Soluble E-cadherin promotes cell survival by activating epidermal growth factor receptor.

High levels of the soluble form of E-cadherin can be found in the serum of cancer patients and are associated with poor prognosis. Despite the possible predictive value of soluble E-cadherin, little is understood concerning its patho-physiological consequences in tumor progression. In this study, we show that soluble E-cadherin facilitates cell survival via functional interaction with cellular E-cadherin. Exposure of cells to a recombinant form of soluble E-cadherin, at a concentration found in cancer patient's serum, prevents apoptosis due to serum/growth factor withdrawal, and inhibits epithelial lumen formation, a process that requires apoptosis. Further, soluble E-cadherin-mediated cell survival involves activation of the epidermal growth factor receptor (EGFR) and EGFR-mediated activation of both phosphoinositide-3 kinase (PI3K)/AKT and ERK1/2 signaling pathways. These results are evidence of a complex functional interplay between EGFR and E-cadherin and also suggest that the presence of soluble E-cadherin in cancer patients' sera might have relevance to cell survival and tumor progression.

Intramural acellular porcine dermal matrix (APDM)-assisted gastrotomy closure for natural orifice transluminal endoscopic surgery (NOTES).

BACKGROUND: A highly reliable and safe means of gastric closure for natural orifice transluminal endoscopic surgery (NOTES) has yet to be developed. The authors have previously described the self-approximating transluminal access technique (STAT) as a means for gastrotomy closure in transgastric surgery. It has yet to be determined whether biologic mesh can be utilized in facilitating gastrotomy closure via STAT. The aim of this study was to determine the feasibility of implanting an acellular porcine dermal matrix (LifeCell) into the STAT tunnel and investigate whether it will become incorporated into the submucosal plane of the STAT tunnel. METHODS: Five pigs underwent transgastric left uterine horn resection utilizing STAT. For closure, the acellular porcine dermal matrix was implanted within the submucosal plane, occluding the seromuscular incision. The mucosal incision was then closed over the matrix with endoscopically placed clips. Necropsy was performed after a 3 week survival period. Histopathological evaluation of the tunnel and matrix was performed. RESULTS: The matrix was successfully implanted in all five animals. Average OR time was 151 ± 68 min. Average time to anchor and embed the matrix within the tunnel was 4 ± 1 and 9 ± 12 min, respectively. There was one duodenal perforation related to a balloon occlusion device. Postoperative course was unremarkable; the average weight gain at 3 weeks was 22 ± 5 lbs. On necropsy, one animal had some protrusion of the matrix at the serotomy, with adhesions to small bowel and liver. Histopathology revealed one clinically insignificant microabscess but otherwise demonstrated local inflammation and fibrovascular ingrowth into the matrix. CONCLUSIONS: The porcine dermal matrix can be successfully implanted within the gastric submucosal plane and evidence of incorporation into the gastric wall by 3 weeks was demonstrated.

Development of tasks and evaluation of a prototype forceps for NOTES.

BACKGROUND AND OBJECTIVES: Few standardized testing procedures exist for instruments intended for Natural Orifice Translumenal Endoscopic Surgery. These testing procedures are critical for evaluating surgical skills and surgical instruments to ensure sufficient quality. This need is widely recognized by endoscopic surgeons as a major hurdle for the advancement of Natural Orifice Translumenal Endoscopic Surgery. METHODS: Beginning with tasks currently used to evaluate laparoscopic surgeons and instruments, new tasks were designed to evaluate endoscopic surgical forceps instruments. RESULTS: Six tasks have been developed from existing tasks, adapted and modified for use with endoscopic instruments, or newly designed to test additional features of endoscopic forceps. The new tasks include the Fuzzy Ball Task, Cup Drop Task, Ring Around Task, Material Pull Task, Simulated Biopsy Task, and the Force Gauge Task. These tasks were then used to evaluate the performance of a new forceps instrument designed at Pennsylvania State University. CONCLUSIONS: The need for testing procedures for the advancement of Natural Orifice Translumenal Endoscopic Surgery has been addressed in this work. The developed tasks form a basis for not only testing new forceps instruments, but also for evaluating individual performance of surgical candidates with endoscopic forceps instruments.

Durability of the self-approximating translumenal access technique (STAT) for potential use in natural orifice translumenal surgery (NOTES).

BACKGROUND: The self-approximating translumenal access technique (STAT) has been shown to provide a safe and reliable means of abdominal access for natural orifice translumenal endoscopic surgery (NOTES). However, the feasibility of using STAT for translumenal organ resection is unknown. This study aimed to evaluate the technical performance of organ resection using STAT, the integrity of the STAT gastric tunnel after organ resection, and the postoperative morbidity of organ resection using STAT. METHODS: In this study, 14 domestic swine underwent transgastric organ resection (7 cholecystectomies, 7 uterine horn resections) followed by sequential removal of two different sizes of standardized specimens. Evaluation of operative injury to the tunnel and difficulty of specimen extraction was performed. After 2 weeks of observation, necropsy was performed for evaluation and documentation of gross findings. RESULTS: The mean operating room time (intubation recovery) was 4.1 h. A tunnel with a mean length of 12 cm and a mean width of 4 cm was created. The tunnel remained fully intact in 14 of 14 animals after organ resection, in 13 of 13 animals after balloon extraction, and in 12 of 14 animals after rigid specimen extraction (1 clinically significant tear occurred). Postoperatively, all the animals gained weight appropriately. Necropsy findings included adhesions (n = 4), bile leak (n = 2), minor lap-port abscess (n = 1), and ventral hernia (n = 1). CONCLUSIONS: Although this study was a limited, prospective, animal survival study without a control arm, it again indicates that STAT allows safe abdominal access, a reliable means of closure, and directed endoscope positioning. Although one significant mucosal tear did occur, this study suggests STAT will tolerate the mechanical forces of peroral transgastric procedures provided the organ resected is small to moderate in size (<8 × 3 cm).

Reliability of gastric access closure with the self-approximating transluminal access technique (STAT) for NOTES.

BACKGROUND: STAT, or the self-approximating transluminal access technique, has been previously described and involves the dissection of a submucosal tunnel for peritoneal or mediastinal access from the esophagus and stomach. The objective of this study was to assess the safety and reliability of gastric access and closure in a porcine experience using STAT for natural orifice transluminal endoscopic surgery (NOTES). METHODS: A review of the experience using STAT access tunnels for intraperitoneal access was performed in 39 female pigs at a university animal lab. All animals underwent a predetermined NOTES surgical procedure using a STAT transgastric access tunnel based on a specific protocol. Details of the procedure, complications, and clinical course were documented. Necropsy was performed at 2 weeks. The main outcome measurements were clinical or necropsy evidence of gastrostomy site leak or inadequate access site closure. RESULTS: STAT was successful in providing safe peritoneal access in all animals. The width of the tunnel ranged from 1.5 to 5.5 cm and the length was up to 27 cm. There was no evidence of gastrostomy site leak in any animals. One animal required a single laparoscopic suture to help with tunnel closure. CONCLUSION: STAT provides safe transgastric access and allows secure closure of the gastrotomy site.

Transgastric organ resection solely with the prototype R-scope and the self-approximating transluminal access technique.

BACKGROUND: The self-approximating transluminal access technique (STAT) has been demonstrated to provide safe transluminal access and in-line endoscope positioning to target abdominal organs during natural orifice transluminal endoscopic surgery (NOTES). To date, organ resection with NOTES has typically required percutaneous assistance. We hypothesized that the in-line positioning and partial stability provided by STAT would allow single-access NOTES procedures if a multiarticulated endoscope could be used. OBJECTIVE: Assessment of single-site NOTES, using STAT and a prototype, multi-articulated endoscope. DESIGN: Animal survival study. SETTING: Penn State Hershey Medical Center Research Laboratories. INTERVENTIONS: Thirteen pigs underwent NOTES using a prototype endoscope with 2 articulated channels, a grasping forceps, and an insulated-tip needle-knife. The gallbladder was dissected using a fundus down technique, and hemoclips and a detachable loop were placed on the cystic duct and artery before removal. After a 2- to 3-week observation period, animals were euthanized and necropsy performed. RESULTS: All target organs were successfully resected without laparoscopic assistance. Significant complications were 2 perforations (1 caused by a prototype duodenal occlusion device and 1 caused by enterotomy during cholecystectomy) and 1 entrapment of the small bowel with an endoloop. Postoperatively, all animals gained weight appropriately with 1 killed on postoperative day 12 because of lethargy (cystic duct leak/biloma). LIMITATIONS: This is a limited animal survival study without control arm. CONCLUSIONS: The combination of the R-scope and STAT does allow effective, single-site NOTES procedures; however, although the R-scope provides improved tissue manipulation and visibility, the complications incurred here suggest that further improvements in devices and technique will be required for safe and effective single-site NOTES procedures.

Na,K-ATPase beta1-subunit increases the translation efficiency of the alpha1-subunit in MSV-MDCK cells.

The Na,K-ATPase consists of an alpha- and beta-subunit. Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK) express low levels of Na,K-ATPase beta(1)-subunit. Ectopic expression of Na,K-ATPase beta(1)-subunit in these cells increased the protein levels of the alpha(1)-subunit of Na,K-ATPase. This increase was not due to altered transcription of the alpha(1)-subunit gene or half-life of the alpha(1)-subunit protein because both alpha(1)-subunit mRNA levels and half-life of the alpha(1)-subunit protein were comparable in MSV-MDCK and beta(1)-subunit expressing MSV-MDCK cells. However, short pulse labeling revealed that the initial translation rate of the alpha(1)-subunit in beta(1)-subunit expressing MSV-MDCK cells was six- to sevenfold higher compared with MSV-MDCK cells. The increased translation was specific to alpha(1)-subunit because translation rates of occludin and beta-catenin, membrane and cytosolic proteins, respectively, were not altered. In vitro cotranslation/translocation experiments using rabbit reticulocyte lysate and rough microsomes revealed that the alpha(1)-subunit mRNA is more efficiently translated in the presence of beta(1)-subunit. Furthermore, sucrose density gradient analysis revealed significantly more alpha(1)-subunit transcript associated with the polysomal fraction in beta(1)-subunit expressing MSV-MDCK cells compared with MSV-MDCK cells, indicating that in mammalian cells the Na,K-ATPase beta(1)-subunit is involved in facilitating the translation of the alpha(1)-subunit mRNA in the endoplasmic reticulum.

Dose response curves for microwave ablation in the cardioplegia-arrested porcine heart.

INTRODUCTION: Microwave ablation has been used clinically for the surgical treatment of atrial fibrillation, particularly during valve procedures. However, dose- response curves have not been established for this surgical environment. The purpose of this study was to examine dosimetry curves for the Flex 4 and Flex 10 microwave devices in an acute cardioplegia-arrested porcine model. METHODS: Twelve domestic pigs (40-45 kg) were acutely subjected to Flex 4 (n = 6) and Flex 10 (n = 6) ablations. On a cardioplegically arrested heart maintained at 10-15(o)C, six endocardial atrial and seven epicardial ventricular lesions were created in each animal. Ablations were performed for 15 s, 30 s, 45 s, 60 s, 90 s, 120 s, and 150 s (65 W, 2.45 GHz). The tissue was stained with 2,3,5-triphenyl-tetrazolium chloride and lesions were sectioned at 5 mm intervals. Lesion depth and width were determined from digital photomicrographs of each lesion (resolution +/- .03 mm). RESULTS: Average atrial thickness was 2.88 +/- .4 mm (range 1.0 to 8.0 mm). 94% of ablated atrial sections created by the FLEX 4 (n = 16) and the FLEX 10 (n = 16) were transmural at 45 seconds. 100% of atrial sections were transmural at 90 seconds with the FLEX 10 (n = 14) and at 60 seconds with the Flex 4 device (n = 15). Lesion width and depth increased with duration of application. CONCLUSION: Both devices were capable of producing transmural lesions on the cardioplegically arrested heart at 65 W. These curves will allow surgeons to ensure transmural ablation by tailoring energy delivery to the specific atrial geometry.

Na-K-ATPase regulates tight junction permeability through occludin phosphorylation in pancreatic epithelial cells.

Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.

Microwave ablation for atrial fibrillation: dose-response curves in the cardioplegia-arrested and beating heart.

BACKGROUND: Microwave ablation has been used to replace the traditional incisions used in the surgical treatment of atrial fibrillation. However, dose-response curves have not been established in surgically relevant models. The purpose of this study was to develop dose-response curves for the Flex 10 (Guidant, Inc) microwave device in both the acute cardioplegia-arrested heart and on the beating heart. METHODS: Twelve domestic pigs (40 to 45 kg) were subjected to microwave ablation in either the arrested (n = 6) or beating heart (n = 6). The cardioplegia-arrested heart was maintained at 10 degrees to 15 degrees C while six atrial endocardial and seven right ventricular epicardial lesions were created in each animal. On the beating heart, six right atrial and seven ventricular epicardial lesions were created. Ablations were performed for 15, 30, 45, 60, 90, 120, and 150 seconds (65 W, 2.45 GHz). The tissue was stained with 2,3,5-triphenyl-tetrazolium chloride, and sectioned at 5-mm intervals. Lesion depth and width were determined from digital micrographs. RESULTS: Mean atrial wall thickness was 2.8 mm (range, 1 to 8 mm). In the arrested heart, 94% of atrial lesions were transmural at 45 seconds and 100% were transmural at 90 seconds. In the beating heart, only 20% of atrial lesions were transmural despite prolonged ablation times (90 seconds). Ventricular lesion width and depth increased with duration of application, and were similar on the arrested and beating hearts. CONCLUSIONS: Microwave ablation produces linear dose-response curves. Transmural lesions can be reliably produced on the arrested heart, but not consistently on the beating heart.

HPAF-II, a cell culture model to study pancreatic epithelial cell structure and function.

OBJECTIVES: Epithelial cells have distinct apical and basolateral plasma membrane domains separated by tight junctions. This phenotype is essential for the directional transport functions of epithelial cells. Here we characterized a well-differentiated pancreatic epithelial cell line to establish a useful model for understanding the mechanisms involved in the regulation of junctional complexes, polarity, and disease processes in the pancreas. METHODS: Immunofluorescence of cell junction marker proteins and electron microscopy were used to determine the presence of tight junctions, adherens junctions, and desmosomes. The functionality of tight junctions was tested by transepithelial resistance measurements and transepithelial permeability studies of nonionic molecules. Tight junction function in polarity was determined by laser scanning confocal microscopy. RESULTS: Immunofluorescence analysis in HPAF-II cells revealed tight junction localization of ZO-1, occludin, and claudin-4; adherens junction localization of E-cadherin and beta-catenin; and desmosomal localization of desmocollin. Transmission electron microscopy showed the presence of tight junctions, adherens junctions, and des-mosomes, and freeze-fracture electron microscopy revealed the presence of distinct anastomosing tight junction strands. Transepithelial electrical resistance and permeability measurements revealed functional tight junctions. In addition, 3-dimensional images of the monolayer generated by laser scanning confocal microscopy revealed that HPAF-II cells show polarity. Immunoblotting and RT-PCR analyses revealed high expression levels of E-cadherin and Na,K-ATPase beta-subunit but low levels of the transcription factor Snail in HPAF-II cells compared with MiaPaCa-2 cells. CONCLUSION: The HPAF-II cell line is a well-differentiated human pancreatic carcinoma cell line that should be useful as a model for studies aimed at understanding epithelial polarity, regulation of junctional complexes, and disease processes in pancreas.

Na,K-ATPase inhibition alters tight junction structure and permeability in human retinal pigment epithelial cells.

Na,K-ATPase regulates a variety of transport functions in epithelial cells. In cultures of human retinal pigment epithelial (RPE) cells, inhibition of Na,K-ATPase by ouabain and K(+) depletion decreased transepithelial electrical resistance (TER) and increased permeability of tight junctions to mannitol and inulin. Electrophysiological studies demonstrated that the decrease in TER was due to an increase in paracellular shunt conductance. At the light microscopy level, this increased permeability was not accompanied by changes in the localization of the tight junction proteins ZO-1, occludin, and claudin-3. At the ultrastructural level, increased tight junction permeability correlated with a decrease in tight junction membrane contact points. Decreased tight junction membrane contact points and increased tight junction permeability were reversible in K(+)-repletion experiments. Confocal microscopy revealed that in control cells, Na,K-ATPase was localized at both apical and basolateral plasma membranes. K(+) depletion resulted in a large reduction of apical Na,K-ATPase, and after K(+) repletion the apical Na,K-ATPase recovered to control levels. These results suggest a functional link exists between Na,K-ATPase and tight junction function in human RPE cells.