43.07 Is Omega-3 Synergistic with Glucagon-Like Peptide-2 in Intestinal Failure?

Posted on January 18, 2016

A. Karmaker1,2, C. M. Costanzo1,2, M. Z. Schwartz1,2 1St. Christopher’s Hospital For Children,Surgery,Philadelphia, PA, USA 2Drexel University College Of Medicine,Philadelphia, Pa, USA

Introduction: Previous studies have demonstrated that glucagon-like-peptide-2 (GLP-2) enhances mucosal mass and function in residual intestine after massive small bowel resection (MSBR). Luminal omega-3 fatty acid (OM-3) reduces inflammation and may have a growth factor-like effect on small intestine (SI). However, there are few studies that have investigated the effects of OM-3 on bowel adaptation after MSBR. This study was designed to evaluate OM-3 alone and in combination with GLP-2, to determine a potential synergistic effect on intestinal growth and function using a rat model of intestinal failure.

Methods: 60 adult female Sprague-Dawley rats underwent 80% MSBR and were divided into 4 groups (N=15/group): Group 1: IV Saline (Control)+regular feeds; Group 2: IV GLP-2+regular feeds; Group 3: IV Saline+OM-3 enriched feeds; and Group 4: IV GLP-2+OM-3 enriched feeds(GO). 5 animals per group were sacrificed at 7, 14, and 28 days. SI mucosa was harvested. DNA and protein content were measured as markers of mucosal mass. Galactose and Glycine absorption were measured in a 10 cm segment of remaining SI using an in-vivo closed, recirculation technique at 28 days. Statistical analysis was done by ANOVA with post-hoc Tukey’s HSD test.

Results:7 Days: DNA was increased in all treatment groups compared to control(p<0.05) but not when compared to each other. Protein was increased in all treatment groups compared to control(p<0.05). Both OM-3 and GO groups had increased protein compared to GLP-2 only group(p<0.05) but not compared to each other.14 days: OM-3 and GO groups had increased DNA compared to control(p<0.05). Both OM-3 and GO groups had increased DNA compared to GLP-2 group (p<0.001) but not compared to each other. Protein was increased in all treatment groups compared to control(p<0.05). The OM-3 group had increased protein compared to the GLP-2 group(p<0.05), but GO group did not have increased protein compared to GLP-2 or OM-3 groups(p>0.05). 28 days: DNA was increased in all treatment groups compared to control(p<0.05). Both OM-3 and GO groups had increased DNA compared to GLP-2 group(p<0.005) but not compared to each other. The OM-3 and GLP-2 groups had increased protein when compared to control(p<0.05) and each other (p<0.005). All treatment groups had increased galactose and glycine absorption compared to the control group(p<0.005) but not compared to each other (Fig. 1).

Conclusion:Individually, GLP-2 and OM-3 are very effective in enhancing the adaptive process by increasing mucosal mass and function, at all three time points. More importantly clinically, GLP-2 and OM-3 increase substrate absorption in a rat model of intestinal failure. However, the combination does not show any synergism.

43.08 Attenuated Expression of Nuclear Receptor Family NR4A in Familial Diverticulitis

Posted on January 18, 2016

C. S. Choi1, Y. Imamura Kawasawa3, G. S. Yochum4, L. R. Harris1, S. M. Deiling1, W. A. Koltun1 1Penn State Hershey Medical Center,Division Of Colon & Rectal Surgery, Department Of Surgery, Penn State University College Of Medicine,Hershey, PA, USA 3Penn State University College Of Medicine,Genome Sciences Facility Institute For Personalized Medicine,Hershey, PA, USA 4Penn State University College Of Medicine,Department Of Biochemistry & Molecular Biology,Hershey, PA, USA

Introduction: There is increasing evidence that diverticulitis has a significant genetic predisposition. We sought to identify possible genes involved in diverticulitis by performing RNA-Seq on a group of youthful surgical diverticulitis patients with a positive family history (FD) compared to 1) a group of typical elderly diverticulitis patients (ED) without family history and 2) control patients without evidence of diverticulosis (C).

Methods: The FD group consisted of five surgically managed patients under the age of 50 (mean age 46, 3M/2F) each patient having at least one first-degree relative with diverticulitis. The ED group consisted of five surgically managed patients over age 65 (mean age 72, 3M/2F) without a family history of diverticulitis. Full thickness bowel wall from patients in both FD and ED groups were obtained from both diseased (Ds) and adjacent normal (Nl) sigmoid colon. The C group had single normal samples from five patients (mean age 50, 2M/3F) who required sigmoid resection (endometriosis: 2, polyp/cancer: 3), without diverticulosis. Total RNA was extracted from fresh-frozen tissue specimens, quality confirmed with RNA Integrity Number > 7.0, converted to cDNA and barcoded for next generation sequencing. Pooled libraries were loaded onto an Illumina HiSeq 2500 and run for 50 cycles using the single-read protocol setting. Quality filtered sequencing reads were de-multiplexed and aligned to the human reference genome (hg38) using Tophat. RPKM (Reads Per Kilobase per Million mapped reads) values were calculated after applying GC-content and quantile normalizations using an R package ‘cqn.’ An R package ‘limma’ identified differentially expressed genes between tissue groups based on empirical Bayes moderated t-statistics and associated P-values.

Results: 58 genes were differentially expressed (p<0.015) between FD-Nl vs. ED-Nl and/or C. Based on biological function and eliminating non-coding transcripts and pseudogenes, 3 genes of the same nuclear receptor superfamily (NR4A1, NR4A2, NR4A3) clustered, sharing similar expression profiles. Expression of these genes was significantly decreased in FD-Nl compared to ED-Nl and C groups. In addition, these gene transcripts were reduced in FD-Ds vs. ED-Ds. There was no significant difference between Ds and Nl tissues in either FD or ED.

Conclusion: Expression of NR4A family of nuclear receptors, which are modulators of immunity via the NF-κB pathway and known to be altered in several inflammatory states such as atherosclerosis and psoriasis, was decreased in both diseased and normal sigmoid of FD vs. ED patients. This suggests a possible genetic/molecular mechanism for youthful onset FD.

43.03 Epigallocatechin-3-gallate Upregulates mir-221 to Inhibit Osteopontin-Dependent Hepatocyte Injury

Posted on January 18, 2016

M. L. Arffa5, M. A. Zapf5, A. N. Kothari1, V. Chang5, G. N. Gupta1, X. Ding1, M. M. Al-Gayyar2,4, W. Syn3, N. M. Elsherbiny4, P. C. Kuo1, Z. Mi1 1Loyola University Chicago Stritch School Of Medicine,Department Of Surgery,Maywood, IL, USA 2University Of Mansoura,Department Of Clinical Biochemistry,Mansoura, DAKAHLIA, Egypt 3Institute Of Hepatology,London, LONDON, United Kingdom 4University Of Tabuk,Department Of Pharmaceutical Chemistry,Tabuk, TABUK, Saudi Arabia 5Loyola University Chicago Stritch School Of Medicine,Maywood, IL, USA

Introduction: Osteopontin (OPN) expression is upregulated in liver injury and promotes fibrosis. Epidemiological studies suggest that green tea consumption is associated with lower incidence of liver disease. Epigallocatechin-3-gallate (EGCG) is a polyphenol found in green tea that abrogates hepatic fibrosis. Our previous results show that EGCG reduces OPN protein expression by increasing mRNA degradation. EGCG upregulates expression of multiple miRNAs in HepG2 cells, including miRNA 221. In Silico analysis identified the 3’UTR of OPN mRNA as a potential target for miRNA 221. Since miRNA expression varied depending on EGCG treatment protocols, we also identified miRNA 181a and 10b as candidates. We investigated whether these miRNAs mediate EGCG dependent OPN mRNA decay and their role both in vitro using HepG2 cells and in vivo using a rat model of thioacetamide (TAA) liver injury and fibrosis.

Methods: HepG2 cells were treated with EGCG [0.02-20µg/ml], and real time-PCR (RT-PCR) was used to quantify miRNA expression. Antagomirs and mimics were transfected. OPN protein was quantified by ELISA. MicroRNA was extracted from rat liver tissue and quantified by RT-PCR. Immunohistochemical (IHC) staining was performed for trichrome, p53, ki67, and OPN. IHC profiler was used to analyze OPN staining.

Results: EGCG treatment [0.02-20 µg/ml] of HepG2 cells increased expression of miRNAs 221, 181a, and 10b (p<0.05), with 221 having the highest expression. 2µg/ml of EGCG was chosen for downstream experiments because it is physiologically relevant. 2µg/ml EGCG decreased OPN protein levels by ~0.6 fold (p<0.01) while antagomir inhibition of only miRNA 221 reversed this effect (p<0.01). However, all three mimic miRNAs caused a ~0.6-fold decrease in OPN protein levels (p<0.01). Hepatic tissue from TAA rats treated with EGCG had upregulated expression of all three miRNAs (p<0.05). IHC analysis showed that TAA treated rats developed mild to moderate steatohepatitis and fibrosis. Rats simultaneously treated with EGCG showed no signs of liver damage. TAA rats had increased expression of ki67, p53, and OPN, indicating increased hepatocyte proliferation, apoptosis, and fibrosis, respectively, while EGCG+TAA rats did not have increased expression.

Conclusion: In in vitro and in vivo models of TAA induced hepatocyte injury, EGCG inhibits OPN dependent injury and fibrosis by primarily upregulating miRNA221 to accelerate OPN degradation. EGCG may have utility as a protective agent in settings of liver injury.

43.04 Cellular mechanism of increased anti-tumor efficacy of paclitaxel-loaded polymer films

Posted on January 18, 2016

K. J. Hachey1, R. Liu1, M. Grinstaff2, Y. Colson1 1Brigham And Women’s Hospital,Thoracic Surgery,Boston, MA, USA 2Boston University,Departments Of Biomedical Engineering And Chemistry,Boston, MA, USA

Introduction: Curative resection for the treatment of sarcoma is a significant challenge due to tumor size and anatomic complexity, resulting in 5-year locoregional recurrence (LRR) rates of over 50% and relative resistance to systemic chemotherapy and radiation. We previously demonstrated that local treatment with paclitaxel (Pax)-loaded polymer films after partial tumor resection in a murine xenograft model of human chondrosarcoma resulted in improved LRR and survival compared to adjuvant systemic Pax. This study aims to characterize mechanisms of cytotoxicity induced by Pax films that may explain improved outcomes with local drug delivery.

Methods: Pax drug effect was assessed in the human chondrosarcoma cell line CS-1 at 1000nM and 10nM Pax, which approximate in vivo concentrations previously measured in local tissues one day after treatment with Pax films and systemic Pax, respectively. In vitro incubation with Pax for 30hr continuous or 4hr pulse dose (with additional 26hr incubation in fresh media) was perfomed to evaluate response to Pax as a function of dose and duration. CS-1 monolayers treated with these regimens were harvested at 30hrs for cell cycle analysis or inoculation into Nu/J mice (n=7/group) to assess differences in tumor volume and disease-free survival (DFS).

Results: Treatment with 1000nM pax for both 4hr pulse dose and 30hr continuous incubation in vitro induced a predominant G2/M peak on flow cytometric analysis by 30hrs, with a decline in p53 expression below control levels. In contrast, cell cycle profiles and p53 levels in the 10nM group remained similar to controls even after 30hrs. When treated monolayers were harvested at 30hrs and injected in vivo, treatment with 1000nM Pax resulted in improved DFS with both treatment durations (fig). The 4hr and 30hr 1000nM Pax-treated groups showed statistically significant reductions in measured tumor volume at 28 days post-treatment compared to 10nM groups treated for the equivalent duration (p=0.01 and p=0.007, respectively).

Conclusion: CS-1 monolayers exposed to 1000nM Pax, as demonstrated during Pax film treatment in vivo, undergo a G2/M arrest that correlates with significantly reduced in vivo tumor growth and improved DFS. In contrast, monolayers treated with 10nM Pax, as demonstrated following systemic Pax in vivo, appear relatively resistant to G2/M accumulation, or ‘escape’ from mitotic arrest, resulting in inferior DFS. Cell death during a p53-independent G2/M arrest, with microtubule damage and mitochondrial collapse, is a hallmark of cytotoxicity at high Pax concentrations but both dose and duration appear important for improved efficacy as achieved by polymer films in vivo compared to equivalent bolus dose Pax given systemically.

43.05 Resveratrol Promotes EGFR Expression and Enhances Fatty Liver Regeneration in a Murine Model

Posted on January 18, 2016

D. P. Milgrom1, X. Jin2, Y. Jiang1, L. Koniaris1, T. Zimmers1 1Indiana University,Department Of Surgery,Indianapolis, IN, USA 2Thomas Jefferson University,Department Of Surgery,Philadelphia, PA, USA

Introduction: Repair and regeneration in fatty liver is associated with increased hepatocellular injury and increased risk for perioperative mortality. We set out to identify a pharmacologic agent that induces epidermal growth factor receptor (EGFR) and improves hepatic regeneration in the setting of fatty liver and obesity.

Methods: Diet-induced obese mice were injected with resveratrol or DMSO control. After being subjected to 70% hepatectomy they were necropsied and liver samples were evaluated with the use of immunohistochemistry, western blot assay, and liver EchoMRI.

Results: Resveratrol was found to induce liver EGFR mRNA, protein, and phosphorylation of EGFR. Resveratrol increased PCNA levels without increasing liver mass, due to concomitant reduction in hepatic lipid content. Resveratrol did not increase serum ALT, AST, or bilirubin in normal or obese mice. Resveratrol ameliorates liver injury and accelerated regeneration after partial hepatectomy, while reducing macrosteatosis and presence of apoptotic hepatocytes. Resveratrol reduced mortality in obese mice that underwent extended 80% hepatectomy.

Conclusion: Resveratrol increases EGFR expression, decreases hepatic lipid content, and promotes hepatocyte proliferation and recovery after hepatectomy. This indicates that further study into resveratrol and other EGFR restoring therapy is warranted for potential benefit in preventing liver failure and dysfunction in the setting of fatty liver surgery.

42.10 The role of thymic tolerance in neonatal gene therapy

Posted on January 18, 2016

D. T. Lee1,2, C. Woo2, C. Tran2, G. S. Lipshutz2 1Mount Sinai School Of Medicine,Department Of Surgery,New York, NY, USA 2University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA

Introduction: Gene therapy is a promising strategy for treatment of inherited genetic disorders. Its efficacy, however, is limited by a potential immune response against transgene encoded neoantigens and the consequent diminution of therapeutic protein levels. Prior studies have shown that neonatal exposure to recombinant viral vector and transgene proteins can result in immunologic tolerance and persistent transgene expression. In this study we sought to determine if central tolerance plays a role in the mechanism of neonatal tolerance via thymic deletion.

Methods: An adeno-associated viral vector (AAV) was developed that expressed secreted ovalbumin (OVA) and green fluorescent protein (GFP) and was administered to C57BL/6 pups at birth; control mice received no virus. OVA and anti-OVA levels were serially obtained for over 1 year. Vaccination with OVA administered subcutaneously was performed. At 6 weeks and 1 year euthanasia was performed and viral DNA was quantified using qPCR. OT-2 mice that express CD4+ transgenic T cell receptors specific for OVA peptide received either no virus at birth or were administered AAV-OVA. OT-2 mice crossed with transgenic mice expressing membrane bound chicken ovalbumin on all cells (CAG-OVA) were injected with AAV-OVA neonatally. Thymi were removed for flow cytometry analysis as adults.

Results: A single intravenous dose of AAV administered to C57BL/6 pups led to life-long expression of ovalbumin in wild type mice (>1 year) without a humoral immune response; this persisted after vaccination with adjuvant. Quantitative PCR revealed that the highest viral copy number levels after 1 year were expressed in the liver. OT-2 mice that received no injection were found to have 81.3% CD4+ thymocytes when examined as adults, while OT-2 mice crossed with CAG-OVA mice were found to have 10.1% of their thymocytes CD4+. OT-2 mice administered AAV-OVA at birth demonstrated 69.6% of their thymocytes as CD4+ (p=0.01 compared to OT-2).

Conclusion: These studies show that neonatally administered AAV-OVA in C57BL/6 mice results in long term protein expression and tolerance to OVA. After vaccination challenge with a purified OVA protein, serum OVA levels persisted without significant humoral response. In order to better understand if central tolerance plays a role in this mechanism of neonatal tolerance, thymic subpopulations of OT2 transgenic mice that received no virus at birth were compared to OT2 mice crossed with CAG-OVA mice (allowing the earliest post-conception exposure to membrane-bound OVA). There was significant reduction of CD4+ positive cells (81.3% vs. 10.1%, p <0.001) suggesting that early exposure to OVA led to negative selection during immunologic ontogeny. However, OT2 mice that received neonatally administered AAV-OVA demonstrated 69.6% CD4+ thymocytes as adults, suggesting that thymic deletion has little if any role in the mechanism of neonatal tolerance.

43.01 Mouse Notch1 is a Novel Aryl Hydrocarbon Receptor Driven Gene in Hepatotoxicity

Posted on January 18, 2016

P. Carney1, M. Nukaya1, G. D. Kennedy1 1University Of Wisconsin,School Of Medicine And Public Health, Department Of Surgery,Madison, WI, USA

Introduction:
Notch signaling is a critical pathway in determination of cell fate in various tissues, specifically in the context of inflammation. A recent study in our lab revealed that Notch signaling is essential for generation of innate lymphoid cells (ILCs) producing interleukin (IL)-22, which protect the intestinal mucosa from infection. Interestingly, induction of the Notch signaling pathway and subsequent production of IL-22 was found to be dependent on the aryl-hydrocarbon receptor (AHR). Because the AHR is well characterized in liver inflammation, we hypothesize that Notch gene activation in the liver is driven by AHR expression. To examine our hypothesis, we investigated (1) which Notch isoform (Notch1, Notch2, Notch3, Notch4) is regulated by AHR and (2) whether the Notch gene is controlled by canonical or non-canonical AHR signaling pathway.

Methods:
To examine the AHR-dependency on the regulation of Notch genes, we used hepatocyte-specific AHR deletion mice. Mice were injected with a single intraperitoneal (i.p.) dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (64-100 μg/kg total body weight) dissolved in DMSO, or DMSO alone. 5-7 days after the single i.p. injection, mice were sacrificed and liver samples were removed for total RNA extraction. We then measured gene expression of Notch1, Notch2, Notch3, and Notch4 by qualitative rt-PCR. To determine if Notch gene induction occurs through canonical or non-canonical AHR signaling, we used hepatocyte-specific AHR nuclear translocator (ARNT) and AHR-interaction protein (AIP) deletion mouse models.

Results:
In mouse livers of wild-type (WT, C57BL/6) mice, the expression of Notch1 gene was significantly increased (8 fold, p<0.05) by TCDD, while Notch2, Notch3 and Notch4 were not significantly induced (Notch2; p=0.605, Notch4; p=0.252, and Notch3 was not detectable.). The Notch1 gene induction by TCDD is not observed in hepatocyte-specific AHR deletion mice. In addition, the increase of Notch 1 expression was not detected in both hepatocyte-specific AHR nuclear translocator (ARNT) and AHR-interaction protein (AIP) deletion mouse models.

Conclusion:
Our results suggest that the mouse Notch1 gene is a novel AHR-driven gene that acts through the canonical AHR signaling pathway. Notch1 gene expression is significantly induced in the liver by TCDD treatment in wild-type controls of all three mouse lines (AHR, Arnt, and Ara9 deletion models), while its expression cannot be induced in the absence of these canonical AHR pathway genes. In the context of human disease, Notch1 activity is important in the maintenance of hepatocyte homeostasis and irregular Notch signaling is most likely implicated in liver inflammation.

43.02 The Role of Circulating DNA in Pancreatic Cancer and Potential for DNA Scavengers as Therapeutics

Posted on January 18, 2016

J. E. McDade1, J. Naqvi2, J. Lee2, D. S. Pisetsky3,4, B. A. Sullenger2, T. V. Brennan2, R. R. White2 1Duke University Medical Center,Durham, NC, USA 2Duke University Medical Center,Department Of Surgery,Durham, NC, USA 3Duke University Medical Center,Department Of Rheumatology And Immunology,Durham, NC, USA 4Duke University Medical Center,Department Of Medicine,Durham, NC, USA

Introduction: Circulating cell-free DNA (cfDNA) is known to correlate with tumor stage in several different cancer types. Levels of cfDNA are increased in the serum of pancreatic cancer (PC) patients, and increasing evidence shows cfDNA may play a role in mediating disease progression. Degradation of cfDNA using DNase I reduces pancreatic cancer cell invasion in vitro and reduces metastasis in a PC mouse model. These data indicate that cfDNA is more than a biomarker and may also be a target for therapy. For this purpose, we propose to use nucleic acid binding polymers (NABPs) as a more clinically feasible approach to reverse the effects of cfDNA. Our lab has previously demonstrated that NABPs can bind and reverse the effects of cfDNA in vitro and improve outcomes in various inflammatory conditions. Our hypothesis is that cfDNA will increase PC cell invasion in vitro, and that NABPs can ‘scavenge’ cfDNA and block this effect.

Methods: Human PC cell lines Panc-1 and BxPC-3 were used for the following experiments. Cells were seeded in serum-free media onto a Matrigel-coated Transwell membrane with 8μm pores and allowed to invade for 24hr. Invaded cells were fixed, stained with 5% crystal violet, and the average number of invaded cells per image was counted. We examined the effects that various cfDNA sources had on invasion and investigated the co-administration of PAMAM-G3, a commercially available NABP, to assess its potential as a therapeutic agent.

Results: CpG ODN 2006 (4μM), a synthetic unmethylated DNA oligonucleotide, potently stimulates Panc-1 and BxPC-3 invasion. Sources of DNA in the serum of PC patients include direct secretion from immune cells, or release from dying cancer cells. To emulate these settings, we investigated naturally occurring sources of cfDNA and found that conditioned media (CM) containing high levels of cfDNA released from irradiated PC cells stimulate BxPC-3 invasion. PAMAM-G3 (20μg/mL) blocks the effects of both CpG and endogenous cfDNA from CM and reduces PC cell invasion back to baseline levels (Fig A). In a separate experiment, purified mitochondrial DNA (mtDNA), which contains a higher proportion of unmethylated CpG sequences than genomic DNA, also stimulates PC cell invasion at 20μg/mL and may be the component of cfDNA that is mediating this effect (Fig B).

Conclusion: CfDNA-containing media and purified mtDNA stimulate PC cell invasion in vitro, and the effects of cfDNA can be blocked by PAMAM-G3. These data provide evidence that cfDNA may be an important mediator of disease progression in PC and suggest a potential therapeutic application for NABPs.

42.07 Milk Fat Globule-Epidermal Growth Factor-Factor VIII Attenuates Sepsis-induced Acute Kidney Injury

Posted on January 18, 2016

C. Cen1, M. Aziz1,2, W. Yang1,2, J. Nicastro1, G. Coppa1, P. Wang1,2 1Hofstra North Shore LIJ School Of Medicine,Department Of Surgery,Manhasset, NY, USA 2The Feinstein Institute For Medical Research,Center For Translational Research,Manhasset, NY, USA

Introduction: Acute kidney injury (AKI) is most commonly caused by severe sepsis in critically ill patients, and it is associated with high morbidity and mortality. The pathophysiology of sepsis-induced AKI includes direct inflammatory injury, endothelial cell dysfunction, and apoptosis. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is a secretory glycoprotein with a known role in the enhancement of apoptotic cell clearance and regulation of inflammation. We hypothesize that MFG-E8 can protect the kidneys from injury caused by sepsis.

Methods: Sepsis was induced in 8-week-old male C57BL/6 mice by cecal ligation and puncture (CLP). Recombinant mouse MFG-E8 (rmMFG-E8) or PBS (vehicle) was injected intravenously at a dosage of 20 µg/kg body weight at time of CLP (n=4-5 mice/group). After 20 h, serum and renal tissue were harvested for various analyses. The renal injury markers blood urea nitrogen (BUN) and creatinine were determined by enzymatic and chemical reactions, respectively. The levels of mRNA and protein were measured by real-time qPCR and ELISA, respectively.

Results: At 20 h after CLP, serum levels of BUN and creatinine were both increased in the vehicle group compared to the sham group (BUN: 39.7 ± 6.6 vs. 17.3 ± 1.7 mg/dL; creatinine: 1.5 ± 0.49 vs. 0.29 ± 0.03 mg/dL; p<0.05). In contrast, animals treated with rmMFG-E8 had a reduction in BUN and creatinine by 31% and 57%, respectively. Levels of mRNA and protein of pro-inflammatory cytokine IL-6 were reduced by 22% and 24%, respectively, in rmMFG-E8-treated mice compared to vehicle mice. The mRNA levels of pro-inflammatory cytokine TNF-α were also reduced by 48% in rmMFG-E8-treated mice compared to the vehicle. The mRNA levels of macrophage inflammatory protein-2 (MIP-2), a chemokine, were reduced by 47% in mice treated with rmMFG-E8 compared to the vehicle group. In addition, the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) mRNA was downregulated by 27% in rmMFG-E8-treated mice compared to the vehicle animals (p<0.05).

Conclusion: Treatment with rmMFG-E8 reduces renal tissue injury induced by sepsis through inhibiting the production of pro-inflammatory cytokines and chemokines, as well as through decreasing the activation of endothelial cells. Thus, MFG-E8 may have a therapeutic potential for treating AKI induced by severe sepsis.

42.08 Inhibition of PAD4 Attenuates CitH3 Production and Mortality in a Rodent Model of Lethal Endotoxemia

Posted on January 18, 2016

Y. Liang1, E. Chen1, B. Liu1, Y. Wang1, W. He1, J. Zhang2, E. Chen2, Y. Li1, V. Nikolian3, H. B. Alam1 1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Internal Medicine,Ann Arbor, MI, USA 3University Of Michigan Health System,Ann Arbor, MI, USA

Introduction: The peptidylarginine deiminases (PADs) are a family of enzymes (PADs 1–4 and 6) that catalyze the conversion of arginine to citrulline in proteins (e.g., histones). We have previously shown that citrullinated histone H3 (CitH3) serves as a potential diagnostic biomarker in a mouse model of lethal lipopolysaccharides (LPS) shock. However, it is not clear whether treatment with YW3-56, a specific PAD4 inhibitor, would be beneficial. The present study was designed to test the hypothesis that administration of YW3-56 could attenuate LPS-induced CitH3 production, and improve survival.

Methods: Three experiments were carried out. In experiment I: HL-60 neutrophilic cells were treated with LPS (1 µg/ml) in the presence or absence of YW3-56 (5 µmol/L) for 6 hours. Neutrophil extracellular traps (NETs) were collected and analyzed by Western blots with antibodies against histones CitH3, and H3. The protein bands were quantified by densitometry and expressed as mean values ± standard error of mean (n=3). Moreover, human umbilical vein endothelial cells (HUVEC) were grown in Transwell® plates and treated with the NETs (10 µg/ml) derived from LPS only or LPS+YW3-56. Cell permeability was assessed by measuring leakage of FITC-labeled dextran. In experiment II, male C57BL/6J mice were randomly assigned to the following three groups with intraperitoneal injection (ip) of different reagents (n=8-10/group): (1) Dimethyl sulfoxide (DMSO, 1µl/g mouse body weight), (2) LPS (35 mg/kg) + DMSO, and (3) LPS + YW3-56 (5 mg/kg) dissolved in DMSO (ip, post LPS). Survival was monitored for10 days. In experiment III, mice were subjected to the same treatments as in experiment II, and blood and tissue samples were collected 1 and 3 hours after LPS or YW3-56 treatment. CitH3 and cytokines were measured by Western blot and ELISA, respectively.

Results: YW3-56 significantly suppressed CitH3 production and NET formation in the HL-60 cells, and decreased cell permeability in the HUVEC compared to LPS only (p=0.015). In an in vivo study, DMSO treatment had no impact on the CitH3 levels in the blood or survival. LPS injection (35 mg/kg, ip) significantly increased the levels of CitH3 in the circulation by 1 hour and was associated with 100% mortality within 2 days. YW3-56 treatment significantly attenuated the CitH3 levels and improved the survival (Figure 1, p<0.0001).

Conclusion: We have demonstrated for the first time that inhibition of PAD4 with YW3-56 can significantly attenuate LPS-induced CitH3 production and improve the survival in a mouse model of lethal endotoxic shock.

42.09 Consumption of Aspartame Contributes to the Development of Metabolic Syndrome

Posted on January 18, 2016

S. S. Gul1, R. A. Hamilton1, T. Phupitakphol1, L. Wei1, S. K. Hyoju1, D. Hu1, W. Zhang1, M. H. Gharedaghi1, H. Huo1, K. P. Econompolous1, S. A. Morrison1, S. Hamarneh1, R. Hodin1 1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA

Introduction: Diet sodas, despite being calorie-free, cause minimal weight loss. Aspartame (ASP) is a non-nutritive sweetener, used in sugar-free food with the objective of reducing obesity rates. In the gut, it is metabolized into aspartic acid, phenylalanine (PHE) and methanol. PHE inhibits intestinal alkaline phosphatase (IAP), a gut enzyme shown to prevent metabolic syndrome in mice. This led us to hypothesize that the aspartame in diet sodas could be the reason why there is minimal weight loss associated with consumption of sugar-free drinks. This study aimed to assess whether consumption of aspartame could promote the development of metabolic syndrome in mice fed a high fat diet.

Methods: IAP was added to solutions of diet soda and controls of regular soda and water to study the effects of ASP on IAP in vitro. pH settings were adjusted to imitate the different acidic environments along the intestinal canal. IAP activity was measured through pNPP assays after 1-12 hours. For the acute in vivo model, 6 week old C57BL/6 male mice were anesthetized, and a 6 cm closed loop section of the small intestine was created with 2,0 nylons. ASP, PHE or water (controls) was injected in the loops before closing the abdomen. After 3 hours, mice were sacrificed and luminal contents from the loop were harvested and IAP activity was assessed using pNPP assays. The chronic in vivo model used 6 week old C57BL/6 male mice that were fed either a chow or a high fat diet (HFD), along with tap water (controls) or water + ASP (150mg/kg/day) for 18 weeks. Mice were monitored for weight gain, food and water intake. GTT was conducted before sacrifice.

Results: In the in vitro study, IAP activity was significantly lower (p<0.05) in solutions containing ASP compared to controls without ASP at pH 3-6. In the bowel loop model, IAP activity was significantly (p<0.05) reduced in mice that received ASP compared to controls. At 18 weeks of the chronic study, mice in the HFD+ASP group had gained more weight compared to HFD+water group (42.38±3.12 vs 48.05±1.61; p=0.0001). Glucose levels were significantly higher in the HFD+ASP group after 16 hours of fasting (p=0.02), and in both ASP groups after 6 hours of fasting (p=0.02 and p=0.006 respectively). Although no significance was reached, a trend was seen indicating that mice receiving ASP had lower luminal IAP activity compared to the controls (p=0.07). Serum TNF-alpha was increased in both ASP groups (p=0.005), and was highest in HFD+ASP (p=0.01), indicating higher level of inflammation in groups receiving ASP.

Conclusion: IAP has been proven to halt the inflammatory changes leading to the metabolic syndrome caused by the increase in endotoxin levels (LPS) associated with a high fat diet. This protective effect by IAP could be inhibited by phenylalanine – one of aspartame’s metabolites – perhaps explaining the observation that diet drinks are not associated with the weight loss that people expect.

42.05 Peptide Nanofibers Stimulating Non-inflammatory Immune Responses as Wound Healing Scaffolds

Posted on January 18, 2016

Y. Vigneswaran1, H. Han1, J. Collier1 1University Of Chicago,Surgery,Chicago, IL, USA

Introduction:
Potent antibody and T cell responses elicited by wound healing materials are often assumed to be counterproductive to wound healing, but this is not well studied. Recently, we observed self-assembling peptide nanofiber materials incorporated with specific epitopes can raise strong immune responses without detectable inflammation at sites of delivery. We hypothesized that by engaging this specific non-inflammatory immune response, these materials could be compatible with normal healing and even enhance healing.

Methods:

In mice, an excisional dermal wound healing model was used to study the effects of active immune responses. We used the self-assembling peptide Q11 (QQKFQFQFEQQ) and the OT-II antigen from ovalbumin (T cell and B cell epitope) with either Freund's adjuvant (inflammatory peptide) or without additional adjuvant (non-inflammatory peptide). Mice were naive with no specific antibodys to the material or immunized against the peptides to raise strong antibody responses. Wound healing rates were grossly measured over time in the presence of either the inflammatory or non-inflammatory peptide scaffold (n=20), skin was harvested at various time points for immunohistochemistry (n=18) and lymphocytes isolated for flow cytometry analysis (n=18).

Results:
In the presence of equivalent antibody responses immunized mice had faster healing rates when treated with non-inflammatory peptide compared to inflammatory peptide (p<0.05). The inflammatory peptide treatment had faster healing when mice were not immunized compared to the immunized mice (p<0.05). However the non-inflammatory peptide had no significant difference in healing rates with or without immunization. Immunohistochemistry demonstrated significantly greater recruitment of CD3+ cells to the wound bed by day 17 when immunized (p<0.01). With the non-inflammatory peptide treatment, the ratio of CD4+ to CD8+ cells isolated from the wound beds decreased by day 10 regardless of antibody response (p<0.05). Yet inflammatory peptide treatment showed no significant change in CD4+/CD8+ ratio and at day 10 this ratio was significantly higher than that observed with the non-inflammatory peptide. Additionally cytokine staining of these CD4+ lymphocytes demonstrated higher Th1/Th2 ratio with the inflammatory peptide at all time points compared to the non-inflammatory peptide (p<0.05).

Conclusion:
Here we report that immunogenicity of the material itself is not always detrimental for wound healing. At the cellular level these non-inflammatory peptide nanofibers appear to promote a pro-healing immune phenotype by accelerating the progression of the dominant T cell phenotype from CD4+ to CD8+ cells in the wound and a Th2 slanted response throughout the duration of healing when compared to other immunogenic material. This elicited immune response by peptide nanofibers may provide a unique opportunity to engage the immune response for a more productive wound healing environment.

42.06 Efficient generation of targeted mutation using nuclease-directed homologous recombination.

Posted on January 18, 2016

J. R. Butler1, G. R. Martens1, J. M. Ladowski1, B. W. Estes1, Z. Wang1, P. Li1, M. Tector1, A. Tector1 1Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA

Introduction: Nuclease-based genome editing has rapidly sped the creation of new models of human disease. Moving forward, these techniques also hold great promise for the future of cell-based therapies for cancer, HIV and immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to employ non-integrating selection and nuclease-directed homologous recombination to better control the process of genetic modification. The results have immediate application to the creation of animal models of surgical disease.

Methods: CRISPR/Cas9 directed mutagenesis with a single-guide RNA target was designed to target the GGTA1 locus of the porcine genome. A bisistronic vector expressing a single-guide RNA, Cas9 protein, and GFP was employed to increase plasmid-delivered mutational efficiency. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths to the mutational event site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by sanger sequence and restriction enzyme digest.

Results: Bisistronic expression of a fluorescence protein on the Cas9 vector created a non-integrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5 to 82% (Figure 1A). Cotransfection with homologous DNA oligonucleotides (Figure 1B) increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic events (Figure 1C). Single-strand DNA (ssDNA) was twice as efficient as double strand DNA (dsDNA). Furthermore, these oliogos were able to effect controlled insertion with an efficiency of up to 37%. (Figure 1D-E)

Conclusion: A non-integrating selection strategy based on bisistronic fluorescence expression can increase the mutational efficiency the CRISPR/Cas9 system by greater than 2,500%. The precision of this system can be increased by the addition of a very short homologous template sequence. Together these strategies may be employed to efficiently control mutational events at unprecedented levels. This system can better utilize the potential of nuclease-mediated genomic editing.

42.01 SARS – Oncolytic JX-594-based Vaccinia Virus Treatment Of Colorectal Liver Metastases

Posted on January 18, 2016

R. V. Dave1,2, F. Errington-Mais1, E. West1, P. Selby1, G. J. Toogood2, A. Melcher1 1Targeted And Biological Therapies Group,Leeds Institute Of Cancer And Pathology,Leeds, YORKSHIRE, United Kingdom 2Department Of Hepatobiliary And Transplant Surgery,St James University Hospital,Leeds, YORKSHIRE, United Kingdom

Introduction: There is still a need for a novel treatment modality for colorectal cancer (CRC) liver metastases (CRLM). We examined the ability of JX-594-based vaccinia viruses (JXVV) to preferentially replicate in and kill CRLM, and induce immune-mediated tumour cytotoxicity by activation of natural killer (NK) cells.

Methods: The vaccinia virus was genetically manipulated to encode for granulocyte macrophage colony stimulating factor (JXVV-GM-CSF-fLuc) and green fluorescent protein (JXVV-GM-CSF-GFP). Viability assays and Enzyme Linked Immunoabsorbent Assay were used to confirm tumour cell killing and production of inflammatory cytokines when CRC cell lines were infected with JXVV. Viral replication was investigated by plaque assay and using a novel ex-vivo ‘tissue core’ method from freshly resected liver tissue. Induction of the innate immune response was measured by flow-cytometric analysis of JXVV-treated NK cells from blood, liver and lymph nodes, and anti-tumour cytotoxicity investigated by chromium release.

Results: We demonstrated direct lysis of CRC cell lines by treatment with JXVV, with greater lysis and replication (up to 250-fold) in cells with upregulated surface EGFR. JXVV treatment resulted in substantial expression of GMCSF and induction of inflammatory cytokines within the tumour microenvironment, and inhibition of anti-inflammatory and pro-angiogenic cytokines. Ex vivo infection of CRLM with JX-594-GFP-GMCSF resulted in tumour-specific GFP and GMCSF expression, which was not seen in normal liver tissue resected around the tumour. Treatment of NK cells with JX-594-GMCSF led to activation, degranulation and increased cytotoxicity against CRC cell targets. This was dependant on Interferon alpha production and the presence of CD14+ve monocytes, which acquired an antigen-presenting phenotype (CD86+veCD11c+veClassIIDR+ve). It was also observed that JX-594-GFP-GMCSF expressed GFP within monocytes, but did not replicate or express transgenes within lymphocytes, further implicating monocytes in JXVV-mediated NK cell activation.

Conclusion: Oncolytic Virotherapy holds promise as a novel treatment modality. Direct tumour-specific lysis and transgene expression and the induction of tumour-specific innate immunity means that it may provide a two-pronged attack against tumour cells whilst sparing normal tissue.

42.02 Nafamostat mesilate enhances effect of gemcitabine + nab-paclitaxel therapy for pancreatic cancer.

Posted on January 18, 2016

T. Horiuchi1,2, H. Shiba1, N. Saito1,2, Y. Shirai1,2, R. Iwase1, K. Haruki1, Y. Fujiwara1, K. Furukawa1, T. Uwagawa1, T. Ohashi2, K. Yanaga1 1The Jikei University School Of Medicine,Department Of Surgery,,Tokyo, , Japan 2The Jikei University School Of Medicine,Department Of Gene Therapy, Research Center For Medical Science,Tokyo, , Japan

Introduction: Pancreatic cancer has a poor prognosis among visceral cancers, with an overall 5-year survival rate of around 5%. Recently, gemcitabine plus nab-paclitaxel therapy was reported to be superior to gemcitabine alone in patients with advanced pancreatic cancer. However, the tumor resistance to chemotherapy is common and activation of nuclear factor-kappa B (NF-κB) plays a key role in attenuation of anti-tumor effect of chemotherapy in pancreatic cancer. We previously reported that nafamostat mesilate, a synthetic serine protease inhibitor, inhibited NF-κB activation and induced antitumor effects for pancreatic cancer. We hypothesized that nafamostat mesilate downregulates the activity of NF-κB and improves therapeutic outcome of pancreatic cancer.

Methods: In vitro, we assessed NF-κB activity, cell viability, induction of caspase cascade, and quantification of apoptosis of human pancreatic cancer cell lines (PANC-1, MIA PaCa-2, ASPC-1) in the following five groups: 1) gemcitabine and nab-paclitaxel, 2) nafamostat mesilate alone, 3) triple combination (gemcitabine, nab-paclitaxel and nafamostat mesilate), or 4) vehicle as control. In vivo, we established orthotopic pancreatic cancer model in mice by injection of PANC-1 cells to the pancreas. At six weeks after injection, the animals were treated with nafamostat mesilate 3 times a week (Nafamostat mesilate group), with gemcitabine plus nab-paclitaxel once a week (Chemotherapy group), or with combination of nafamostat mesilate 3 times a week and gemcitabine plus nab-paclitaxel once a week (Triple combination group). In Control group, only vehicle of gemcitabine and nafamostat mesilate were injected at the same time course.

Results:In vitro, NF-κB activity in Chemotherapy group was higher than that in Control group, and NF-kB activity was significantly surpressed in Nafamostat mesilate group and Triple combination group as compared with Control group and Chemotherapy group, respectively (PANC-1: p<0.001, MIAPaCa-2: p<0.001, ASPC-1: p<0.001). Cell viability in Triple combination group was lower than that in Chemotherapy group. Cleaved caspase-3 and 8 level in Triple combination group was the greatest in the 4 groups. Apoptosis in Triple combination group was significantly more frequent than that in Chemotherapy group. In vivo, tumor size in Tripe combination group was smaller than those in other groups. NF-κB activation of the excised tumor in Triple combination group was significantly inhibited in comparison with that in Chemotherapy group (p<0.01). Cleaved caspase-3 level and the number of apoptotic cells in the Triple combination group were the greatest in the 4 groups.

Conclusion:Combination chemotherapy of gemcitabine plus nab-paclitaxel with nafamostat mesilate exerts enhanced anti-tumor effect against orthotopic pancreatic cancer model in mice.

42.03 A Novel Dual Chamber Stent to Maintain Organ Perfusion in a Porcine Model of the Cardiac Death Donor

Posted on January 18, 2016

B. W. Tillman1,4, Y. Chun3, T. Richards1, T. Maul3, N. L. Liang1, A. Tevar2 1University Of Pittsburgh,Division Of Vascular Surgery, Department Of Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh,Starzl Transplant Institute, Department Of Surgery,Pittsburgh, PA, USA 3University Of Pittsburgh,Department Of Bioengineering,Pittsburgh, PA, USA 4University Of Pittsburgh,McGowan Institute For Regenerative Medicine,Pittsburgh, PA, USA

Introduction: The single most significant obstacle facing organ transplantation remains the critical shortage of donor organs. While donation after cardiac death (DCD) has demonstrated significant growth, these organs have inferior outcomes compared to other types of donation. During the ‘agonal period’, after life support is discontinued, malperfusion of organs leads to ischemic organ damage. As a result of the tenet that cardiac death should not be expedited, previous approaches could not be used until after cardiac death and after organ injury. In contrast to the global withdrawal of life support in DCD, among non-donors end of life care is determined by the family for individual organ systems. We have designed a dual chamber organ perfusion stent (OPS) to reduce ischemic injury by isolation of the visceral vessels and to perfuse only those organs with oxygenated blood while the heart fails on its own terms. This differs from all prior strategies because this removable stent would be placed by a needlestick femoral access and would allow the failing heart to continue to perfuse the rest of the body while protecting against ischemic injury of the visceral organs.

Methods: Prototype OPS were constructed from commercial stent components and final versions were custom made from nitinol and electrospun polyurethane. An OPS (n=5) was evaluated in a porcine model compared to non-stented controls. Paired venous and arterial OPS were deployed. Next, venous blood from the viscera in the external chamber was oxygenated and cycled to the external chamber of the arterial stent. Blood from the heart perfused the body through the large central lumen of the arterial OPS. DCD conditions were simulated for one hour with profound hypotension (SBP < 50 mm Hg).

Results: Invasive cardiac monitoring revealed no significant change in heart rate, mean arterial pressure, mixed venous oxygen, or venous pressure during stent deployment (P< 0.05). A small decrease was observed in the cardiac output consistent with exclusion of the visceral blood flow. Visceral blood flow of over 650 mL/min was achieved with the OPS and oxygen pressures were increased nearly 5 fold compared to agonal control animals. Control organs demonstrated severe malperfusion, contrasting to the well perfused OPS organs.

Conclusion: This pilot study suggests that an OPS offers: 1) rapid endovascular delivery 2) minimal impact on cardiac physiology 3) improved oxygen delivery to organs. Our early results suggest this novel stent design may increase availability of donor organs without impacting the natural cardiac death of the consented donor. Further development of this approach may ensure that nearly every consented donor successfully donates lifesaving organs.

42.04 Nanofiber Scaffold for Recreation of the Abdominal Wall in Rattus norvegicus II: Histopathology

Posted on January 18, 2016

L. Fluke1, R. Restrepo1, B. Hoagland1, J. Stephenson1 1Naval Medical Center Portsmouth,General Surgery,Portsmouth, VIRGINIA, USA

Introduction:

Full thickness soft tissue defects from congenital absence or traumatic loss are difficult to surgically manage. Healing requires cell migration, organization of an extracellular matrix (ECM), inflammation, and wound coverage.

We have previously shown that Poly(L-lactide) (PLLA) nanofibrous scaffolds enhance cell infiltration in vitro and in vivo in wound healing models, and we have demonstrated that the PLLA scaffold provides adequate strength to serve as a replacement for a full thickness abdominal wall defect in the rat model. Our current study compares aligned and unaligned nanofibrous scaffolds to a commonly used absorbable mesh, Vicryl, and permanent mesh, Gortex. We hypothesized that aligned electrospun PLLA nanofibrous grafts best facilitate native tissue ingrowth, providing physiologic strength necessary for repair of soft tissue defect while serving as a scaffold to guide tissue regeneration.

Methods:
Abdominal wall defects were created in 72 rats, 6 rats per mesh per time frame, followed by underlay implantation of Vicryl, Gortex, aligned or unaligned PLLA Nanofiber mesh. Abdominal walls were harvested at 2, 12, and 24 weeks. Histopathologic semiquantative analysis was graded for cellular infiltration, multinucleated giant cells (MNG), vascularity, and tissue organization in a blinded fashion by an expert reviewer using a validated scoring system. Mean scores were compared and analyzed with non-parametric Mann-Whitney Wilcoxon testing.

Results:
Both aligned PLLA and Vicryl showed improved cellular infiltration at 2 weeks versus unaligned PLLA and Goretex. At 12 weeks, only the aligned PLLA had persistent cellular infiltration of the graft, while both aligned and unaligned PLLA grafts showed the presence of multinucleated giant cells (MNG). The presence of MNG’s decreases in the aligned PLLA graft by 24 weeks. There was no significant difference in graft vascularity at any timepoints. Vicryl graft had significant disruption at 2 and 12 weeks resulting in moderately organized connective tissue by the end of the study. Meanwhile, both aligned and unaligned PLLA grafts were broken down with less densely organized connective tissue, or scar.

Conclusion:
PLLA graft maintains structural integrity until at least 12 weeks and exhibits substantial tissue replacement at 24 weeks. Vicryl and PLLA grafts have similar tissue responses; however the Vicryl mesh has substantial degradation at 2 weeks and results in more dense scarring. These results demonstrate that PLLA Nanofiber mesh offers early strength comparable to Gortex but breaks down, replaced with cellular growth creating a favorable option in management of complex surgical wounds or native soft tissue defects.

« Previous Page