHETIC VALVE ENDOCARDITIS

Bruce W. Lytle, M.D., Brian P. Priest, M.D.*, Paul C. Taylor, M.D.*, Floyd D. Loop, M.D., Robert W. Stewart, M.D.*, Patrick M. McCarthy, M.D.*, Derek Muehrcke, M.D.* and Delos M. Cosgrove, M.D.

Cleveland, Ohio

From 1975 through 1992, we reoperated on 146 patients for the treatment of prosthetic valve endocarditis (PVE). Inclusion in the study stopped in 1992 to allow follow-up of all patients. PVE was considered early (<1 year after operation) in 46 cases, active in 103 cases, and the extent of the infection was prosthesis only, 66, annulus, 46, and cardiac invasion, 34. Surgical techniques evolved in the direction of increasingly radical debridement and reconstruction with biological materials. All patients were treated with prolonged postoperative antibiotic therapy.

There were 19 (13%) in-hospital deaths. Univariate analyses demonstrated trends toward increasing risk for patients with active PVE and extension of infection beyond the prosthesis; however, the only variables with a significant (P<0.05) association with increased in-hospital mortality confirmed with multivariate testing were impaired LV function, heart block, major organ system failure and identification of organisms in the pathology specimen. During the study period, mortality decreased from 20% (1975-1984) to 10% (1984-1992). For hospital survivors the mean length of stay was 25 days.

Follow-up (mean interval 62 months) documented late survival of 82% at 5 and 60% at 10 postoperative years. Increasing age was the only factor associated (P=0.006) with late mortality. Nineteen patients needed at least one further operation; reoperation-free survival was 75% at 5 and 50% at 10 postoperative years. Fever in the immediate preoperative period was the only factor associated with decreased late reoperation-free survival (P=0.049).

PVE remains a serious complication of valve replacement but the in-hospital mortality of reoperations for PVE has declined. With extensive debridement of infected tissue and postoperative antibiotic therapy the extent and activity of PVE does not appear to have a major impact on late outcome and the majority of patients with PVE survive for 10 years after surgery.

*By invitation


44. THE ADDITIONAL HOSPITAL COSTS GENERATED IN THE MANAGEMENT OF COMPLICATIONS OF PACEMAKER AND DEFIBRILLATOR IMPLANTATIONS

T. Bruce Ferguson, Jr., M.D., Candice Lilley Ferguson, M.S.P.H.* and Patricia Crimmins-Reda, R.N., M.S.N.*

St. Louis, Missouri

The rapid approach of capitated reimbursement mandates that providers examine their practice patterns associated with all surgical procedures. As part of this effort, documentation of 1) the complications associated with these procedures; and 2) the additional hospital costs associated with the management of these complications, is critical for comprehensive fiscal accountability. This study analyzed the fully-loaded hospital costs generated in the management of the most common surgical complications - infection, lead-related problems and hematoma formation requiring evacuation - associated with pacemaker and non-thoracotomy implantable defibrillator therapies.

Between July 1989 and September 1994 a total of 1, 031 pacemaker (PM) and 331 implantable defibrillator (ICD) procedures were performed by a cardiac surgeon in a tertiary-level teaching hospital setting. Over the past two years, 105 of these ICD procedures involved non-thoracotomy (NT-ICD) systems. Because of the commonly presumed similarities between PM and NT-ICD procedures, these two groups will be examined in this analysis.

There were no deaths associated with the 1, 136 procedures. The overall in-house PM infection rate was 0.58% (6/1, 031). An additional nine PM patients were transferred from outside institutions for definitive therapy of infected systems. The in-house PM lead reposition/replacement rate was 4.1% (37/902 procedures involving lead placement), performed from 1-78 days post-initial implant. Four PM patients developed pocket hematomas requiring evacuation; all were on systemic anticoagulation for medical problems. The overall in-house infection rate for NT-ICD was 0.95% (1/105). Two patients, both on systemic anticoagulation, developed pocket hematomas requiring reexplora-tion; both required lead configuration modifications due to elevated defibrilla-tion thresholds (DFTs) at the second procedure. Two patients had ICD system malfunctions, and four other patients (3.8%) had lead fractures (N=2) or dis-lodgements that necessitated surgical reexploration.

The additional fully-loaded hospital costs were documented as additional length of stay (LOS) in-hospital, ICU requirement, OR procedure costs, device costs and additional pharmacy, administrative and miscellaneous costs directly associated with the complication (data are expressed as mean ± SD):

Procedure

Complication

#Pts

Mean Age

Mean LOS

Added Costs

(Yrs)

(Days)

($)

PM

Infection

15

63.4 ± 17.4

17.3 ± 7.0

30, 765 ± 12, 984

Lead-Related

37

65.1 ± 15.3

2.4 ± 1.8

4, 595 ± 1, 734

Hematoma

4

63.5 ± 16.5

5.0 ± 4.7

7, 596 ± 4, 998

NT-ICD

Infection

1

52

16

63, 830

Lead-Related

4

47.3 ± 17.6

3.5 ± 0.6

13, 056 ± 6, 091

Hematoma/High DFTs

2

46.5 ± 24.7

11.0 ± 4.2

30, 592 ± 8, 994

System Malfunction

2

69.5 ± 10.6

2.5 ± 2.1

7, 978 ± 5, 032

Thus management of these common complications of PM and NT-ICD therapies is expensive. For all Medicare patients with complications in this study, costs exceeded reimbursement, suggesting that similar shortfalls would occur under a capitation scheme. The study demonstrates that accurate identification of the costs associated with complications of interventional procedures is feasible. This information is critical to a complete understanding of the financial impact of surgical procedures in a capitated reimbursement environment.

10:50 am INTERMISSION

*By invitation


11:10 am SIMULTANEOUS SCIENTIFIC SESSION D

ADULT CARDIAC SURGERY
Grand Ballroom, Sheraton Boston Hotel

Moderators: D. Glenn Pennington, M.D.

Robert L. Hardesty, M.D.

45. A PROSPECTIVE RANDOMIZED TRIAL OF EARLY EXTUBATION AFTER CORONARY ARTERY BYPASS

Charles M. Peniston, M.D., C.M.*, Davy Cheng, M.D.*, Jacek Karski, M.D.*, Zhao Sun, M.A.*, Susan Armstrong, M.Sc.* and

Tirone E. David, M.D.

Toronto, Ontario, Canada

Early extubation within the first few hours after cardiac surgery has been proposed as a safe method of cost containment by reducing ICU-and hospital stay. Early extubation has been possible in most cardiac patients by modifying the anaesthetic. All patients who had this modified anaesthetic were retrospectively reviewed to determine the predictors of failure of early extubation. Stepwise logistic regression analysis revealed that age > 65 years, female gender, moderate or severe left ventricular function and urgent operations were associated with a lower probability of early extubation. We then undertook a prospective study to determine if early extubation is safe and reduces the length of ICU and hospital stay after coronary artery bypass (CAB).

Methods: After institutional approval, 89 patients undergoing elective CAB were randomized to either conventional anaesthesia (CONVENTIONAL) (induction with fentanyl 50 meg/kg; maintenance with isoflurane and midazo-lam 0.1 mg/kg/hr) and assisted ventilation until next morning after surgery, or modified anaesthesia (EARLY EXTUBATION) (induction with fentanyl 15 meg/kg, isoflurane and propafol 2-4 mg/kg/hr) and early extubation (1 to 6 hours post-op). Continuous ST analysis was performed with pre-op baseline to 48 hrs postop. CK and CK-MB were measured at 6, 12, 24, 36, and 48 hrs after X-clamp release. Arterial and venous blood gases were measured at 240 mins after extubation. Respiratory patterns and shunt fraction (Qs/Qt) were monitored for 24 hours after extubation. Unless indicated data are expressed as mean ± S.D. and were compared by Student's t-test.

RESULTS

CONVENTIONAL

EARLY EXTUBATION

Number of patients

45

44

Age

61 ± 11

58 ± 9

Number of grafts

3.2 ± 1.0

3.5 ± 1.0

Incidence of ST changes

39%

50%

Maximum CK-MB (IU/L)

25

18

Pulmonary shunt (Qs/Qt)

23 ± 9%

13 ± 6%*

Assisted ventilation (hrs)

18.9 ± 1.9

3.8 ± 1.1*

Apnea incidence

30%

22%

ICU discharge criteria (hrs)

25.6 ± 10.1

7.0 ± 1.8*

Hospital stay (days)

9.1 ± 3.8

6.9 ± 1.4*

*indicates p<0.05 between groups

Data from 784 patients undergoing CAB or valve procedures were also examined. ICU length of stay was reduced from 2.5±4.7 days to 1.43±0.9 days (p<0.0001) and hospital stay was reduced from 10.6±7.5 days to 8.3±4.2 days (p<0.0001).

Conclusion: These results indicate that early extubation after elective CAB is safe and reduces the length of ICU and hospital stay.

*By invitation


46. ANTI-ISCHEMIC AND ANTI-ARRHYTHMIC PROTECTION BY PERIOPERATIVE INFUSION OF NIFEDIPINE AND ETOPROLOL IN PATIENTS UNDERGOING ELECTIVE AORTOCORONARY BYPASS SURGERY

Bruno K. Podesser, M.D.*, Severin S. Schwarzacher, M.D.*, Werner Zwolfer, M.D.*, Friedrich Peschl, M.D.*, Thomas Binder, M.D.*, Ernst Wolner, M.D. and Rainald Seitelberger, M.D.*

Vienna, Austria

A randomised study was performed on 70 patients undergoing elective coronary bypass procedure to examine whether the combined perioperative 24-hour infusion of nifedipine (10 ug/kg/h) and metopropol (12 ug/kg/h) reduces the incidence of perioperative myocardial ischemia and arrhythmias (group NM, n=34). The control group (group N, n=36) received nifedipine only. Repeated assessments of serum enzyme levels (CK, CK-MB) and 12-lead-ECG together with a 3-channel Holier monitoring over 48h were used to classify perioperative myocardial ischemia (transient ischemic event, myocardial infarction) and supraventricular and ventricular arrhythmias.

The two groups did not differ with respect to their demographic data, extracorporeal circulation, aortic cross-clamping time, or number of distal anastomosis. No perioperative myocardial infarction in either group was detected. However, a significantly lower incidence of transient ischemic events was observed in the NM group as compared to the N group (3% versus 11%; p<0.05). In addition, there was a tendency towards lower CK-MB-levels and peak-values of CK- and CK-MB-enzymes in the NM group. With regard to perioperative dysrhythmias, there was a significantly lower incidence of sinus tachycardia (9%) and atrial flutter/fibrillation (6%) in the NM group as compared to the N group (33% and 27%, p<0.05). In addition, postoperative heart rate was lower in the NM group starting from the 6th hour after opening the aortic cross-clamp.

In conclusion, the combined perioperative infusion of nifedipine and metoprolol is superior in preventing perioperative myocardial ischemia and decreasing the incidence of supraventricular arrhythmias as compared to a single-drug regimen with nifedipine.

*By invitation


47. TRANSPLANT CANDIDATES' CLINICAL STATUS RATHER THAN RIGHT VENTRICULAR FUNCTION DEFINES NEED FOR UNIVENTRICULAR VS. BIVENTRICULAR SUPPORT

Robert L. Kormos, M.D, Si M. Pham, M.D.*, Thomas A. Gasior, M.D.*, Srinivas Murali, M.D.*, Brack G. Rattier, M.D., Ph.D.* and Hartley P. Griffith, M.D.

Pittsburgh, Pennsylvania

We have studied our experience since 1988 in 31 patients who required a mechanical circulatory bridge to transplantation who also had biventricular failure (mean RV ejection fraction [RVEF] = 12%) to better define the need for biventricular or total artificial heart support vs univentricular support (LVAS). Clinical factors including preoperative inotropic need, fever without detectable infection, radiographic diffuse pulmonary edema, postoperative blood transfusion and RV thickness were compared with hemodynamic parameters including cardiac index, RVEF, CVP, mean PAP, and total pulmonary resistance for their ability to predict need for mechanical or high dose inotropic support for the RV. Patients were grouped as to the need for RV support following LVAS implantation: None (I)=14, inotropic drugs (II)=7, and RV mechanical support (III)=10. There were no differences in preimplant hemodynamic variables. Groups II and III had a significantly lower mixed venous oxygen saturation (39.2% vs 52.4% in Group I; p<0.001), a greater level of inotropic need (p<0.02), greater impairment of mental status, and a lower ratio of RVEF to inotropic need (0.37 vs 0.56 for Group I; p<0.02) before LVAS implantation. A significant discriminator between Groups II and III was the presence of a fever without infection within 10 days of LVAS implant (14% in Group II vs 80% in Group III). Group III had more patients with preimplant pulmonary edema on chest radiograph and a greater requirement for postoperative blood transfusion (6 units cells in Group II vs 38 in Group III). RV thickness at LVAS explant was 0.87 cm in Group III vs. 44 cm in Group II (p<0.05). Transplantation rates after bridging were 100% in Group I, 71 % in Group II and 50% in Group III. Clinical factors which reflect preimplant degree of illness and perioperative factors which result in impairment of pulmonary blood flow or reduced perfusion of the right ventricle after LVAS implantation are now felt to be more predictive of the need for additional RV support than preimplant measures of RV function or hemodynamics.

12:10pm ADJOURN

*By invitation


9:30 am SIMULTANEOUS SCIENTIFIC SESSION E

GENERAL THORACIC SURGERY

Republic Ballroom, Sheraton Boston Hotel

Moderators: Victor F. Trastek, M.D.

Martin F. McKneally, M.D.

48. RESULTS AND PROGNOSTIC FACTORS IN

RESECTIONS OF PRIMARY TRACHEAL TUMORS

J.F. Regnard, M.D.*, P. Fourquier, M.D.* and Ph.Levasseur, M.D.*

Paris, France

Sponsored by: Peter C. Pairolero, M.D., Rochester Minnesota

From 1970 to 1993, 208 patients with primary tracheal tumors were evaluated in a multicentric retrospective study including 26 centers. Ninety-four patients had a squamous cell carcinoma of the trachea, 4 had adeno-carcinoma, 65 had adenoid cystic carcinoma and 45 patients, miscellaneous tumors. The following resections were performed: tracheal resection with primary anastomosis, 146; partial tracheal resection, 19; carinal resection, 24; and tracheo-laryngeal resection, 19. In average, the length of the resected specimen was 38.5 mm (10-60). Postoperative complications occurred in 41% and postoperative mortality was 10.5%. Postoperative morbidity and mortality were statistically correlated (PO.05) to the length of the resection, to the need of a laryngeal release, to the type of resection and to the histologic type. Follow-up was complete for all but 10 patients with a mean follow-up of 5 years. Fifty-nine percent of the patients with tracheal cancers and 43% of the patients with adenoid cystic carcinomas had postoperative radiotherapy.

In complete resection, the 5 and 10-year actuarial survival were respectively 55% and 30% in tracheal cancers, 82% and 25% in adenoid cystic carcinomas. Long-term survival was altered by the occurrence of second primary cancer (ORL, lung) in the group of patients with tracheal cancers and by late metastases in adenoid cystic carcinomas. Postoperative radiotherapy did not improve survival in the group of patients with tracheal cancer when the resection was complete, even if nodes were involved. However, in case of incomplete resection, postoperative radiotherapy did not improve survival in the group of patients with tracheal cancer when the resection was complete, even if nodes were involved. However, in case of incomplete resection, postoperative radiotherapy improved survival (P<0.02). For adenoid cystic carcinomas, the actuarial survival seemed better in complete resection than in incomplete resection but the difference was not statistically significant (PO.20). Postoperative radiotherapy did not improve survival in adenoid cystic carcinoma, even in incomplete resection.

Surgery is the treatment of choice of primary tracheal tumors.

*By invitation


49. THE IMPORTANCE OF SURGICAL STAGING IN THE TREATMENT OF MALIGNANT PLEURAL MESOTHELIOMA

Valerie W. Rusch, M.D. and Ennapadam Venkatraman, Ph.D.*

New York, New York

Progress in the therapy of malignant pleural mesothelioma (MPM) is hindered by the lack of an adequate staging system and controversy about prognostic factors. This personal surgical series was analyzed to determine the value of a new TNM staging system proposed by the International Mesothelioma Interest Group (IMIG), and to examine factors that could affect the design of future clinical trials.

Methods: Thoracotomy was done in previously untreated patients (pts) if CT scans showed technically resectable tumor confined to one hemitnorax. Pleurectomy/decortication (P/D) was used in pts with minimal visceral pleural tumor, extrapleural pneumonectomy (EPP) in pts with more locally advanced disease. Complete resection was defined as no gross residual tumor. Adjuvant therapy was given as required by serial clinical trials. Pts were followed by chest and abdomen CT scans every 3 months after death. Prognostic factors were examined by log rank and Cox regression analyses.

Results: From 10/83 to 7/94 there were 131 consecutive thoracotomies, 101 resections, 72 complete. EPP:P/D=50:51. Men:women=108:23. Median age=63 years (range=32-80). Operative mortality 5/131 pts (3.8%), 3/50 EPP (6%). Most frequent morbidity: atrial arrhythmias, 14/131 pts (11%), 11/50 EPP (22%). 95/131 tumors were epithelial. 51/89 pts (57%) having node dissections had (+) nodes, 45 (50%) N2.

Survival

Operation

T status

N status

Stage

Histology

EPP

P/D

Tl

T2

T3

T4

NO

Nl-3

I

II

III

Epi

Non-Epi