64.08 Penicillin Induces Colonic H,KATPase via Nitric Oxide Precursors: Novel Target for Diarrheal Control

Posted on January 31, 2019

V. Norz1,2, T. Spingler1,2, T. M. Gisinger1,2, V. M. Baratta2, M. J. Barahona2, J. Ollodart2, D. Mulligan2, J. P. Geibel2  1Paracelsus Medical University,Medicine,Salzburg, SALZBURG, Austria 2Yale University School Of Medicine,Surgery,New Haven, CT, USA

Introduction: Antibiotic-associated diarrhea (AAD) is a well-known complication of antibiotic administration, although the pathogenesis is not clearly elucidated. Symptoms range from mild gastrointestinal disturbances to fulminant gastroenteritis. Surgical patients who receive perioperative antibiotics are at higher risk. Antibiotic-associated diarrhea is thought to be due to an imbalance in the host microbiome, leading to pathogen overgrowth and increased intestinal secretion. We propose a novel pathway of AAD, whereby Penicillin G stimulates intestinal H, KATPase. Activation of H,KATPase leads to intraluminal fluid loss. Here, we demonstrate that L-arginine, a nitric oxide (NO) precursor, works synergistically with Penicillin G to activate H,KATPase. We corroborate this by showing how an inhibitor of the NO pathway, L-NAME, N (ω)-nitro-L-arginine methyl ester, eliminates the secretory effect of L-arginine and Penicillin G.

Methods: Rat distal colons were harvested and placed in solution for crypt isolation. Glands were maintained in a thermostatically controlled perfusion chamber and loaded with a pH indicator dye 2',7'-Bis(2-carboxyethyl)-5(6)- carboxyfluorescein,acetoxymethyl ester (BCECF) to measure intracellular pH in real time. Proton extrusion, a measure of acid secretion of the individual cells, was monitored by observing the recovery of pH, as previously described. A higher pH recovery rate indicates a higher rate of cellular secretion, i.e. diarrhea. Rat colonic crypts cells were perfused with the following solutions: 1.) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES + 5 mM Penicillin G sodium salt (Control) 2.) HEPES + 10 mM L-arginine 2.) HEPES + 5 mM Penicillin G + 10 mM L-arginine 3.) HEPES + 5 mM Penicillin G + 30 μM L-NAME. Statistical analysis of data was carried out with Graphpad Prism 7.0 software.

Results: The mean rate of pH recovery in the control was 0.00623 ± 0.00026 ΔpHi/min, p 0.0004. L-arginine administration increased H,KATPase activity 0.00097 ± 0.00011 Δ  pHi/min, p 0.0002. L-arginine + Penicillin G administration led to a greater, statistically significant increase in H-KATPase activity (Figure 1, 0.00883 ± 0.00051 ΔpHi/min, p 0.0004). Exposing crypts to a combination of L-NAME and Penicillin G provoked a significant reduction in H,KATPase activity (0.00292 ± 0.0001 ΔpHi/min, p 0.0022).          

Conclusion: It is well-known that antibiotic exposure disrupts the microbiome, leading to diarrhea. We propose a novel mechanism of antibiotic-associated diarrhea through Penicillin G’s activation of the H,KATPase. Maximum fluid secretion was found with Penicillin G and L-arginine, a nitric oxide precursor. In contrast, fluid secretion was significantly decreased when tissues were exposed to L-NAME, a known NO pathway inhibitor. This study may provide new therapeutic opportunities to address AAD in clinical settings by modulation of the colonic H,KATPase.
 

64.07 "Natural Brush Border Enzyme IAP Blocks Gut Inflammatory State after Severe Burn Injury"

Posted on January 31, 2019

F. Adiliaghdam1, P. M. Cavallaro1, M. Najibi1, Y. Liu1, L. Rahme1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery/ Harvard Medical School,Boston, MASSACHUSETTS, USA

Introduction: Our previous data has shown that severe burn injury induces gut barrier dysfunction and leads to a gut-derived systemic inflammatory response.We hypothesized that there is a vicious cycle occurring in the gut after acute burn injury which progressively damages the gut barrier and transforms the gut into an inflammatory organ.Furthermore, we investigated how targeting this pro-inflammatory state with supplemental intestinal alkaline phosphatase (IAP) can prevent this vicious cycle.

Methods: Mice were subjected to a 30% total body surface area burn +/- burn site super-infection induced by injection of Pseudomonas aeruginosa into the dermis. IAP was given by gastric gavage.(2000unit)The pro-inflammatory characteristics of intestinal contents was measured in vitro by Caco2 trans-well epithelial electrical resistance (TEER).Primary mouse macrophages were incubated with ileocecal contents to evaluate the immune response.Peritoneal macrophages were evaluated for inflammatory gene expression.

Results: Our data suggest that burn injury leads to an increased expression of pro-inflammatory cytokines such as TNF, IL-6, IL-1B,and lipocalin in the intestine(P<0.01).Burn injury increased peritoneal macrophage pro-inflammatory cytokine expression compared to sham treatment(TNF-a and IL-6, 3-fold increase,P<0.01).Incubation of ileocecal contents of burned mice with Caco2 monolayer negatively affected the monolayer integrity and downregulated the tight junction protein(TJP)expression.There was a significant higher inflammatory response to the ileocecal content of burn-injured mice in both epithelial cells and macrophage cells compared to sham-treated ones(P<0.01).IAP supplementation after burn decreased the intestine inflammation(3-fold decrease,P<0.01).Peritoneal macrophages isolated from IAP-treated mice showed less inflammatory gene expression(P<0.01).Also, IAP treatment was associated with an improved monolayer integrity(3-fold improvement in TEER drop,P<0.05).IAP-treated ileocecal content caused a less inflammatory response in both epithelial cells and primary macrophages(P<0.01).Ex-vivo incubation of burn-injured ileocecal content with IAP decreased the pro-inflammatory characteristics of the contents(3-fold with primary macrophages,4-fold with epithelial cells,P<0.05).Furthermore, IAP treatment improved TJPs expression in in-vitro and in vivo burn models(ZO-1,P<0.05 and Occludin,P<0.01).Interestingly,the Pseudomonas strain(PA14) which was injected subcutaneously into the burn site was detectable in the ileum and stool of the burn infected mice, confirming the existence of a vicious inflammatory cycle in the gut after burn.

Conclusion:Acute burn injury causes an inflammatory response in the gut.IAP supplementation as a natural,anti-inflammatory enzyme significantly decreases gut inflammation and improves gut barrier function,and can represent a novel therapy to prevent a gut-induced systemic inflammation.

64.06 The Potential Role of B Cells in Mitigating Metabolic Disease After Sleeve Gastrectomy

Posted on January 31, 2019

D. A. Harris1, R. Subramaniam1, T. Brenner1, A. Tavakkoli1, E. G. Sheu1  1Brigham And Women’s Hospital,General Surgery,Boston, MA, USA

Introduction:  Immune dysregulation in obesity causes insulin resistance. Sleeve Gastrectomy (SG) leads to diabetes remission through weight-loss dependent and independent mechanisms. We developed a model of SG in both lean and obese mice to characterize the weight-loss dependent and independent, global and organ-specific, immune cell changes following SG.

Methods:  C57Bl/6J mice were divided into 4 groups: lean sham and SG (n=10,9); diet induced obese sham and SG (n=7,11). Weights and food intake were measured daily. Oral glucose tolerance testing (OGTT) was performed at 2 weeks. B, T, myeloid, NK, NKT, innate lymphoid cell (ILC) populations from jejunal, ileal, cecal, liver, and splenic tissues were profiled at 4 weeks using a 31-antibody panel and time-of-flight mass cytometry (CyTOF). ViSNE analysis and t-tests were used for comparisons.

Results: SG enhanced glucose tolerance in obese and lean mice (OGTT: p<0.01, 30/60 min), but only led to weight loss in obese mice. CyTOF analysis demonstrated a 15% and 10% reduction in splenic CD19+CD11BnegCD21+CD23+ B cells in obese (p=0.002) and lean (p=0.05) SG mice, respectively, compared to shams (figure 1). Further, there was a 14% reduction in mature IGM+IGD+ B cells in both obese (p=0.03) and lean (p=0.02) SG animals. Interestingly in the jejunum of obese, SG animals, there is a conserved, 63% reduction in the same CD21+CD23+ B cell population (p=0.009).

Further, there were population level changes that were unique to the obese phenotype. SG led to an increase in multiple 11B+ B cell subsets including a 2-fold increase in CD21-CD23+ (p=0.022) and a 4-fold increase in CD21+CD23- (p=0.01) B cells compared to shams.

Changes were also seen across myeloid populations. There was a 5.6- and 4.8-fold increase in splenic neutrophils in obese (p=0.06) and lean (p=0.025) SG compared to shams. In obese SG there was a 4.3-fold increase in cecal neutrophils as well. Finally, there was an increase in splenic, M2 (arginase 1+) macrophage polarization in all tested macrophage populations in obese SG animals (p<0.05).

There were no sustained T, NK, NKT, or ILC population changes in spleen, intestine, or liver in either lean and obese SG groups compared to their respective shams.

Conclusion: SG induces both weight-loss dependent and independent immune cell changes. There is a conserved reduction in total and follicular B cells among these changes, which are associated with improved glucose handling and are independent of weight-loss. B cells have been implicated in diet-induced obesity and diabetes. Thus, their downregulation may play a central role in mediating metabolic improvements following surgery.
 

64.05 Sleeve Gastrectomy with Ileal Transposition Increases Peptide YY and Improves Weight Loss in Mice

Posted on January 31, 2019

L. Ying1, G. Breuer1, M. Hubbard1, J. Hwa1, G. Nadzam1, K. Martin1  1Yale University School Of Medicine,New Haven, CT, USA

Introduction: Sleeve gastrectomy with ileal transposition (SGIT) is superior to sleeve gastrectomy (SG) for promoting weight loss in rat and porcine models, and for preventing weight gain in a mouse model of diet-induced obesity. However, it is unknown if SGIT is superior to SG for promoting weight loss and lowering blood glucose in obese mice, and its weight loss mechanism is unclear.

Methods: SGIT was performed on a Pilot Cohort of 5 C57Bl/6J mice (7-8 weeks old), which were followed for 10 weeks to test viability. Whole transcriptome sequencing (RNAseq) was performed on the transposed ileal segments of these mice to identify highly expressed genes potentially responsible for weight loss. Next, SGIT, SG, or sham surgery (SH) was performed on a Study Cohort of 16-week old obese C57Bl/6J mice (40-45 grams, n=12 each). Prior to surgery, mice were grouped to match initial weight and fasting blood glucose. After surgery, mice were fed a low-fat diet and weighed weekly. 4 weeks after surgery, food intake was measured by weighing food over 4 days. 6 weeks after surgery, fasting blood glucose was re-measured. Additionally, after administering a liquid diet bolus via oral gavage, postprandial serum Peptide YY was measured after 15-minutes, 30-minutes, 1-hour, and 2-hours with competitive enzyme immunoassay.

Results: The overall mortality in the Study Cohort was 0%. In the Study Cohort, SGIT mice lost significantly more weight than SG or SH mice (6-week weight in grams±standard error of mean (sem): SH: 35.7±1.1, SG: 31.9±0.7, SGIT: 25.2±0.9). 4 weeks after surgery, SGIT mice consumed significantly less food than SG or SH mice (daily food intake in grams±sem: SH: 3.8±0.3, SG: 2.4±0.4, SGIT: 1.9±0.5). Fasting blood glucose (mg/dl±sem) was not statistically different between SG (77.8±8.6) and SGIT (87.7±7.7) mice 6 weeks after surgery, but both were significantly lower compared to SH mice (134.8±3.7). RNAseq of the transposed ileum from the Pilot Cohort revealed high expression of the ileal brake Peptide YY (PYY). Consequently, postprandial serum PYY was measured 6 weeks after surgery in the Study Cohort. Fasting serum PYY was not significantly different between the three groups. However, after administering a bolus of liquid diet, serum PYY rose more rapidly in SGIT mice than in SG or SH mice (graphic).

Conclusion: In this study, we show that SGIT is superior to SG for promoting weight loss, and similarly effective for lowering fasting blood glucose. SGIT mice also consume less food than SG or SH mice. An early release of Peptide YY, confirmed directly via serum measurement, provides a potential mechanistic explanation for the enhanced weight loss observed in SGIT mice.

64.04 Anti-IL-6 receptor Monoclonal Antibody Robustly Ameliorated Postoperative Adhesion Formation in Mice.

Posted on January 31, 2019

J. Fujimoto1, N. Uyama1, H. Tsutsui1, W. Songtao1, M. Sudo1, E. Hatano1, J. Fujimoto1  1Hyogo College of Medicine,Surgery,Nishinomiya, HYOGO, Japan

Introduction: After abdominal surgeries more than 90% of patients reported to have adhesion formation.  It often results in significant morbidity, and treatment of these morbidity costs approximately $1.3 billion per year.  We have developed an experimental mouse model of abdominal adhesion, and revealed that IFN-g and PAI-1 played a pivotal role in adhesion formation by regulating the coagulation fibrinolysis system (Nat Med 2018).  Recently, we have performed RNA profiling and pathway analysis in this mouse model and found that IL-6 is the key molecule for adhesion formation.  In response to IL-6, IFN-g, PAI-1, TGF-b were up-regulated which stimulated the peritoneal mesothelial cells to produce collagen fibers.  Thus, we administrated anti-IL-6 receptor monoclonal antibody (IL-6R Ab) to the mouse to examine its preventive effect on the adhesion formation.

Methods: We induced intestinal adhesion using BALB/c mouse by cecal cauterization as previously reported.  Adhesions strongly connected the cecum to the large bowel, the abdominal wall or both at day 7. We used (1) IL-6 receptor antibody: IL-6R Ab (MR16-1) or (2) IL-6 ligand antibody: anti-IL-6 monoclonal antibody (MP5) to the adhesion mouse models.  PBS or rat IgG was injected to the adhesion mouse models as control groups.  These antibodies or PBS/rat IgG was injected intraperitoneally 24 hours before the cauterization.  Adhesion score (0: no adhesion to 5: very thick vascularized adhesion) was examined at day 7, and immuno- histopathological study and molecular analysis were performed between day 0 and day 7.  Moreover, mice treated with PBS or MR16-1 were injured by skin biopsy to examine wound healing.  The repair rate of the wound was monitored every day until day 7.

Results:A single injection of MR16-1 significantly reduced intestinal adhesion (N=11, score: 1.9±0.49) compared to PBS group (N=10, score: 4.9±0.1, p=0.003)  and rat IgG group (N=4, score: 5.0, p=0.00002).  A single injection of MP5 did not reduce the adhesion score (N=4, score: 5.0).  MR16-1 treated mice had significantly lower expression of IFN-g, IL-6, CXCL2, collagen 1a1, and TGF-b1 mRNA in their ceca.  Histopathological analysis revealed that MR16-1 treated mice had significantly reduced fibrotic change and the neutrophil infiltration.  The skin wound of the first biopsy day were 19.68 mm2 and 19.2 mm2 in rat IgG treated and MR16-1 treated mice, respectively.  They were reduced to 1.6mm2 and 1.07mm2 at day 7, respectively.  Thus, wound healing progressed normally in MR16-1 treated mice.

Conclusion:We revealed that IL-6R Ab strongly prevents intestinal adhesion after surgery in mice.  Human IL-6R Ab, Tocilizmab/ Actemra R is available,
IL-6R Ab treatment presented here may eventually be translated into a useful clinical regimen for the preventing adhesion formation in patients undergoing surgery.

 

64.03 Cerium Oxide Nanoparticle with MircoRNA 146a Delivered via Zwitterionic Gel Improves Skin Strength

Posted on January 31, 2019

S. A. Hilton1, C. Zgheib1, L. C. Dewberry1, M. M. Hodges1, S. Singh3, S. Seal3, G. Sener2, M. Krebs2, K. W. Liechty1  1University of Colorado,Laboratory For Fetal And Regenerative Biology, Department Of Surgery,Aurora, CO, USA 2Colorado School of Mines,Department Of Chemical And Biological Engineering,Golden, CO, USA 3University of Central Florida,Department Of Material Science Engineering, AMPAC And NSTC Center,Orlando, FL, USA

Introduction

Impairments in wound healing and wound strength are a significant clinical problem in diabetic wounds.  We have previously shown that local injection of cerium oxide nanoparticles conjugated to MicroRNA-146a (CNP-miR146a) improves diabetic wound healing in a murine model through decreased inflammation and improved angiogenesis. We hypothesized that topical delivery of CNP-miR146a in a zwitterionic gel would be more clinically relevant, result in improved healing and improved wound strength.

 

Methods

12 week old female mice that are breed homozygous diabetic (Db/Db) were used. A single 8mm full thickness punch wound was made on the dorsal neck skin of each mouse. Zwitterionic gels were prepared by dissolving [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) and 2-hydroxyethyl methacrylate (HEMA) in water. To load CNPs into the zwitterionic cryogels, CNP-miR146a was added in the gelation solution. Polymerization was initiated using APS and TEMED, then the reaction mixtures were poured into a plastic mold with inner diameter of 0.5 cm and polymerized at -20 ºC for 24 hr. Cryogels were thawed at room temperature. To remove unreacted monomers and other unbound ingredients, the cryogels were washed with PBS several times.

 

Wounds were treated with one time administration of zwitterionic gel only (n=5) or gel impregnated with CNP-miR146a (n=5, ~10ng  Wounds were photographed over time to closure, and animals were euthanized 4 weeks after wound closure for biomechanical testing. A dumbbell shaped sample was taken from cranial to caudal on each mouse with the healed wound in the center. The Instron 5942 testing unit with Bluehill 3 Software was used for examining maximum load, extension, tensile strain.

 

Results

Mice wounds treated with CNP-miR146a gel demonstrated a significant improvement in time to complete wound healing. Untreated diabetic mouse wounds typically heal at day 22-24 post healing. Wounds treated with control gel healed at day 20 and wounds treated with CNP-miR146a impregnated gel healed at day 14 (P-value=0.002). Wounds also showed improved strength after healing with increased maximum load of 3.24N compared to 2.04N (P-value = 0.03). Elastic modulus measures resistance to being deformed when stress is applied. We see improved modulus with CNP-146a gel compared to control gel – 22.26MPa compared to 14.68MPa (P-value = 0.02). Tensile stress at maximum load is also improved – 1.63MPa in control gel compared to 2.59 in treatment gel (P-value = 0.03).

 

Conclusions

Diabetic mice wounds treated with zwitterionic gel impregnated with CNP-miR146a demonstrated improved time to complete wound healing, strength, elasticity, and resistance to stress after healing. This study shows feasibility of topical delivery of a therapeutic for diabetic wound healing via zwitterionic gel, as well as no adverse effects on wound strength.

64.02 Post-operative Ileus is Reduced by Pharmacologic and Genetic Inhibition of Toll-like Receptor 4

Posted on January 31, 2019

M. L. Kovler1, C. P. Sodhi1, M. R. Ladd1, A. Werts1, W. B. Fulton1, T. Prindle1, S. Wang1, Y. Yamaguchi1, D. J. Hackam1  1Johns Hopkins University School Of Medicine,Baltimore, MD, USA

Introduction:

Post-operative ileus (POI) occurs frequently after abdominal surgery and leads to increased morbidity and prolonged hospitalization. Inflammation within the gut wall triggered by intestinal manipulation is recognized as an inciting event in post-operative ileus, suggesting that the innate immune receptor toll-like receptor 4 (TLR4) may be involved. We have recently identified a novel TLR4 inhibitor, C34, which is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which is safe and effective in a variety of pre-clinical studies of gut inflammation. We now hypothesize that TLR4 activation mediates POI, and that pharmacologic TLR4 inhibition with C34 will reduced this postoperative condition.

Methods:

Post-operative ileus was induced by standardized small bowel manipulation in adult wild-type (Tlr4WT) and TLR4 knockout (Tlr4-/-) mice, which we previously generated. Twenty-four hours after manipulation, GI transit was measured by treating mice with fluorescent dye by oral gavage. After 60 minutes, mice were sacrificed, and gastrointestinal motility was expressed as small intestinal transit (%) = the fluorescent pass distance/the total length of small intestine x 100. GI mucosal inflammation was characterized by quantitative RT-PCR measuring expression of TNFa, IL-1b, Lipocalin-2, iNOS, RORC, and FOXP3 from ileal segments. The mice in the control group did not undergo any operation. Motility and inflammatory indices were measured in mice treated with lipopolysaccharide (LPS), the main TLR4 ligand. To evaluate the effects of pharmaceutical inhibition of TLR4, the same experiments were conducted in Tlr4WT mice after intraperitoneal injection with C34. Comparisons were by student’s t-test with p<0.05.

Results:

In Tlr4WT and Tlr4-/- control mice, the degree of GI motility was similar (Tlr4WT: 100% vs. Tlr4-/-: 95.7% p=.41), and the addition of C34 to unperturbed Tlr4WT mice had no effect on motility (saline: 90% vs C34: 93%, NS). By contrast, Tlr4WT mice treated with the TLR4 agonist LPS (dose 3mg/kg) showed decreased gut motility (saline: 87.5% vs. LPS 60.4%, p<0.05) and also significantly increased gut inflammation as represented by TNFa level (saline: 1.39 vs LPS 10.31, p<0.05). The induction of post-operative ileus resulted in significantly reduced transit (Tlr4WT: 44.6%, Tlr4-/-: 57.9% p<0.05) and increased inflammation (Tlr4WT: 21.3, Tlr4-/-: 9.91 p<0.05), which were milder in Tlr4-/- mice. Strikingly, treatment of mice with the TLR4 inhibitor C34 significantly reduced inflammation and improved motility after intestinal manipulation, consistent with reversal of this post-operative complication.

Conclusion:
 

These results prove that experimental post-operative ileus is mediated through a TLR4-dependent inflammatory pathway and reveal that a novel TLR4 inhibitor can attenuate inflammation and dysmotility.

64.01 Remote Ischemic Conditioning to Decrease Postoperative Complications After Major Abdominal Surgery

Posted on January 31, 2019

J. Rosado1, A. Alvarez1, K. P. Oberoi1, G. Dikdan1, S. R. Pentakota1, S. Husain2, B. Koneru1  1Rutgers New Jersey Medical School,Surgery,Newark, NEW JERSEY, USA 2Rutgers New Jersey Medical School,Genomics Center,Newark, NJ, USA

Introduction:  Remote ischemic conditioning (RIC) modulates inflammation after ischemia reperfusion, and may decrease postsurgical inflammation and complications. Here we present preliminary data regarding peripheral blood leukocyte gene expression in subjects from a prospective randomized a phase II clinical trial of RIC in progress (NCT03234543).

Methods: Adults undergoing abdominal surgery (duration > 2 hours, hospital stay > 2 days) are randomized (1:1) to receive either RIC or sham intervention immediately before surgery and on postoperative days 1 and 2. Each RIC intervention comprises 3 cycles of 5/5 minutes of inflation/deflation of mid-thigh cuff in one lower extremity. Inflation pressures were 250 mmHg for the first and systolic pressures plus 50 mmHg for the others. Sham interventions comprise cuff inflation pressure of 20 mmHg.  Primary outcome is 30-day complications measured as comprehensive complications index. Peripheral blood was collected for plasma and RNA before (R0) and 1hr post surgery (R1), and 1 hour after 2nd and 3rd interventions (R2 and R3). Leukocyte mRNA transcripts were examined in R0 and R3 samples of both groups using RNA sequencing (30 million reads) for global expression profiles (n=6 each; fold change >1.5 and false discovery rate p<0.05) and RT-PCR for cytokine transcripts (IL-1β, IL-6, IL-8, IL-10, and TNF-α; n=8 each; Wilcoxon p< 0.05).

Results: 45 of planned 100 subjects (23/22 RIC/No RIC groups) were enrolled. At baseline (R0), mRNA profiles significantly differed between two groups in only 14 transcripts, and none were related to inflammation. 122 transcripts had significant differential expression between two groups at R3. 39/122 were transcripts of immunoglobulins, and expression levels of all were increased in No RIC. Importantly, TMED7, LGALS2, GZMH, and IL32 transcripts encoding proteins, which promote inflammation via IL-1 signaling, macrophage M1 phenotype, granzyme, and p38 kinase and NF-kb pathways, respectively were increased in No RIC. Transcripts of RELL1, IL1RAP, FKBP5, which encode member of TNF-a receptor family, IL-1 receptor accessory protein, and a cellular glucocorticoid responsiveness protein, respectively were decreased in No RIC.  RT-RCR data showed a trend towards increased expression of IL-10 in No RIC that did not reach significance (p=0.10).

Conclusion: Preliminary results from this novel trial suggest that remote ischemic conditioning in the perioperative period down regulates expression of leukocyte proinflammatory genes. Further studies of gene expression in additional subjects and other time points and studies of plasma acute phase proteins and complement components are in progress. Also, the associations between the molecular data and the primary outcome will be examined. 

 

44.20 Cartilage Oligomeric Matrix Protein (COMP): A Potential Biomarker in Early Onset Colon Cancer.

Posted on January 31, 2019

N. P. Omesiete1, J. Jandova1, K. Memeh1, V. Nfonsam1  1University of Arizona,Surgery,Tucson, AZ, USA 2University of Arizona,Surgery,Tucson, AZ, USA

Introduction:
There has been a steady rise in the incidence of early onset colon cancer (EOCC); age <50. Recent studies suggest that EOCC may be a biologically distinct disease from late onset CC (LOCC). Our previous study showed COMP is upregulated in EOCC compared to LOCC patients and its elevation is associated with tumor aggressiveness and poor survival. The aim of this study was to evaluate COMP as a potential plasma biomarker and to assess its correlation with carcinoembryonic antigen (CEA) level in patients with EOCC.

Methods:

Fresh frozen plasma samples were obtained from our Colorectal biorepository belonging to 16 patients with early and late onset CC (8 patients in each cohort). The samples were matched for stage of disease. An ELISA assay using Human COMP Quantikine ELISA Kit purchased from R&D Systems was performed using 50 ul of 100X diluted plasma sample according to the manufacturer’s protocol. COMP was detected in all the samples of patients with colon cancer.  A quantitative analysis was performed to determine the levels of COMP in each sample by normalizing the COMP protein to total protein. This was accomplished using A Pierce BCA protein assay kit from Thermo scientific. Analysis was performed to compare the levels of COMP between both groups and their CEA levels pre-treatment.

Results:

There were a total of 16 patients (8 males and 8 females). Mean age was 42 years for EOCC and 70 years for LOCC. The patients were propensity matched for stages. There was 1 stage I; 2 stage II; 3 stage III and 2 stage IV in each cohort. COMP was detected in all 16 serum samples. Mean COMP levels was 140 pg of COMP/ug of total protein in EOCC and 20 pg of COMP/ug of total protein in LOCC, indicating a seven fold difference between the cohorts, p <0.005. There was also a positive correlation between COMP and the pre-treatment CEA levels for each stage of disease in each cohort. This was however not statistically significant. 

Conclusion:
Our findings suggest that COMP is differentially and significantly elevated in EOCC with good correlation with an established biomarker CEA. COMP may be useful as a biomarker in EOCC.

44.19 Anti-Claudin-1 Conjugated to a Near Infrared Fluorophore Targets Colon Cancer in Nude Mouse Models

Posted on January 31, 2019

H. M. Hollandsworth1,2,5, T. M. Lwin1,2,5, S. Amirfakhri1,2,5, F. Filemoni1,2,5, D. Stupack2, S. K. Batra3, R. M. Hoffman1,2,4,5, P. Dhawan3, M. Bouvet1,2,5  1University Of California – San Diego,Department Of Surgery,San Diego, CA, USA 2University Of California – San Diego,Moores Cancer Center,San Diego, CA, USA 3University of Nebraska Medical Center,Department Of Biochemistry,Omaha, NE, USA 4AntiCancer, Inc.,San Diego, CA, USA 5VA San Diego Healthcare System,Surgical Services,San Diego, CA, USA

Introduction:
Colorectal cancer remains one of the most prevalent cancers in the United States and is the third leading cause of cancer related death. Claudins are tight junction proteins which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated in the development of colon cancer. Tumor cells express a significantly higher amount of Claudin when compared with normal colonic mucosa.   We postulated that Claudin-1 may be a useful target in near infrared imaging and fluorescence guided surgery for colorectal cancers.

Methods:
We conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 different human colon cancer lysates was performed with Claudin-1-IRDye800CW. Animal imaging was initially performed with LI-COR Pearl Trilogy imaging system on subcutaneous and orthotopic colon tumors with 75 mcg Claudin-1-IRDye800CW 72 hours after injection. A dose-response study was then completed on subcutaneous cell line models. Subcutaneous implantation of LS174T on the bilateral flanks of nude mice was performed. Three groups of mice were administered increasing doses of Claudin-1-IRDye800CW: 12.5, 25, and 50 micrograms reconstituted in 100 microliters PBS, via tail vein injection. 3 mice were used as controls (no treatment, antibody alone, and 1 dye alone). In vivo imaging was performed at 24, 48, and 72 hours after administration of the conjugated dye. The mice were then euthanized and laparotomy was performed to assess for enhancement of internal organs.

Results:
Western blotting revealed that 9 out of 10 colon cancer xenografts expressed varying amounts of Claudin-1.  All tumors demonstrated strong and specific fluorescence labeling at 800 nm wavelength, even with the lowest dose of 12.5 micrograms. A representative image of mouse with an orthotopic LS174T colon tumor treated with 75 mcg of Claudin-1-IRDye800CW is shown in the Figure below. The control mice that did not receive treatment and received Claudin-1 antibody alone did not have any signal on the colon or tumor. The mouse that received IRDye800CW alone had diffuse signal that did not localize to tumors.

Conclusion:
Claudin-1 is a useful target for near infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.  
 

44.17 Intestinal Alkaline Phosphatase Protects Against Radiation Induced Gut Barrier Dysfunction

Posted on January 31, 2019

P. Cavallaro1, F. Adiliaghdam1, Y. Liu1, M. Kaleko1, C. Furlan Freguia1, R. Hodin1  1Massachusetts General Hospital,General Surgery,Boston, MA, USA

Introduction: Radiation injury induces gut barrier dysfunction and local intestinal inflammation, a clinical entity known as radiation enteritis/colitis. The brush border enzyme intestinal alkaline phosphatase (IAP) has been shown to maintain the gut mucosal barrier and attenuate local and systemic inflammation in multiple mouse models of colitis. We wanted to determine if supplemental IAP can prevent radiation induced gut barrier dysfunction and local inflammation, and therefore be a potential therapy for radiation enteritis/colitis.

 

Methods: Adult WT C57BL/6J mice were subjected to whole body irradiation (IR) to a total of 850 or 1400 rads. Mice were administered either 100 U/mL IAP or vehicle in their drinking water starting 4 days prior to IR injury and then continued until mice were sacrificed 4 days after injury. Control “sham” mice received no IR injury. Gut barrier function, tissue and systemic inflammation were measured.

 

Results:There were no difference in food and water intake among any of the groups. Gut barrier function was first measured by FITC-dextran flux from intestinal lumen to systemic blood. Vehicle treated IR mice had a radiation dose dependent increase in FITC flux across the gut barrier compared to sham (10-fold increase in 850 rad group, 30-fold increase in 1400 rad group). Permeability to FITC was almost completely attenuated in IAP treated mice in both groups (p < 0.001). Quantitative PCR of tight junction proteins demonstrated a significant decrease in ileal ZO1, ZO3, and occludin that was partially restored by IAP supplementation in both IR groups. Quantitative PCR also showed a significant decrease in colonic ZO1, ZO3, and occludin, again partially restored by IAP. Similarly, Western blot analysis showed a significant decrease in TJPs in IR treated mice, restored with IAP therapy. Next, local inflammation was assessed by measuring cytokine expression. Relative ileal and colonic TNF-α expression was increased 10-fold in the 850 rad group and 30-fold in the 1400 rad group compared to sham mice and was decreased by 50-70% when treated with IAP (p <0.001). Systemic inflammation was measured by serum ELISA which showed a two-fold increase in TNF-α and IL-6. Again, this response was attenuated by IAP. Interestingly, there was no difference in serum LPS between groups.

 

Conclusion:Radiation-induced gut barrier dysfunction and both local and systemic inflammation can be attenuated with supplemental IAP. This may be a novel approach to the treatment and prevention of radiation enteritis/colitis.

44.16 Characterizing Ileal and Colonic Barrier Permeability in Farnesoid X Receptor Knock Out Mice

Posted on January 31, 2019

M. Mallicote1, C. Gayer1  1Children’s Hospital Los Angeles,Los Angeles, CA, USA

Introduction: Activation of the farnesoid X receptor (FXR) has been reported to decrease gut permeability in colonic murine injury models like DSS colitis, protecting against intestinal bacterial translocation. However, we have shown that FXR knock out (FXR-KO) mice have attenuated barrier dysfunction when challenged with LPS, which affects the small bowel much more than the colon. These observations suggest that FXR may function differently in the small versus large bowel. We hypothesize that FXR-KO animals will have increased intestinal barrier permeability in the colon while less barrier permeability in the small intestine.

 

Methods: Four cm segments of terminal ileum and proximal colon were collected from WT and FXR-KO mice. Luminal contents were gently flushed out and replaced with a fluorescein isothiocyanate-dextran (FITC) solution with or without 2 mg/ml LPS. Both ends of the intestinal segments were then sealed with 2-0 silk ties. Each sealed intestinal segment was then placed in 2 ml of phosphate-buffered saline and barrier leakage was assessed by measuring FITC levels in PBS at 0, 1, 2, 4, 8, 16, and 24-hour time intervals. Two-way repeated measures ANOVA was performed as appropriate.

 

Results: Ileal barrier permeability in FXR-KO mice was significantly increased at 24 hours compared to WT mice at baseline. However, when challenged with LPS, the ileal barrier permeability was attenuated in FXR-KO mice but was worsened in WT mice at 24 hours. No difference was seen in colonic barrier permeability in FXR-KO mice when compared to WT controls. When treated with LPS, WT animals showed significantly increased barrier permeability at 8 hours, while FXR-KO animals did not show a change in barrier function

 

Conclusions: These data suggest that FXR affects the intestinal barrier differently in the small intestine versus the colon, both at baseline and in response to inflammatory stimuli. Understanding these differential effects is crucial if we are to target FXR in diseases such as Crohn’s disease that affects both the small bowel and colon.

44.15 Genetic sub-clones in rectal cancer respond differentially to neoadjuvant therapy

Posted on January 31, 2019

L. Frydrych1, L. H. Maguire2, P. Ulintz3, A. Bankhead4,8, J. K. Greenson5, C. J. Sifuentes3, E. Fearon6,7, K. M. Hardiman2  1University Of Michigan,Department of Surgery,Ann Arbor, MI, USA 2University Of Michigan,Colorectal Surgery,Ann Arbor, MI, USA 3University Of Michigan,Bioinformatics Core,Ann Arbor, MI, USA 4University Of Michigan,Biostatistics,Ann Arbor, MI, USA 5University Of Michigan,Pathology,Ann Arbor, MI, USA 6University Of Michigan,Internal Medicine,Ann Arbor, MI, USA 7University Of Michigan,Human Genetics,Ann Arbor, MI, USA 8University Of Michigan,Computational Medicine And Bioinformatics,Ann Arbor, MI, USA

Introduction:  Recommendations for treatment of locally advanced rectal cancer include chemotherapy, radiation and surgery. Response to neo-adjuvant therapy is variable. We have previously shown rectal cancers are made up of multiple genetically distinct sub-clones.  Unique sub-clones within primary tumors may harbor mutations which contribute to treatment resistance, relapse, and inter-patient variations in response to standard-of-care neo-adjuvant therapy. Analysis of the influence of neoadjuvant therapy on the molecular evolution of rectal cancer may provide insight about mechanisms of resistance and highlight new therapeutic approaches for patients.

Methods: Rectal cancer patients with an indication for neo-adjuvant chemoradiotherapy were identified at a tertiary referral center. Primary tumor biopsies from multiple discrete geographic locations were obtained prior to treatment. At the time of surgical intervention after standard-of-care chemoradiotherapy, tissue from the treated tumor was obtained and analyzed. Pre- and post- treatment specimens were subjected to whole exome sequencing followed by confirmatory deep sequencing to define somatic mutations. Copy number variation was assessed in all samples using Oncoscan SNP arrays. Genomic data were analyzed using Pyclone to identify sub-clonal tumor populations that differed in frequency after neo-adjuvant chemoradiotherapy. Recurrent drivers of resistance were identified in the sub-clones across tumors. Using Hotnet2, pathway analysis was performed on integrated resistant gene mutation and copy number alteration data.

Results: 32 samples were obtained from 10 patients. Pyclone identified 2-9 individual subclonal populations per tumor. Neoadjuvant therapy resulted in substantial change in the relative proportions of individual sub-clones within the primary tumor. Resistant sub-clones recurrently (>30%) contained mutations in TP53, APC, ABCA13, ITIH5, MUC16, PTEN, THSD4, and TNS1. Recurrent copy number variations for selected chromosome regions were seen in cancers after therapy. Pathway analysis revealed significantly altered pathways in resistant tumor specimens associated with RNA splicing and processing, regulation of transcription, APC-mediated pathways, mTOR signaling, fatty acid biosynthesis, and histone modification.

Conclusion: Intra-tumoral heterogeneity is evident in pre-treatment rectal cancer and sub-clonal populations are selectively modified by treatment. Resistant sub-clones demonstrate high frequencies of somatic mutation in multiple known tumorigenesis driver genes, and intra-tumoral heterogeneity persists post-treatment. Our analyses reveal complex and dynamic genomic architectures in pre- and post-treatment rectal cancers. These data indicate that novel treatment strategies must take into account a changing and heterogeneous tumor mutational profile when attempting to target rectal cancers.
 

44.14 Penicillin Prevents Rat Colonic Ischemia, Validating its Use in Ischemic Gastrointestinal Disease

Posted on January 31, 2019

T. M. Gisinger1,2, V. M. Baratta2, M. Barahona2, J. Ollodart2, D. Mulligan2, J. P. Geibel2,3  1Paracelsus Medical University,Department Of Medicine,Salzburg, SALZBURG, Austria 2Yale University School of Medicine,Department Of Surgery,New Haven, CT, USA 3Yale University School of Medicine,Department Of Cellular And Molecular Physiology,New Haven, CT, USA

Introduction: Ischemic colitis (IC) is the most common type of intestinal ischemia and arises when the colonic blood supply does not meet cellular metabolic demands. Though clinical evidence is lacking, many patients with IC are nonoperatively managed with empiric antibiotics. It has been proposed that antibiotics mitigate ischemia by reducing bacterial translocation and preventing breakdown of the epithelial barrier. In this study, we demonstrate that colonic tissue perfused with Penicillin G is more resilient to ischemic injury than tissues not exposed to the drug. These findings suggest a new clinical management paradigm of preventing ischemic conditions of the gastrointestinal tract.

Methods: Colon segments from rats were obtained and perfused with an ex-vivo intestinal perfusion device. The perfusion device consists of concentric chambers that contain the bowel segments perfused at 37°C. FITC-Inulin, fluorescein isothiocyanate, was used to assess the ischemic conditions of the intestinal grafts in real-time; a drop in fluorescence (FITC-Inulin concentration) is indicative of cellular ischemic injury. Intestinal segments were perfused with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES-Ringer. To create an ischemic environment, the HEPES-Ringer was pre-saturated with 100% N2 and perfused on extraluminal surface of all rat colonic segments. The intraluminal components of experimental colons were perfused with 5 mM Penicillin G, whereas control segments were not.  

Results: Control (without Penicillin G) distal colon samples showed a significant decrease in FITC-inulin fluorescence compared with experimental (with Penicillin G) distal colons, (26.98 ±  5.035 μM FITC vs 43.62 ± 1.569 μM FITC, respectively p 0.0083, Figure 1). This indicates that Penicillin G minimizes colonic fluid secretion, which is a marker for cell death. A similar trend was seen with proximal colon rat segments (42.7 ± 1.984 μM vs. 33.28 ± 3.455 μM FITC, p 0.0356).

Conclusion: Patients with ischemic colitis are often clinically treated with antibiotics, though the pathophysiological basis of their use is not well-proven. Our study shows that Penicillin G exposure prolongs colonic viability under ischemic conditions. This result will help further guide clinical management of ischemia in the gastrointestinal tract. Further investigation of the precise mechanism by which Penicillin G mitigates ischemia needs to be conducted.

 

44.13 Hernia Repair Outcomes Enhanced with Both Doxycycline and Antioxidant Therapy

Posted on January 31, 2019

C. Totten1, J. Tharappel1, J. S. Roth1  1University Of Kentucky,General Surgery/Surgery/Medicine,Lexington, KY, USA

Introduction: Incisional hernia is one of the most common complications of abdominal surgery and repairs are associated with significant recurrence rates. Mesh repairs are associated with the best outcomes, but failures are not uncommon. Doxycycline, an antibiotic with antioxidant properties, has been demonstrated to enhance mesh hernia repair outcomes with associated increases in Collagen deposition and improved tensio-metric strength.  This study compares the outcomes of incisional hernia repair with doxycycline administration and the antioxidant tempol.

Methods: 28 male Sprague Dawley rats were assigned to 4 groups: Control (C), Doxycycline (D), Tempol (T), and Doxycyline/Tempol (D/T). Animals underwent midline laparotomy with excision of 1x4cm strip of the midline fascia and repair with a 5 x 6 cm polypropylene mesh as an intraperitoneal underlay. Animals were administered saline, doxycycline (30mg/kg), tempol (20mg/kg), or both beginning one day prior to operation and then daily for 8 weeks.  Abdominal wall was harvested at 8 weeks with analysis of tensio-metric strength and biochemical analysis.

Results:Animals were survived for 8 weeks.  The tensiometric strength of the mesh to fascial interface was increased in the experiemental groups compared to control. (C-15.29, D-24.45, T-24.35, D/T-19.36 ) [Fig 1.].  Collagen 1 deposition was increased and Collagen 3 deposition was decreased in each of the experimental groups relative to control. MMP-2 and MMP-9 was decreased in doxycycline treatment groups.

Conclusion:The strength of a polypropylene hernia repair is enhanced with the administration of doxycycline and tempol.  Dual therapy with doxycycline and tempol provided no benefit over treatment with a single agent.  Treatment with these agents is associated with increased Collagen 1/3 ratios.  The benefits of antioxidant treatment following hernia repair are similar to treatment with doxycycline. The mechanism of antioxidant mediated collagen-1 increases are not well understood and require further study.  In light of the high frequency of incisional hernia repair failures, this study has implications for improving outcomes following ventral hernia repair through the use of either doxycycline or antioxidant therapy.
 

44.12 Penicillin’s Protective Effect on Colonic Ischemia is Mediated by H,KATPase

Posted on January 31, 2019

T. M. Gisinger1,2, V. M. Baratta1, M. J. Barahona1, J. Ollodart1, D. Mulligan1, J. P. Geibel1,3  1Yale University School Of Medicine,Department of Surgery,New Haven, CT, USA 2Paracelsus Medical University,Department of Medicine,Salzburg, SALZBURG, Austria 3Yale University School Of Medicine,Department of Cellular and Molecular Physiology,New Haven, CT, USA

Introduction: Ischemic colitis is one of the most common types of intestinal ischemia. Currently, many patients with ischemic colitis are treated with empiric antibiotics, even though the mechanism of action is not fully understood. Previously, we established that Penicillin G has a protective effect from ischemia in the rat colon. In this study, we demonstrate that the protective effect is mediated through stimulation of the colonic H,KATPase, independent of the Nitric Oxide (NO) pathway.

Methods:  The colonic segments were harvested from Sprague Dawley male rats and perfused with an ex-vivo intestinal perfusion device. Each colon was maintained at 37°C and perfused both from the luminal and basolateral side. FITC-Inulin, fluorescein isothiocyanate, was used to assess the ischemic conditions of the colonic grafts in real-time. Colonic segments were perfused with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES-Ringer solution. To create an ischemic environment, HEPES-Ringer was pre-saturated with 100% N2 and exposed to the extraluminal components of all colonic segments. For the experimental colonic tissues, the intraluminal compartments were perfused with 5 mM Penicillin G and 10 μM SCH-28080, a known colonic H,KATPase inhibitor. The intraluminal components of the control group were exposed to 5 mM Penicillin G. To test the NO-dependent mechanism, we used L-NAME, N(ω)-nitro-L-arginine methyl ester, a NO synthesis inhibitor. The intraluminal compartments of the experimental tissue were exposed to 30 μM L-NAME with 5 mM Penicillin G, while control tissues were exposed to 5 mM Penicillin G.

Results: The colon samples exposed to Penicillin G and SCH-28080 exhibited a significant decrease in FITC-Inulin fluorescence, compared with the control colonic tissue exposed to Penicillin G, (36.39 ± 2.721 μM FITC-Inulin vs 43.62 ± 1.569 μM FITC-Inulin, respectively, p 0.0401, Figure 1). We observed no statistically significant difference in the FITC-Inulin concentration between tissues exposed to L-NAME with Penicillin G versus tissues exposed to only Penicillin G.

Conclusion: Our study unveils the mechanism of Penicillin G’s protective effect from ischemia. Our results indicate that Penicillin G’s protective effect against ischemia acts by stimulating the colonic H,KATPase and is not NO-dependent. Therefore, Penicillin G not only has its well-known antimicrobial properties, but also appears to modulate a transport protein on the colonic cell membrane. A better understanding of Penicillin G’s effects on colonic tissue may help further guide the clinical management of ischemia in the gastrointestinal tract.

 

44.11 Nanoparticle activation of the Calcium Sensing Receptor prevents Ischemic Injury in the Rat Intestine

Posted on January 31, 2019

M. J. Barahona1, M. Finotti1,4, J. Ollodart1, V. M. Baratta1, T. M. Gisinger1,2, G. Caturegli1,4, R. M. Maina1,4, F. D’Amico1,4, D. Mulligan1, J. P. Geibel1,3  1Yale University School Of Medicine,Department Of Surgery,New Haven, CT, USA 2Paracelsus Medical University,Department of Medicine,Salzburg, SALZBURG, Austria 3Yale University School Of Medicine,Department of Cellular and Molecular Physiology,New Haven, CT, USA 4University of Padua,Department Of Transplantation And Hepatobiliary Surgery,Padua, PADUA, Italy

Introduction:  The intestine is one of the most susceptible organs to ischemia making it extremely difficult to transplant. There is a need for the development of innovative methods to preserve intestinal viability. New efforts are focused on creating particle based delivery systems in the range from 10-1000 nm, or collectively known as nanoparticles. Formulation of nutraceuticals into nanoparticles and nanocomplexes can better facilitate delivery and cellular uptake in colonic systems. Here, we examined how calcium nanoparticle perfusion targeting the Calcium Sensing Receptor (CaSR) could reduce intestinal ischemic damage in the mammalian colon.

Methods:  Small intestinal segments from Sprague-Dawley male rats were obtained and perfused with an ex-vivo intestinal perfusion device. Segments were perfused with and without calcium nanoparticles in an induced ischemic environment, 100% N2 and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), compared to small intestine segments perfused without an ischemic environment (HEPES). Fluid secretion or absorption of the intestine was measured by fluorescein isothiocyanate-inulin (FITC-Inulin). Using FITC-Inulin concentration we assessed the ischemic conditions (decreased fluorescence) of the perfusate through the intestinal grafts in real-time. 

Results: Small intestinal segments exposed to 100% N2 developed a significantly greater degree of ischemic damage when compared to intestine perfused with normal HEPES buffer, p <0.0001. In this nitrogen-induced ischemic environment, the presence of 1.0 mM, 2.5 mM and 5.0 mM of calcium carbonate nanoparticles prevented the damage (increased fluid secretion, p <0.0001, Figure 1). Intestinal segments exposed to nitrogen and nanoparticles resisted ischemia to a greater extent than segments exposed to normal HEPES p <0.0001.

Conclusion: Calcium carbonate nanoparticles targeting the CaSR can mitigate ischemic damage in the small intestine. These results suggest that nanoparticles may be a novel therapeutic vector for reducing ischemic and inflammatory injury. This suggests that nanoparticle activation of CaSR would be an important prophylactic therapy to improve organ viability.  

 

44.10 Duration of Doxycycline Treatment Impacts Hernia Repair Strength

Posted on January 31, 2019

C. Totten1, J. Tharappel1, J. S. Roth1  1University Of Kentucky,General Surgery/Surgery/Medicine,Lexington, KY, USA

Introduction: Incisional hernia formation compliates up to 30% of abdominal operations. Doxycycline, an inhibitor of MMPs, can enhance the hernia repair strength, increase collagen 1 depositon and reduce collagen 3 deposition. The optimal timing and duration of treatment is not well understood.  The present study evaluates the effect of duration of doxycycline treatment on hernia repair strength and collagen deposition in an animal model.

Methods: 42 male Sprague Dawley rats underwent hernia creation and repair with a polypropylene mesh. Animals were randomly assigned into 6 groups: (1) 16 weeks of doxycycline treatment with 8 week additional survival  (24 weeks total) (2) 16 weeks of saline treatment and 8 weeks additional survival (3) 8 weeks of doxycycline treatment and 8 weeks additional survival (4) 8 weeks of saline treatment and 8 weeks additional survival, (5) 16 weeks of doxycycline treatment with no additional survival, (6)16 weeks of saline treatment and no additional survival. After the assigned treatment and interval times,  abdominal walls with implanted mesh were harvested and tensiometric testing and biochemical analyses were performed.

Results:42 animals underwent hernia repair with survival. Animals administered doxycycline for 8 weeks demonstrated no improvement in hernia repair strength relative to controls ( Group 1- 20.46 N vs. Group 2 -18.85 N, NS) . Animals receiving 16 weeks of doxycycline with 24-week total post hernia repair survival time demonstrated enhanced abdominal wall strength relative to control (Group 3 -18.55 N vs Group 4 – 15.95 N, p=.06). Animals treated continuously with doxycycline for 16 weeks up until the time of necropsy demonstrated improved abdominal wall strength (Group 5 – 23.77 N vs. Group 6 – 20.63 N, p=.008). Collagen 1 deposition was increased and MMP-2 was decreased in the animals treated with doxycycline for 16 weeks.

Conclusion:In an experimental model of hernia repair, doxycycline administration for 16 weeks was associated with enhanced tensiometric strength and greater collagen 1 deposition than placebo whereas 8 weeks of doxcycyline therapy was not effective. In light of this finding, future studies evaluating the impact of doxycycline upon hernia repair outcomes should utilize longer durations of therapy.  Future studies are needed to ascertain the benefits of doxycycline in hernia repair beyond 16 weeks duration. Translational studies utilizng doxycycline are required to evaluate the impact upon clinical outcomes. 

 

44.09 Mechanisms mediating stress-induced vulnerability to gastrointestinal inflammation

Posted on January 31, 2019

B. Vickers1, C. Graham2, A. Chakraborti2, A. Moon2, J. Bibb2, G. Kennedy2  1Rhodes College,Memphis, TN, USA 2University Of Alabama at Birmingham,Gastrointestinal Surgery,Birmingham, Alabama, USA

Introduction:  Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a chronic and relapsing inflammatory disorder of the intestine. Approximately 1 million Americans are currently diagnosed with IBD and as many as 70,000 new cases are diagnosed each year. One of the main factors contributing to IBD is stress, which has become a rampant problem in America and is considered a high-ranking health concern. Unfortunately, the relationship between IBD and stress has not been thoroughly investigated and the contributions of stress to IBD pathology are not well understood. We hypothesize that stress decreases susceptibility to intestinal inflammation.

Methods:  To better understand the pathways that connect stress and IBD, C57Bl/6 mice were administered a Chronic Unpredictable Stress (CUS) regimen followed by 7 consecutive days of  a subthreshold dose of  Dextran Sodium Sulfate (DSS), a chemical inducer of colitis. After completion of CUS +/- DSS, animals were sacrificed, fecal samples were collected, and RNA was isolated from the proximal colon.  

Results: To assess the effects of CUS and/or DSS on colon inflammation, an Enzyme-Linked Immunosorbent Assay (ELISA) was performed to quantify Lipocalin-2 (LCN-2) protein levels in fecal samples. We found that the LCN-2 levels in the CUS + DSS mice were significantly higher than control. Additionally, Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) was used to quantify mRNA transcript levels of three pro-inflammatory cytokines (IL-1β, IL-6, TNFα) in the proximal colon. Interestingly, CUS + DSS mice showed a significant increase in TNFα over control or either treatment alone.  

Conclusion: We found that CUS and DSS alone failed to induce inflammation, but the combination of CUS and DSS showed a significant increase in colon inflammatory mediators suggesting that chronic stress may lower gut inflammatory threshold thereby increasing susceptibility to colitis.  In the future, we plan to assess CUS in an IL10-/- colitis model as well as in the context of high fat/high carbohydrate diet. 

44.08 Increased expression of long Non-coding RNA H19 is Associated with Colon Adenocarcinoma Recurrence

Posted on January 31, 2019

S. J. O’Brien1, C. Fiechter1, M. Paas1, A. Rochet1, S. Galandiuk1  1University Of Louisville,Price Institute Of Surgical Research, The Hiram C. Polk Jr. M.D. Department Of Surgery,Louisville, KY, USA

Introduction:

Colorectal adenocarcinoma is the fourth leading cause of cancer-related death in the United States. Non-coding RNAs have recently been identified as critical mediators of tumor biology. Long non-coding RNAs (lncRNAs) are diverse in their mechanisms but are known mediators of tumor progression; H19 is a well characterized lncRNA involved in the regulation of P53 and in cancer progression. The aim of this study was to identify the association of tumor H19 expression with recurrence-free and overall survival in a colon cancer data set.

Methods:

The clinical dataset from The Cancer Genome Atlas colon adenocarcinoma data set was downloaded using FirebrowseR. The normalized expression of H19 from the associated RNA-seq data set was downloaded using cBioportal. Univariable and multivariable cox proportional regression analysis were used to identify an association between H19 expression in cancer tissue at the time of resection and recurrence-free, and overall survival. 

Results:

Four hundred and eighteen patients were included in this study. Median age was 69 years (IQR 58-75) Two hundred and nineteen patients (52%) were men. Most tumors were located in the sigmoid colon (135/418- 32%).  Two hundred and thirty-nine patients (57%) had stage 1 or 2 cancers. Lymphatic invasion was present in 36% patients (152/418). The median expression of H19 in the data set defined high or low expression groups. The only difference between groups was that high H19 expression was associated with stage 3 and 4 disease (p=0.048). There was no difference in the overall survival between the low and high H19 groups (Log Rank=0.481). High H19 expression was associated with reduced recurrence-free survival (Log-Rank=0.007) (Figure 1). On univariable regression analysis, high H19 expression, stage 3 or 4 disease, and lymphatic invasion were associated with disease recurrence (Hazard ratio=1.861, 95%CI:1.181-2.932, p=0.007, HR=2.498, 95%CI: 1.622-3.847, P<0.001, and HR= 1.928, 95%CI: 1.233-3.014, p=0.004 respectively). On multivariable regression analysis, only high H19 expression (HR=1.686, 95%CI: 1.046-2.720, p=0.032) and stage 3 or 4 disease (HR=2.888, 95%CI: 1.317-3.976, P=0.003) remained statistically significant predictors of disease recurrence.

Conclusion: 

H19 was associated with advanced stage of tumor disease and is a significant predictor of recurrent cancer on multivariable analysis. The results of this study are in keeping with in vitro studies in that H19 has a number of oncogenic mechanisms of action, including increased proliferation and mediation of epithelial-mesenchymal transition. As it is a critical molecule in cell regulation, it may have both prognostic and therapeutic uses in the future.

 

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