WEDNESDAY MORNING, MAY 6, 1998
7:00 a.m. GENERAL THORACIC SURGERY
FORUM SESSION
Ballroom C, Hynes Convention
Center
Moderators: Larry R. Kaiser, M.D.
David J. Sugarbaker, M.D.
F9. HUMAN
PLEURAL MESOTHELIOMAS CONTAIN SIMIAN VIRUS-40 (SV40) REGULATORY AND LARGE TUMOR
(T) ANTIGEN DNA SEQUENCES.
Harvey I. Pass, M.D., Jessica S.
Donington, M.D.*, Peter Wu, M.D.*, Paola Rizzo, Ph.D.*, Ronald Kennedy, Ph.D.*
and Michele Carbone, M.D, Ph.D.*
Detroit, Michigan; Washington,
DC; Bethesda, MD;
Chicago, Illinois and Oklahoma
City, Oklahoma
A cohort (20%) of mesothelioma
(meso) patients will not have an exposure to asbestos. Recently, a DNA tumor
virus (SV40) has been shown to cause hamster mesos, and we previously described
SV40-like DNA amino terminus sequences in 29 of 48 mesos (Oncogene 9:1781-90,
1994). We analyzed an additional 42 mesos to determine (1) whether our
initial observations were durable (2) if other regions of the SV40 genome were
present and (3) whether meso patients exhibited an immune response to SV40. METHODS:
Genomic DNA was extracted from snap frozen meso tumor samples and from the
SV40-induced hamster meso tumor H9A. PCR primers were used to amplify various
SV40 large T antigen (T-ag) regions including a 105 bp amino terminus fragment,
a 281 bp carboxy terminus fragment, and a 310 bp fragment of the enhancer
promoter region. Endonuclease digestions were used to verify the expected
product. SV40 T-ag specificity and liter of human serum antibodies were
examined in 31 mesothelioma patients using an enzyme-linked immunosorbent assay
(ELISA). RESULTS: 30 of the 42 (71%) samples amplified T-ag amino
sequences, and specificity was verified by Southern hybridization. Thirteen of
42 samples (31%) amplified the appropriate size fragment for the carboxy terminus,
and digestion with BsaB I matched that of H9A. Twenty of 42 samples (48%)
amplified SV40 regulatory sequences and Fok I digestion matched that of the
hamster control tumor. Sequence analysis (4 patients) revealed 100% homology
with the regulatory region of SV40 strain 776. Compared to non-SV40 exposed
controls, the frequency of serum antibodies to T-ag in meso was significantly
greater (P2 = 0.045). CONCLUSIONS: These data suggest an
association between the SV40 virus and human mesothelioma which could be
exploited for diagnostic/therapeutic options including early detection and
potential vaccination strategies.
*By invitation
F10. EVIDENCE THAT DIFFUSION CAPACITY LIMITS
LUNG VOLUME REDUCTION SURGERY IN A RABBIT MODEL OF EMPHYSEMA.
John C. Chen, M.D.*, Dan L. Serna,
M.D.*, Ledford L. Powell, M.D.*, Henry E. Aryan, B.S.*, Robert J. McKenna,
M.D., Arthur Gelb, M.D.* and Matthew Brenner, M.D.*
Orange and Irvine, California
Purpose: Lung volume
reduction surgery (LVRS) has been suggested to improve lung compliance and
expiratory flows in patients with obstructive emphysema. Endpoints for optimal
resection volumes are not known. Spirometric improvements may have to be
weighed against reduction in gas exchange surfaces as larger volumes of lung
tissue are removed. The purpose of this study was to evaluate the effects of
LVRS on lung diffusion capacity (DLCO), pulmonary compliance, and
airway flow in a rabbit model of emphysema.
Methods: Forty-nine
rabbits were induced with 15,000 units of elastase by aerosolization through an
endotracheal tube. Emphysema was confirmed histologically 5 weeks following
induction. Single breath DLCO, static pressure volume relationships,
and forced expiratory flows (FEF 33%) were measured at baseline prior to
induction, preoperatively at 4 weeks following induction, and 1 week
postoperatively following sham sternotomy (n = 10) or bilateral upper lobe LVRS
(n = 39).
Results: Transpleural
pressures with 60 cc insufflation above FRC increased with resection of larger
volumes of lung tissue (Graph 1, ANOVA p = 0.000). Comparison of changes in DLCO
reveal diminishing diffusing capacity with increasing amounts of lung volume
resected (Graph 2, p = 0.063). In contrast, early expiratory flow improved most
in the rabbits with 2.3-3 grams of lung tissue resected (Graph 3, p = NS).
Conclusions: Static lung
compliance continues to improve with escalating volumes of lung resected.
Decreased compliance and increased airway flow following LVRS in this animal
model parallel findings in clinical studies. These findings support the
hypothesis that mechanisms of improved elastic recoil and airway resistance
contribute to patient improvement. Our findings suggest that there exists an
optimal resection volume range for improvement in
early expiratory flow and DLCO, which may help to explain clinical response. Supported by
ALA #CI-030-N,CTRDRP #6RT-0158, DOE #FG03-91ER61227.
*By invitation
F11. ADJUVANT TREATMENT OF REFRACTORY LUNG
TRANSPLANT REJECTION WITH EXTRACORPOREAL PHOTOPHORESIS.
C.T. Salerno, M.D.*, Soon J. Park,
M.D.*, David M. Kulick, M.D.*, Nathan S. Kreykes, B.S.*, Marshall I. Hertz,
M.D.* and R. Morton Bolman, III, M.D.
Minneapolis, Minnesota
Photophoresis (PPE) is a
technique in which a patient's leukocytes, removed by apheresis, are exposed to
ultraviolet-A light after pretreatment with 8-methoxypsoralen, a photosensitive
drug activated by long-wavelength ultraviolet light. The irradiated mononuclear
suspension is subsequently re-infused to the patient. PPE has been used for the
treatment of autoimmune diseases, T cell lymphoma and the treatment of acute
rejection in heart transplant recipients. PPE appears to induce specific
suppression of both cellular and humoral rejection. The altered lymphocytes
produce a suppressor response that targets unirradiated T cells of similar
clones in a mechanism that is not yet elucidated. To date there have been few
published reports detailing the use of PPE in lung transplant recipients. We
have used PPE, in addition to standard anti-rejection chemotherapy in 7 lung
transplant recipients with refractory rejection since 1992. All 7 patients had
progressively worse graft function and were BOS grade 3 prior to the initiation
of photophoresis. There were 3 women and 4 men. Two patients had a pre-transplant
diagnosis of COPD, one a-1 antitrypsin deficiency, one cystic fibrosis, one
bronchiectasis and two primary pulmonary hypertension. Prior to developing
refractory rejection all patients had been treated with three drug
immunosuppression, and anti T-cell therapy. The mean time from transplant to
the initiation on PPE was 16 months (range 7-32). The mean number of treatments
was 6 (range 3-12). RESULTS: Five of six patients subjectively improved after
PPE therapy. In these 5 patients PPE was associated with a stabilization in the
measured FEV1. In two patients there was biopsy proven reversal of
rejection following PPE. With a mean follow up of 28 months (range 2-53), all 7
patients are alive and well. Three patients required re-transplant at a mean
period of 19 months (range 15-21) after completion of the PPE treatments. Four
patients have remained stable post PPE with only subtle changes in their
immunosuppressive therapy. There were no PPE related complications. We believe
that this treatment is a safe option for patients with refractory lung
allograft rejection when increased immunosuppression is contraindicated or
ineffective.
*By invitation
F12. BILE ACIDS INDUCE CYCLOOXYGENASE-2 AND
PROSTAGLANDIN SYNTHESIS: A POTENTIAL MECHANISMOF ESOPHAGEAL CARCINOGENESIS.
Fan Zhang, M.D., Ph.D.*, Andrew J.
Dannenberg, M.D.* and Nasser K. Altorki, M.D.
New York, New York
Bile reflux has been implicated
in the genesis of esophageal cancer in animals and humans but the underlying
mechanism(s) remains unclear. A large body of data from a variety of
experimental systems suggest that cyclooxygenase-2 (Cox-2), the inducible form
of Cox, is important in tumor formation because it catalyzes the synthesis of
prostaglandins (PCs) from arachidonic acid, inhibits apoptosis and bioactivates
chemical carcinogens. We investigated, therefore, the effects of bile acids,
known endogenous promoters of colon cancer, on the expression of Cox-2 and
synthesis of PCs in a human Barrett's adenocarcinoma cell line. Treatment with
the unconjugated dihydroxy bile acids, chenodeoxycholate (CD) and deoxycholate
(DC), resulted in about a 10-fold increase in the production of PGE2
Enhanced synthesis of PGE2 was associated with a marked increase in
levels of Cox-2 mRNA (Fig. 1) and Cox-2 protein (Fig. 2) with maximal effects
at 8-12 h and 12-24 h, respectively. In contrast, neither cholic acid nor
conjugated bile acids affected levels of Cox-2 or synthesis of PGE2.
Bile acid-mediated induction of Cox-2 was blocked by
inhibitors of protein kinase C activity, including calphostin C and
staurosporine. Bile acids were also potent inducers of Cox-2 in esophageal
squamous cell carcinoma cell lines. These results may explain, in part, the
mechanism by which bile reflux contributes to the pathogenesis of esophageal
cancer.
*By invitation
F13. LIPOSOME-MEDIATED GENE TRANSFER IN RAT
LUNG TRANSPLANTATION: A COMPARISON BETWEEN THE IN VIVO AND EX
VIVO APPROACH.
Carlos H. Boasquevisque, M.D.*, Bassem Mora, M.D.*,
Mariano Boglione, M.D.*, Jonathan S. Bromberg, M.D.*, Ronald K. Scheule,
Ph.D.*, Joel D. Cooper, M.D. and G. Alexander Patterson, M.D.
St. Louis, Missouri; Ann Arbor,
Michigan;
Framingham, Massachusetts
Background: We have
recently studied the pattern of in vivo and ex vivo liposome-mediated
gene transfer to rat lung isografts using the reporter gene chlorarnphenicol
acetyl transferase (CAT). We also demonstrated that ex vivo transfection
of TGF(3-1 cDNA into rat lungs improved allograft function. In this study we
compared the efficacy of in vivo and ex vivo transfection in rat
lung transplantation. Methods: Animals were divided into two groups
according to the transfected cDNA. (1) CAT: Fischer rats underwent isogeneic
left lung transplant and were divided into 6 groups (n = 4). In group I grafts
were infused ex vivo with 660 µg of liposome-CAT cDNA and transplanted
immediately (1-1/2 hours of exposure at 10°C). In group II animals underwent
the same procedure but the exposure time was 10 hrs. In group III (in vivo),
donors received an intrajugular injection of 1320 µg of CAT cDNA. Left
lungs were harvested after 1-1/2 hours at 10°C and transplanted. In group IV
grafts were transfected in vivo, harvested after 10 hours and preserved
for 10 hours at 10°C. In groups V and VI, grafts were transfected in vivo and
ex vivo respectively with CAT cDNA not complexed to liposomes. The
exposure time was 1 1/2 hours in both groups. In all groups recipients were
sacrificed at two days post transplant for CAT assay. (2) TGFβ-1
Brown-Norway rats served as donors and Fischer rats as recipients. Animals were
divided into 4 groups. In group I (in vivo, n = 7) donors received an
intrajugular injection of 1320 ng of TGFβ-1 sense cDNA complexed to
liposomes. Left lungs were harvested 3 hours later. In group II (n = 7) grafts
were harvested and then infused ex vivo with 660 µg of TGFβ-1 sense
cDNA complexed to liposomes. In group 111 (n = 5) grafts were harvested and
then infused ex vivo with 660 µg of TGFβ-1 anti-sense complexed
liposomes. In group IV (n = 5) lung grafts were harvested and transfected ex
vivo with TGFβ-1 sense cDNA not complexed to liposomes. All grafts
were preserved for 3 hours at 10°C prior to transplantation. Recipients were
sacrificed on postoperative day 5 for arterial oxygenation and histologic
assessment of rejection score. Results: (1) CAT -Transfection was
clearly superior in the ex vivo subgroup. Transfection was similar for
both exposure times. CAT cDNA alone was inefficient either in vivo or ex
vivo. (2) TGFβ-1 oxygenation was superior in the ex vivo liposome-TGFβ-1
sense group in comparison to the in vivo liposome TGFβ-1 sense
group and ex vivo liposome TGFβ-1 anti-sense group. Allograft
function was superior in both groups treated ex vivo and in vivo with
TGFβ-1 sense-liposomes compared to TGFβ-1 sense cDNA alone. Rejection
scores were not significantly different between the ex vivo liposome-sense
versus in vivo liposome-sense subgroups. However rejection scores were
superior after liposome-sense transfection compared to liposome-anti-sense and
cDNA alone groups, Conclusions: (1) using current liposome
technology the ex vivo approach is superior to the in vivo approach
in experimental lung transplantation. (2) Infusion of cDNA not complexed to
liposomes was sufficient to provide transgene expression but not at levels high
enough to produce a functional effect.
*By invitation
F14. INDUCTION CHEMOTHERAPY, SURGICAL
RESECTION AND RADIOTHERAPY IN PATIENTS WITH MALIGNANT PLEURAL EFFUSION,
MEDIASTINO-SCOPY NEGATIVE(STAGE IIIB) NON-SMALL CELL LUNG CANCER.
Scott J. Swanson, M.D.*, Michael
T. Jaklitsch, M.D.*, Steven J. Mentzer, M.D., Malcolm M. DeCamp, M.D.*, William
G. Richards, Ph.D.*, Raphael Bueno, M.D.* and David J. Sugarbaker, M.D.
Boston, Massachusetts
Objective:The
purpose of this study was to determine the feasibility, morbidity and mortality
of multimodality therapy including extrapleural pneumonectomy in patients with
malignant pleural effusion and negative mediastinal lymph nodes (by
mediastinoscopy) with a diagnosis of non-small cell lung cancer (NSCLC) (stage
IIIB).
Rationale:Stage
IIIB NSCLC on the basis of malignant pleural involvement has a guarded
prognosis with average survival of 3-6 months in most series regardless of
therapy. A subset of these patients at presentation do not manifest
hematogenous or mediastinal lymphatic metastases but only local regional
pleural spread. Clinically, these patients can be confused with patients with
malignant pleural mesothelioma for whom multimodality therapy may extend
survival. Induction chemotherapy followed by surgery has been shown to have a
survival benefit in several recent series of patients with stage IIIA disease.
Surgical resection in early stage malignant mesothelioma has been shown to be
feasible. Radiotherapy has been demonstrated to decrease local recurrence
following surgery in NSCLC. A combination of the strategies used to treat
malignant mesothelioma and stage IIIA NSCLC might be applicable to a select
group of patients with stage IIIB (malignant pleural involvement) NSCLC.
Methods:Between
1994 and 1997, 12 consecutive patients with stage IIIB NSCLC on the basis of
malignant pleural involvement were selected to undergo a multimodality
treatment regimen. Patients who demonstrated adequate functional reserve (ECOG
ps 0-2), who had no significant medical comorbidites and had adequate pulmonary
reserve to tolerate a pneumonectomy (predicted postoperative FEV 1 ≥ 1)
were screened for signs of metastatic disease. Each patient had a bone scan,
head ct scan and chest ct scan that included the liver and adrenal glands.
Patients without signs of hematogenous metastases underwent mediastinoscopy to
rule out mediastinal nodal involvement. In those without N2 or N3 disease,
induction chemotherapy was carried out with 3 cycles of a platinum-based
regimen. Twelve patients without clinical or radiographic signs of progression
went on to have an extrapleural pneumonectomy including prosthetic
reconstruction of the hemidiaphragm and pericardium. Seven patients had
post-operative external beam radiotherapy. Perioperative data and followup
information was gathered from our prospective thoracic database
Results: This
cohort was made up of 6 males and 6 females with a median age of 61.5 years
(range 41-69), median follow-up 8.5 months (range 3-25). Histological
evaluation of the specimens revealed 9 adenocarcinomas and 3 squamous cell
carcinomas. Median length of stay was 7.5 days (range 6-80). Seven patients
(58%) received red blood cell transfusions (median 1.5 units, range 0-6). There
were no perioperative deaths and 7 (58%) patients had complications (6- atrial
fibrillation, 1- aspiration pneumonia, 1- vocal cord paresis). Median survival
in the N1 node positive subgroup is 24 months (n = 5). The median survival has
not been reached in the node-negative subgroup (n = 7), (p = NS).
Conclusion: Multimodality
therapy including surgical resection for stage IIIB NSCLC is feasible in
selected patients. This pilot study would suggest that aggressive local
regional strategies may be the subject of larger clinical trials in patients
with stage IIIB NSCLC.
*By invitation
F15. PIG LUNG PRE-PERFUSION PREVENTS LUNG
HYPERACUTE REJECTION IN THE PIG-TO-HUMAN COMBINATION.
Paolo Macchjarini, M.D., Ph.D.*,
Rafael Oriol, M.D.*, Robert Rieben, Ph.D.*, Nicolao Bovin, Ph.D.* and Philipe
Dartevelle, M.D.
Le Plessis and Villejuif,
France; Berne, Switzerland; Moscow, Russia
Background: The clinical
use of pig lungs is currently limited by hyperacute rejection (HAR), triggered
by the binding of human anti-αGal xenoreactive natural antibodies (hXNA)
to endothelial xenograft cells. Objective: To test the relative effects
of pig organ pre-perfusion and specific depletion of anti-αGal hXNA in
preventing HAR in the pig-to-human lung combination. Methods: Large
White pig (20-25 kg) left lungs were harvested and ex-vivo continuously
ventilated and reperfused, using a neonatal oxygenating system, with either
whole human blood pre-perfused trough donor pigs right lung (group I), liver
(group II), spleen (group III) or human plasma filtrated on an immunoabsorbent
column with α-galactose (Galαl-3Galβ(CH2)3NH2;
Bdi) (group IV). Each study group included 6 animals. Results: Pre-perfusion
of human blood through pig organs or the αGal column achieved a similar 89
± 4% reduction of anti-αGal hXNA in the four groups. All pre-perfusing
organs displayed HAR after 15 ± 3 min. Following ex-vivo reperfusion,
group I lung xenografts had a significantly (p<0.001) longer functional
survival than groups II, III and IV xenografts, and were the only
histologically normal 5 hours upon reperfusion.
|
Groups
|
2 min
|
30 min
|
60 min
|
180 min
|
Xenograft
|
|
|
PVR
|
AVO2
|
PVR
|
AVO2
|
PVR
|
AVO2
|
PVR
|
AVO2
|
survival
|
|
Group I
|
15 ± 4
|
24 ± 2
|
25 ± 10
|
24 ± 4
|
18 ± 3
|
20 ± 2
|
17 ± 4
|
17 ± 2
|
300 ± 54
|
|
Group II
|
24 ± 7
|
18 ± 4
|
39 ± 20
|
16 ± 7
|
29 ± 11
|
12 ± 5
|
27 ± 8
|
13 ± 3
|
145 ± 40
|
|
Group III
|
15 ± 6
|
21 ± 6
|
48 ± 36
|
17 ± 7
|
39 ± 23
|
9 ± 1
|
-
|
-
|
50 ± 15
|
|
Group IV
|
28 ± 15
|
21 ± 8
|
61 ± 24
|
18 ± 9
|
47 ± 21
|
16 ± 7
|
-
|
-
|
78 ± 45
|
|
Data are
presented as mean ± standard deviation; pulmonary vascular resistance
(PVR: mm
Hg/ml/min.); arteriovenous oxygen difference (AVO2: mlO2/100
ml).
|
Human blood reperfusing group I
xenografts had a significantly (p<0.05) lower: i) fall in white blood
cells, clotting factors, total circulating immuno-globulins; ii) total
(CH100) and membrane attack complex (C5b-9) complement activation; and in) hemolysis.
Conclusions: We provide evidence that specific depletion of
anti-αGal hXNA alone incompletely protects pig lungs from HAR. It is
speculated that the complete protection afforded by lung pre-perfusion relates
to a better removal of other critical humoral or cellular elements provoking
HAR.
*By invitation
F16. ADENOVIRAL-MEDIATED P53 GENE TRANSFERS
TO NON-SMALL CELL LUNG CANCERS.
David Weill, M.D.*, Allan N.
Shulkin, M.D.*, Juliette L. Wait, M.D.*, Milissa Tuzzolino, R.N.*, John A.
Osborne, M.D, Ph.D.*, John J. Nemunaitis, M.D.* and Michael J. Mack, M.D.
Dallas, Texas
Introduction The p53 gene,
a tumor suppressor gene, is often rendered nonfunctional in human non-small
cell lung cancer (NSCLC) by mutation or deletion. By restoring the expression
of p53, it is postulated that tumor growth could be suppressed. We performed a
Phase I study using an adenoviral vector expressing wildtype p53 (Ad-p53) to
demonstrate the feasibility of safely delivering the gene and to document any
observed antitumor activity.
Materials and Methods A
replication-defective adenoviral vector containing wildtype p53 gene was
constructed for intralesional injection into NSCLC that had documented p53
mutations. All patients were refractory to conventional treatment including
cisplatin chemotherapy (n = 8) or external beam radiation (n = 3). Patients
were enrolled into one of two treatment arms: treatment with Ad-p53 alone or
treatment with Ad-p53 plus cisplatin. The Ad-p53 injections were performed
monthly in patients with bronchoscopically accessible tumors. The study
treatments continued as long as there was no tumor progression or unacceptable
adverse effects. A maximum of 6 treatments was given. Results The Ad-p53 was
injected into 11 patients with primary NSCLC. Doses of Ad-p53 ranged from 1 x 106
to 1 x 1011 plaque forming units. Following the injections, p53
expression was documented in 8 of the 9 evaluable patients. Toxicity after
injection of Ad-p53 was minimal and included minor complications associated
with the endobronchial biopsy procedure. During the course of treatment, 2
patients developed pneumonia which was likely secondary to the obstructing
tumors.
In 6 of the 9 evaluable patients, endobronchial
obstruction was relieved as determined by serial bronchoscopic imaging. Disease
remained stable in 2 patients and progressive in 1. The average survival of
patients enrolled in the study was 139.3 + days (range 16-292+).
Conclusion We demonstrated
that Ad-p53 can be safely delivered intralesionally to patients with NSCLC.
Toxicity was minimal, and transgenic expression was achieved. Furthermore,
apoptosis as measured by relief of endobronchial obstruction was observed in
most patients.
*By invitation
F17. A SELECTIVELY REPLICATING ADENOVIRUS
(ONYX-015) LYSES NON-SMALL CELL LUNG CANCER CELLS THAT LACK FUNCTIONAL p53.
David M. Jablons, M.D.*, Liang
You, M.D, Ph.D.*, Adam Sampson-Johannes, M.S.*, David Kirn, M.D.* and Frank
McCormick, Ph.D.*
San Francisco and Richmond,
California
Sponsored by: Frank Hanley,
M.D., San Francisco, California
Loss or mutation of p53 tumor suppressor gene is a
common genetic abnormality in many human tumors including lung cancer. p53
mutations and loss of heterozygosity have been detected in more than 50% of
lung cancers. p53 protein prevents replication of damaged DNA in normal cells
and promotes apoptosis of cells with abnormal DNA. p53 mutations are frequently
associated with poor prognosis in tumor patients. ONYX Pharmaceuticals has
genetically designed a tumor-targeting adenovirus (a replication competent E1B-deleted)
which only replicates in cells that lack functional p53 gene and therefore
kills tumor cells specifically. In an in vitro study, this mutant adenovirus
ONYX-015 has been shown to kill cervical carcinoma cells, colon carcinoma
cells, glioblastoma ceils and pancreatic adenocarcinoma ceils lacking
functional p53. It was also demonstrated that this virus caused a significant
reduction in tumor size and caused complete regression of 60% of the tumors
induced by p53-defi'cient human cervical carcinoma cells in nude mice. In this
study, we tested the cytotoxicity of this mutant adenovirus against two
non-small cell lung cancer (NSCLC) cell lines that lack functional p53 gene
(NCI-H522 and NCI-HI703) using cytopathic effect (CPE) assays. NCI-H522 is a
lung adenocarcinoma cell line with a missense mutation at codon 285 (GAG-»AAG)
and NCI-HI703 is a squamous lung carcinoma cell with a single base deletion at
codon 191 (CCT->CT) of p53 gene. Loss of heterozygosity of the p53 gene was
found in both NCI-H522 and NCI-HI703 cells. We have demonstrated that the virus
can lyse those cells but not NSCLC cells with functional p53 (NCI-H2304) or a
mesothelioma cell line (MS-1). In replicate experiments, ONYX-015 virus lysed,
in a dose-dependent fashion, NCI-H522 and NCI-HI703 cells. Five days after
infection, 50% of the cells were lysed at multiplicities of infection (MOI) of
1 plaque-forming unit (PFU) per cell; 9 days after refection, at the MOI of 1
and 0.1, 100% and 50% of the cells were lysed respectively. No signs of cell
lysis were noticed in NCI-H2304 and MS-1 at MOI of 0.1 and 1. PFU per cell 9
days after infection. Wild-Type adenovirus served as control for effective
infection. We are currently in the process of testing this virus in combination
with chemotherapy in these NSCLC cell lines as well as in fresh human lung
tumor cells. It is hoped that with subsequent pre-clinical studies ONYX-015 can
be advanced to phase I clinical trials for the treatment of patients with
advanced lung cancer.
*By invitation
F18. MDM-2 EXPRESSION: AN ALTERNATIVE
MECHANISM FOR p53 INACTIVATION IN ESOPHAGEAL ADENOCARCINOMA.
Robert Soslow, M.D.*, Liang Ying,
M.D.* and Nasser K. Altorki, M.D.
New York, New York
Several immunohistochemical studies have shown that
the p53 protein is expressed in 60-70% of esophageal adenocarcinomas. Since
mutations of this tumor suppressor gene are present in approximately 40% of
tumors, it is presumed that p53 stabilization and expression may develop as a
result of mechanisms other than gene mutation. Amplification and expression of
the mdm-2 gene, a known regulator of p53 activity, can result in inactivation
of the p53 gene with stabilization of its protein product and loss of its tumor
suppressor function.
In this study we evaluated the incidence of p53
abnormalities as well as the expression of the mdm-2 protein product in 16
esophageal adenocarcinomas. Routine immunohistochemical studies were performed
following standard antigen retrieval methods and interpreted using a previously
described immunoreactive score. Tumor DNA was obtained by microdissection,
amplified and sequenced for mutations in the p53 hot spot regions (exons 5-8).
P53 expression was observed in 12 of 16 (75%) cases while p53 mutations were
detected in 7 of 16 (43%). This does not exclude the possibility of mutations
in exons other than 5-8. Overall, mdm-2 expression was present in 8 of 16
tumors. Moderate or strong expression of mdm-2 was observed in 4 tumors none of
which had detectable p53 mutations.
We conclude that in esophageal adenocarcinoma (1.)
Discordance of p53 immunohistochemistry and mutations occurs in 20-30% of cases
and (2.) mdm-2 expression may be responsible for loss of p53 tumor suppressor
function in 25% of esophageal adenocarcinomas.
*By invitation
7:00 a.m. CARDIAC SURGERY FORUM SESSION
Ballroom B, Hynes Convention
Center
Moderators: Fred A. Crawford, Jr., M.D.
Edward D. Verrier, M.D.
F19. AN EXPERIMENTAL MODEL OF SMALL INTESTINE
SUBMUCOSA AS A GROWING VASCULAR GRAFT FOR CONGENITAL CARDIAC SURGERY.
MonicaC. Robotin-Johnson, M.D.,
F.R.A.C.S.*, Paul E. Swanson, M.D.*, David C. Johnson, M.D., F.R.A.C.S.* and
James L. Cox, M.D.
St. Louis, Missouri and
Washington, DC
The ideal vascular graft for use in children with
congenital heart defects should not only be biocompatible, nonthrombogenic and
present no infectious risk, but should grow at the same rate as the recipient.
We have tested autologous small intestine submucosa
(SIS) as a superior vena cava (SVC) interposition graft in 11 piglets, with a
mean weight of 10.7 kg. The grafts were prepared from segments of autologous
jejunum, rendered nonthrombogenic by bonding with heparin. The SVC from the
level of the azygous vein to the SVC-right atrial junction, measuring a mean
length of 9.9 mm and circumference of 25.9 mm was replaced. There was one early
and one late death, not related to the SVC replacement. At 90 days the 9 long
term survivors had a mean weight increase of 630% and the grafts increased in
length by 147% [p<0.03] and in circumference by 184% [p<0.001], paralleling
the growth of the native cava. All 11 grafts were patent and free of thrombus
at the time of explantation. Cavograms showed no anastomotic stricture or
aneurysm formation in 7 of 9 cases. The luminal surface of all grafts was
smooth, shiny and indistinguishable from that of the native cava. The light
microscopy showed a loosely textured cellular collagen framework, with a dense
capillary network and complete luminal coverage by cells resembling endothelial
cells. Electron microscopy confirmed that a complete endothelial cell layer was
present.
In conclusion, SIS provides a collagen framework
that becomes remodelled, keeps up with the growth of the recipient and acquires
a nonthrombogenic endothelial surface. This makes it potentially very well suited
as material for pulmonary artery reconstruction or Fontan operations in small
children.
1996-97 AATS Graham Fellow
*By invitation
F20. MEMANTIN IS A POTENT EXCITATORY AMINO
ACID BLOCKER FOR PREVENTING SPINAL CORD INJURY IN A RABBIT MODEL.
Marek P. Ehrlich, M.D.*, Erich
Knolle, M.D.*, Ruxandra Ciovica, M.D.*, Peter Boeck, M.D.*, Martin Grubenwoger,
M.D.*, Ricarda Konetschny, M.D.*, Fabiola Cartews-Zumelzu, M.D.*, Edvin R.
Turkof, M.D.*, Ernst Wolner, M.D. and Michael Havel, M.D.*
Vienna, Austria
Introduction: This study
was conducted to investigate the effect of memantin, a noncompetitive
N-methyl-D-aspartate receptor antagonist, on the neurological outcome of spinal
cord ischemia and reperfusion after aortic occlusion.
Materials and methods: New
Zealand white rabbits (3 - 4.5 kg) were anesthetized and spinal cord ischemia
was then induced for 40 minutes by infrarenal aortic occlusion. Animals were
randomly assigned into three groups. Group A animals (n = 8) received
intraaortic memantin infusion (20 mg/kg) after aortic clamping. Group B animals
(n = 8) were pretreated with memantin infusion (20 mg/kg) 45 minutes prior to
aortic occlusion. Group C (n = 8) was the control group, in which no
pharmacological intervention was applied. Neurological status was assessed at
12, 24, 36 and 48 hours after operation and scored using the Tarlov system (4
normal, 0 paraplegia). Lumbar spinal root stimulation potentials (SRS) as well
as electrical transcranial motor evoked potentials (MEP) from lower limb
muscles were monitored pre-, intra- and postoperatively. After sacrifice at 48
hours, the spinal cord was fixed and studied histopathologically.
Results: All measured
potentials disappeared shortly after aortic cross-clamping. MEP did not
correlate with clinical findings. Quantitative analysis of SRS showed, that
potentials in both memantin treated groups regained activity earlier compared
to the placebo group. Histologic examination of spinal cords from group A and B
rabbits revealed only a few abnormal motorneurons, whereas spinal cords from
the control group had evidence of extensive cord injury with prominent lysis of
Nissl substance, destruction of nuclear chromatin and vacuolization of anterior
horn motorneurons. Median values for neurological observations from each group
are reported below:
|
Variable
|
12 Hours
|
24 Hours
|
36 Hours
|
48 Hours
|
|
Control
(n= 8)
|
0
|
0
|
0
|
0
|
|
Intraaortal(n
= 8)
|
2
|
2.5
|
3
|
3
|
|
Systemic
(n= 8)
|
3
|
4
|
4
|
4
|
|
control
vs. intraaortal
|
P = 0.001
|
P = 0.0002
|
P = 0.0006
|
P = 0.0006
|
|
control
vs. systemic
|
P = 0.00022
|
P = 0.0002
|
P = 0.0002
|
P = 0.0002
|
Conclusion: Memantin
significantly reduced neurological injury secondary to spinal cord ischemia and
reperfusion after aortic occlusion at 40 minutes in the rabbit model.
F21. MODULATION OF MYOCARDIAL PERFUSION AND VASCULAR
REACTIVITY BY PERICARDIAL bFGF: IMPLICATIONS IN THE TREATMENT OF INOPERABLE
CORONARY ARTERY DISEASE.
Roger J. Laham, M.D.*, Motohisa
Tofukuji, M.D., Ph.D.*, Michael Simons, M.D.* and Frank W. Sellke, M.D.
Boston, Massachusetts
Endothelium-dependent relaxation and perfusion are
reduced in the collateral-dependant myocardium. To examine the modulating
effects of pericardial fluid basic-fibroblast growth factor (bFGF) on
endothelium-dependent responses and expression of inducible (iNOS) and endothelial
(eNOS) nitric oxide synthases in the collateral-dependant myocardium, ameroid
occluders were placed around the left circumflex (LCx) artery of pigs. After 3
weeks, LCx occlusion was confirmed with angiography. Saline containing 30 µg (n
= 6) or 2 mg (n = 6) bFGF or no bFGF (control, n = 6) was injected into the
pericardial space. Four weeks later myocardial blood flow was determined with
colored microspheres, endothelium-dependent coronary microvascular responses to
ADP (10 µM) and endothelium-independent responses to nipride (100 µM) were
examined, and protein and gene expressions of iNOS and eNOS were determined
(Western analysis, PCR) in the ischemic LCx and non-ischemic LAD regions.
Vascular response = % relaxation of precontract diameter.
|
Group
|
ADP
|
Nipride
|
Blood
Flow
|
(ml/min/g)
|
|
LAD-control
|
60 ± 6*
|
82 ± 6
|
1.7 ± 0.2*
|
*p<0.05
vs LCx-control
|
|
LCx-control
|
25 ± 7
|
82 ± 4
|
0.9 ± 0.1
|
(ANOVA,
Scheffe's test)
|
|
LCx-bFGF-30ug
|
44 ± 6*
|
86 ± 4
|
1.2 ± 0.1*
|
|
|
LCx-bFGF-2mg
|
70 ± 7*
|
82 ± 3
|
1.3 ± 0.1*
|
|
Both iNOS protein and mRNA were increased 3.3 ± 1
fold in the LCx compared to the LAD territory, whereas eNOS expression was
similar in both regions. This suggests that the decreased endothelium-dependent
relaxation in the collateral-dependent circulation may be due to increased iNOS
expression and NO-induced inhibition of eNOS. bFGF improves endothelium-
dependent relaxation and blood flow in the collateral-dependent myocardium.
These findings may have implication regarding the cause of altered blood flow
regulation in chronically ischemic myocardium and in the treatment of patients
with inoperable coronary disease with the injection of bFGF into the
pericardial space.
*By invitation
F22. ANGIOGENESIS ACCOMPANIES IMPROVED
PERFUSION IN REGIONS OF HIBERNATING MYOCARDIUM FOLLOWING TRANSMYOCARDIAL LASER
REVASCULARIZATION.
G. Chad Hughes, M.D.*, Alan P.
Kypson, M.D.*, James D. St. Louis, M.D.*, Brian H. Annex, M.D.*, Timothy R.
DeGrado, Ph.D.*, R. Edward Coleman, M.D.*, James E. Lowe, M.D. and Kevin P.
Landolfo, M.D.*
Durham, North Carolina
Background. The
mechanism for clinical improvement following transmyocardial laser
revascularization (TMR) is unknown, although preliminary work in normal
myocardium suggests that angiogenesis may play a role. The purpose of this
study was to describe the quantity and nature of the neovascularization
accompanying improved myocardial perfusion following TMR in a model of
hibernating myocardium.
Methods. Five adult
mini-swine (n = 5) underwent left circumflex coronary artery (LCx) occlusion to
reduce resting blood flow by 90% as documented by ultrasonic flow probe
measurement. Two weeks post-occlusion, the animals underwent positron emission
tomography (PET) to document the presence of ischemic, viable myocardium in the
LCx distribution. TMR was then performed using a holmium:YAG (n = 3) or CO2
(n = 2) laser with 20 channels placed at 1 cm intervals in the ischemic region.
Six months post-TMR, repeat PET was performed. Animals were then sacrificed,
and their hearts harvested for histology as well as immunohistochemical
analysis to identify the presence of endothelial cells.
Results. Mean
myocardial blood flow by PET in the ischemic LCx distribution increased from
0.37 ± 0.16 to 0.60 ± 0.13 ml/g/min (p<0.05) at 6 months post-TMR. There was
no change in control septal flow from pre- to post-TMR (0.73 ± 0.38 to 0.64 ±
0.15 ml/g/min). Histologic examination of the lased LCx area revealed many
neovessels located predominantly at the periphery of the TMR channels. These
vessels were present in a highly disorganized pattern consistent with
angiogenesis. The presence of endothelial ceils within the neovessels was
confirmed with the endothelial cell specific antibodies anti-von Willebrand
factor and anti-human tie-2 (TEK). Quantitative analysis revealed the lased
ischemic regions contained a mean of 26.0 ± 8.8 microvessels per 200x field,
compared with a mean of 5.1 ± 2.0 in control non-lased regions (p = 0.0003).
Conclusions. Angiogenesis
occurs in the channel regions following TMR in hibernating myocardium and
accompanies improved regional perfusion. These findings strongly suggest that
angiogenesis is the mechanism of increased blood flow following TMR in ischemic
myocardium.
*By invitation
F23. TIME-COURSE OF FUNCTIONAL RECOVERY AFTER
CORONARY BYPASS SURGERY IN PATIENTS WITH CHRONIC LEFT VENTRICULAR ISCHEMIC
DYSFUNCTION.
Jean-Louis J. Vanoverschelde,
M.D., Ph.D.*, Christophe Depre, M.D., Ph.D.*, Bernhard L. Gerber, M.D.*, Marcel
Borgers, Ph.D.*, William Wijns, M.D.*, Jacques A. Melin, M.D., Ph.D.* and
Robert A. Dion, M.D.*
Brussels, Belgium
Sponsored by: Bruce W. Lytle,
M.D., Cleveland, Ohio
Background Chronic left
ventricular (LV) ischemic dysfunction, a condition often referred to as
myocardial hibernation, is associated in humans with ultrastructural
alterations of the myocytes, including the loss of myofilarnants and the
accumulation of glycogen. Given the severity of these structural changes,
contractile function is unlikely to resume immediately upon revascularization.
Methods and results We studied
32 patients with coronary disease and chronic LV ischemic dysfunction
undergoing bypass surgery. Dynamic Positron Emission Tomography with 13N-ammonia
and 18F-deoxyglucose to assess myocardial perfusion and glucose
metabolism was performed in 29 patients. Coronary bypass surgery was
subsequently performed with the use of the left internal mammary artery to
graft the left anterior descending coronary artery. All other co-diseased
vessels were also revascularized. On average, each patient received 2.9 anastomoses,
of which 1.3 were constructed with arterial conduits. In every patient, a
peroperative transmural biopsy was harvested from the center of the
dysfunctional area, to quantify the increase in extracellular matrix and the
presence of structurally altered cardiomyocytes. LV function was serially
measured by digitized 2D-echocardiography before and again 10 days, 2 months
and 6 months after revascularization. The time-course of recovery of regional
function was estimated from the monoexponential decrease in dysfunctional wall
motion score. At the 6 months followup, 19 patients had improved LV function
while 13 patients showed persistent dysfunction. Before revascularization,
reversibly dysfunctional segments had higher myocardial blood flow (83 ± 29 versus
60 ±21 ml·(min·100g)-1, p<0.01), higher glucose uptake (40 ±14
versus 21 ±9 µmol·(min·100g)-1, p<0.05) and less increase in
extracellular matrix (25 ± 17 versus 46 ± 17%, p = 0.01) than segments with
persistent dysfunction. The extent of functional recovery correlated with
myocardial blood flow (r = 0.84) and the increase in extracellular matrix (r =
-0.60). In patients with reversible dysfunction, the return of segmental
function was progressive and followed a monoexponential time-course with a time
constant of 23 days (range 6-78 days). The rate of recovery correlated best
with the proportion of altered cardiomyocytes in the biopsy (r = 0.83).
Conclusions. The present study indicates that the
recovery of regional and global LV function after successful revascularization
is progressive and follows a monoexponential time-course which is influenced by
the extent and severity of the structural changes affecting the cardiomyocytes.
*By invitation
F24. MYOCYTE ENDOTHELIN EXPOSURE DURING
CARDIOPLEGIC ARREST EXACERBATES CONTRACTILE DYSFUNCTION WITH REPERFUSION.
R. Brent New, M.D.*, Jeffrey S.
Mandel, M.D.*, Angela C. Sampson, B.A.*, Christopher A. Kerr, B.S.*, Rupak
Mukherjee, Ph.D.*, Fred A. Crawford, Jr., M.D. and Francis G. Spinale, M.D.,
Ph.D.*
Charleston, South Carolina
Background: While
transient left ventricular (LV) dysfunction can occur following cardioplegic
arrest (CA), the contributory mechanisms for this phenomenon remain
incompletely understood. Institution of cardiopulmonary bypass and CA results
in neurohormonal system activation which includes increased release of the
vasoactive peptide, endothelin (ET). Past studies have suggested that ET can
influence LV myocyte contractility. Therefore, this project tested the
hypothesis that exposure of LV myocytes to ET during simulated CA, would have
direct effects on contractile processes with subsequent reperfusion.
Methods: LV porcine
myocytes were randomly assigned to 3 groups: (1) Control: Normothermic (37°C)
cell media (n=156); (2) Cardioplegia: simulated CA (K+ 24 mEq/L, 4°C
x 2hrs) followed by reperfusion and rewarming with cell media (n = 73); (3)
Cardioplegia and ET: simulated CA in the presence of ET (200 pM) followed by
reperfusion with cell media and ET (n = 54). Myocyte contractility was measured
following reperfusion by videomicroscopy.
Results: Myocyte
shortening velocity was reduced following simulated CA compared to controls (62
±3 vs 80 ± 3 µm/s, respectively p<0.05) and was further reduced with CA and
ET exposure (49 ± 3 µm/s, p<0.05). Myocyte velocity of relengthening, which
reflects sarcomere cross-bridge release rates and calcium resequestration, was
reduced after CA compared to controls (51 ± 3 vs 77 ± 3 µm/s, respectively,
p<0.05) and was reduced to a greater degree with CA and ET exposure (41 ± 3
µm/s, p<0.05).
Conclusions: The unique
findings of the present study demonstrated that exposure of the LV myocyte to
ET during simulated CA, directly contributed to contractile dysfunction
following reperfusion. A contributory molecular mechanism for the effect of ET
with CA may be alterations in myocyte active relaxation processes. These
findings suggest that increased ET levels which occur in the setting of cardiac
surgery directly influence myocyte contractility, which in turn contributes to
the transient LV dysfunction following cardioplegic arrest.
*By invitation
F25. ROLE OF ENDOTHELIN-1 AND ENDOTHELIN-1
RECEPTOR BLOCKADE IN PLACENTAL DYSFUNCTION AFTER FETAL CARDIAC BYPASS.
Doff B. McElhinney, M.D.*, V.
Mohan Reddy, M.D.*, Hiranya A.
Rajasinghe, M.D.*, John R. Liddicoat, M.D.*, Karen Hendricks-Munoz, M.D.*,
Jeffrey R. Fineman, M.D.* and Frank L. Hanley, M.D.
San Francisco, California
Fetal cardiac bypass (FCB) causes placental
dysfunction, characterized by increased placental vascular resistance (PVR),
decreased placental blood flow (PBF), hypoxia, and acidosis. A variety of
factors have been found to contribute to the development of this placental
dysfu nction, but its exact mechanisms remain unclear. Vasoactive factors
produced by the vascular endothelium, such as nitric oxide and endothelin-1
(ET-1), are important regulators of placental vascular tone. To study the role
of the vascular endothelium in placental dysfunction related to FCB, we studied
3 groups of fetal sheep. In the first (n:7), we determined placental
hemodynamic responses pre- and post-FCB to an endothelium-dependent vasodilator
(acetylcholine), an endothelium-independent vasodilator (sodium nitroprusside),
and ET-1, a vasoactive polypeptide produced by the endothelium. Controls (n =
7) received the same vasoactive substances but were not exposed to FCB. In the
second group (n = 5), an ET-1 receptor blocker (PDQ123) was administered and
placental hemodynamics were measured before and after FCB. Results were compared
with control fetuses that did not receive PDQ123 in order to assess the effect
of ET-1 receptor blockade. In the third group (n = 5), no medications were
given and plasma ET-1 levels were measured before and after FCB, then compared
with controls that did not undergo FCB. Before FCB, exogenous ET-1 decreased
PBF by 8.7% and increased PVR by 9.3%. After bypass, however, ET-1 decreased
PBF by 47% and increased PVR by t06%. In addition, there was a significant
attenuation of the placental vascular relaxation response to acetylcholine
after FCB, with a decrease in PVR of 14%, compared with 20% pre-FCB. The
response to sodium nitroprusside was not significantly altered by FCB. In
fetuses that received the ET-1 blocker, PVR increased from 0.32 ± 0.03 U
pre-FCB to 0.41 ± 0.07 after bypass, which was significantly less than in
control animals (0.31 ± 0.04 pre-FCB, 0.51 ± 0.14 post-FCB). Similarly, PBF
decreased significantly more in control animals (from 172 ± 29 ml/min/kg to 116
± 36) than in fetuses receiving ET-1 receptor blocker (from 168 ± 31 to 140 ±
35). Plasma ET-1 increased significantly in fetuses exposed to FCB, but did not
change in control animals. This study demonstrates that FCB causes a
significant increase in plasma ET-1 and impairs the placental vascular response
to endothelium-dependent modulators of vascular tone. ET-1 receptor blockade
substantially reduces post-FCB placental dysfunction. This and other
pharmacologic or physiologic interventions aimed preserving endothelial
function may be effective means of optimizing fetal outcome after FCB.
1995-97 TSFRE Research
Fellow
*By invitation
F26. PHOSPHODIESTERASE INHIBITORS PREVENT THE
SYSTEMIC INFLAMMATORY RESPONSE SYNDROME.
Koh Takeuchi, M.D.*, Pedro J. del
Nido, M.D, Dimitrios N. Poutias, B.S.* and Francis X. McGowan, Jr., M.D.*
Boston, Massachusetts and
Bethesda, Maryland
The systemic inflammatory response syndrome (SIRS)
is an important cause of multiple organ dysfunction after cardiopulmonary
bypass; its magnitude and consequences are particularly great in neonates and
infants. We and others have previously found that certain phosphodiesterase
inhibitors (PDI) interfere with inflammatory signaling pathways at several
points. The present study tested the hypothesis that two clinically used PDIs,
amrinone (AMR) and vesnarinone (VES), would decrease the SIRS.
A well-characterized, severe SIRS
model of rabbit endotoxemia was used. Rabbits received S. lyphi endotoxin
(lipopolysaccharide, LPS; 0.2 mg/kg iv bolus); LPS + AMR (1.0 mg/kg bolus + 10
mg/kg/min infusion); LPS + VES (3 mg/kg bolus only-t½ =~8 hours); or vehicle
alone. Systemic effects were assessed by mortality, fever, and acidosis.
Indices of the inflammatory response included plasma cytokine and
myeloperoxidase (MPO) concentrations. Pulmonary involvement was assessed by
changes in pulmonary vascular resistance (PVR) and lung MPO. Myocardial
function was quantified in excised, Langendorff-perfused hearts; myocardial
calcium cycling and contractile protein calcium sensitivity were measured using
fluorescence spectroscopy. Plasma hepatocellular enzyme concentrations (SGPT,
SGOT) served as markers of liver injury. Myocardial protein kinase C (PKC) and
inducible nitric oxide synthase (iNOS) activity were used as indices of
cytokine signaling. Measurements were made 0, 1, 2, and 6 hours after LPS.
Results are summarized in the Table as mean ± sem, N = 6-8 each. *P<0.05 vs
control; #P<0.05 vs LPS alone.
|
|
Mortality
(%)
|
Tmax
|
HCO3-
|
MPO
|
TNF
|
iNOS
|
PDF
|
|
Control
|
0
|
37 ± 0.6
|
21 ± 3
|
155 ± 21
|
< 10
|
< 25
|
88 ± 7
|
|
LPS
|
60*
|
39 ± 0.2*
|
10 ± 1*
|
350 ± 55*
|
480 ± 30*
|
140 ± 30*
|
57 ± 8*
|
|
LPS+AMR
|
20
|
40 ± 0.6*
|
14 ±3*
|
220 ± 40#
|
210± 25#
|
60 ± 15#
|
97 ± 12#
|
|
LPS+VES
|
9#
|
38 ± 0.1#
|
18±3#
|
200 ± 30#
|
50 ± 10#
|
30 ± 5#
|
92 ± 7#
|
|
Tmax (0C;
HCO3-, MPO, U/ml; TNF, pg/ml; iNOS, pmol/hr/mg protein;
POP, peak developed LV pressure, mmHg
|
Thus, VES, which has unique ion
channel and gene expression effects unrelated to its PDI properties, was
particularly effective in SIRS suppression. VES also protected against
LPS-induced increases in cytokines, hepatocellular enzymes, myocardial PKC
activity, and PVR; the protective effects of AMR upon these indices were
significant, but less than VES. VES prevented LPS-induced reductions in
myocardial calcium-activated contractile force, diastolic relaxation, and
diastolic calcium removal. Neither VES nor AMR exacerbated LPS-induced
hypotension. VES also prevented LPS-induced TNF production in cultured
macrophages and cytokine-stimulated iNOS production in cultured neonatal
cardiomyocytes. The beneficial effects of VIES and AMR in vivo were not shared
by dobutamine, and the in vitro effects of VES were not mimicked by a
cell-permeable cyclic AMP analog. These results indicate that certain
phosphodiesterase inhibitors have multiple and potent effects upon inflammatory
activation and signaling pathways. The mechanism does not appear to be due to
elevations in cyclic AMP. These compounds, which are used clinically for their
inotropic and vasodilating properties, may be useful to limit inflammatory
activation and signaling cascades during pediatric CPB, as well as other states
that are associated with inflammatory cytokine production.
*By invitation
F27. IN VIVO IMAGING OF APOPTOSIS DURING
CARDIAC ALLOGRAFT REJECTION USING RADIOLABELED ANNEXIN V.
Patrick W. Vriens, M.D.*, Francis
G. Blankenberg, M.D.*, Eric R. Davis, M.D.*, Gerald J. Berry, M.D.*, Bruce A.
Reitz, M.D., H. William Strauss, M.D.* and Robert C. Robbins, M.D.*
Stanford, California
Introduction We
hypothesized that technetium 99m labeled annexin V, a new radioactive tracer of
apoptotic cell death, can be utilized to monitor cardiac allograft rejection.
Annexin V is a human protein that binds to phosphatidyl serine, a phospholipid
that is selectively expressed on the cell surface, during the early stage of
apoptosis.
Methods Untreated ACI rats
served as recipients of heterotopically placed, allogeneic PVG rat, or
syngeneic ACI rat cardiac grafts. Function of grafts was assessed by daily
palpation. Annexin V was derivatized with technetium 99m hydrazinonicotinamide
(99mTcHYNIC), and injected i.v. one hour prior to imaging at day 3, 4, and 5
after transplantation. Region of interest image analysis was used to quantify
uptake of the radiopharmaceutical. Histopathologic analysis and TUNEL staining
were performed at each time point after imaging. To investigate whether uptake
of 99mTcHYNIC-annexin V decreased after treatment of rejection, recipients of
allogeneic grafts were treated daily with Cyclosporin A (CSA, oral, 10 mg/kg),
starting at day 4, and imaged at day 4, 7, and 11 after transplantation.
Results Allogeneic PVG
cardiac grafts showed a readily visualizable 50% increase in uptake of
99mTcHYNIC-annexin V at day 3, a 160% increase at day 4 and a 274% increase at
day 5 after transplantation in untreated ACI rats (Fig. 1) (n = 6 for each time
point), compared to syngeneic hearts (Fig. 2)
(n = 3 for each time point) (P<0.01). Histopathology showed grade 1, grade
1.5 and grade 2.3 rejection at these time points, respectively. Apoptotic
nuclei were identified by TUNEL staining in myocytes and mononuclear
infiltrates of rejecting allogeneic grafts, but not of syngeneie grafts.
Palpable contractions of allogeneic grafts ceased at day 7, indicating complete
rejection (n = 6). When CSA treatment was started at day 4, rejection could be
reversed, and allogeneic grafts remained functional. Uptake of
99mTcHYNIC-annexin V in treated animals decreased to 62% at day 7 and 0% at day
11 (n = 6).
Conclusion Uptake of
radiolabeled annexin V correlates with histopathological grades of acute
rejection in cardiac allografts. When rejection is reversed by CSA treatment,
uptake decreases to levels comparable to syngeneie grafts. Imaging of apoptosis
using radiolabeled annexin V may enable detection of acute cardiac allograft
rejection, and may provide a new tool to monitor rejection.
*By invitation
F28. SKELETAL MUSCLE VENTRICLES FROM LEFT
VENTRICULAR APEX TO AORTA, EXPERIENCE UP TO 37WEEKS: STEP TOWARDS CLINICAL
APPLICATION.
Frank A. Baciewicz, Jr., M.D.*,
Gregory A. Thomas, M.D.*, Kevin A. Greer, M.D.*, Robert L. Hammond, Ph.D.*,
Huiren Lu, M.D.*, Steven Bastian, M.S.* and Larry W. Stephenson, M.D.
Detroit, Michigan
Skeletal muscle ventricles (SMVs) were constructed
from the latissimus muscle and lined with autogenous pericardium in 6 dogs.
After 3 weeks of vascular delay and 6 weeks of electrical conditioning, SMVs
were connected from the left ventricular apex with a valved conduit and then
from the SMV to the descending aorta with a second-valved conduit. The SMV was stimulated
during diastole at a 1:2 ratio with the heart. SMV contraction increased
femoral pressure by 23% at 33 Hz and 28% at 50 Hz, resulting in 32% and 33% of
the total cardiac output being pumped by the SMV. Data at implant (n = 6) is
shown below:
|
|
FAPaug
|
FAPdia
|
CAPaug
|
CAPdia
|
Qsmv
|
QAV
|
Qtotal
|
|
OFF
|
59 ± 4
|
51 ± 5
|
62 ± 3
|
55 ± 3
|
604 ± 173
|
2976±434
|
3580 ± 556
|
|
33Hz
|
73 ± 8*
|
61 ± 5*
|
72 ± 5*
|
61 ± 5*
|
1147 ± 223*
|
2395 ±319*
|
3542 ± 454
|
|
50Hz
|
76 ± 7*
|
62 ± 5*
|
75 ± 4*
|
67 ± 4*
|
1162 ± 212*
|
2247 ± 308*
|
3454 ± 408
|
|
(FAP &
CAP. fenorai and carotid arterial pressure, aug = augmented, dia = diastolic,
QSMV: flow through SMV, QAV flow through
aortic valve, Qtotal = Qsmv + QAV,*
= P<0.05 by
ANOVA).
|
The dogs survived 6, 17, 30, 72
and 263 days in circulation; one dog is alive at 160 days. Three deaths,
including that at 263 days were related to complications of indwelling
monitoring devices and not directly to the SMV. In the two longest surviving
dogs, SMV function remained stable over time. During propranolol induced heart failure,
SMV contraction augmented diastolic pressure 89% at 33Hz and 96% at 50 Hz. At a
1:2 contraction ratio with the heart, SMV assist increased systemic perfusion
(Qsmv QAV) 23%
at 33 Hz and 64% at 50 Hz. At a 1:1 contraction ratio, systemic perfusion was
increased further by 25% at 33 Hz and 114% at 50Hz. This model of skeletal
muscle assist is the most hemodynamically effective that we have tested, and
now appears capable of functioning long-term.
*By invitation
9:30 a.m. SIMULTANEOUS SCIENTIFIC SESSION
A-2 CONGENITAL HEART DISEASE
Ballroom A, Hynes Convention
Center
Moderators: John E. Mayer, Jr., M.D.
John A. Waldhausen, M.D.
40. VENO-VENOUS
MODIFIED ULTRAFILTRATION AND CIRCULATING CYTOKINES: A PROSPECTIVE RANDOMIZED
STUDY.
Ugursay Kiziltepe, M.D.*, Azhar
Hossain, M.D.*, Daniel Remick, M.D.*, Samuel Barst, M.D.*, Jeffrey P. Gold,
M.D. and Hani A. Hennein, M.D.*
Atlanta, Georgia; New York, New
Hyde Park and Bronx, New York; Ann Arbor, Michigan
Discussant: J. William Gaynor, M.D., Philadelphia, Pennsylvania
Background: Cardiopulmonary
bypass (CPB) is associated with the production of both proinflammatory (IL-6,
IL-8 and TNFcc) and antiinflammatory (IL-10) cytokines, and a resultant
systemic inflammatory response. Arterio-venous modified ultrafiltration has
been shown to be associated with a reduction in total body water and improved
hemodynamic and hemostatic functions. Veno-venous modified ultrafiltration
(VVMUF) is a further modification of the technique with the potential advantage
of not reducing effective cardiac output. We tested the hypothesis that VVMUF
reduces extracellular body water and removes circulating cytokines following
CPB.
Methods: Thirty-seven
children undergoing open surgical correction of congenital heart defects were
randomly assigned to VVMUF or controls. Inferior and superior vena cava
cannulas were used as the inflow and outflow of the VVMUF circuit. VVMUF was
performed for 10 min after weaning from CPB. IL-1P, IL-6, IL-8, IL-10 and TNF-a
plasma levels measured at seven time points before, during and after CPB.
Results: Both groups were
similar in terms of age (6.48 ± 5.38 years in VVMUF vs. 6.27 ± 5.37 years in
control, p: not significant (n.s.)). Mean cross-clamp times were 604-35 minutes
(rain) in VVMUF and 41 ± 23 rain in control group, p:n.s. Mean bypass duration
was 98 ± 56 min in VVMUF and 92 ± 50 min in control group, p:n.s. Minimum
temperatures were 28.8 ±2.1 C in VVMUF and 28.4 ±4.4 C in control group, p:n.s.
VVMUF resulted in a significant removal of extracellular fluid (980 ± 601 cc)
and rise in hematocrit levels (from 28.27 ± 3.64% to 34 ± 3.64%, p<0.05 ).
There was no mortality in either group. All 5 cytokines measured rose during
and following bypass in both groups. A significant reduction of IL-lβ
levels (from 11.3 ± 39.7 pg/ml to 4.4 ± 20.9 pg/ml) followed VVMUF. Changes in
cytokines other than IL-lβ could not be demonstrated.
Conclusions: Veno-venous modified ultrafiltration is
a safe and effective
method of removing extracellular volume following CPB. VVMUF
is
associated with a significant reduction in IL-lβ levels
and may therefore
reduce the deleterious effects of
CPB by diminishing systemic inflammatory
response.
*By invitation
41. TOTAL REPAIR OF PULMONARY ATRESIA WITH
VENTRICULAR SEPTAL DEFECT AND MAJOR AORTOPULMONARY COLLATERALS: AN INTEGRATED
APPROACH.
Adriano Carotti, M.D.*, Roberto M.
Di Donate, M.D.*, Cosimo Squitieri, M.D.*, Paolo Guccione, M.D.* and Glauco
Catena, M.D.*
Rome, Italy
Sponsored by: Aldo R.
Castaneda, M.D., Guatemala City, Guatemala
Discussant: Frank L. Hanley,
M.D., San Francisco, California
Background: Predicting
postrepair ratio of the peak systolic pressure in the right ventricle (pRV) to
that in the left ventricle (pLV) may be of absolute prognostic value for
patients undergoing total repair of pulmonary atresia, ventricular septal
defect and major aortopulmonary collaterals (PA.VSD.MAPCAS). To this purpose,
we currently rely on 2 novel parameters: 1) preoperative total neopulmonary
index (TNPAI = CAI [total MAPCA index] + PAI [pulmonary artery index]); 2) mean
pulmonary artery pressure changes during an intraoperative flow study,
according to Reddv M. et al.
Patients and methods: Since
January 1994, 15 patients (age mean±SD: 64 ± 54 months) with PA.VSD.MAPCAS were
managed according to TNPAI: a preoperative value of ≥ 150 mm2/m2
was indication for total repair. Seven patients with hypoplastic pulmonary
arteries and TNPAI < 150 mm2/m2 first underwent
palliative conduit right ventricular outflow tract reconstruction (RVOTR),
followed by secondary one-stage, midline total unifocalization and VSD closure.
The other 8 patients with preoperative TNPAI > 150 mm2/m2 (absent
pulmonary arteries m 2 cases) underwent single-stage unifocalization and
repair. The VSD was closed in all cases. In 9, the decision to close the VSD
was based on an intraoperative pulmonary flow study. In one case the VSD had to
be reopened due to hypersystemic pRV.
Results: The 7 patients
who initially underwent RVOTR had a significant increase of PAI (from 46 ± 26
to 194 ± 74 mm2/m2 p<0.0001) within 22 ± 6 months from
the palliation. Of the total group of 15 patients, repair was successful in 14,
with a postrepair pRV/pLV ratio of 0.47 ± 0.1. There was one hospital death due
to hypersystemic pRV, despite a reassuring intraoperative
