3.01 Intraperitoneal Application of Intestinal Alkaline Phosphatase Decreases Post-Operative Adhesions in Mice

Posted on December 22, 2014

S. K. Hyoju1, S. Morrison1, M. Gharedaghi1, M. Mohamed1, S. S. Gul1, M. Najibi1, T. Phupitakphol1, A. Osmani1, K. Economopoulos1, S. Hamarneh1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA

Introduction: Damage to the peritoneum initiates an inflammatory response leading to the formation of adhesions which subsequently cause significant morbidity in some patients.  Intestinal alkaline phosphatase (IAP) is a gut enzyme capable of detoxifying various inflammatory mediators such as lipopolysaccharide, lipoteichoic acid, CpG DNA and nucleotide triphosphates, and could therefore play a role in decreasing adhesion formation. This study aimed to test the anti-inflammatory effects of IAP on the development of post-operative adhesions in mice.

Methods: C57BL/6 mice were used in this study and underwent midline laparotomy. Six peritoneal buttons were created by pinching and ligating the peritoneum with 5-0 silk sutures. The buttons were subsequently cut and cauterized. The cecum was exteriorized and abraded with sterile dry gauze and returned to the abdominal cavity. At designated time points during the procedure, a total of, 500µl of either IAP (5000U) or vehicle was applied over the peritoneal cavity. On the 21st postoperative day, mice were euthanized and adhesions from abdominal viscera to each button were scored based on number, tenacity, and extent of the adhesions. The final adhesion score for each mouse was calculated by the summation of these scores. The investigators were blinded to the treatment groups. In a second series of experiments, this procedure was repeated, and the animals were instead euthanized on the 1st post operative day. Peritoneal buttons were then excised and homogenized, and the resulting supernatant was collected for cytokine analysis.Student’s t test was used to analyze the data

Results:The mice treated with IAP had significantly lower adhesion scores compared to vehicle-treated animals (16.4 ± 6.8 vs. 27.8 ± 8.7, P = 0.04).Peritoneal button homogenates from IAP-treated mice had significantly lower IL-1β level and TNF-α levels compared to vehicle-treated animals (64.57±7.4 vs. 111.31±18.9 pg/mg tissue protein, P=0.0006; 6.76±1.69 vs. 10.5±3.07 pg/mg tissue protein, P=0.04) respectively.

Conclusion:Intraperitoneal application of IAP at the time of laparotomy could represent a novel approach to the prevention of post-operative adhesions.

 

3.02 PRP Enhances Posterior Lumbar Spinal Fusion Using a Composite Engineered Scaffold In a Rabbit Model

Posted on December 22, 2014

J. L. Van Eps1,4, J. S. Fernandez-Moure1,4, F. J. Cabrera4, S. J. Minardi4,5, B. Aghdasi2,4, A. Tampieri4,5, E. Tasciotti4, B. K. Weiner2,3,4  1Houston Methodist Hospital,Department Of Surgery,Houston, TX, USA 2Houston Methodist Hospital,Department Of Orthopedics & Sports Medicine,Houston, TX, USA 3Weill Cornell Medical College,New York, NY, USA 4Houston Methodist Research Institute,Department Of Nanomedicine, Surgical Advanced Technology Lab,Houston, TX, USA 5Istituto Di Scienza E Tecnologia Dei Materiali Ceramici (ISTEC),Department Of Bio-Ceramic And Bio-Hybrid Composites,Faenza, FAENZA, Italy

Introduction:

With increased implementation of spinal instrumentation and fusion, an emphasis to develop biologic alternatives to the morbidity of autografting or prosthetic implants has emerged. Additionally, existing platforms utilizing bone morphogenic proteins (BMPs) are limited by potential side effects and alternatives are needed. Autologous platelet-rich plasma (PRP) is a universally-available source of growth factors with currently untapped potential for bone regeneration.

Methods:

A proprietary magnesium-hydroxyapatite/collagen 70/30 wt% composite scaffold was implanted in sixteen New Zealand White rabbits randomly assigned to two experimental groups undergoing L5-L6 intertransverse posterior spinal fusion – scaffold alone (S1) or scaffold + PRP (S2). Autologous PRP was isolated preoperatively for S2 rabbits via double-centrifugation of whole blood (8mL), standardized platelet concentration to 5×106/mL and activation with a solution of 10%CaCl and 300U thrombin, and used to soak scaffolds pre-implantation. The left anatomic side received decortication alone, serving as an internal control (IC). DynaCT imaging and post hoc bone volume quantification was performed at 2, 4, and 6 weeks postoperatively, and differences in experimental side/IC bone volume were compared using thresholds of 200 and 500 Hounsfield units (HU) – known correlates to trabecular and cortical bone respectively. Postoperative specimens were harvested, plastic embedded and sectioned, H&E stained, and visualized under microscopy to confirm fusion of new and native bone.

Results:

Both groups displayed significantly more osteogenesis on the experimental side compared to IC (p<0.05), confirming scaffold osteoconductivity, and earlier, more robust osteogenesis was seen in S2 vs. S1 rabbits by dynaCT imaging and volumetric analysis (Figure 1). This trend for trabecular bone formation was witnessed as early as 2 weeks (p<0.05) and became more pronounced at 4-6 weeks (p<0.01). The near 2-fold greater trabecular bone formation translated into more mature bone formation at 6 weeks as well, as Group S2 rabbits displayed significantly more cortical bone at 4 and 6 weeks postoperatively (p<0.05). Histological analysis confirmed more robust fusion of native and new bone and bridging intertransverse osteogenesis in S2 rabbits.

Conclusion:

This work confirms the feasibility of bone regeneration and spinal fusion using solely biocompatible materials and autologous biologic products. Additionally, the greater overall osteogenesis and earlier mineralization afforded by PRP support its clinical utility and potential abrogation for usage of synthetic moieties such as BMPs – or at minimum offer a safer, diminished required dose – in the future.

3.03 Oncolytic Recombinant Vaccinia Virus GLV-2b372 Efficiently Kills Hepatocellular Carcinoma

Posted on December 22, 2014

J. W. Ady1, C. Johnsen1, K. Mojica1, J. Heffner1, D. Love1, A. Pugalenthi1, J. Belin1, J. R. Aguilar5, N. Chen5, Y. A. Yu5, A. Szalay5, Y. Fong4  1Memorial Sloan-Kettering Cancer Center,Surgery,New York, NY, USA 2University Of California – San Diego,3Department Of Radiation Medicine And Applied Sciences, Rebecca & John Moores Comprehensive Cancer Center,San Diego, CA, USA 3University Of Würzburg,4Department Of Biochemistry, Rudolph Virchow Center For Experimental Biomedicine, And Institute For Molecular Infection Biology,Würzburg, BAVARIA, Germany 4City Of Hope National Medical Center,Surgery,Duarte, CA, USA 5Genelux,Research And Development,San Diego, CALIFORNIA, USA

Introduction:  Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with limited treatment options and poor prognosis. Therefore, there is a great need to develop novel therapies for HCC.   Oncolytic viruses that specifically infect, replicate within, and kill cancer cells represent an emerging and promising platform for cancer therapy.   In this study we assess at the ability of GLV-2b372, a novel nonattenuated vaccinia virus derived from the Lister 1.1.1 strain, to kill HCC.

Methods:  The infectivity, viral replication and cytotoxicity of GLV-2b372 was assayed in 4 human HCC cell lines.  Huh-7 flank xenografts were generated in athymic nude mice.  Mice were treated with intratumoral injections of GLV-2b372.  Biodistribution of viral presence in animals treated with GLV-2b372 was assessed.

Results: Infectivity increased in a time and concentration dependent manner in all cell lines. All cell lines supported efficient replication of virus. Flank xenografts treated with GLV-2b372 demonstrated complete erradication of tumor in 75% of animals with significant tumor reduction as compared to controls (p < 0.05).  Biodistribution confirmed sustained intratumoral presence of virus at 2 weeks post infection, with rapid clearance of tumor from all other tissues. 

Conclusion: Our results demonstrate that the novel oncolytic vaccinia virus GLV-2b372 selectively infects and replicates in HCC tissue.  We demonstrate infectivity and killing of HCC both in vitro and in vivo.  These findings indicate that GLV-2b372 is a potent oncolytic vector that could serve as a promising platform for oncolytic cancer immunotherapy.  

 

3.04 Traumatic Brain Injury Alters Circulating Microparticles which then Impact Coagulation

Posted on December 22, 2014

E. F. Midura1, C. C. Caldwell1, M. D. Goodman1  1University Of Cincinnati,Cincinnati, OH, USA

Introduction:  Traumatic brain injury (TBI) is known to result in a sub-acute, post-traumatic, hypercoagulable state, however the pathophysiology behind this alteration is poorly understood. Platelet-based contribution to clot is known to decrease over time following trauma and microparticles have also been shown to be changed after TBI. Whether platelets and microparticles interact to influence coagulation has not been determined. Using a murine model, we hypothesized that microparticle and platelet contributions to clot formation would be altered after TBI. 

Methods:  An established weight-drop model was used to induce TBI in anesthetized mice. Sham mice underwent anesthesia without TBI. Blood samples were collected 24 hours after injury, and circulating microparticle and platelet counts determined. Thromboelastometry (ROTEM) was used to evaluate changes in hemostasis. ROTEM was then repeated on sham and post-injury blood treated with normalized concentrations of microparticles isolated from sham mice (sham microparticles), TBI mice (TBI microparticles), or microparticle-free saline. A microparticle pro-coagulant assay was used to compare activity in sham and TBI mice.

Results: One day after TBI, we observed a decrease in overall microparticle concentrations (3.6 vs. 1.9 x 108 microparticles/ml, p < 0.001). Overall platelet counts did not change after injury. ROTEM demonstrated a reduced platelet contribution to hemostasis 24 hours after TBI compared to sham (62.9% vs. 48.0%, p < 0.01). When TBI microparticles were added to sham blood, a significant decrease in platelet contribution to clot was seen compared to saline (Figure 1). Conversely, when sham microparticles were added to blood from injured mice, there was a normalization of platelet contribution to clot formation when compared to saline (Figure 1). Furthermore, when microparticle pro-coagulant activity was assayed, TBI microparticles had increased activity compared to sham microparticles (3.28 vs. 1.72, p = 0.04).

Conclusion: Our data demonstrate that after TBI, 1) circulating microparticles are decreased, 2) platelet contribution to clot formation is decreased despite unchanged platelet count, and 3) TBI microparticles independently augment post-traumatic clot formation. Post-TBI microparticles may therefore be responsible for the altered platelet role in coagulation and the development of a post-traumatic hypercoagulable state.

 

3.05 Controlled Release of Nitric Oxide Enhances Gemcitabine Cytotoxicity in Pancreatic Adenocarcinoma

Posted on December 22, 2014

J. Fernandez-Moure1,2, D. Kirui1, J. Van Eps1,2, N. Dhanani1,4, F. Cabrera1, M. Ferrari1,3, E. Tasciotti1  1Houston Methodist Research Institute,Nanomedicine,Houston, TX, USA 2Houston Methodist Hospital,Surgery,Houston, TX, USA 3University Of Texas Health Science Center At Houston,Houston, TX, USA 4Texas A & M Health Science Center College Of Medicine,Bryan, TX, USA

Introduction:

Gemcitabine (GEM) is the first-line treatment for pancreatic adenocarcinoma (PAC). Despite such broad use, intrinsic and acquired chemoresistance is common. Loss of the tumor suppressor SMAD4 is present in more than 50% of PAC and is known to play a role in chemoresistant mechanisms. Nitric oxide (NO) is the predominant species responsible for the cytotoxic action of macrophages against cancer cells yet localized delivery is difficult given the short half-life and volatility. Silica (Si) nanoparticle mediated local delivery has been shown to be an effective strategy for NO delivery and controlled release. We sought to study the effects of locally delivered NO on GEM mediated PAC cytotoxicity and the potential role of SMAD4 in this effect. We hypothesized that NO would enhance the cytotoxicity of GEM in a SMAD4 independent manner.

 

Methods:

NO-Silica nanoparticles (NO-Si) were synthesized via a co-condensation of tetraethoxysilane with aminoalkoxysilane under high pressure nitrous oxide for 3 or 4 days. Particle characterization included scanning electron microscopy, dynamic light scattering, and Fourier transform infrared spectroscopy. NO release was measured using a chemiluminescence nitric oxide analyzer. A SMAD4 negative PAC cell line (SMAD4-) was made using lentiviral knockdown of Panc1 PAC cells and confirmed by western blot. Panc1 and SMAD4- cells were then treated with gemcitabine (100nm to 30μm), 30 mg NOSi particles (NOSihi or NOSilo), or both for 72 hours. Cell viability was then quantified by MTS cell proliferation assay.

 

Results:

Half-life of NOSi NO release is 3.5±0.6 hours. NOSilo maximum concentration of NO release was 750ppb/mg and NOSihi was 2250ppb/mg. NOSilo alone reduced cell viability by 30% and 35% in SMAD4 and Panc1 respectively. When combined with GEM, NOSilo led to a significant reduction in cell viability of both cell lines at all concentrations used. The greatest effect was seen at 1μm where cytotoxicity was increased by 50% in Panc1 and 70% in SMAD4-. NOSihi led to >90% reductions in cell viability in Panc1 and no difference was seen when combined with GEM. NOSihi alone led to only 70% reduction in SMAD4- cells. When combined with GEM, cytotoxicity was improved to 90% at GEM concentrations as low as 5μm.

 

Conclusion:

We have demonstrated the in vitro dose dependent cytotoxic effects of Si nanoparticle NO controlled release on PAC. When combined with GEM there is a synergistic effect resulting in improved cytotoxicity seen in both Panc1 and SMAD4- PAC cells with a differential pattern of cell death seen at high concentrations of NO. These findings suggest not only that NOSi is useful chemosensitizing agent but that SMAD4 may play a role in its synergism with GEM. Creation of a novel class of therapeutics where local controlled release of NO is used as a method of chemosensitization may lead to a paradigm shift in not only treating PAC but all solid tumors where chemoresistance is common.

 

22.08 Impact of the 2011 ACGME Duty Hour Reform on Surgical Outcomes and Resident Exam Performance

Posted on December 22, 2014

R. Rajaram1,2, J. Chung2, A. Jones3, M. Cohen1, A. Dahlke2, L. Hedges4, C. Ko1,5, J. Tarpley6, F. Lewis3, D. Hoyt1, K. Bilimoria1,2  1American College Of Surgeons,Division Of Research And Optimal Patient Care,Chicago, IL, USA 2Northwestern University,Surgical Outcomes And Quality Improvement Center, Department Of Surgery And Center For Healthcare Studies In The Institute For Public Health And Medicine, Feinberg School Of Medicine,Chicago, IL, USA 3American Board Of Surgery Inc,Philadelphia, PA, USA 4Northwestern University,Institute For Policy Research And Department Of Biostatistics,Chicago, IL, USA 5University Of California – Los Angeles And VA Greater Los Angeles Healthcare System,Department Of Surgery,Los Angeles, CA, USA 6Vanderbilt University Medical Center,Department Of Surgery,Nashville, TN, USA

Introduction:  In 2011, the Accreditation Council for Graduate Medical Education (ACGME) furthered restrictions to existing resident duty hour requirements. However, it remains unknown whether these policies improved patient care or resident education or worsened them due to decreased continuity of care. The objectives of this study were to assess if implementation of the 2011 ACGME resident duty hour reform was associated with improvements in (1) surgical patient outcomes or (2) resident exam performance.
 

Methods:  General surgery patients at teaching and non-teaching hospitals participating in the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP; 2009/10 to 2012/13 academic years) were identified. Using a difference-in-differences approach, we evaluated outcomes in teaching hospitals compared to non-teaching hospitals for two years prior to and after the duty hour reform. We examined 6 patient outcomes with our primary outcome measure being Death/Serious Morbidity. Additionally, categorical general surgery resident performance on the American Board of Surgery in-training exam (ABSITE) and scores for first-time examinees on the written and oral board certification exams were compared with trend tests for this same time period.

Results: There were 204,641 patients were identified from 23 teaching (n=102,525) and 31 non-teaching (n=102,116) hospitals. The 2011 ACGME duty hour reform was not associated with a significant change in Death/Serious Morbidity (OR 1.06, 95% CI 0.94-1.20) or other adverse outcomes in teaching hospitals. Moreover, similar results were found when comparing adverse outcomes in teaching hospitals pre-reform to post-reform years 1 and 2 separately. A subgroup analysis of high-risk patients suggested that duty hour reform was significantly associated with an increase in Death/Serious Morbidity in the first year after the policy (OR 1.16, 95% CI 1.01-1.33) but not in the second year (OR 0.95, 95% CI 0.81-1.12). Written board exam pass rates improved significantly (83.0% to 87.9%, P < 0.001); however, there were no differences in ABSITE scores or oral board exam pass rates during this time period.

Conclusion: Implementation of the 2011 ACGME duty hour reform was not associated with improved general surgery patient outcomes or changes in resident ABSITE or oral board scores. Given concerns about continuity of care and resident education, consideration should be given to revising existing duty hour requirements.

 

22.09 Toll Like Receptor 4 (TLR4) Regulates the Adaptive Response of Autophagy in Intestinal Stem Cells

Posted on December 22, 2014

S. Shaffiey1,2, H. Jia1, C. Sodhi1, K. Thadikona1, M. Good3, M. Neal2, Y. Yamaguchi1, S. Mielo1, T. Prindle1, D. J. Hackam1,2  1Children’s Hospital Of Pittsburgh Of UPMC,Pediatric Surgery,Pittsburgh, PA, USA 2University Of Pittsburg,General Surgery,Pittsburgh, PA, USA 3Children’s Hospital Of Pittsburgh Of UPMC,Neonatal Medicine,Pittsburgh, PA, USA

Introduction:   Autophagy is an adaptive response to cellular stress important in the pathogenesis of many disease states. The stress response of intestinal stem cells (ISC) has been incompletely studied. Our lab has previously elucidated the importance of TLR4 signaling in both autophagy and intestinal stem cell proliferation. We now focus on linking the role of autophagy in the intestinal stem cells with TLR4 following stress conditions. 

Methods:  Enteroids were cultured following exposure to radiation or LPS in vivo or following two days of in vitro culturing from C57/Bl6 (WT), mice lacking the critical autophagy protein ATG7 selectively in the intestinal villi (ATG7ΔIEC), and mice lacking TLR4 selectively in labeled intestinal stem cells. Whole mount imaging was performed and mRNA isolated for qt-PCR. WT, ISC (LGR5 or Bmi1) labeled, ISC labeled cells lacking TLR4, TLR4ΔIEC and ATG7ΔIEC strains (n=8/group) of mice underwent endotoxemia and radiation models at different dosages. BrdU was injected at the time of injury to evaluate differentiation from the stem cell compartment following stress. Tissue was collected for immunohistochemistry and qt-PCR for ISC, autophagy, and proliferative markers.

Results: Improved proliferation was seen from ATG7ΔIEC, TLR4ΔIEC and labeled ISC missing TLR4 enteroids compared to WT under homeostatic conditions. However, autophagy deficient enteroids showed increased apoptosis following LPS or radiation treatment both when injury occurred in vivo or in vitro. Interestingly qt-PCR also showed an exaggerated cytokine response (iNOS, IL6) in these same groups. Imaging confirmed the inability to undergo autophagy with decreased LC3 staining in apoptotic enteroids.  In vivo, stem cell and proliferative mRNA levels were markedly increased in TLR4ΔIEC and ATG7ΔIEC mice at baseline, but were unable to increase following endotoxemia models compared to WT mice. Following radiation models a robust increase in autophagy was noted in WT mice by mRNA levels but lacking  in mice missing TLR4 from the intestinal stem cells and TLR4ΔIEC.  BrdU staining showed impaired migration from the stem cell compartment in both endotoxemia and radiation models in TLR4ΔIEC, TLR4 missing from the ISC and ATG7ΔIEC mice compared to WT mice. Repeated models showed the effects to be dose dependent. Linage tracing showed following stress models ISC’s lacking TLR4 are unable to recapitulate the intestinal villi with decreased levels of LC3 staining in the stem cell compartment.

Conclusion: Autophagy through TLR4 signaling is an important mediator of the intestinal stem cell stress response. Our findings suggest that pharmacologic regulation of autophagy pathways may provide novel therapeutic approaches for diseases of impaired healing including radiation or bacterial injury. 

 

22.10 Acid Sphingomyelinase Inhibition Decreases Lung Injury after Transfusion with Stored Blood

Posted on December 22, 2014

R. S. Hoehn1, P. L. Jernigan1, E. F. Midura1, J. M. Sutton1, C. C. Caldwell1, M. J. Edwards1, E. Gulbins1,2, T. A. Pritts1  1University Of Cincinnati,Surgery,Cincinnati, OH, USA 2University Of Duisburn-Essen,Molecular Biology,Essen, ESSEN, Germany

Introduction:  Transfusion of human packed red blood cell units (pRBCs) is ideal for resuscitation after hemorrhage. However, studies have shown that use of aged pRBCs are associated with increased morbidity and mortality compared to fresh units. Our laboratory and others have demonstrated that pRBC storage results in accumulation of microparticles that cause increased lung inflammation after transfusion. Ceramide, a sphingolipid formed by the enzyme acid sphingomyelinase (Asm), is present in cell membranes and mediates a variety of cell signaling and membrane changes in red blood cells, but its role in microparticle formation is unknown. We hypothesized that Asm inhibition would lead to decreased ceramide accumulation and microparticle formation in pRBCs, with a resultant decrease in lung injury after hemorrhage and resuscitation.

Methods:  Human and murine pRBCs were obtained and treated with increasing concentrations of Amitriptyline (AT), a specific Asm inhibitor, or vehicle, then stored under standard blood banking conditions. At intervals, microparticles were isolated from pRBC units and quantified with Nanoparticle Tracking Analysis. Asm activity and ceramide concentration were measured in stored erythrocytes and microparticles. Mice underwent hemorrhage and resuscitation with equal numbers of microparticles from AT- and vehicle-treated pRBC units. Lungs were collected from shocked mice and assessed for inflammatory cytokines and histology.

Results: During storage, pRBC units demonstrated significantly increased microparticle formation, ceramide accumulation, and Asm activity. Human and murine pRBCs treated with AT formed fewer microparticles in a dose-dependent manner (Figure 1A). AT-treated pRBCs and microparticles contained less ceramide and Asm activity. After hemorrhage, mice resuscitated with microparticles isolated from vehicle-treated pRBCs had significantly elevated lung levels of pro-inflammatory cytokines including macrophage-derived chemokine (Figure 1B), IL-1b, IL-6, KC, MCP-1, MIP-1a, and MIP-2 as well as increased lung damage as determined by histology. Resuscitation with equal numbers of microparticles from AT-treated pRBCs resulted in significantly decreased lung injury.

Conclusion: Amitriptyline treatment of pRBC units leads to decreased Asm activity, ceramide formation, and microparticle accumulation. Asm inhibition caused alterations in the microparticles that led to decreased acute lung injury following hemorrhage and resuscitation. Asm inhibition represents a novel opportunity to mitigate harmful effects of resuscitation with stored pRBCs.

 

21.01 CXCL12 Reverses Hypercapnia-Induced Suppression of Epithelial Cell Migration and Lung Healing

Posted on December 22, 2014

J. A. Kanter1, H. Sun2, M. M. DeCamp1, P. Sporn2, J. I. Sznajder2, A. Bharat1  1Northwestern University,Division Of Thoracic Surgery, Feinberg School Of Medicine,Chicago, IL, USA 2Northwestern University,Division Of Pulmonary And Critical Care Medicine, Feinberg School Of Medicine,Chicago, IL, USA

Introduction:  Poor healing after lung surgery is manifested by prolonged air leaks (PAL). PAL can affect over 50% of patients with emphysema and 25% of those with normal lungs. Although PAL remains the predominant cause of morbidity and mortality following lung surgery, its pathogenesis remains unknown. Here we determined whether high carbon dioxide levels (hypercapnia) in the pleural cavity leads to poor lung healing and PAL.

Methods:  Human data was collected from an IRB-approved prospective clinical trial on patients undergoing lung resection. Intrapleural gas analysis was performed on the air leaking into the chest drainage tube using spectroscopy. Cultures were performed in chambers with adjustable CO2 levels. Cell migration was determined by scratch wound and transwell migration assays using serum-depleted medium to limit cell proliferation. Western blot and gene/RNA arrays were performed to analyze the chemokine CXCL12 and the GTPase protein Rac1.

Results:

Human: Intrapleural CO2 levels showed a strong correlation with duration of air leaks in patients undergoing lung resection (n=120, r=0.07, p<0.001). Patients with PAL had a three-fold higher level of intrapleural CO2 compared to those without (p<0.001). Intrapleural hypercapnia was associated with a 2.5-fold decreased pleural CXCL12 levels (p<0.001). Decreasing intrapleural CO2 concentration using supplemental oxygen and extrapleural suction resulted in a two-fold reduction of the duration of air leaks (n=20, p=0.001).

Laboratory: Hypercapnia (15-20%) impaired cell migration and wound healing of primary human and murine alveolar epithelial cells as well as cell lines A549 (human) and MLE12 (murine), independent of cell proliferation. Although total intracellular Rac1 was preserved, both CXCL12 and activated Rac1 were significantly reduced in hypercapnia. Exogenous CXCL12 restored intracellular activated Rac1 levels and reversed the inhibition of cell migration in hypercapnia. CXCL12 antagonist AMD-3100 or CXCL12-antibodies blocked the effects of exogenous CXCL12 in hypercapnia and suppressed cell migration in normocapnia.

Conclusion: Intrapleural hypercapnia suppresses migration of alveolar epithelial cells that are crucial for repair following lung injury. This is likely due to suppression of intracellular Rac1 activation, which is known to trigger cell migration. Further, levels of CXCL12 that can activate Rac1 are suppressed by intrapleural hypercapnia. Hence, modulation of intrapleural CO2 levels and/or CXCL12 might promote lung healing following surgery.
 

21.02 Thrombelastography is Superior to Trauma Scoring Systems as a Predictor of Massive Hemorrhage

Posted on December 22, 2014

D. Burneikas2, E. E. Moore1,2, M. P. Chapman2,3, H. B. Moore1,2, E. Gonzalez1,2, C. Silliman2,4, A. Banerjee2  1Denver Health Medical Center,Aurora, CO, USA 2University Of Colorado Denver,Aurora, CO, USA 3Georgia Health Sciences University,Augusta, GA, USA 4Children’s Hospital Colorado,Aurora, CO, USA

Introduction: Massive blood transfusion is required in roughly 3% of civilian and 8% of military trauma patients and requires mobilization of enormous hospital and personnel resources to administer. Thus, accurate prediction of the need for massive transfusion in severely injured trauma patients is highly desireable from both a logistical and patient outcome standpoint. To this end, numerous trauma scoring systems have been developed to predict massive transfusion, such as the Trauma-Associated Severe Hemorrhage (TASH) and Assesment of Blood Consumption (ABC) score. These scores are cumbersome to calculate in the chaotic setting of an emergent trauma, therefore we sought to determine if admission assessment of the patient's Rapid thrombelastogram (Rapid TEG) could provide an easier and more reliable predictive parameter for massive hemorrhage and transfusion. 

Methods:  61 consectutive trauma patients of our highest level of activation and deemed likely to require a massive transfusion by the attending surgeon received an admission Rapid TEG. Their ABC and TASH scores were calculated, as well as binary criteria based on the Resusciatation Outcomes Consortium vital sign inclusion criteria and our center's newly implemented MTP entrance trigger criteria. Reciever operating characteristic (ROC) plots were constructed for these scoring systems with respect to massive hemorrhage (defined as 10 or more units of PRBCS in the first 6 hours or death from massive hemorrhage prior to that time) as the binary outcome variable. ROC plots were also constructed for the Rapid TEG parameters: ACT, Alpha Angle, MA and LY30. Lastly, an ROC curve for Global TEG Assessment parameter was constructed based upon an abnormal finding in any of the three most specific TEG parameters for predicting massive hemorrhage (Alpha Angle, MA and LY30) in order to reflect the TEG tracing as a whole. The merits of these various predictive methodologies were compared according the area under their respective ROC curves.

Results: The area under the ROC curves for the scoring systems was generally very poor (ABC 0.50, TASH: 0.58) and the vital sign and MTP criteria no better (0.53). The TEG parameters generally performed better as predictors of massive hemorrhage based upon the are under their ROC curves (ACT: 0.63, Angle: 0.68 MA: 0.72, LY30: 0.71), skewed toward higher specificity. TH=he composite Global TEG parameter based on Angle, MA and LY30 improved sensitivity markedly and brought the overal area under the curve to 0.74

Conclusion: Admission Rapid TEG is a superior predictor of massive transfusion requirement to existing trauma scoring systems. It is particularly effective when teh TEG tracing is examined as a whole, rather than relying on any one parameter.

 

21.03 Oxidative Stress Induces Neutrophil Extracellular Traps in a TLR4- and PHOX-Dependent Mechanism

Posted on December 22, 2014

A. B. Al-Khafaji1, D. Miller2, H. Huang1, S. Tohme1, A. Tsung1  1University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA 2Beth Israel Deaconess Medical Center,Department Of Medicine,Boston, MA, USA

Introduction: Neutrophils accumulate in the liver after ischemia-reperfusion injury and contribute to inflammation-associated damage. Although intra-hepatocellular sources release reactive oxygen species after I/R, more significant to injury is neutrophils’ oxidative burst observed 6-24h after reperfusion. Neutrophils have recently been shown to extrude select intracellular contents to form a Neutrophil Extracellular Trap (NET). Stimulation of Toll-like receptors (TLRs) initiates a signaling cascade that includes activation of NADPH Oxidase (PHOX), a required step in NET formation. Superoxide has previously been shown to signal for neutrophil activation and increased proinflammatory cytokine production through TLR4, but it is unknown whether it also induces NETs. We hypothesize that in settings of non-infectious inflammation, such as oxidative stress, reactive oxygen species, specifically superoxide, induce NET formation through TLR4.

Methods: WT and TLR4KO neutrophils were treated with xanthine oxidase and its substrate hypoxanthine to generate extracellular superoxide. We inhibited xanthine oxidase by allopurinol and inhibited PHOX by diphenylene iodonium (DPI). We also performed neutrophil depletion and adoptive transfer of TLR4KO neutrophils followed by liver I/R.

Results: In vitro, WT neutrophils exposed to superoxide demonstrated elevated levels of citrullinated Histone H3, a specific NET marker, by western analysis; however, TLR4KO neutrophils expressed only basal cit-H3 despite superoxide treatment. Superoxide-inhibited neutrophils (allopurinol treatment) or PHOX-inhibited (DPI treatment) also expressed only basal cit-H3 compared to treatment with phorbol-myristate-acetate (PMA; positive control) in both WT and TLR4KO neutrophils. Thus, the activity of both TLR4 and PHOX are necessary to complete citrullination of histone H3 and chromatin decondensation. Additionally, superoxide exposure generated characteristic NET fibers in WT neutrophils, but not TLR4KO, as visualized qualitatively by immunofluorescence microscopy and measured quantitatively by mean cytoplasmic spot intensity of extracellular DNA. In vivo, MPO-DNA ELISA, a specific marker for NETs, revealed decreased NET formation in mice adoptively transferred with TLR4KO neutrophils, correlating with protection from liver injury by ALT assay.

Conclusion: In summary, our study demonstrates that superoxide induces NETs, and that WT TLR4 and functional PHOX are required for this process. During liver I/R, ROS can stimulate NETs through TLR4, while its absence precludes NET formation correlating with reduced liver inflammation and injury. Furthermore, our finding that extracellular superoxide induces NETs expands upon previous findings that superoxide activates neutrophils and increases proinflammatory cytokine production. This suggests that products of oxidative stress and damage-associated signals released in liver I/R act via ligand-receptor interactions to promote NET formation.

21.04 Staging Studies are of Limited Utility for Newly Diagnosed Clinical Stage I-II Breast Cancer

Posted on December 22, 2014

A. Linkugel1, J. Margenthaler1, A. Cyr1  1Washington University,General Surgery/College Of Medicine,St. Louis, MO, USA

Introduction:   For patients diagnosed with clinical Stage I-II breast cancer, treatment guidelines recommend against the routine use of radiologic staging studies in the absence of signs or symptoms suggestive of distant metastasis. However, these tests continue to be used for many early-stage breast cancer patients. This study aims to determine the utilization and yield of these studies at a National Comprehensive Cancer Network (NCCN) member institution.

Methods:   Female patients presenting with AJCC 7th Edition clinical stage I-II invasive breast cancer between 1998 and 2012 at Siteman Cancer Center, an NCCN member institution, were identified in a prospectively maintained institutional surgical database. Patients treated with neoadjuvant chemotherapy were excluded. Charts were reviewed to verify clinical stage and to document staging studies performed within six months of diagnosis.  Staging studies of interest included computed tomography (CT) of the chest, abdomen, and/or pelvis, bone scan, and positron emission tomography (PET).  Results of staging studies and additional diagnostic studies or procedures were recorded.  Descriptive statistics were used for the analysis.

Results:  A total of 3291 patients were included in the analysis (2044 were stage I and 1247 were stage II). Of these, 882 (27%) received CT of the chest, abdomen, and/or pelvis; bone scan; or PET within 6 months of diagnosis. A total of 691/882 (78%) received chest CT, 705/882 (80%) abdominal/pelvic CT, 704/882 (80%) bone scan, and 70/882 (8%) PET. Of these 882 patients, 312 were stage I (15% of the stage I cohort) and 570 were stage II (46% of the stage II cohort). Of the 882 patients imaged, 194 (22%) required additional imaging (x-ray, CT, bone scan, sonogram, or PET) and/or biopsies to follow-up abnormalities seen on the staging studies. However, only 11 of those 194 (6%) were confirmed to have metastatic disease (1.2% of the 882 imaged patients, 0.33% of the total study cohort). Of these 11 patients, one was clinically stage I at presentation, and 10 were stage II. Metastatic sites identified included lung (n=3), bone (n=4), liver (n=1), and a combination of sites (n=3). Numbers of patients determined to have metastatic disease were too small for comparative analysis.

Conclusions:  The identification of distant metastasis among clinical Stage I-II patients in this study was rare (0.33% of the total cohort). Even among patients judged appropriate for staging studies (CT, bone scan, and/or PET), only 1.2% were diagnosed with metastatic disease. These findings suggest that even at an NCCN member institution, staging studies are overused and lead to additional procedures in over 20% of patients.

21.05 Prevalence and Impact of Admission Hyperfibrinolysis in Severely Injured Pediatric Trauma Pateints

Posted on December 22, 2014

I. N. Liras1, B. A. Cotton1, J. C. Cardenas1, M. T. Harting1  1University Of Texas Health Science Center At Houston,Houston, TX, USA

Introduction:  Hyperfibrinolysis (HF) on admission is associated with increased mortality in adult trauma patients. Several studies have demonstrated that 9% of severely injured adults present to the emergency department (ED) with HF. The purpose of the current study was to (1) define HF in pediatric patients and a relevant cut-point for therapeutic intervention (if any), (2) identify the prevalence of HF in severely injured pediatric patients, and (3) determine if HF on admission is as lethal a phenomenon as it is in adults. 

Methods:  Following IRB approval, we identified all pediatric trauma admissions (≤17 years old) that met highest-level trauma activation criteria between 01/2010 and 12/2013. Fibrinolysis rates were determined using LY-30 by rapid thrombelastography (rTEG),which represents the percent reduction of the maximal clot amplitude (fibrinolysis) 30 minutes after such amplitude is achieved. HF was defined a priori as initial LY-30 inflection point that translated to a doubling of mortality. Two previous studies in adults demonstrated an inflection point of ≥3%; where mortality doubled from 9 to 20%. We began by identifying a relevant inflection point to define HF and its prevalence, followed by univariate analysis to compare HF and non-HF patients. Finally, a purposeful logistic regression model was developed to evaluate predcitors of mortality in severely injured pediatric patients. 

Results: 819 patients met study criteria. LY-30 values were plotted against mortality. A distinct inflection point was noted at ≥3%, where mortality doubled from 6 to 14%. Of note, mortality continued to increase as the amount of lysis increased, with a 100% mortality demonstrated at an LY-30 ≥30% (compared to 77% in adults).  Using LY-30 ≥3%, patients were stratified into HF (n=197) and non-HF (n=622), with prevalence on admission of 24%. With the exception of HF patients being younger (median 11 vs. 15 years; p<0.001), there were no differences in demographics, scene vitals or injury severity scores between the groups.  On arrival to the ED, HF patients had a lower systolic blood pressure (median 118 vs. 124 mmHg) and lower hemoglobin (median 12.2 vs. 12.7 g/dL); both p<0.001). Controlling for age, arrival vital signs, admission hemoglobin and injury severity (ISS), logistic regression identified admission LY30 ≥3% (OR 6.2, 95% CI 2.47-16.27) as an independent predictor of mortality.

Conclusion: Similar to adults, admission HF appears to reach a critical threshold at LY30 ≥3% in pediatric patients. Admission HF in pediatric patients occurs more frequently than in adults (24 vs. 9%) but is similarly associated with a doubling in mortality (6 to 14%). Admission LY-30 ≥3% carries a 6-fold increased likelihood of mortality in severely injured pediatric patients. HF on admission may serve to rapidly identify those injured children and adolescents likely to benefit from hemostatic resuscitation efforts and to guide anti-fibrinolytic therapy. 

21.06 Increased Malignancy Rates in Surgical Patients with Incidentally-Discovered Thyroid Nodules

Posted on December 22, 2014

A. R. Marcadis1, M. Rodriguez1, S. Liu1, B. Wang1, J. I. Lew1  1University Of Miami Miller School Of Medicine,Division Of Endocrine Surgery,Miami, FL, USA

Introduction: With the advent of better imaging technology and its widespread utilization in the clinic setting, incidental thyroid nodules are often discovered while evaluating patients for unrelated disease. If the risk of underlying thyroid malignancy in such incidental nodules is common, further evaluation is required. However, if thyroid cancers are exceedingly rare in such incidentally discovered thyroid nodules, further costly evaluations can be avoided. The purpose of this study is to compare the rate of malignancy in incidentally-discovered thyroid nodules (IDTN) by imaging to nonincidentally-discovered thyroid nodules (NDTN) in surgical patients.

Methods: A retrospective review of prospectively collected data of 1370 patients who underwent thyroidectomy at a single institution was performed. Before surgical resection, all patients underwent surgeon-performed ultrasound (SUS) and fine-needle aspiration (FNA). Patients who had IDTN by imaging studies for unrelated reasons (n=536) and patients who had NDTN (n=834) were further stratified according to age, gender, FNA results, SUS characteristics and final pathology. Rates of malignancy in IDTN and NDTN were calculated and statistical significance was determined by  two-tailed Z-test.

Results: Of 1370 patients, 536 presented with IDTN by imaging and 306 (57%) were found to have malignancy on final pathology. Of patients with IDTN found to have malignancy, 92% (n=283) had papillary thyroid cancer, 5% (n=14) medullary thyroid cancer, and 3% (n=9) follicular thyroid cancer. Of the patients with NDTN, 401 (48%) were found to have malignancy following surgery with 44% (n=366) papillary thyroid cancer (PTC), 2.2% (n=18) medullary thyroid cancer, 2% (n=14) follicular thyroid cancer, and <1% anaplastic thyroid cancer (n=3). The rate of PTC on final pathology for IDTN was statistically higher than the rate of PTC found in NDTN (p<0.05). Patients with IDTN had statistically higher rates of microcalcifications (34%), hypoechogenicity (60%), and irregular borders (35%) by SUS compared to patients with NDTN (p<0.05). There was also a significantly higher rate of patients with IDTN who had all three SUS features together (14%) compared to patients with NDTN (10%) (p<0.05). There was no significant difference in percentage of malignant FNA results between patients with IDTN and NDTN.

Conclusion: For IDTN, malignancy rates may be higher than expected in surgical patients. Furthermore, certain characteristics of SUS may help predict malignancy in IDTN. The high rate of malignancy suggests that total thyroidectomy by an experienced surgeon should be strongly considered when managing such patients with IDTN.

 

21.07 GelE/sprE are critical for Enterococcus faecalis-induced anastomotic leak in a rat model

Posted on December 22, 2014

J. N. Luo1, B. A. Shakhsheer1, R. Klabbers2, A. Zaborin1, N. Belogortseva1, O. Zaborina1, J. C. Alverdy1  1The University Of Chicago Pritzker School Of Medicine,Department Of Surgery,Chicago, IL, USA 2Radboud University Nijimegen Medical Centre,Department Of Surgery,Nijimegen, GELDERLAND, Netherlands

Introduction:  Anastomotic leak following colorectal surgery is a dreaded complication of which the cause remains unknown. Our laboratory has recently established that high collagenase producing strains of Enterococcus faecalis (E. faecalis) “bloom” in the gut following anastomotic injury and are both necessary and sufficient to cause anastomotic leak in rats.  Here we performed a mutational analysis to define the roles of gelE and sprE – co-regulated genes that control: adherence, penetration and collagenase production – on anastomotic leak.  We used a double knockout mutant lacking gelE and sprE derived from E. faecalis V583 (a vancomycin-resistant clinical isolate). We hypothesize that mutants deficient in both genes (ΔgelEΔsprE) would be attenuated in their capacity to cause leak in rats. 

Methods:  Adult rats (n=24) underwent colon resection followed by recto-sigmoid anastomosis. Following anastomotic construction, 5cc of 107 CFU/ml of either the double mutant deficient in both gelE and sprE (ΔgelEΔsprE), or the mutant complemented with gelE and sprE (ΔgelEΔsprE/gelE+sprE- termed VT07) were administered via rectal enema to inoculate the anastomosis. On postoperative day six, all rats were sacrificed and evaluated for evidence of anastomotic leak via previously established criteria. Anastomotic tissues were harvested and separated into mucosal, serosal, and perianastomotic samples for bacterial species identification via culture and phenotype analysis (collagenase activity).
 

Results: Anastomotic leakage was observed to be significantly greater in rats rectally inoculated with the complemented strain VT07 that produces high collagenase compared to its matched double mutant ΔgelEΔsprE that produces no collagenase (p<0.01). Leaks were characterized by dense perianastomotic adhesions, perianastomotic abscesses, and occasional gross anastomotic dehiscence.  The most severe leaks were identified to be associated with high adherence of E. faecalis to the mucosa, penetration into the serosa, high collagenase activity, and culture positivity of perianastomotic tissues. Dense mucosal colonization alone was not associated with leak. 
 

Conclusion: High collagenase producing E. faecalis present at and within anastomotic tissues appears to play a critical role in the pathogenesis of anastomotic leak. Although various genes are likely to confer its pathogenic potential at healing anastomotic tissues, we show in this study that gene products and pathways regulated by the gelE/sprE system that control adherence, penetration, and collagenase/protease production are likely to play a key role.  Because E. faecalis is a ubiquitous commensal organism in humans, and only certain strains of it are associated with anastomotic leak, a more complete understanding of the microbial pathogenesis and phenotypic expression could enable us to better predict leak and thus improve patient management

21.08 Treatment of Necrotizing Enterocolitis with Enteral Intestinal Alkaline Phosphatase

Posted on December 22, 2014

B. Biesterveld1, N. Heinzerling2, R. Rentea2, S. Welak3,4, K. Fredrich2, D. Gourlay2,5  1Medical College Of Wisconsin,Milwaukee, WI, USA 2Medical College Of Wisconsin,Surgery,Milwaukee, WI, USA 3Medical College Of Wisconsin,Pediatrics,Milwaukee, WI, USA 4Children’s Hospital Of Wisconsin,Neonatology,Milwaukee, WI, USA 5Children’s Hospital Of Wisconsin,Pediatric Surgery,Milwaukee, WI, USA

Introduction:  Necrotizing enterocolitis (NEC) is the most common surgical emergency of the neonate. Previously, we demonstrated intestinal alkaline phosphatase (IAP) activity to be decreased in a neonatal rat model of NEC.  Furthermore, enteral IAP supplementation before the onset of NEC prevented NEC development, reduced intestinal inflammation and maintained gut barrier function. It is not known if IAP given after NEC onset can reverse the course of the disease. We hypothesized that enteral IAP given after induction of NEC would not be able to reverse intestinal injury.

Methods:  Sprague Dawley pups were delivered 1-day preterm followed by NEC induction with LPS added to formula and hypoxia exposure. All pups received NEC stressors on days 0 and 1. They were subsequently divided into 4 groups: continued NEC stressors (NEC), continued NEC stressors with enteral IAP (NEC+IAP), withdrawal of NEC stressors then formula fed (NEC then FF) or withdrawal of NEC stressors then formula fed with supplemental IAP (NEC then IAP) on days 2,3. Control pups were spontaneously delivered and dam-fed. All pups were then sacrificed on day 4. NEC severity was scored based on H&E stained terminal ileum sections, and AP activity was measured using a colorimetric assay. IAP and IL-6 expression were measured using RT-PCR.

Results: Intestinal AP activity in the NEC group was significantly decreased 0.18 U/mg compared to controls of 0.57 U/mg (p<0.001). Discontinuation of NEC stressors after 2 days (NEC then FF) was associated with increased AP activity to 0.36 U/mg (p<0.01 vs. NEC). IAP supplementation in matched groups, NEC+IAP and NEC then IAP, did not impact AP activity. IAP mRNA expression followed a similar trend as activity, with significantly decreased IAP expression in NEC which significantly increased with withdrawal of NEC stressors on day 2. Similarly, the NEC group had significantly worse mean injury score (2.25 vs. 0.67 in control (p<0.01).  Discontinuation of NEC stressors on day 2 resulted in improved intestinal injury scores to 1.14 compared to continued NEC stressors (p<0.01).  While, IAP supplementation (NEC then IAP) significantly decreased IL-6 expression two-fold compared to NEC (p<0.05), it did not decrease the intestinal injury when compared to matched groups in NEC vs. NEC+IAP (p=0.50) and NEC then FF vs. NEC then IAP (p=0.54).

Conclusion: This is the first work to demonstrate that the current clinical treatment, to remove the source of NEC, improves intestinal damage and this is associated with a return of IAP expression and activity. When used as a rescue treatment after NEC onset, IAP decreased intestinal inflammation though did not impact the severity of histologic injury. Given the often rapid progression of NEC, and our previous work demonstrating the ability of early IAP supplementation to prevent NEC development, this data supports the use of IAP as a preventive therapy given to all those identified as at risk early in life.

 

21.09 Delivery of Monocyte Lineage Cells in a Biomimetic Scaffold Enhances Tissue Repair

Posted on December 22, 2014

G. G. Walmsley1,2, M. S. Hu1, K. Weiskopf2, R. C. Rennert1, M. Januszyk1, Z. N. Maan1, D. Duscher1, K. Senarath-Yapa1, A. J. Whittam1, R. Tevlin1, D. A. Atashroo1, I. L. Weissman2, H. P. Lorenz1, G. C. Gurtner1, M. T. Longaker1,2  1Stanford University School Of Medicine,Department Of Surgery, Division Of Plastic & Reconstructive Surgery,Stanford, CA, USA 2Stanford University School Of Medicine,Institute For Stem Cell Biology And Regenerative Medicine,Stanford, CA, USA

Introduction:
Macrophages are thought to play a critical regulatory role in many stages of wound healing, including angiogenesis, reepithelialization, and remodeling. Evidence for the importance of macrophages in these processes comes from experiments demonstrating impaired wound healing in mice following DTR-based ablation of macrophages, genetic knockout of G/M-CSF, or administration of anti-macrophage antiserum. We have previously shown that transplantation of macrophages into excisional wounds on wild type (FVB/NJ) mice significantly increases the rate of wound healing. Here, we expand the analysis to include diabetic wound healing and monocyte transplantation.

Methods:
Macrophages derived from the bone marrow of L2G (FVB-Tg(CAG-luc,-GFP)L2G85Chco/J) were seeded on pullulan-collagen hydrogels and transplanted onto splinted excisional wounds on the dorsum of diabetic (FVB.BKS(D)-Leprdb/ChuaJ) mice. Human monocytes isolated from drawn blood were similarly transplanted on pullulan-collagen hydrogels onto splinted excisional wounds on the backs of immunodeficient nude (Foxn1nu) mice. Histologic analysis allowed for in vivo tracking of the survival, localization, and phenotype of transplanted macrophages and monocytes. Microfluidic single-cell gene expression analysis of transplanted L2G macrophages (GFP+Luc+) FACS-isolated on the basis of GFP expression from cutaneous wounds provided further insight into macrophage phenotype and behavior during wound healing.

Results:
L2G macrophage-seeded hydrogels improved wound healing compared to un-seeded hydrogel controls on days 4-20 (*p<0.01) in diabetic mice. The average time for complete wound healing was 17.2 days in the macrophage group versus 20.3 days in the control group (*p<0.001). IVIS imaging revealed survival of transplanted macrophages in diabetic wounds through day 20 of wound healing. Microfluidic single-cell gene expression analysis revealed that macrophages transplanted into wounds displayed a predominantly M2 phenotype after being in the wound environment for 24 hours. Human monocyte-seeded hydrogels significantly improved healing compared to un-seeded control hydrogels. The average time to complete healing was 17.8 days in the monocyte group versus 21 days in the control group (*p<0.005). Histologic analysis of monocyte treated wounds showed that transplanted monocytes differentiate in vivo to a predominantly M2 phenotype after 48 hours in the wound environment. Importantly, scar size and quality was not affected in wounds receiving either monocyte or macrophage transplant as compared to controls.

Conclusion:
Here we demonstrate that by increasing the number of monocyte lineage cells in the wound site above physiologic levels in diabetic and nude mice the rate of wound healing can be significantly accelerated with no adverse impact on the quality of repair. These findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.

21.10 Metabolic Derangement of Coagulation: A Likely Suspect For Post Operative Bleeding

Posted on December 22, 2014

G. D. Wiener1, H. B. Moore1, P. Lawson1, E. Gonzalez1, M. P. Chapman1, A. P. Morton1, A. Sauaia1, A. Banerjee1, E. E. Moore2  1University Of Colorado Denver,Aurora, CO, USA 2Denver Health Medical Center,Aurora, CO, USA

Introduction: Recently it has been appreciated that bile acids can promote degradation of fibrin sealant.  The concept of metabolites changing coagulation may explain persistent localized bleeding from the liver despite normalization of systemic coagulation.  We hypothesize that bile acid impairs whole blood clot formation and promotes fibrinolysis.

Methods: Blood was collected from healthy volunteers (n=6) into citrated tubes.  Taurcholic acid (TUCA) was titrated in vitro into whole blood with a range from 250 µM to 1000 µM.  This range was selected as previous literature indicated that systemic TUCA levels could exceed 600 µM.  Whole blood mixtures were assayed using thrombelastography (TEG) to quantify clot strength (MA) and degree of fibrinolysis (LY30).  Tranexamic acid (TXA) was used to block plasmin mediated fibrinolysis.  Statistical analysis used SPSS software.  Correlations between TEG parameters and concentration of TUCA were analyzed using Spearman's Rho Test, and the Wilcoxon test was used for pair-wise comparisons.

Results: Clot strength had a negative correlation to dose of TUCA (Spearman’s Rho = -0.677, p<0.001).  Median whole blood MA was 56.25 (IQR 53.25-58.50), which decreased to a median MA of 42.00 (IQR 38.00-48.50) at the highest dose of TUCA.  Ly30 had a positive correlation to dose of TUCA (Spearman’s Rho = 0.702, p<0.001).  Median whole blood Ly30 was 1.35 (IQR 0.8-1.73), which increased to a median Ly30 of 4.0 (IQR 2.85-10.3) at the highest dose of TUCA.  At a dose of 750 µM TUCA, TXA reduced Ly30 from 7.7 (IQR 5.7-14.8) to 2.9 (IQR 1.9-3.3), p=0.028, but did not have a significant effect on MA (p=0.249).  At the highest dose of 1000 µM TUCA, TXA did not significantly reduce Ly30 (p=0.658) and MA was unchanged by TXA at this dose (p=0.144).

Conclusion: Bile acid has a dose response in reducing clot strength and promoting fibrinolysis.  This is consistent with previous literature that bile has clot degradation properties.  The reduction in clot strength also suggests platelet inhibition, which is not correctable by TXA.  This metabolic effect on coagulation warrants further investigation, as localized areas of the body, particularly the liver, with high levels of bile acid may be at risk for post-operative bleeding.

22.01 A Novel System for Supplemental Funding of Surgical Graduate Medical Education

Posted on December 22, 2014

M. R. Dimon1, B. H. Ahmed1, P. Pieper1, B. Burns1, J. J. Tepas1  1University Of Florida College Of Medicine – Jacksonville,General Surgery,Jacksonville, FL, USA

Introduction:

In July 2014 the Institute of Medicine released a review of the governance and financing of Graduate Medical Education (GME).  It concluded that major changes to GME financing were needed to redesign the system to reward desired performance and reshape the physician workforce to better meet the nation’s needs.  A 10 year transition period was recommended during which the role of Medicare in GME funding could be altered, phasing out the current system.  Anticipating significant changes in funding, we investigated an alternate funding system based on resident reimbursement for the provision of care.  We aim to show that resident billing of assistant surgeon fees in appropriate cases could generate significant income to support a GME program.

Methods:

The Department of Surgery Business Group Manager provided CPT codes for procedures performed by the General/Acute Care surgical services with resident involvement at our institution between July 1, 2011 and June 30, 2012. For each code, the charge and total instances were provided. CPTs allowing an assistant fee were identified using the Searchable Medicare Physician Fee Schedule, and this fee was calculated as 20% of the primary charge. This fee was multiplied by the number of CPT instances, resulting in the total potential resident contribution for each code. These were summed to determine the total potential resident contribution to GME funding.

Results:

A total of 515 unique CPTs were billed for a total of 6,578 cases with resident involvement, of which 2,552 (39%) were reimbursable.  The allowable CPTs generated $1,882,854 in billable assistant charges.  The top 50 most frequently performed CPTs resulted in 4,247 procedures, 65% of the total.  Within the top 50, 1362 CPTs (32% of the top 50, 21% of the total) were reimbursable.  Of the total billable assistant charges, $963,227.00 (51%) occurred in the top 50 most frequent procedures.

Conclusion:

Assistant surgeon billing would average $67,244.76 per resident when evenly distributed within our program.  This compares to our current Medicare Direct and Indirect GME funding level of $142,635.78 per resident.  Use of this system could therefore provide 47% of our current CMS funding.  While this would not entirely fund our GME program, it would be a significant source of added funding should changes in the current Medicare funding levels lead to a decrease in monies directed towards surgical training. Moreover, the obvious skew in distribution of procedures in which 21% (billable portion of the top fifty procedures) drives 51% of charges suggests that this system could monitor resident experience and possibly guide a more balanced operative experience.  Performance and competency based credentialing of residents could be used to ensure appropriate personnel are involved with cases billing for assistant fees; furthermore, reimbursements could be adjusted based on quality metrics to provide a tool for transformational change in GME and patient outcomes.

22.02 Heme oxygenase-2 Protects In Hemorrhage/Resuscitation Via Regulation of Hypoxic Responses

Posted on December 22, 2014

J. Luclano1, B. Kautza1, P. Waltz1, B. Zuckerbraun1,2  1University Of Pittsburg,Pittsburgh, PA, USA 2VA Pittsburgh Healthcare System,Pittsburgh, PA, USA

Introduction: Tissue and cellular hypoxia are a component of many disease processes, including shock states and ischemia/reperfusion insults.  Heme oxygenase (HO) enzymes, which are the rate limiting enzymes in the breakdown of heme to free iron, biliverdin, and carbon monoxide (CO), are critical to the maintenance of cellular homeostasis.  HO enzymes have been implicated in the sensing of oxygen levels and the regulation of oxygen consumption. Additionally, mitochondrial responses are critical to changes in oxygen sensing.  Mitochondria act as critical rheostats within a cell to orchestrate cellular responses to various stimuli, including hypoxia. The purpose of these investigations was to test the hypothesis that HO-2 protects againts hemorrhagic shock or hypoxia induced injury.  Furthermore that HO-2 serves as a critical regulator of hypoxic responses in hepatocytes via modulation of mitochondrial signaling.

Methods: C57BL/6 male mice were hemorrahged to a MAP of 20 mmg for 30 minutes and then resuscitated with 2X the maximum shed blood volume with lactated Ringers.  Some mice were pretreated with control siRNA or HO-2 specific siRNA 72 hours prior to  hemorrhage and resuscitation.  Primary hepatocytes were harvested and cultured from C57BL/6 or gp91phox-/- mice.  Cells were exposed to standard culture conditions (5%CO2, and 21% O2) or hypoxia (5%CO2, 1%O2) for 0-12 hours.  In some experiments hepatocytes were pretreated with control siRNA, HO-1 siRNA, or HO-2 siRNA.  Additionally, tin protoporphyrin (SnPP) was utilized as a pharmacological inhibitor of HO.  Western blots or immunohistochemistry for HO-1, HO-2, hypoxic signaling proteins, mitochondrial fusion pathways, and oxidative phosphorylation proteins were performed.  Mitochondrial reactive oxygen species were measured by mitosox fluorescence.  ANOVA was used for statistical analysis and significance wsa assumed a s P<0.05.

Results:  HO-2 siRNA pretreatment effectively limited expression of HO-2 in the liver and exacerbated organ injury ( serum ALT increased 2.5+/-0.9 fold over control siRNA, shocked mice, P<0.05). Additionally, there were significant increases in serum markers of inflammation (IL-6 and TNF-alpha).HO-2 but not HO-1 was expressed in hepatocytes at baseline and was localized within mitochondria.  SnPP or HO-2 siRNA, but not HO-1 siRNA inhibited hypoxia induced mitochondrial ROS.  Furthermore, hypoxia increased HIF1a protein levels, and this was inhibited by HO-2 siRNA or SnPP.  Furthermore, 12 hours of hypoxia exposure led to increased expression of proteins of oxidative phosphorylation and this was inhibited by HO-2 siRNA.

Conclusion:  HO-2 limits organ injury and inflammation in hemorrhagic shock and resuscitation.  This may be through the influence on hypoxia induced mitocohndrial signaling.  Understanding critical adaptive responses in hypoxic type of insults are critical to impriving care and the development of future therapeutics.

 

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