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Engineered tissue using transfected cells inhibits tumor growth
EIGHTH ANNUAL SURGICAL RESIDENT & FELLOW RESEARCH PRESENTATION DAY
Abstracts 1 Atrial fibrillation in the era of off pump coronary artery bypass Ascioti AJ, Khabbaz K, Carpino P . From the Division of Cardiothoracic Surgery, New England Medical Center, Boston, Massachusetts. Purpose: Atrial fibrillation remains one of the most common postoperative complications in cardiac surgery. Historically, this arrhythmia was thought to be associated with cardiotomy, cardioplegic arrest, and extracorporeal circulation. Despite its frequency, the origin of atrial fibrillation remains unclear. In recent years, beating heart surgery has emerged as an acceptable alternative to myocardial revascularization with cardiopulmonary bypass. It is thought that the less-invasive nature of off-pump coronary artery bypass (OPCAB) results in an overall reduction in morbidity, especially postoperative arrhythmias, although this remains controversial. In an attempt to characterize atrial fibrillation within the on- and off-pump patient, we have undertaken a retrospective analysis of 248 individuals (on-pump, n ⫽ 101; off-pump, n ⫽ 147) who underwent isolated myocardial revascularization within our institution from November 1999 to October 2000. Results: The incidence of atrial fibrillation was lower in the off-pump cohort (17.1% vs 32.7%, p-value 0.006). Postoperative length of stay was also lower in the off-pump cohort (7.6 days vs 9.7 days, p-value 0.003). Analysis of the subgroup of patients who developed atrial fibrillation, both on-pump and offpump, did not reveal any statistically significant difference in duration of arrhythmia, need for antiarrhythmics/anticoagulation, discharge rhythm, or length of stay. Conclusions: Morbidity and hospitalization because of atrial fibrillation is reduced through the use of OPCAB. Once a patient develops atrial fibrillation, however, the postoperative course is similar regardless of the method of bypass. 2 Colonization-dependent expression of inducible nitric oxide synthase by the gastrointestinal epithelium Mitchell ME,* Lyons CR,† Closs EI,† Park M,† Cunningham JM.† From the Departments of *Surgery and †Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Purpose: Disruption of the gastrointestinal tract with bacterial contamination is responsible for a host of surgical conditions. The mechanisms responsible for bacterial resistance of the gastrointestinal (GI) tract have not been fully elucidated. In other tissues, the bactericidal activity of activated macrophages is dependent in part on the expression of inducible nitric oxide synthase (iNOS), the enzyme that catalyzes the synthesis of nitric oxide (NO) from L-arginine and molecular oxygen. Several reports have identified iNOS expression in GIderived epithelial cell lines; however, a systematic in vivo study of iNOS expression in the GI tract has not been performed. It is our hypothesis that iNOS plays a role in the GI mucosal barrier to bacterial and parasitic invasion. Methods: To test this hypothesis, we examined the in vivo expression of iNOS in the GI tract of normal balb/c, germ-free, and ␣-interferon ⫺/⫺ mice using RNAse protection and a sensitive in situ hybridization assay. Results: Inducible nitric oxide synthase expression was strongly localized to the epithelium of the GI tract. Increasing steady state levels of iNOS RNA were observed in each more distal segment of the GI tract of normal mice from the stomach (1⫻), duodenum (4.1⫻), jejunum (6.3⫻), caecum (9.1⫻), to the colon (10.5⫻), paralleling the degree of bacterial colonization. Consistent with our hypothesis, iNOS mRNA and enzyme activity were not detected in the GI tract of germ-free mice, but rapidly appeared after removal from the germ-free environment or injection with Escherichia coli LPS. Moreover, the pattern of iNOS expression in the GI tract of ␣-interferon ⫺/&munis; mice was not detectably changed compared with ⫹/⫹ littermates. LPS-dependent induction
of iNOS expression and production of NO in GI epithelial cells were confirmed using the colon carcinoma-derived cell line, CMT-93. In addition to the GI epithelium, LPS-induced accumulation of iNOS mRNA was also observed in the epithelium covering the renal papilla and preferentially in the periportal region of hepatic lobules. Conclusions: Inducible nitric oxide synthase is expressed by the GI epithelium of normal mice, and the degree of expression throughout the GI tract parallels the degree of bacterial colonization. Furthermore, iNOS expression is absent in germ-free mice but is reinstated when these mice are removed from their germfree environment or treated with LPS. These results are consistent with the hypothesis that nitric oxide produced by these tissues in response to bacterial colonization contributes to the barrier that prevents bacterial and parasitic invasion. 3 Engineered tissue using transfected cells inhibits tumor growth Stephen AE, Masiakos PT, Segev DL, Vacanti JP, Donahoe PK, MacLaughlin DT. From the Massachusetts General Hospital, Boston, Massachusetts. Purpose: We propose combining tissue engineering and gene therapy to create a living device for the ongoing production and delivery of biological response modifiers to control tumor growth. Most ovarian carcinomas are derived from Mullerian structures. We have previously shown that Mullerian inhibiting substance (MIS) causes regression of ovarian tumor cell lines. We now demonstrate the creation of living tissue that secretes bioactive MIS in vitro and in vivo and inhibits the growth of an ovarian cancer cell line in vivo. Methods: Chinese hamster ovary cells transfected with the human MIS gene (CHO-B9 cells) were seeded onto a highly porous mesh of polyglycolic acid fibers. The concentration of MIS secreted into the media was measured serially using a sensitive MIS enzyme-linked immunosorbent assay and an in vitro MIS-specific bioassay. Two days later, the mesh was implanted in the right ovarian pedicle of severe combined immunodeficient mice, serial MIS was measured in each animal, and inhibition of growth of the IGROV-1 human ovarian cancer cell line was tested in vivo. Results: CHO-B9 cells seeded on the mesh secreted large quantities of immunoactive MIS in vitro. Mullerian inhibiting substance was detected in mouse sera within 3 days of implantation of the mesh in vivo, and supraphysiologic levels were seen at 7 to10 days (n ⫽ 40). The growth of the IGROV-1 tumor cell line was significantly inhibited in the animals with CHO-B9 impregnated mesh (n ⫽ 30) versus controls (n ⫽ 30; p ⬍ 0.01). Conclusions: Transfected cells on biodegradable mesh can produce high levels of complex bioactive molecules for use as chemotherapeutic agents. The inhibition of ovarian cancer by MIS indicates that this delivery system could be used for this and other inhibitory proteins not otherwise amenable to scale up for clinical use. Use of cryopreserved cadaver vein allograft for hemodialysis access precludes renal transplantation because of allosensitization. 4 Estrogen does not inhibit the vascular injury response in estrogen receptor alpha- and beta-deficient mice Pare GJ, Karas RH, Aronovitz M, Schulten H, Barnatan M, Mendelsohn ME. From the New England Medical Center, Boston, Massachusetts. Purpose: Estrogen receptors mediate the direct effects of estrogen in target tissues. Two estrogen receptors have been identified (ER␣ and ER), and both are expressed in the vasculature. Methods: It has been shown previously that estrogen inhibits the response to vascular injury in both female ER␣ knockout (ER␣KO) and female ER knockout (ERKO) mice to the same extent as in wild-type mice. Mice have
CURRENT SURGERY • © 2001 by the Association of Program Directors in Surgery Published by Elsevier Science Inc.
0149-7944/01/$20.00
557
Abstracts 1 Atrial fibrillation in the era of off pump coronary artery bypass Ascioti AJ, Khabbaz K, Carpino P . From the Division of Cardiothoracic Surgery, New England Medical Center, Boston, Massachusetts. Purpose: Atrial fibrillation remains one of the most common postoperative complications in cardiac surgery. Historically, this arrhythmia was thought to be associated with cardiotomy, cardioplegic arrest, and extracorporeal circulation. Despite its frequency, the origin of atrial fibrillation remains unclear. In recent years, beating heart surgery has emerged as an acceptable alternative to myocardial revascularization with cardiopulmonary bypass. It is thought that the less-invasive nature of off-pump coronary artery bypass (OPCAB) results in an overall reduction in morbidity, especially postoperative arrhythmias, although this remains controversial. In an attempt to characterize atrial fibrillation within the on- and off-pump patient, we have undertaken a retrospective analysis of 248 individuals (on-pump, n ⫽ 101; off-pump, n ⫽ 147) who underwent isolated myocardial revascularization within our institution from November 1999 to October 2000. Results: The incidence of atrial fibrillation was lower in the off-pump cohort (17.1% vs 32.7%, p-value 0.006). Postoperative length of stay was also lower in the off-pump cohort (7.6 days vs 9.7 days, p-value 0.003). Analysis of the subgroup of patients who developed atrial fibrillation, both on-pump and offpump, did not reveal any statistically significant difference in duration of arrhythmia, need for antiarrhythmics/anticoagulation, discharge rhythm, or length of stay. Conclusions: Morbidity and hospitalization because of atrial fibrillation is reduced through the use of OPCAB. Once a patient develops atrial fibrillation, however, the postoperative course is similar regardless of the method of bypass. 2 Colonization-dependent expression of inducible nitric oxide synthase by the gastrointestinal epithelium Mitchell ME,* Lyons CR,† Closs EI,† Park M,† Cunningham JM.† From the Departments of *Surgery and †Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Purpose: Disruption of the gastrointestinal tract with bacterial contamination is responsible for a host of surgical conditions. The mechanisms responsible for bacterial resistance of the gastrointestinal (GI) tract have not been fully elucidated. In other tissues, the bactericidal activity of activated macrophages is dependent in part on the expression of inducible nitric oxide synthase (iNOS), the enzyme that catalyzes the synthesis of nitric oxide (NO) from L-arginine and molecular oxygen. Several reports have identified iNOS expression in GIderived epithelial cell lines; however, a systematic in vivo study of iNOS expression in the GI tract has not been performed. It is our hypothesis that iNOS plays a role in the GI mucosal barrier to bacterial and parasitic invasion. Methods: To test this hypothesis, we examined the in vivo expression of iNOS in the GI tract of normal balb/c, germ-free, and ␣-interferon ⫺/⫺ mice using RNAse protection and a sensitive in situ hybridization assay. Results: Inducible nitric oxide synthase expression was strongly localized to the epithelium of the GI tract. Increasing steady state levels of iNOS RNA were observed in each more distal segment of the GI tract of normal mice from the stomach (1⫻), duodenum (4.1⫻), jejunum (6.3⫻), caecum (9.1⫻), to the colon (10.5⫻), paralleling the degree of bacterial colonization. Consistent with our hypothesis, iNOS mRNA and enzyme activity were not detected in the GI tract of germ-free mice, but rapidly appeared after removal from the germ-free environment or injection with Escherichia coli LPS. Moreover, the pattern of iNOS expression in the GI tract of ␣-interferon ⫺/&munis; mice was not detectably changed compared with ⫹/⫹ littermates. LPS-dependent induction
of iNOS expression and production of NO in GI epithelial cells were confirmed using the colon carcinoma-derived cell line, CMT-93. In addition to the GI epithelium, LPS-induced accumulation of iNOS mRNA was also observed in the epithelium covering the renal papilla and preferentially in the periportal region of hepatic lobules. Conclusions: Inducible nitric oxide synthase is expressed by the GI epithelium of normal mice, and the degree of expression throughout the GI tract parallels the degree of bacterial colonization. Furthermore, iNOS expression is absent in germ-free mice but is reinstated when these mice are removed from their germfree environment or treated with LPS. These results are consistent with the hypothesis that nitric oxide produced by these tissues in response to bacterial colonization contributes to the barrier that prevents bacterial and parasitic invasion. 3 Engineered tissue using transfected cells inhibits tumor growth Stephen AE, Masiakos PT, Segev DL, Vacanti JP, Donahoe PK, MacLaughlin DT. From the Massachusetts General Hospital, Boston, Massachusetts. Purpose: We propose combining tissue engineering and gene therapy to create a living device for the ongoing production and delivery of biological response modifiers to control tumor growth. Most ovarian carcinomas are derived from Mullerian structures. We have previously shown that Mullerian inhibiting substance (MIS) causes regression of ovarian tumor cell lines. We now demonstrate the creation of living tissue that secretes bioactive MIS in vitro and in vivo and inhibits the growth of an ovarian cancer cell line in vivo. Methods: Chinese hamster ovary cells transfected with the human MIS gene (CHO-B9 cells) were seeded onto a highly porous mesh of polyglycolic acid fibers. The concentration of MIS secreted into the media was measured serially using a sensitive MIS enzyme-linked immunosorbent assay and an in vitro MIS-specific bioassay. Two days later, the mesh was implanted in the right ovarian pedicle of severe combined immunodeficient mice, serial MIS was measured in each animal, and inhibition of growth of the IGROV-1 human ovarian cancer cell line was tested in vivo. Results: CHO-B9 cells seeded on the mesh secreted large quantities of immunoactive MIS in vitro. Mullerian inhibiting substance was detected in mouse sera within 3 days of implantation of the mesh in vivo, and supraphysiologic levels were seen at 7 to10 days (n ⫽ 40). The growth of the IGROV-1 tumor cell line was significantly inhibited in the animals with CHO-B9 impregnated mesh (n ⫽ 30) versus controls (n ⫽ 30; p ⬍ 0.01). Conclusions: Transfected cells on biodegradable mesh can produce high levels of complex bioactive molecules for use as chemotherapeutic agents. The inhibition of ovarian cancer by MIS indicates that this delivery system could be used for this and other inhibitory proteins not otherwise amenable to scale up for clinical use. Use of cryopreserved cadaver vein allograft for hemodialysis access precludes renal transplantation because of allosensitization. 4 Estrogen does not inhibit the vascular injury response in estrogen receptor alpha- and beta-deficient mice Pare GJ, Karas RH, Aronovitz M, Schulten H, Barnatan M, Mendelsohn ME. From the New England Medical Center, Boston, Massachusetts. Purpose: Estrogen receptors mediate the direct effects of estrogen in target tissues. Two estrogen receptors have been identified (ER␣ and ER), and both are expressed in the vasculature. Methods: It has been shown previously that estrogen inhibits the response to vascular injury in both female ER␣ knockout (ER␣KO) and female ER knockout (ERKO) mice to the same extent as in wild-type mice. Mice have
CURRENT SURGERY • © 2001 by the Association of Program Directors in Surgery Published by Elsevier Science Inc.
0149-7944/01/$20.00
557