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A Sprayable Hyaluronate/Carboxymethylcellulose Based Adhesion BarrierReduces Remote Intraabdominal Adhesion Formation and Does Not ImpairIntestinal HealingHolly K. Sheldon, Melanie L. Gainsbury, Michael R. Cassidy, M. Jude Colt, Rubina L.Corazzini, Olga L. Syrkina, Keith E. Greenawalt, Thomas H. Jozefiak, Arthur F. Stucchi,James M. Becker
Background: Intraabdominal adhesions are a significant source of postoperative morbidity.While bioresorbable solid physical barriers such as modified hyaluronate/carboxymethylcel-lulose (HA/CMC) film (Seprafilm - SF) are effective in preventing adhesions, their efficacyis limited to the site of application. The aim of this study was to compare the effectivenessof modified HA/CMC sprayable powder (Sepraspray - SS) and SF in preventing adhesionsnot only to sites of direct application, but also to remote peritoneal defects to which a barrierwas not directly applied. Methods: Adhesion reduction was assessed in a rat ischemic buttonmodel and also in a rabbit cecal-sidewall injury model. Intraabdominal adhesions wereinduced in 30 rats by creating 3 peritoneal sidewall ischemic buttons on each side of amidline incision. SS (5 mg/button) or SF (1 cm2/button) was applied intraoperatively overthe 3 ischemic buttons on one side of the midline only. Adhesions were induced in 50rabbits by cecal abrasion with the concurrent creation of a 3 cm x 5 cm sidewall defectdelineated with silk sutures and knots. Operated control animals received no SS or SF. Onday 7 adhesions were scored in rats as the percent of buttons with attached adhesions andin rabbits as the % of the sidewall defect covered by adhesions. To assess the effect of eitherSS or SF on intestinal healing, an additional 27 rats underwent a colonic transection distalto the cecum, which was repaired with an end-to-end anastomosis. SS or SF was appliedcircumferentially to the anastomosis site. The anastomosed colonic sections were removed7 days later and their integrity assessed by burst pressure and tensile strength measurements.Results: The direct application of both SS and SF significantly (p<0.04) reduced adhesionformation compared with controls in both efficacy models (Table). While SF had no remoteeffects on adhesion formation in either model, SS significantly (p<0.01) reduced adhesionformation to ischemic buttons to which the powder was not directly applied. In rabbits, SSreduced remote adhesion formation to the sidewall defect by 70% (p<0.1). In the anastomosishealing model, neither SF nor SS affected intestinal anastomotic burst pressure (Control:240 ± 8.2 vs. SS 215 ± 19.0 vs. SF: 216 ± 27.2 mm Hg) or tensile strength (Control: 2.4± 0.2 vs. SS: 2.3 ± 0.2 vs. SF: 2.2 ± 0.2 Newton). Conclusions: While both SS and SF havecomparable adhesion reduction efficacy where applied, SS was additionally effective inreducing adhesion formation at remote sites of peritoneal injury to which SS was not applieddirectly. These data suggest that SS may have widespread efficacy throughout the peritoneumin reducing adhesion formation without compromising intestinal wound healing.
NOD2 Mutation Results in Altered Wound Healing in Epithelial CellsLisa S. Poritz, Leonard R. Harris
Introduction: A mutation in the NOD2 gene has been linked to terminal ileal Crohn's Disease(CD). CD is characterized by repeated mucosal injury and healing. No work has been doneon the effect of the NOD2 mutation on wound healing. We have stably transfected the IEC-18 cell line (ileal cells) with wildtype and the c-insertion mutation of the NOD2 gene. Wehave previously characterized these cells and have found marked disruption of the tightjunction proteins in the cells carrying the NOD2 mutation. The hypothesis for this studyis that there will be altered wound healing in the cells with the NOD2 mutation. Methods:IEC-18 cells were stably transfected with wild-type human NOD2 (NOD2 cell line) or the
S-1025 SSAT Abstracts
c-insertion mutation (3020InsC cell line). Western blot and PCR was used to identify NOD2.Cells were then grown in 6 well plates for 72 hours until confluent. The monolayer wasthen wounded with a plastic pipette tip in an X formation. 24 or 48 hours later the cellswere stained Hema 3 stain kit and viewed with a phase contrast microscope. Duplicate,unwounded, monolayers were used to measure cell proliferation using a coulter counter,n=8. Results: Human NOD2 protein was detected by Western blot and PCR in the NOD2and 3020InsC cell lines, but not the parental cell line. There was a significant increase inproliferation in the NOD2 cell line compared to the parental cell line (p<0.05). There wasno significant change in proliferation in the 3020InsC cell line compared to parental andNOD2 cell lines. The figure shows monolayer wound healing for the three cell lines at 24hours. The wounds in the NOD2 cell line healed the quickest. They were nearly closed at24 hours and undectable at 48 hours. The parental cell line had intermediate healing. Therewas minimal to no wound healing in the 3020InsC cell line at 24 hours or 48 hours. Inaddition, the 3020InsC cells seemed to bunch up in a “ridge” at the edge of the woundsuggesting migration into the wound was impaired. This “ridge” was not seen in the NOD2or parental cell lines. Conclusions: 1. Wound healing was markedly retarded in the 3020InsCcell line compared to the parental cell line while the NOD2 cell line healed more quicklythan the parental cell line. 2. The increase in proliferation seen in the NOD2 cell line maycontribute to the improved wound healing in this cell line. However, there was no changein proliferation in the 3020InsC cell line to explain the lack of wound healing in the3020InsC cell line. 3. The disruption in the tight junction complex we have previouslydescribed in the 3020InsC cell line may contribute to the delay in wound healing. 4. Lackof wound healing may have implications in the mucosal injury and repair seen in CD.
Left: IEC-18 parental, Middle: NOD2 Wildtype, Right: 3020InsC mutant
The Micro-RNA 192 as an Effective Response Prediction Factor in theMultimodality Therapy of Locally Advanced Esophageal CancerDaniel Vallbohmer, Peter P. Grimminger, Christoph Wandhoefer, Jan Brabender, UtaDrebber, Elfriede Bollschweiler, Arnulf H. Hölscher, Ralf Metzger, Magarethe Odenthal
Background: Neoadjuvant multimodality treatment is frequently applied to improve thepoor prognosis associated with locally advanced esophageal cancer. However, only patientswith a major histopathologic response to neoadjuvant therapy seem to have a significantsurvival benefit. We have shown in a recent pilot study using microarray-technique that theexpression profile of microRNAs depends significantly on the histopathologic response ofpatients with locally advanced esophageal cancer undergoing multimodality treatment. Thisstudy aimed to validate these identified single microRNAs by real-time PCR. Patients andMethods: Eighty-eight patients with locally advanced esophageal cancer (cT2-4, Nx, M0)were included in the study. All patients received neoadjuvant chemoradiation (cisplatin, 5-FU,45 Gy) and subsequently underwent transthoracic en bloc esophagectomy. Histomorphologicregression was defined as major histopathological response when resected specimens con-tained less than 10% vital residual tumor cells (major response: 34 patients; minor response:54 patients). Intratumoral microRNA was isolated from pretherapeutic tissue biopsies andcorresponding surgical specimens. Based on the microarray results, the amplification profileof dysregulated microRNAs was analyzed and the microRNAs 192, 194 and 622 wereselected for the further analysis of the validation population. Results: The expression of allthree microRNAs was significantly reduced during neoadjuvant therapy, showing lower levelsin post-therapeutic tumor samples (p<0.001). Furthermore, the pre-therapeutic intratumoralexpression of microRNA 192 was significantly correlated with histopathologic response:patients with a major response had a significantly higher intratumoral microRNA 192 amountcompared with patients having a minor response (p=0.01). Moreover, by using an expressioncut-off value of 0.63, the sensitivity, specificity and accuracy of pre-therapeutic microRNA192 for assessment of histopathologic response was 96%, 82% and 88% respectively (p=0.05). Conclusion: Our data support the role of micro RNA 192 as a predictive marker fortherapy response in the multimodality therapy of patients with locally advanced esophagealcancer. In a multi-institutional trial we will now try to confirm these results.
Diazoxide, a Opening mitoKATP, Reduces Liver Damage Secondary toIschemic/Reperfusion InjuryMateus A. Nogueira, Ana Maria M. Coelho, Sandra N. Sampietre, Nilza A. Molan, LuizAugusto C. D'Albuquerque, Marcel C. Machado
Background/Aim: Previous studies have demonstrated that diazoxide protects heart fromischemia/reperfusion injury however there are no prior studies of the role of diazoxide onliver ischemic reperfusion injury. In the present study we evaluated the effect of diazoxideon local and systemic liver ischemia/reperfusion (I/R) process. Methods: Wistar male ratsunderwent partial liver ischemia performed by clamping the pedicle from medium and leftanterior lateral segments during an hour under mechanical ventilation. They were dividedinto 2 groups: Control Group (n=26): rats received saline and Diazoxide Group (n=26): ratsreceived IV diazoxide (3.5mg/kg) 15 minutes before liver reperfusion. Four and 24 hoursafter reperfusion, blood were collected for determinations of AST, ALT, TNF-α, IL-6, andIL-10. Liver tissues were assembled for histologic analysis, malondialdehyde (MDA) content,and mitochondrial oxidation and phosphorylation. Pulmonary vascular permeability andmyeloperoxidade (MPO) were also determined. Results: Four hours after reperfusionDiazoxide Group presented elevation of AST, ALT, TNF-α, IL-6 and IL-10 serum levels
significantly lower than Control Group (p<0.05). A significant reduction on liver MDAcontent and on mitochondrial dysfunction were observed in Diazoxide Group compared toControl Group (p<0.05). No differences in pulmonary vascular permeability and MPOactivity were observed between groups. Twenty four hours after reperfusion Diazoxide Groupshowed a reduction of AST and ALT serum levels when compared to Control group (p<0.05).Conclusion: Diazoxide attenuates liver ischemia/reperfusion injury probably by a mechanismrelated to mitochondrial function preservation during liver ischemia.
Pterostilbene Induces Mitochondrially-Derived Apoptosis in Pancreatic CancerCells by Increasing MnSOD Activity and Release of Cytochrome C and Smac/DIABLODenise E. McCormack, Debbie E. McDonald, David W. McFadden
BACKGROUND: We have previously shown that Pterostilbene (3, 5- dimethoxy-4-hydro-xystilbene), a compound found in blueberries, inhibits cell proliferation and promotesapoptosis in pancreatic cancer In Vitro by induction of mitochondrial membrane depolariza-tion and caspase 3/7 activation. To further investigate the role of mitochondria in Pterostil-bene-induced apoptosis in pancreatic cancer, we examined its effects on manganese superox-ide dismutase (MnSOD) activity, Cytochrome C and Smac/DIABLO release. The mitochon-drial enzyme MnSOD plays a critical role in regulation of cancer cell proliferation throughan unknownmechanism. Smac/DIABLO is amitochondrial protein that potentiates apoptosis.Both Smac/DIABLO and Cytochrome C have been shown to exit mitochondria and enterthe cytosol during apoptosis. We hypothesized that Pterostilbene would increase MnSODactivity and cytosolic levels of Cytochrome C and Smac/DIABLO in a dose dependent manner.METHODS: MIA and PANC-1 cell lines were treated with 25 micromolar and 50 micromolarconcentrations of Pterostilbene for 48hrs and quantitative MnSOD activity was measuredby superoxide dismutase ELISA assay. In separate experiments, MIA and PANC-1 cell cellswere treated with 25 micromolar, 50 micromolar and 75 micromolar concentrations ofPterostilbene for 24hrs and cytosolic extracts were analyzed for Cytochrome C and Smac/DIABLO utilizing ELISA protocols. One way ANOVA and Tukey post-hoc analysis wereused for statistical analysis. RESULTS: Pterostilbene increased enzymatic activity of MnSODin both cell lines in a dose dependent manner (p <0.01). Pterostilbene treatment increasedcystosolic levels of Cytochrome C in MIA cells at the 25 micromolar (p < 0.05) and 75micromolar concentrations in PANC-1 cells (p < 0.05). Cystosolic levels of Smac/DIABLOincreased in both MIA and PANC-1 cells with treatment at the 75 micromolar concentrationof Pterostilbene (p <0.01). CONCLUSION: We have previously demonstrated that Pterostil-bene, a natural plant-derived stilbene, inhibits pancreatic cancer In Vitro through activationof the mitochondrial apoptosis pathway. MnSOD, an inducible mitochondrial enzyme thatconverts superoxide anion to hydrogen peroxide, has an essential role in regulation ofpancreatic cancer cell proliferation. The results of our current study demonstrate for thefirst time that Pterostilbene increases MnSOD activity in pancreatic cancer cells. In addition,Pterostilbene increases cytosolic levels of Cytochrome C and Smac/DIABLO in both celllines, confirming mitochondrially derived apoptotic cell death. Further studies are ongoingto elucidate the intricate relationship between intrinsic apoptosis, MnSOD activity, andpancreatic cancer cell death upon Pterostilbene treatment.
Systemic Inflammation With Multiorgan Dysfunction is the Cause of Death inMurine Pancreatic Duct Ligation-Induced Acute PancreatitisZuobiao Yuan, David Meyerholz, Deborah Williard, Erik Twait, Kempuraj Duraisamy,Isaac Samuel
Existing animal models of acute pancreatitis (e.g., cerulein, choline-deficient ethionine-supplemented diet) do not resemble gallstone pancreatitis as the etiologies are not analogous.Distal pancreatic duct ligation (PDL)more closely resembles early events in gallstone pancreat-itis. We recently developed a novel murine model of PDL-induced acute pancreatitis associ-ated with substantial mortality. Using this model, we previously showed that specific inhibi-tion of the stress kinase ERK with In Vivo gene modulation significantly improves survival.In the present study we determine the cause of death in our murine model by serialexamination of multiple parameters in three groups: a) Acute pancreatitis group had PDL;b) Hepatic obstruction group had bile duct ligation (BDL) without PDL; c) Sham operationgroup. The mice were observed for 15 days post-operatively. BDL and Sham controls hadno mortality. Close to 100% mortality was seen in PDL-induced acute pancreatitis withmost deaths occurring between day 2 and day 4. Characteristics of micewith acute pancreatitisincluded the following (ANOVA; P<0.05): ERK activation in the pancreas and distant organs;pancreatic neutrophil infiltration and acinar cell necrosis maximal on day 2; increased plasmaIL-1β and TNF-α levels on day 2, that peaked on day 3, in parallel with worseninghypotension and bradycardia; bronchoalveolar lavage (BAL) fluid neutrophil count and IL-1β level, and plasma aspartate aminotransferase (AST) level, also peaked on day 3; pulmonaryneutrophil infiltration and plasma creatinine level peaked on day 4.. Liver injury evidencedby raised AST after hepatic obstruction was exacerbated by PDL. Increased plasma IL-1βand TNF-α on day 2 after BDL subsided thereafter. Although BDL was also associated withpulmonary neutrophil infiltration it was not associated with increased IL-1β or neutrophilsin BAL fluid. BDL-induced renal tubular damage was not associated with raised plasmacreatinine. Our findings indicate that the high mortality rate seen in PDL-induced acutepancreatitis in mice is associated with progressive systemic increase of inflammatory cytokinelevels, cardiovascular instability, acute lung injury, liver injury, and renal dysfunction,suggesting that systemic inflammation with multiorgan failure is the proximate cause ofdeath in this experimental model. Lung and renal injury as observed by morphology afterhepatic obstruction alone is not associated with systemic inflammation or death. In conclu-sion, systemic inflammation with multiorgan dysfunction causes death in pancreatic ductligation-induced acute pancreatitis in mice. This experimental model is a useful analogyof severe gallstone pancreatitis to investigate disease pathogenesis and to evaluate noveltherapeutic strategies.
A Promising Novel Target in Pancreatic Cancer: HuR Modulates Multiple CoreSignaling Pathways Required for Pancreatic TumorigenesisVanessa A. Talbott, Koree Ahn, David W. Rittenhouse, Nathan G. Richards, AgnesWitkiewicz, Eugene P. Kennedy, Myriam Gorospe, Charles J. Yeo, Jonathan R. Brody
Introduction: Twelve core signaling pathways with 540 overexpressed individual genes haverecently been identified as critical for the development of pancreatic ductal adenocarcinoma(PDA) (Science 2008, 321:1801-1806). The mechanism of overexpression for nearly all(99%) of the identified up-regulated genes in pancreatic tumorigenesis is unknown. Weexplored the hypothesis that post-transcriptional gene regulation may be a powerful alternat-ive process in which these up-regulated genes were being disrupted. A key component ofthis regulatory process is Human antigen R (HuR), which can modulate gene expression bybinding to mRNAs that encode for tumor-promoting proteins in cancer cells. Previously,we discovered that HuR is a key marker for poor pathologic features in PDA and is apredictive marker for gemcitabine-based chemotherapy. Methods: Using a bioinformaticapproach, we identified putative HuR targets from the 540 overexpressed genes in PDA(Nucl. Acids Res. 2001, 29:246-254, PNAS 2004, 101:2987-92). HuR binding to mRNA-targets was validated by PCR-based analysis in ribonucleoprotein immunoprecipitatedHuR:RNA complexes. Further validation of expression of HuR target genes was performedby quantitative PCR analysis and immunoblotting. Results: We identified 60 putative targets(11.1%) for HuR regulation among the overexpressed genes in PDA. In comparison, geneticand epigenetic alterations contribute only 1% and 1.8% respectively to the proposed mechan-isms by which these 540 genes are disrupted in PDA. Ten of the 60 putative target geneswhich are a part of 5 of the 12 core signaling pathways in PDA, including K-Ras, wereexperimentally validated as specific HuR targets. Conclusion: HuR is an unprecedentedregulatory protein of at least 5 critical signaling pathways in PDA. Targeting multiple corepathways in PDA through silencing HuR expression may be a potent therapeutic strategyto treat this disease.
Changes in Neurotransmission via Alpha- and Beta-Receptors DuringPostoperative Ileus in Rat Circular Jejunal MuscleMichael S. Kasparek, Brigitte Goetz, Bernhard Stoklas, Petra Benhaqi, Mario H. Mueller,Martin E. Kreis
Background Our aim was to: 1) investigate the role of α and β-receptors in control ofcontractile activity in rat circular jejunal muscle; 2) explore changes in adrenergic neuro-transmission via these receptors during postoperative ileus (POI); 3) determine if thesechanges are paralleled by intramural inflammation and delayed intestinal transit. MethodsMuscle strips (n=8/rat) from 6 male Sprague Dawley rats per group were studied in organchambers. Groups: Naïve controls (NC), sham controls (SC) 24h after laparotomy, rats 12h(P12h), 1 (P1d), 3 (P3d), and 7days (P7d) after laparotomy and standardized small bowelmanipulation to induce POI. After spontaneous contractile activity (g/mm2/min) wasrecorded, dose-response curves for phenylephrine (αa; α-agonist; 10-8-3x10-6M) and iso-prenaline (βa; β-agonist; 3x10-10-10-7M) were established. Responses were repeated withtetrodotoxin (TTX; blocking enteric nerves; 10-6M), after precontraction with bethanechol(3x10-6M), and with phentolamine (α-anatagonist; 10-5M), or propranolol (β-antagonist;5x10-6M). Intestinal transit was studied by charcoal transit (% of small bowel passed).Histology of jejunal whole mounts was performed for myeloperoxidase positive cells (MPO),macrophages, and mastcells (cells/mm2).Data: mean±SEM. Results Spontaneous contractileactivity was increased in SC, P12h, and P7d (NC 1.3±0.3; SC 3.7±0.9; P12h 4.5±1.5; P7d3.6±1.0; p<0.05), but not in P1d (1.7±0.4) and P3d (1.8±0.5; p=NS). αa and βa inhibitedspontaneous contractile activity dose-dependently in all groups (p<0.05). InNC, TTX reducedαa-induced inhibition (p<0.05), but TTX had no effect on αa-responses in POI groups (p=NS). In contrast, TTX did not affect βa-response in NC (p=NS), but increased responses inPOI groups (p<0.05). αa and βa-induced inhibition was reduced in P12h, P3d, and P7dand in all POI groups, respectively (p<0.05). Precontraction had no effect on αa and βa-responses (p=NS). Effects of αa and βa were blocked by phentolamine or propranolol(p<0.05). Intestinal transit was delayed in all POI animals and recovered over time (NC53±3; P12h 22±2; P7d 44±2%; p<0.05), but was unaffected in SC (50±5%; p=NS). MPOpositive cells and mastcells increased postoperatively and peaked in P1d (NC 14±2; P1d763±48) and P12h (NC 9±1; P12h 700±79; both p<0.05), respectively; no effect was observedin SC (56±37 and 30±12, respectively; p=NS). Macrophages peaked in P3d (NC 367±41;P3d 1306±178; p<0.05); counts in SC and P12h were similar to NC (SC 395±82; P12h706±19; p=NS). Conclusion Contractile activity can be inhibited predominantly via muscularα- and β-receptors. However, during POI long lasting changes in balance of muscular andneuronal α- and β-receptors occur that might participate in pathophysiology of POI. Thesechanges are paralleled by intramural inflammation and impaired intestinal transit. DFG KA2329/5-1
Central Vagal Activation During Early Postoperative Ileus in the MouseMia Karpitschka, Mario H. Mueller, Michael S. Kasparek, Jorg Glatzle, Martin E. Kreis
Introduction: Postoperative ileus (POI) involves reflex inhibition of intestinal motility anda subsequent intestinal inflammatory response that is characterized by efferent vagal modula-tion. However, the role of central vagal afferents in the early phase of POI is unknown. We,therefore, aimed to explore central vagal afferent nerve activation early during POI. Materialand Methods: C57BL/6 mice were vagotomized 3-4 days prior to ileus experiments, whilecontrol animals received a sham operation without vagotomy. For ileus experiments, laparo-tomy was either followed by standardized small bowel manipulation to induce POI orsham treatment for control. Then, after 3h or 9h, the brain was removed, fixed and Fos-immunohistochemistry was performed to determine neuronal activation in the vagal nucleusof the solitary tract (NTS) and the area postrema. Each subgroup contained an n of 6. Datawere analyzed by two-way ANOVA. Results: The number of Fos-positive neurons in the