71.04 A Deacytlase Enzyme, SIRT3, Plays a Major Role in Macrophage Inflammation and Wound Healing

Posted on January 26, 2017

A. E. Boniakowski1, A. Kimball1, A. Joshi1, S. Kunkel1, K. A. Gallagher1  1University Of Michigan,Ann Arbor, MI, USA

Introduction: Wound healing in chronic inflammatory diseases, such as diabetes, is impaired due to failed resolution of inflammation.  Innate immune cells, particularly macrophages, play a significant role in the establishment of a regulated inflammatory response during wounding.  Macrophage function is dictated by metabolism, which alters gene expression.  The mitochondrial deacytlase enzyme, SIRT3, was recently shown to suppress inflammatory signaling and decrease reactive oxygen species; therefore we investigated the role of SIRT3 in inflammation in wound macrophages.

Methods: SIRT3 knockout (KO) mice and their littermate controls were either fed a normal diet or a high fat diet (HFD) for 12 weeks.  They subsequently underwent 4mm hindlimb wounds, and CD3-CD19-NK1.1-CD11b+ (macrophages) were isolated from mouse wounds on days 1, 3, 5, and 7 using magnetic bead sorting. SIRT3 expression was quantified by qPCR. 

Results: Utilizing a murine model of wound healing, CD3-CD19-NK1.1-CD11b+ (macrophages) isolated from wounds demonstrated a significant three fold increase in SIRT3 gene expression during the intermediate stage (day 3-5) of wound healing.  Additionally, when we examined wound macrophages from our diet-induced obese (DIO) model of diabetes, we found that they did not upregulate SIRT3. To directly examine the role of SIRT3 in diabetic wound healing, we wounded our SIRT3KO on HFD and compared them to HFD littermate controls. We found our SIRT3KO animals on HFD demonstrated significantly delayed wound healing compared to HFD controls. (Fig 1)

Conclusion: SIRT3 is important in macrophages for normal wound healing and is decreased in macrophages from wounds of diabetic mice. These results suggest an important role for SIRT3 in regulating inflammation in wound healing.

 

71.03 Diabetic MSCs Promote Endothelial Cell Invasion through Unique Angiogen Pathways

Posted on January 26, 2017

A. D. Morris1, H. Li1, L. Brewster1,2  1Emory University Hospital,Division of Vascular Surgery,Atlanta, GA, USA 2Atlanta VA Medical Center,Division of Vascular Surgery,Atlanta, GA, USA

Introduction: Diabetes increases the risk of cardiovascular disease and major amputation. Mesenchymal Stem Cells(MSCs) have robust regenerative potential, but concerns remain that MSCs from diabetic patients or under diabetic conditions may limit therapeutic effect. The objective of this project was to compare healthy MSCs(hMSCs) and diabetic MSCs(dMSCs) angiogenic activity under standard and high glucose conditions.

Methods: MSCs were harvested from 4 patients (2 healthy and 2 diabetic). Human aortic endothelial cells (HAECs) (20,000 cells), HAECs with hMSCs(10,000/10,000 cells) or HAEC with dMSC(10,000/10,000 cells) were formed into pellets, suspended in fibrin gel, and cultured in 5mM, 20mM, or 40mM glucose media. Cell invasion was captured by daily microscopy for 3 days. To obtain secretome, hMSCs and dMSCs were cultured under 5mM glucose, 20mM glucose, or 40mM glucose in 10% FBS media for 24 hours. Secretome analysis was performed using a commercial microarray for 20 individual angiogenic factors. Densitometry using positive control as reference allowed comparison of relative secretion.

Results: High glucose environments did not significantly inhibit HAEC invasion at any time point. At day 3, co-culture with hMSCs improved HAEC invasion under high glucose conditions(20mM and 40mM, p= 0.004 and p=0.02) but not in 5mM glucose media(p=.24). dMSC co-culture also improved HAEC invasion at day 3 (p<0.001 for all conditions). On day 3, dMSCs encouraged HAEC invasion more than hMSCs in 5mM glucose and 40mM glucose, and equivalently in 20mM glucose (p<0.01,p<0.01,p=0.14). hMSC co-culture improved HAEC invasion in 20mM glucose compared to 5mM and 40mM glucose conditions(Day 2:p=0.034,p<0.001, Day 3:p=0.029,p=0.013). On day 1, 40mM glucose inhibited HAEC invasion in dMSC co-culture compared to 5mM glucose(p=0.002), but that difference did not persist on day 2 or day 3(p=0.366, p=0.176).

Compared to hMSCs, dMSCs secreted increased levels of CXCL-1(p=0.002). Secretion of CXCL-1 was an average of 6.5 times greater by dMSCs versus hMSCs. Additionally, 20mM glucose environments stimulated secretion of CXCL-1 compared to 5mM and 40mM glucose in dMSCs(p=0.043, p=0.021).

Conclusions: We have shown that addition of hMSCs and dMSCs are capable of improving cell invasion. Diabetic MSCs are not inferior to hMSCs and may have superior function in certain instances. While dMSC and hMSC are functionally similar, dMSCs secrete the unusual angiogen CXCL-1 at higher levels. We theorize that CXCL-1 may serve as an alternate pathway for encouraging endothelial function under diabetic conditions. With future in vivo experiments, we hope to show dMSCs retain their function by alternate mechanisms and dMSCs can be used in clinical trials with equal efficacy.

71.02 Identifying Lost Surgical Needles with Visible and Near Infrared Fluorescent Light Emitting Coating

Posted on January 26, 2017

E. P. Ward1, J. Yang3, J. Delong1, J. Wang2, N. Mendez4, C. Barback5, S. Horgan1, W. Trogler3, A. Kummel3, S. Blair1  1University Of California – San Diego,General Surgery,San Diego, CA, USA 2University Of California – San Diego,Nanoengineering,San Diego, CA, USA 3University Of California – San Diego,Chemistry,San Diego, CA, USA 4University Of California – San Diego,Material Science,San Diego, CA, USA 5University Of California – San Diego,Radiology,San Diego, CA, USA

Introduction: The consequences of retained foreign bodies (RFB) are significant for all types of medical procedures, but in laparoscopic surgery, RFB such as lost surgical needles may cause a minimally invasive surgery (MIS) to be converted to an open surgery. MIS relies on an endoscopically placed camera for navigation and visual localization of a small item, such as a needle, can be daunting and time consuming. A dual-purpose film to coat surgical needles was developed to augment localization of needles under UV (black light) for open procedures and near infrared (NIR) light for MIS cases using specialized fluorescent laparoscopes.

Methods: Epoxy was used as the matrix for dansyl chloride (DC) and indocyanine green (ICG) as visible and NIR labels, respectively, in a single film.  The needles were coated via dip coating with methanol cosolvent and subsequently cured at room temperature to form a clear polymer film with tunable thickness ranging from 10 to 30 um. With UV excitation at 390 nm, DC emits green fluorescence at 520 nm. With 980 nm NIR excitation, ICG dye emits NIR light above 1000 nm visible with specialized laparoscopes. IACUC approved open and laparoscopic surgeries were simulated in New Zealand white rabbits. In the laparoscopic setting, 26 needles were searched for with a standard camera by a surgeon and 26 with an NIR sensitive laparoscope. The surgeon was blinded to needle location. In the open laparotomy setting, 26 needles were searched for with standard light and 26 were searched for with a UV light. Control needles not located within the maximum 300 s were searched with the assistance of the corresponding NIR or UV light. Time to identification was evaluated for statistical significance, p<.05. 

Results:All 52 dual dye coated needles searched utilizing the NIR camera (n=26) or UV light (n=26) were located within 300 s. 9 needles in both control settings were unable to be located within 300 s (p=0.0006). The mean time to locate control needles in the open surgery and laparoscopic surgery was statistically 2-3x longer than the time to localization utilizing the dye as an adjunct (p=.0027 for open, p<.001 for laparoscopic, Table 1). Overall the dual dye resulted in greater reducuction in time required to locate the needles in laparoscopic surgery compared to open surgery (p=.0006).

Conclusion:The incorporation of a dual-dye coating on surgical needles shows potential to improve the efficiency of locating RFB and may minimize the need to convert a MIS procedure to an open surgery.  Although a benefit was quantified for open surgery the greatest benefit was observed in MIS. Dual-dye coating of surgical needles has potential to decrease the time to localize lost surgical needles and may reduce the risk of RFB.

71.01 Integrin αVβ3 as a Target to Increase Endothelial Cell Recruitment Under Shear Stress Conditions

Posted on January 26, 2017

E. S. Lee1, K. Samadzadeh1, A. Afkhami1, A. Rona1, D. Hao2, A. Wang2  1Sacramento Veterans Affairs Medical Center,Research Service,Mather, CA, USA 2University Of California – Davis,UC Davis Medical Center,Sacramento, CA, USA

Introduction:  Endothelial cell seeding has been shown to improve long term vascular graft patency and minimize intimal hyperplasia. Previous work has shown endothelial cells wash away after exposure to shear stress conditions. We hypothesize that the use of a novel ligand targeting αVβ3 can improve upon endothelial cell recruitment and adhesion under shear stress conditions.  The purpose of this study is to compare the effects of shear stress in vitro on cell recruitment and adhesion between an αVβ3 Ligand and other conventional ligand substrates.

Methods:  Channels in a BioFlux200 48 well plate were treated with Avidin (20 μg/mL) + Biotin (2 μM) and at various concentrations of the αVβ3 ligand or a fibronectin analog arginylglycylaspartic acid (RGD) (0.05 μM, 0.2 μM, 0.5 μM, 2.0 μM, and 5.0 μM) to determine optimal standard concentration. All channels were treated with phosphate-buffered saline (PBS), Avidin (20 μg/mL), RGD (2 μM) or the αVβ3 ligand (2 μM). Human umbilical vein endothelial cells (HUVECs) from commercial stock of low passage were suspended in media and seeded into channels at a concentration of 1×106 cells/mL under 2 dyn/cm2 shear stress for 2 hours at 37 C. Following cell seeding, the channels were fixed using 3.7% PFA and imaged using an Olympus IX81 fluorescent microscope to determine recruited cell count.

Results: Under shear stress, greater cell recruitment was seen from the αVβ3 ligand treatment (2.62×105 ± 1.02×105) when compared to RGD treatment (1.13 x105 ± 1.03×104) in static concentrations (Figure 1, p = 0.03). Cell adhesion and recruitment were directly correlated with increasing concentrations of both the αVβ3 ligand and RGD treatments.  Cell recruitment between 2 μM and 5 μM αVβ3 ligand was not significantly different. In concentrations above 2 μM, greater adhesion overall was seen from the αVβ3 ligand, which influenced more cell-cell adhesion and clustering resulting in a vastly greater number of recruited HUVECs (4.16×105 vs. 2.57×106).

Conclusion: Targeting for the integrin αVβ3 increases endothelial cell recruitment and adhesion under shear stress conditions much greater than other accepted standard ligand substrates. Clinical application in targeting for the αVβ3 ligand may assist in improving endothelial cell seeding and thereby decreasing vascular bypass graft complications. 

 

67.01 CHALLENGING THE THRESHOLD FOR INTERVENTION IN BREAST CANCER RELATED LYMPHOEDEMA

Posted on January 26, 2017

C. D. Ness1, I. Buccimazza2, B. Sartorius3, S. S. Maharaj1  1Physiotherapy Dept. Health Sciences, UKZN  2Breast Unit, Dept. of General Surgery, Nelson R Mandela School of Medicine  3UKZN,Discipline of public Health, Bio-medical and statistics (UKZN)

Introduction: Breast Cancer Related Lymphoedema (BRCA Ly) is clinically diagnosed once a 2cm circumferential difference is measured between the affected and unaffected limb, equating to 200ml limb volume difference (LVD).

Aim(s):

1. To determine at what limb volume difference (LVD) the lymphatic system starts to fail (pre-clinical) by using Bio Impedance Spectroscopy (BIS).

 2. To determine whether this correlates with a different circumferential/LVD  measurement?

Methods: This was a prospective study comprising 60 consenting, female BRCA survivors, post adjuvant therapy at the Provincial Oncology Clinic KZN. We included all consenting women up to 18 months post radiation and excluded patients with bilateral BRCA or other primary cancers, previous mantle field radiation. Data collected included epidemiological information extracted from the patient files, circumferential limb measurements with a tape measure and electrical impedance in the subcutaneous space using the LDEX-U400  BIS unit. The study was approved by the UKZN Bio-ethics Research Committee BE250/010

Results: BRCA Ly cut off at ≥200ml performed fairly well as a diagnostic tool for true abnormality based on BIS sensitivity of 67% and specificity of 93% ( AUC 0.8). Using a cut off of ≥100ml based on BIS abnormality scores, performed better with sensitivity but specificity was reduced to 69% (AUC 0.84). Optimal break point in actual continuous volume at169ml performed best in terms of diagnostic capability for abnormal/normal BIS, PPV of 71% and NPV of 93%, 95% CI: 0.86-0.99.

Conclusion: This study shows that the lymphatic systems optimal breakpoint for lymphatic system failure is 169ml LVD, which corresponds significantly to abnormal BIS readings. Using a clinical threshold of 100ml/1cm LVD provides 100%NPV but only 52% PPV versus NPV of 93% and PPV of 71% if 169ml LVD is used as a threshold for intervention of lymphatic drainage therapy. Early intervention of risk reducing strategies for patients improves quality of life, function and reduces costs for patients and stakeholders in a low resource setting.

63.10 The Role of the Etoposide Induced 2.4 Gene Ei24 in Regulating Pancreatic β Cell Function

Posted on January 26, 2017

S. R. Hamarneh1, J. M. Ramirez Decrescenzo1, F. M. Kuehn1, A. R. Munoz1, S. Morrison1, F. Adiliaghdam1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery, General & Gastrointestinal Surgery,Boston, MA, USA

Introduction:  Type 2 diabetes is characterized by insulin resistance, inadequate insulin secretion and declined pancreatic β-cell mass. EI24 is a tumor suppressor gene and has emerged as a regulator of autophagy and inflammatory pathways. We sought to explore the role that EI24 plays in insulin production and β-cell proliferation.

Methods:  We performed expression analysis of the EI24 gene using Gene-ontology data from patients with or without diabetes. To study the role of Ei24 in β-cell function, rat insulinoma cell line INS-1 was incubated with different inflammatory mediators and under nutrient deprivation conditions to study the effect of these stressors on Ei24 levels. Additionally, we studied the effect of Ei24 deletion or activation on inflammation levels, autophagy influx and insulin production in β-cells in vitro. Furthermore, functional analysis was performed after immunoprecipitation of Ei24 protein complexes to elucidate the plausible cellular pathways affected by Ei24 in pancreatic β-cells.

Results: Ei24 expression levels were lower in human β-cells from diabetic compared to non-diabetic patients (-1.27 vs. 1, p=0.0004). In INS-1 cells, inflammatory mediators such as TNF-α, LPS and bacterial contents suppressed Ei24 levels. Furthermore, the Ei24 expression was induced by nutrient availability. Overexpression of Ei24 increased insulin levels (Ei24 WT vs. Ei24-overexpressing Cells, 1.0 ± 0.3 Vs. 2.8 ± 0.45 Relative Expression, p< 0.01) in INS-1 cells in vitro.  Ei24 deletion in β-cells altered the expression of genes involved in β-cell activity such as Ppar-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.24 vs. 0.4 ± 0.056 Relative Expression, p< 0.05), Ppar-γ (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.16 Vs. 7.2 ± 1.0 Relative Expression, p< 0.01) and Pgc1-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.18 Vs. 3.7 ± 0.68 Relative Expression, p< 0.01). Also, EI24 deletion increased inflammatory cytokine levels: TNF-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.08 vs. 63 ± 7.8 Relative Expression, p< 0.001), IL-1β (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.23 Vs. 57 ± 5.5 Relative Expression, p< 0.001) and IL-6 (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.1 Vs. 42 ± 3.8 Relative Expression, p< 0.001) and impaired autophagic flux in β cells in vitro. Functional analysis demonstrated an extended role for Ei24 in β-cell function.

Conclusion: EI24 plays a major role in β-cell function and homeostasis. The EI24 pathway in pancreatic β-cells may represent an important therapeutic target to prevent or treat diabetes in humans.

 

63.09 Blockade of Canonical Notch Signaling in Helper T-Cells Impairs Wound Healing

Posted on January 26, 2017

A. S. Kimball1, A. Boniakowski1, A. Joshi1, M. Schaller1, R. Allen1, P. Henke1, I. Maillard1, S. Kunkel1, K. Gallagher1  1University Of Michigan,Ann Arbor, MI, USA

Introduction: For the past century, the study of wound healing has focused mainly on the role of the innate immune system in maintaining tissue homeostasis. Up to this point, there have been few studies looking directly at adaptive immune cells and their role in promoting tissue repair. In this study, we sought to quantify the presence of T-cells in wounds over time and to evaluate their effects on wound healing by blocking a well-described T-cell signaling pathway.  

Methods: C57BL/6 mice were obtained for general wound healing studies. DNMAMLf/f CD4-Cre+/- mice were obtained to evaluate wound healing in the setting of canonical Notch signaling blockade in CD4+ cells. 4mm punch biopsies were created on the mid-backs of the mice and wound healing was monitored daily using NIH ImageJ software. Wounds were harvested at various time-points for cell isolation and analysis. CD3+ cell volumes were calculated over-time post-injury using analytical flow cytometry.

Results: CD3+ T-Cells are present and dynamic in wounds over-time post-injury, representing ~4% of live cells at day 2 and ~6% of the live cells at day 6. Given the two peaks of T-cell presence in the inflammatory and proliferative phases of wound healing, we sought to probe the relevance of these cells by blocking the well described Notch signaling pathway in CD4+ cells. Wound healing was evaluated in DNMAMLf/f CD4-Cre+  vs. Cre- mice and this demonstrated significantly delayed wound healing in those mice with impeded Notch signaling. 

Conclusion: Contrary to popular dogma, the adaptive immune system plays a not-insignificant role in wound healing and further studies are needed to evaluate the role of T-cells and Notch signaling. These findings are consistent with the impaired wound healing seen in immunocompromised patients and represents an exciting new area of future research.

 

63.08 Menin/MicroRNA-24 Regulatory Axis Drives Hepatic Fibrosis in Mdr2-/- Mice

Posted on January 26, 2017

C. Hall1, L. Ehrlich2, T. Shepperd2, A. O’Brien2, G. Alpini2, S. Glaser2, T. C. Lairmore1  1Scott & White Healthcare,Surgery,Temple, Texas, USA 2Texas A & M Health Science Center College Of Medicine,Bryan, TX, USA

Introduction:
Liver transplantation remains the primary treatment for Primary Sclerosing Cholangitis (PSC), one of several cholangiopathies that result in cholestatic liver disease. Mdr2-/- mice provide an in vivo model of PSC with characteristic biliary inflammation and fibrosis that subsequently develop cirrhosis and hepatic malignancies. Since cholangiocytes express a neuroendocrine phenotype within the liver, we tested the hypothesis that the tumor suppressor protein menin is implicated in the progression of liver fibrosis and that menin expression can be regulated in the liver via MicroRNA-24. 

Methods:
Menin expression was measured in human PSC and Mdr2-/- mice. Twelve-week old Mdr2-/- mice were treated with MicroRNA-24 Vivo Morpholino (miR-24 VM) to knockdown microRNA-24 expression levels. Liver fibrosis was evaluated using sirius red staining, hydroxyproline assay, and qPCR for genes associated with liver fibrosis (fibronectin 1 (FN1), collagen type 1 alpha1 (Col1α1), transforming growth factor-Beta1 (TGF-β1), and alpha-smooth muscle actin (α-SMA)). Intrahepatic bile duct mass was visualized using immunohistochemistry for CK-19, a cholangiocyte specific protein. Studies were replicated in vitro using mouse cholangiocytes and human hepatic stellate cells treated with microRNA-24 hairpin inhibitor and mimic. Menin and microRNA-24 gene expression measured via qPCR.

Results:
Menin gene expression was increased in Mdr2-/- mice and advanced stage human PSC samples. Treatment of Mdr2-/- mice with miR-24 VM increased menin expression, which correlated with increased expression of fibrotic genes (Figure 1). Mice treated with miR-24 VM showed significant increase in peri-ductular fibrosis and bile duct mass. In vitro, inhibition of microRNA-24 significantly increased cholangiocyte expression of FN1, Col1α1, TGF-β1, and α-SMA. 

Conclusion:
The menin/miR-24 regulatory system is implicated in cholestatic liver fibrosis. Inhibition of microRNA-24 increases menin and TGF-β1 expression, subsequently driving hepatic fibrosis and bile duct mass in Mdr2-/- mice, a murine model of PSC. Previous studies in other models of cholestatic liver disease have shown that TGF-β1 drives hepatic fibrosis through increased expression of phosopho-Smads. Modulation of the menin/microRNA-24 axis may provide novel targeted therapies to slow the progression of hepatic fibrosis into cirrhosis by altering TGF-β1 and phospho-Smad expression. 
 

63.07 EP2 Receptor Blockade Decreases Intestinal Barrier Breakdown Following Cecal Ligation and Puncture

Posted on January 26, 2017

J. Golden1, P. Kavarian1, L. Illingworth1, J. Uppuluri1, O. Escobar1, M. Isani1, C. Gayer1, A. Grishin1, H. Ford1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:  Cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are inflammatory mediators that have been implicated in sepsis, inflammation, and intestinal barrier breakdown. Previous work in our lab has shown that COX-2 can be upregulated by its end-product PGE2 in enterocytes. We have identified pro-inflammatory prostanoid receptor, EP2, as a key mediator in this feedback loop leading to upregulation of COX-2 during inflammation. Therefore, we hypothesized that EP2 inhibition would protect against induction of COX-2 and intestinal barrier breakdown in experimental peritonitis.

Methods: Following IACUC approval, C57/Bl6 mice underwent sham operation as a control or cecal ligation and puncture (CLP) to induce experimental peritonitis. Mice were injected intraperitoneally with or without 10mg/kg EP2 receptor antagonist PF-04418948 at time of operation and were orally gavaged with fluorescein isothiocyanate (FITC)-dextran 8 hours later. All mice were sacrificed 12 hours following sham or CLP. Blood samples were analyzed for FITC-dextran to determine intestinal barrier breakdown and terminal ileum was analyzed for COX-2 expression.

Results: CLP led to increased serum FITC-dextran levels and higher intestinal COX-2 mRNA and protein expression compared with sham. PF-04418948 attenuated serum FITC-dextran levels and intestinal barrier breakdown from 4.2±0.8 to 2.0±0.5 fold change from sham (p<0.05) in mice who underwent CLP. PF-04418948 decreased COX-2 mRNA levels in terminal ileal samples from 25±19 to 9±3.3 fold compared with sham mice. Additionally, COX-2 protein levels in terminal ileal samples decreased from 1.8±0.1 to 1.2±0.1 fold sham levels (p<0.05) in CLP mice who received PF-04418948. 

Conclusion: EP2 receptor inhibition protects against intestinal barrier breakdown following cecal ligation and puncture and may inhibit the positive feedback induction of COX-2 via PGE2 activation of EP2. This suggests that EP2 receptor specific inhibition may have important therapeutic implications in the treatment of inflammation and gut-origin sepsis.
 

63.06 Intestinal Alkaline Phosphatase Regulates Gut Barrier Function And Enhances Life Span In Drosophila

Posted on January 26, 2017

F. Adiliaghdam1, A. Tsurumi1,2,3, Y. Dhole1, J. Ramirez1, F. Kuehn1, A. Munoz1, S. Hamarneh1, L. Rahme1,2,3, R. Hodin1  1Massachusetts General Hospital,General Surgery,Boston, MA, USA 2Shriners Hospitals For Children,Boston, MA, USA 3Harvard School Of Medicine,Department Of Microbiology And Immunobiology,Brookline, MA, USA

Introduction: Intestinal barrier dysfunction is considered to be an underlying factor in the pathophysiology of aging.We have previously shown that the gut enzyme intestinal alkaline phosphatase(IAP) maintains a healthy gut mucosal barrier.We therefore hypothesized that IAP could play an important role in aging process.

Methods: The wild-type(WT) Oregon-R Drosophilia Melanogaster was used to study the changes of gut alkaline phosphatase(AP)activity with aging and the effect of IAP supplementation on lifespan.Strain y/w67C23 was used to develop double-cross mutants for the midgut-specific AP genes in D.melanogaster, CG5150 and CG10827, for lifespan analysis. Furthermore,Gut barrier function was assessed using feeding with a blue food dye. Junction protein gene expression (DECad: Drosophila E-Cadherin and dlg1:discs large 1genes) assayed by qPCR from dissected mid-guts of 35 day-old WT and AP double-cross mutants. Three independent trials were done for all experiments.

Results: We found that AP activity in WT D.melanogaster’s midgut decreased with aging (P< 0.0001). In WT flies, oral IAP supplementation caused major lifespan extension compared to vehicle controls (median survival: 53d vs. 47d, P<0.0001). Climbing assay showed that IAP-treated flies climbed better than the vehicle-treated controls (%24 increase at week3, P< 0.0001 and 10% increase at week5, P<0.01). We also found that the double-cross mutants lacking the two AP genes died earlier than the WT-flies (median survival:42d vs. 47d, P<0.0001) and also performed worse in the climbing assay (21% decrease P<0.001). WT-flies showed a significant age-dependent breakdown in the gut barrier and this impairment was much worse in the AP double-cross mutants.(Figure1) Furthermore, AP double-cross mutants had significantly reduced mRNA levels of Junction proteins in the midgut compared to WT flies: DECad (Mutants vs. WT, 0.491± 0.070 Vs.1.0± 0.14 Relative Expression ,P=0.001) and dlg1 (Mutants vs. WT, 0.607± 0.07 Vs.1.0± 0.26 Relative Expression, P<0.01)

Conclusion: Decreasing endogenous levels of IAP is an important factor in age-dependent loss of intestinal integrity.We believe that IAP supplementation could represent a novel therapy to delay aging process.

 

63.05 Endogenous Intestinal Alkaline Phosphatase Modulates Inflammatory Pathways and Homeostasis in the Gut

Posted on January 26, 2017

F. M. Kuehn1, S. R. Hamarneh1, J. M. Ramirez1, A. R. Munoz1, F. Adiliaghdam1, S. A. Morrison1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA

Introduction:  The inability of epithelial cells to cope with various stresses and cellular damage plays a crucial role in the development of many inflammatory diseases in the gut. The exact functions of the brush border enzyme intestinal alkaline phosphatases (IAP) in various cellular pathways are not well understood. We propose that IAP functions as an important modulator of intracellular homeostasis and stress responses in the gut.

Methods: Functional analysis and identification of IAP-binding proteins were performed using Liquid chromatography-tandem mass spectrometry (LC-MS) after immunoprecipitation of IAP complexes from Caco-2 cells in vitro. Furthermore, IAP knockout and overexpressing Caco-2 cells were developed using CRISPR/Cas9 gene editing technique and cell transfection, respectively. Subsequently, the effect of IAP activation or inhibition on inflammatory cytokine levels was measured using qPCR in the IAP-knockout and overexpressing Caco-2 cells. Additionally, the role of IAP in Caco-2 cell survival was assayed after incubation with high doses of TNF-α.

Results: Analysis of IAP protein complexes showed that IAP binds to key modulators of the NFκB, TNF-α and TLR-4 pathways. Overexpression of human IAP significantly reduced mRNA-levels of inflammatory cytokines in Caco-2 cells: TNF-α (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.08 Vs. 0.19 ± 0.1 Relative Expression, p= 0.01), IL-1β (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.4 Vs. 0.47 ± 0.06 Relative Expression, p= 0.001) and IL-8 (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.26 vs. 0.46 ± 0.16 Relative Expression, p= 0.046). IAP deletion increased the expression of TNF-α (IAP WT vs. IAP-KO Cells, 1.0 ± 0.2 Vs. 7.9 ± 0.7 Relative Expression, p= 0.007) and IL-1β (IAP WT vs. IAP-KO Cells, 1.0 ± 0.6 Vs. 11.4 ± 0.1.1 Relative Expression, p= 0.004) and IL-8 (IAP WT vs. IAP-KO Cells, 1.0 ± 0.12 Vs. 15.9  ± 0.8 Relative Expression, p= 0.0013). Furthermore, overexpression of IAP resulted in significantly less inflammation and cytokine production in Caco-2 cells when incubated with inflammatory mediators such as TNF-α (IAP WT vs. IAP-overexpressing Cells, 9.7 ± 1.8 Vs. 1.2 ± 0.46 Relative Expression, p= 0.004), LPS and bacterial contents. Additionally, higher IAP levels significantly increased Caco-2 survival after incubation with TNF-α 20ng/mL for 24 hours (IAP WT vs. IAP-overexpressing Cells, 30.5 ± 7.2 Vs. 90.3 ± 11.1 % Survival, p= 0.018). 

Conclusion: Endogenous IAP functions as a modulator of the stress response and inflammatory pathways in intestinal epithelial cells. Intracellular IAP pathways in the gut may represent an important therapeutic target to prevent or treat a variety of gut inflammatory conditions.

 

63.04 Hepatic Exosomes Regulated by Rab27a Promote Liver Ischemia/Reperfusion (I/R) Injury

Posted on January 26, 2017

M. Yang1,2, Q. DU1, P. R. Varley1, J. Goswami1, R. Wang1, B. Chen1, N. D. Anderson1, D. B. Stolz3, D. A. Geller1  1University Of Pittsburgh,Department OF Surgery,Pittsburgh, PA, USA 2The First Hospital Affiliated To Nanchang University,Department OF Surgery And Department Of Transplantation,Nanchang, JIANGXI, China 3University Of Pittsburgh,Center Of Biology Imaging,Pittsburgh, PA, USA

Introduction:  Exosomes play an important role in cell communication, and Rab27a is a GTPase that has been shown to promote exosome secretion. However, the role of Rab27a and exosomes in liver I/R injury is unknown. We hypothesized that exosome secretion regulated by Rab27a promotes liver I/R injury.

Methods:  70% liver I/R injury mouse model was used. Adenoviral Rab27a shRNA (Ad-Rab27a shRNA) was used to knock down hepatic Rab27a expression. Circulating serum exosomes (Exo) in vivo or murine hepatocyte-secreted exosomes in vitro were isolated with ultracentrifugation. Exosome pellets were verified by TEM and by exosome markers CD63, CD81 and HSP70. Serum Exo concentration was analyzed with CD81 exosome ELISA kit.  Liver damage during I/R was calculated with ALT and liver HE staining. Hypoxia or normoxia primed hepatocyte-secreted exosomes were injected to mice in vivo during warm I/R with/without Rab27a knockdown.

Results: Hepatic Rab27a protein was induced in vivo during liver I/R in a time-dependent manner with strong induction 6 hr after I/R (Fig. A).  Liver I/R induced increase in hepatic Rab27a protein expression was diminished by knockdown of Rab27a in vivo with Ad-Rab27a shRNA (Fig. B, last lane), but not scrambled shRNA. Knockdown of Rab27a resulted in a dramatic reduction in liver damage measured by ALT (Fig. C) and liver necrosis on HE staining (not shown).

During liver warm I/R injury, serum exosomes were increased in a time-dependent manner with maximal serum exosome pellet seen 6-12 hr after I/R (Fig. D). Knockdown of hepatic Rab27 in vivo with Ad-Rab27a shRNA decreased serum exosome concentration (Fig. E). Liver damage in vivo during warm I/R was reduced by Rab27a knockdown (Fig. F, third column), and this protective effect was abrogated by injecting hypoxia primed hepatocyte-secreted exosomes (Fig. F, last column). 

Conclusion: Hepatic Rab27a protein expression was markedly increased in vivo during liver warm I/R injury, and this resulted in increased serum exosome concentrations. Knockdown of Rab27a decreased exosome secretion and liver damage. Hypoxia primed exosomes recapitulated liver damage reversed by Rab27a knockdown. These findings increase our understanding of fundamental exosome cell signaling and regulation during liver I/R injury.
 

63.03 Inflammatory Cytokine Regulation of Extracellular Matrix Results in Attenuated Renal Fibrosis

Posted on January 26, 2017

X. Wang1, P. Duann1, C. Lu1, C. Moles1, H. Li1, M. Fahrenholtz1, M. Rae1, Y. Dhamija1, J. Cheng2, S. Balaji1, S. Keswani1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Baylor College Of Medicine,Medicine,Houston, TX, USA

Introduction:

Renal fibrosis is a pathological characteristic of chronic kidney disease (CKD), which affects nearly 700 million patients globally, and is a product of aberrant inflammation and extracellular matrix (ECM) deposition. Patients with CKD are associated with a three-fold or higher mortality rate compared to the general population. We have previously shown a novel role for interleukin-10 (IL-10) in dermal fibrosis, beyond its accepted anti-inflammatory role. In this role, IL-10 regulates the ECM, specifically hyaluronan (HA), and TGFβ isoforms, which are crucial for regenerative tissue repair. However, the roles of IL-10 and HA in renal fibrosis are not completely elucidated. We hypothesize that IL-10 might regulate HA and TGFβ expression in the kidney, and attenuate renal fibrosis in murine unilateral urethral obstruction (UUO) model. 

Methods:
Primary renal fibroblasts (FB) were isolated from 8-10 week-old male C57BL/6J (WT) mice. IL-10 (200 ng/ml) with or without hyaluronidase (HYAL, 1.5 unit/ml) was added to cultures. HA matrices were analyzed by particle-exclusion assay at 24h. Gene expression of HA synthases 1, 2, and 3 (HAS1-3), hyaluronidases 1 and 2 (HYAL1-2) and TGFβ-1 were assessed by qPCR at 1, 2, 3 and 6 h. 8 weeks C57BL/6J (WT) and IL-10 KO male mice were injected with lenti-IL-10/ lenti-GFP (1×1010 IU) under the kidney capsule. Three days after the injection, unilateral ureteral obstruction (UUO) was performed. UUO/sham kidneys and serum were collected at 14 days after UUO for RNA, ELISA, and immunohistochemical (IHC) analysis. n=3/treatment group; p-values by ANOVA.

Results:
In vitro, IL-10 resulted in an upregulation of HAS-1,2, and 3 expression at 2h after treatment, and a significant downregulation of HYAL 1, 2 and TGFβ-1. IL-10 resulted in a 1.88-fold increase in HA-rich matrix formation at 24h, and the effect was abolished by HYAL treatment (p<0.05). In vivo, IL-10 KO mice demonstrated more fibrosis than WT mice. Lenti-IL-10 treatment resulted in less dilated tubules and decreased kidney fibrosis, as well as reduced α-SMA expression as compared to lenti-GFP treated kidneys in both WT and IL-10 KO mice. The HA level in serum was 1.7-fold higher in lenti-IL-10 treated mice as compared to lenti-GFP treated (p<0.05) (Fig.1).

Conclusion:
Our data demonstrates that IL-10 regulates HA metabolism and TGFβ expression of renal FB in vitro, and is effect of IL-10 is validated in the UUO model. The endogenous IL-10 is essential for normal kidney integrity against excessive fibrosis with UUO injury. This previously unreported mechanism for IL-10 regulation of ECM in the kidney may have a significant impact for future therapies to ameliorate kidney fibrosis.

63.02 Del1 Knockout Affects Bone Cartilage Stroma Progenitor Cells Following Femur Fracture in Mice

Posted on January 26, 2017

T. V. Boyko1,2, O. Marecic1, E. Y. Seo1, C. K. Chan1, T. Leavitt1, M. T. Longaker1, G. P. Yang1,3  1Stanford University,Surgery,Palo Alto, CA, USA 2State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 3VA Palo Alto Healthcare Systems,Surgery,Palo Alto, CA, USA

Introduction:  DEL1 is a secreted protein, which has been shown to be involved in bone fracture healing. In previous experiments we have demonstrated that Del1 gene knockout (KO) mice healed fractures with 15% less bone when compared to wildtype (WT) mice. Increased apoptosis was also seen in fracture calluses of KO mice. Separately, we identified the mouse skeletal stem cell (mSSC) and 7 other unique subpopulations of skeletal progenitor cells that are capable of self-renewal and giving rise to all three components of the skeleton: bone, cartilage and stroma. One subpopulation, the Bone Cartilage Stroma Progenitor Cells (BCSPs), are the primary skeletal stem cell population involved in fracture repair. Following fracture, BCSPs transition to another sub-type, f-BCSPs, that has greater osteogenic potential. We hypothesized that Del1 deletion leads to decreased fracture callus due to an effect on BCSP biology. 

Methods:  Femurs were fractured in KO and WT mice. Fracture calluses were harvested on post-operative day 7. BCSPs, mSSCs and f-BCSPs were isolated by Fluorescent Activated Cell Sorting (FACS) following staining for signature cell surface markers. Antibody staining for Annexin V was used to determine extent of apoptosis. KO and WT BCSPs were cultured in vitro and colony-forming units (CFUs) were counted 14 days after plating. 

Results: FACS analysis revealed that the BCSP populations in KO and WT femurs were equivalent prior to fracture (p=0.498, n=3 each). There is an increase of skeletal progenitors in the callus following fracture, but fewer BCSPs were found in KO mice compared to WT (11,700 cells/1 million events vs 77,706 cells/1 million events, p<0.01, n=4 each). Additionally, mSSCs showed a similar pattern in the KO mice with an attenuated increase after fracture (12,452 cells/1 million events in KO vs 57,451 cells /1 million events in WT, p<0.02, n=4 each). The percentage of apoptotic cells was found to be higher in both BCSPs (23.98% KO vs. 6.56% WT, p<0.001, n=4 each) as well as for mSSCs (14.83% KO vs. 4.74% WT, p<0.002, n=4 each). Following culture in vitro, KO BCSPs showed no difference in proliferation compared to WT, but did form significantly fewer CFUs (16.3 vs. 24 CFUs, p<0.05, n=6 each).  Examining f-BCSPs showed an equivalent percentage in KO compared to WT.

Conclusion: Following fracture, skeletal progenitors expand to create bone. In KO mice, this expansion is attenuated leading to decreased bone formation. BCSPs still transition to a more osteogenic phenotype, but there are just fewer of them. These data suggest DEL1 may have a therapeutic role in promoting fracture healing or regenerating bone.

 

63.01 Obeticholic acid accelerates liver regeneration following portal vein embolization in a rabbit model

Posted on January 26, 2017

P. B. Olthof1, F. Huisman1, K. Van Lienden2, R. F. Van Golen1, M. Heger1, J. Verheij3, F. F. Schaap4, P. L. Jansen4, S. W. Olde Damink4, T. Van Gulik1  1Academic Medical Center,Surgery,Amsterdam, ZUID-HOLLAND, Netherlands 2Academic Medical Center,Radiology,Amsterdam, ZUID-HOLLAND, Netherlands 3Academic Medical Center,Pathology,Amsterdam, ZUID-HOLLAND, Netherlands 4NUTRIM School Of Nutrition And Translational Research In Metabolism, Maastricht University,Surgery,Maastricht, LIMBURG, Netherlands

Introduction: Portal vein embolization (PVE) is used to increase future remnant liver volume in patients scheduled for major liver surgery. The bile salt-activated transcription factor farnesoid X-receptor (FXR) is a key mediator of bile salt signaling, an event implicated in the early phase of liver regeneration following partial hepatectomy. The aim of this study was to evaluate the effect of a potent FXR agonist (obeticholic acid, OCA) on PVE-induced liver hypertrophy.

Methods: Twenty-four rabbits (female, 2.9±0.4kg) were given a daily oral gavage with OCA (10mg/kg) or vehicle starting 7 days pre-PVE until 7 days post-PVE of the cranial liver lobes.  Effectiveness of the embolization procedure (coils, PVA particles) was confirmed by portography. Caudal liver volume (CLV) was analyzed by CT-volumetric analysis at days -7, -1, +3 and +7. Rabbits were sacrificed at day +3 and +7.

Results:Three days after PVE the increase in CLV was 2.0-fold (59.3 ± 19.2% vs. 29.7 ± 16.1, p=0.0013) greater in the OCA group compared to controls. No differences in CLV increase were measured after 7 days. OCA had no effect on volume of the atrophic cranial lobes at the respective time points. Likewise, OCA did not cause spontaneous liver growth, as liver volume before PVE was proportional to body weight increase over the days before PVE.

Conclusion:Obeticholic acid accelerated liver regeneration in a rabbit model of PVE by 2.0-fold over the first 3 days. The ultimate increase in CLV is the same in both groups. OCA treatment has potential in extending resectability as well as the prevention of postoperative liver failure.

 

62.10 Human Stem Cell-Derived Neural Crest Cells Restore Neurons and Glia in Hirschsprung Disease Colon

Posted on January 26, 2017

C. R. Schlieve1, K. L. Fowler1, I. Hajjali1, X. Hou1, T. C. Grikscheit1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:  Disruption of enteric nervous system (ENS) development and function leads to varying degrees of pathology, including Hirschsprung Disease (HD). Impaired migration of enteric neural crest cells (ENCC) within the gastrointestinal tract results in an inability to relax intestinal smooth muscle causing obstructive symptoms. Surgical resection of the aganglionic portion of the colon in HD decreases mortality, but does not completely resolve symptoms. Therefore, restoration of the ENS through cellular transplantation has become a promising area of research for the treatment of enteric neuropathies. We have previously generated ENCC from human pluripotent stem cells (hPSC) and demonstrated their ability to differentiate into diverse classes of neurons and glia. Additionally, injection of hPSC-derived ENCC into the muscular layer of the colon demonstrated extensive migration and rescued mortality in an EDNRB knockout mouse model that mimics HD. However, successful integration of hPSC-derived ENC into human HD colon has not been demonstrated. The purpose of our study was to explore hPSC-derived ENC supplementation to restore ENS components in tissue-engineered colon (TEC) from patients with HD.

Methods:  Multicellular clusters of epithelial and mesenchymal cells, termed organoid units (OU), were derived from colon obtained from patients with HD. Human embryonic stem cell line H9 was exposed to LDN193189, SB431542, and CHIR99021 under defined conditions with the addition of Retinoic Acid to promote differentiation into ENCC. Unsorted ENCC were mixed with HD OU prior to implantation, seeded onto a biodegradable scaffold, wrapped in the omentum of adult NOD/SCID mice, and allowed to mature for 1 month. Implants were analyzed for development of ENS cell types through immunostaining of neurons (Tuj1) and glia (GFAP/s100b). Neurons were evaluated for expression of excitatory marker choline acetyltransferese (ChAT) and inhibitory marker neuronal nitric oxide synthase (nNOS).

Results: Human iPSC-derived ENCC supplementation restored components of the ENS in TEC generated from HD patients. Aganglionosis of HD donor samples was confirmed by intraoperative frozen section and H&E analysis. HD TEC with ENCC co-implantation resulted in intestinal constructs with villi, underlying smooth muscle and the presence of enteric neurons and glia. Excitatory (ChAT/Tuj1) and inhibitory (nNOS/Tuj1) neurons and glia (s100b) were identified within the submucosa and muscular layers of HD TEC supplemented with ENCC and were absent in HD TEC alone. 

Conclusion: Our findings validate a novel approach to restoring human ENS cell types in HD colonic tissue through hPSC-derived ENC supplementation. This advancement is a necessary step toward establishing cellular therapies for future treatment of enteric neuropathies.

 

62.09 Renal Injury in Premature Lambs Supported by the Artificial Placenta

Posted on January 26, 2017

J. S. McLeod1,4, J. T. Church1,2, M. A. Coughlin1,5, E. M. Perkins2, R. H. Bartlett2, R. Rabah3, G. B. Mychaliska1  1University Of Michigan,Department Of Pediatric Surgery,Ann Arbor, MI, USA 2University Of Michigan,Department Of General Surgery,Ann Arbor, MI, USA 3University Of Michigan,Department Of Pediatric And Perinatal Pathology,Ann Arbor, MI, USA 4Detroit Medical Center,Michael And Marian Ilitch Department Of Surgery,Detroit, MI, USA 5Henry Ford Health System,Department Of General Surgery,Detroid, MI, USA

Introduction:
Neonates born <28 weeks gestational age (GA) have exceptionally high morbidity and mortality. A novel approach would be to recreate the intrauterine environment with an artificial placenta (AP), utilizing venovenous extracorporeal life support (VV-ECLS) and avoiding ventilation. The effect of the AP on renal perfusion and development is unknown.  The aim of this study was to evaluate renal injury following AP support, and to identify clinical and laboratory predictors of injury during AP support.

Methods:
Extremely premature lambs at 110-120 days GA (term=145; n=11) were delivered and placed on AP support, with jugular venous drainage and umbilical vein reinfusion.  The lungs remained fluid-filled.  The lambs received parenteral nutrition and voided spontaneously.  Support was continued for a goal of 7 days.  A subset of lambs (n=5) were transitioned to mechanical ventilation after 10-12 days of AP support.  Creatinine (Cr) and blood urea nitrogen (BUN) were checked daily, normalized to birth weight, and the highest value over the last 3 days of life were used in statistical analysis. The renal injury score was based on evaluation of the following histologic findings: tubular dilation, tubular vacuolation, tubular nuclear apoptosis, epithelial necrosis, interstitial hemorrhage, edema and inflammation, tubular casts, and glomerular necrosis. Individual scores ranged from 0-4 based on % involvement of the field (0- No injury; 1- Injury to 25% of field; 2- Injury to 50% of the field; 3- Injury to 75% of the field; 4- Diffuse Injury).  Means were compared using t-test.  Correlation was evaluated using linear regression.  P<0.05 was considered significant.

Results:
Survival was 6 – 14 days. Three of 11 lambs (27%) became anuric during AP support.  Renal injury scores for all lambs ranged from 1-21 with a mean of 8.8. Anuric lambs demonstrated higher renal injury scores than non-anuric lambs, although this did not reach statistical significance (12.3±4.5 vs. 7.5±6.5; p=0.22).   Anuric lambs also exhibited significantly higher BUN/kg in the last 3 days of life (22.1±5.4 vs. 9.2±6.6; p=0.03); this same significant difference was not seen with Cr/kg (0.76±0.6 vs. 0.27±0.29; p=0.29).  BUN/kg correlated with renal injury score (p=0.02) while Cr/kg did not (p=0.20).  

Conclusion:
Extremely premature lambs supported with the AP are vulnerable to renal failure.  These data suggest BUN/kg may predict renal failure from both a clinical and histopathologic standpoint, while Cr/kg was not predictive. Future studies will aim to identify factors contributing to renal failure in AP supported premature lambs.
 

62.08 Enteric Glial-Mediated Enhancement of Intestinal Barrier Integrity is Compromised by Morphine

Posted on January 26, 2017

B. D. Bauman1, A. Louiselle1, E. Zheng1, J. Meng1, S. Roy1, B. Segura1  1University Of Minnesota,Minneapolis, MN, USA

Introduction:
The opioid epidemic is a growing concern for American physicians.   The increased use of prescription opioids has been linked to a growing number of complications.  Side effects from opioid use include nausea, vomiting, and constipation.  Emerging evidence suggests that morphine use may be associated with sepsis although the mechanism is undefined.  Enteric glial cells (EGCs) are the most numerous cell population in the enteric nervous system and contribute to the maintenance of intestinal barrier function through the production of a variety of trophic factors including Glial Derived Neurotrophic Factor (GDNF).  We sought to determine the effect of morphine on enteric glia and hypothesized that morphine contributes to EGC dysfunction and increased gut permeability.

Methods:
We tested our hypothesis using in-vitro methodology including QPCR, cell co-culture in a transwell system, cell staining and microscopy, and electronic cell-impendence sensing (ECIS).  Rat Intestinal Epithelial Cell (IEC-6) and EGC lines were purchased from ATCC.  We measured permeability in the transwell system with 4kD FITC-Dextran and 70kD Rhodamine-Dextran.  Further, production of the tight junction protein, ZO-1, was monitored as a measure of epithelial barrier integrity.  Statistical significance was determined using a student t-test.

Results:
Morphine receptor antibody staining was positive on EGCs.  GDNF RNA expression within EGCs was decreased by morphine treatment.  FITC-dextran permeability was decreased in the co-culture model of unstimulated EGCs while the barrier protective effect of EGC co-culture was lost when EGCs were treated with morphine for 72 hours.  In the ECIS model, barrier integrity was diminished when IEC-6 cells were exposed to culture media from morphine-treated EGCs (EGC-CM), while barrier integrity was maintained with standard EGC-CM.  ZO-1 production was decreased in morphine treated IEC-6 cells yet maintained in the presence of treatment with EGC-CM. 

Conclusion:
EGCs contain receptors for Morphine.  Morphine stimulated EGCs have diminished capability to preserve intestinal barrier function, apparently through decreased production of GDNF, although other trophic factors may be involved.  IEC6 cells have decreased ZO-1 expression in the presence of Morphine.  IEC6 ZO-1 production is restored in the presence of EGC conditioned media stimulation. Further studies are warranted to delineate the role of enteric glial cell function in opioid signaling and sepsis.
 

62.07 Lactobacillus Murinus Protects Against Necrotizing Enterocolitis in Standard Rat Model of NEC

Posted on January 26, 2017

M. Isani1, J. Bowling1, C. Moneme1, K. A. Durairaj1, M. Elizee1, J. Golden1, B. Bell1, L. Illingworth1, G. Anatoly1, H. Ford1  1Children’s Hospital Los Angeles,Los Angeles, CA, USA

Introduction: Necrotizing enterocolitis (NEC) is a devastating disease that affects premature infants. Lactobacillus probiotics have been shown in a number of studies to protect against NEC.However, results of trials remain inconclusive due to the use of variable species and doses. Furthermore, whether the lactobacilli are colonizing the intestine remains unknown.We propose that an efficient probiotic strain is naturally occurring and should not only protect the intestine against NEC, but should be capable of colonizing the GI tract. 

Methods:  Animal experiments were approved and neonates were obtained from timed- pregnant rats and subjected to formula feeding and hypoxia for 4 days. Rat pups were sacrificed on day 4. To enumerate and isolate the lactobacilli, large intestine was plated on agar. After incubation at 37oC, colonies were classified according to morphology and Gram stain. Species identity was established by 16S rRNA sequencing. Three species were identified: L.reuteri, L.murinus, and L.acidophilus. 

Lactobacillus reuteri was re-introduced to newborn rats at 107 and 108 CFU/animal. Dosage for colonization was determined based on titers of L.reuteri in the large intestine. All three species of Lactobacillus were introduced to neonatal rats at the 107 and 108 CFU/animal in the standard rat model. Next, to determine if the Lactobacillus species protect intestine against a known NEC pathogen, Cronobacter muytjensii, we introduced Cronobacter into our NEC model with the first feeding and the lactobacillus species with the second feed. Terminal ileum sections were stained with H&E and examined to determine NEC score. Sections were also immunostained to determine if COX-2 and iNOS levels were changed in the presence of the lactobacillus species and Cronobacter. 

Results: Three species of lactobacilli were isolated from neonatal rat intestine: L.reuteri, L.murinus, and L.acidophilus. Upon re-introduction of L.reuteri at various dosages, we achieved colonization at 107  (p=0.006) and 108 (p=0.0146) CFU/animal. All three species were able to colonize the intestine. However, NEC scores were only lower in groups that received L.murinus (p= 0.0484 at 107 CFU/animal n=25 and p=0.0310 at 108 CFU/animal, n=35). Furthermore, L. murinus was able to protect against NEC when challenged by a known NEC pathogen, Cronobacter muytjensii (p=0.0455 at 108 CFU/animal, n=6). Pups treated with Cronobacter had increased expression of both COX-2 and iNOS; in contrast, those treated with L. murinus had decreased COX-2 and iNOS expression in tissue. 

Conclusion:  L.murinus is a naturally occurring species that is able to colonize the intestine and protect against NEC in the standard rat model. It can also protect against NEC when challenged by known NEC pathogen, Cronobacter muytjensii. L.murinus decreases iNOS and COX-2 expression in tissue and may serve as a preventive and therapeautic treatment against NEC.  

62.06 Inhibition of Necroptosis Attenuates Lung Injury and Improves Survival in Neonatal Sepsis

Posted on January 26, 2017

A. C. Bolognese1,2, W. Yang1,2,3, L. W. Hansen2, J. Nicastro2, G. F. Coppa2, P. Wang1,2,3  1Elmezzi Graduate School Of Molecular Medicine,Manhasset, NY, USA 2Hofstra Northwell School Of Medicine,Department Of Surgery,Manhasset, NY, USA 3The Feinstein Institute For Medical Research,Center For Immunology And Inflammation,Manhasset, NY, USA

Introduction:  Neonatal sepsis represents a unique therapeutic challenge owing to an immature immune system with a hyperactive innate immune response. Necroptosis, a form of programmed cell death, has been identified as an important mechanism of inflammation-induced cell death. Receptor-interacting protein kinase 1 (RIPK1) plays a key role in mediating this process. We hypothesized that inhibition of necroptosis via RIPK1 would be protective in neonatal sepsis.

Methods:  Sepsis was induced in C57BL/6 mouse pups (5-7d) by intraperitoneal injection of adult cecal slurry (CS, 0.175 µg/g body weight, LD100). At 1 h after CS injection, the RIPK1 inhibitor necrostatin 1 stable (Nec-1s, 10 µg/g body weight) or vehicle (5% DMSO in PBS) was administered via retroorbital injection. At 20 h after CS injection, blood and lung tissues were collected for ELISA, PCR, and histologic analysis. For the survival study, pups were monitored for 7 days. 

Results: At 20 h after sepsis induction, pups that received the vehicle showed an increase in serum levels of proinflammatory cytokines IL-1β and IL-6 compared to sham (396.30 vs. 1.58 pg/ml and 41.43 vs. 0.32 ng/ml, respectively). With Nec-1s treatment, serum levels of IL-1β and IL-6 were decreased by 81% and 72%, respectively, compared to the vehicle (IL-1β: 74.26 pg/ml, range 0-344; IL-6:11.69 ng/ml, range 0-32). In the lungs, sepsis induction resulted in a 10-fold increase in IL-1β mRNA levels compared to sham, while Nec-1s treatment decreased these levels to just 4-fold (p<0.05). Expression of the neutrophil chemokines KC and MIP-2 was also increased in the lungs in sepsis (1105- and 516-fold compared to sham, respectively, p<0.05), while Nec-1s treatment reduced these levels by 89% and 77%, respectively, compared to vehicle (p<0.05). The above findings correlated with largely retained normal lung architecture observed on histology after Nec-1s treatment compared to vehicle (Figure). In addition, treatment with Nec-1s resulted in a 29% survival rate compared to no survival in the vehicle-treated septic pups (Figure).

Conclusion: Inhibition of RIPK1 by Nec-1s decreases systemic and pulmonary inflammation, reduces lung injury, and increases survival after cecal slurry injection in neonatal mice. Targeting the necroptosis pathway represents a promising therapeutic strategy for neonatal sepsis.

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