27.07 Protective Effect of Phosphatidylserine Blockade in Sepsis Induced Organ Dysfunction

Posted on January 31, 2019

G. Beattie1, C. Cohan1, W. Brigode1, E. J. Miraflor1, G. P. Victorino1  1University Of California San Francisco – East Bay,Department Of Surgery,Oakland, CA, USA

Introduction:  Phosphatidylserine (PS) is usually an intracellularly oriented membrane phospholipid. Externalized PS on activated or apoptotic cells is a signal for phagocytosis. In sepsis, persistent PS exposure is a signal for activation of the coagulation and inflammatory cascades. As such, PS may be a key molecule in sepsis induced cellular and organ injury. We hypothesize that PS blockade provides a protective effect in sepsis induced organ dysfunction.

Methods:  Anesthetized adult female Sprague-Dawley rats were allocated to control (n=5), sepsis (n=6), or sepsis + PS blockade (n=9) groups. Each group underwent 4-hour intravenous infusion protocols as follows: sepsis was induced using 0.65mg/h of lipopolysaccharide (LPS), PS blockade was performed using diannexin 400mcg/kg dosed at time zero and at two hours, and controls were infused with lactated ringers. Gut dysfunction was evaluated using intravital microscopy to measure microvascular leak (Lp). Lung and renal dysfunction were evaluated by arterial blood gas (ABG) and serum creatinine (Cr) analysis, respectively.  Activated clotting time (ACT) and glomerular fibrin deposition measurements were performed to evaluate coagulopathy. Lp units are listed as x10-7 cm*s-1*cmH2O-1. Data are presented as mean ± standard error of the mean. Statistical analysis was performed with paired t-test and analysis of variance.

Results: In the sepsis group, Lp increased 2-fold from 1.17± 0.03 to 2.62 ± 0.20 (p< 0.01). In the sepsis + PS blockade group, Lp increased over the first two hours of infusion (from 1.16 ± 0.01 to 1.48 ± 0.01), however, unlike the sepsis group, Lp returned to baseline levels at four hours (p < 0.01). Lp in the control group remained unchanged. There was no difference in pulmonary dysfunction in any of the groups (p=0.6). Renal function worsened in the sepsis group with a 59% increase in Cr from 0.53 to 0.88 mg/dL (p=0.01). In the sepsis + PS blockade group, there was a trend toward attenuation of renal dysfunction as Cr levels only increased 40%, from 0.5 to 0.70mg/dL, compared to the sepsis group (p=0.1). Creatinine levels remained stable in the control group. Coagulopathy was observed in the sepsis group as ACT increased 19% (p =0.04) and glomerular fibrin deposition increased compared to controls, (99.5% ± 0.5% glomeruli involvement vs 20% ± 4.6%, p<0.001). There was a protective effect of PS blockade in sepsis as ACT decreased 8% (p=0.03) and glomerular fibrin deposition improved from 99.5% ± 0.5% glomeruli involvement in sepsis alone to 20% ± 9.8% with PS blockade in sepsis (p=0.008).

Conclusion: In sepsis, PS blockade had a protective effect on gut dysfunction and coagulopathy with a trend toward improved renal function. Increased PS exposure during cellular injury may be a key mediator of organ dysfunction and coagulopathy during sepsis. This may provide insights into novel treatment options for septic patients.

 

27.06 Severe Injury Precipitates Prolonged Dysregulation of Circulating von Willebrand Factor and ADAMTS13

Posted on January 31, 2019

W. E. Plautz1, M. R. Dyer2, S. Haldeman2, M. Rollins-Raval3, J. S. Raval3, J. L. Sperry2, B. S. Zuckerbraun2, M. D. Neal2  1University Of Pittsburgh,School Of Medicine,Pittsburgh, PA, USA 2University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA 3University Of North Carolina At Chapel Hill,Department Of Pathology,Chapel Hill, NC, USA

Introduction: Increases in plasma von Willebrand Factor (vWF) levels, accompanied by decreases in its respective metalloprotease ADAMTS13, have been demonstrated in diseases of microvascular injury. We hypothesized that following severe trauma, a burst of ultra-large vWF is released into the bloodstream by damaged endothelium, resulting in a dysregulation of the circulating vWF multimeric composition. We further hypothesized that impaired ADAMTS13 activity would be insufficient to cleave the burst of ultra-large vWF, facilitating organ injury.

Methods: 37 severe trauma patients from a randomized control trial (RCT) on the use of pre-hospital plasma were analyzed for antigen levels of plasma vWF at 0- and 24-hours after admission. Circulating vWF multimeric composition from both time points was determined by vertical agarose gel electrophoresis, followed by a quantitative structural analysis of vWF multimeric length. ADAMTS13 antigen, activity, and antibody levels were obtained by ELISA and FRETS-73 analyses, and the circulating vWF multimeric composition at both 0- and 24-hours was evaluated for a dependence on ADAMTS13 activity. Finally, multivariate analyses were performed with data abstracted from the RCT database and electronic medical records to identify further dependences.

Results: VWF levels were increased in severe trauma patients when compared to healthy controls at presentation (189% (110-263) vs. 95% (74-120)) and persisted through 24-hours (213% (146-257) vs. 132% (57-160)). Ultralarge-vWF forms were elevated at both 0- and 24-hours when compared to pooled normal plasma ((10.0% (8.9-14.3) and 11.3% (9.1-21.2), respectively, vs 0.6%), while the proportion of small multimers concomitantly decreased. The largest vWF forms within trauma patient plasma circulated at 33±4 dimers vs 18±1 dimer in length within pooled normal plasma and were sustained through 24-hours. Trauma patient ADAMTS13 activity was decreased at 0-hours (66% (47-86) vs. 100% (98-100)) and at 24-hours (72.5% (56-87.3) vs 103% (103-103)) when compared to healthy patients, with antigen levels showing congruent trends. Furthermore, within the trauma patient population, the circulating vWF composition demonstrated a significant plasticity within its small multimeric forms at 24-hours that was dependent upon ADAMTS13 activity (Decreased ADAMTS13 Activity: 20.4% (15.0-22.7) vs Normal Activity: 25.8% (22.7-35.2)). ADAMTS13 activity independently predicted the development of coagulopathy, correlating with presentation INR (ρ =-0.63), activated clotting time of thromboelastography (TEG) (ρ=-0.36), and TEG maximum amplitude (ρ=0.36). ADAMTS13 activity also closely correlated with injury severity (ISS) (ρ=-0.34) and blood product transfusion (ρ =-.45).

Conclusion: Severe traumatic injury dysregulates ADAMTS13 and its target, vWF, contributing to a distorted vWF multimeric profile, persistently altered hemostasis, and the development of coagulopathy.

27.05 Herpes Virus Entry Mediator (HVEM): A Novel Potential Mediator of Trauma Induced Immunosuppression

Posted on January 31, 2019

M. E. Wakeley1, S. F. Monaghan1, W. G. Cioffi1, A. Ayala1, D. S. Heffernan1  1Brown University School Of Medicine,Surgery,Providence, RI, USA

Introduction: Trauma induces significant immune-deficiency associated with secondary infection, long term organ dysfunction and an increased risk of death. Lymphocyte loss and dysfunction plays a central role in adverse trauma related outcomes, and co-inhibitory molecules have emerged as key drivers of immune dysfunction in critical illness.  HVEM, a TNF family transmembrane receptor is known to mediate host immunity at mucosal barriers in both stimulatory and inhibitory ways. Altered expression of HVEM and one of its ligands BTLA, are associated with poor outcomes and increased nosocomial infections in critically ill septic patients. Given infections are a driving force in poor long-term trauma outcomes, we hypothesize critically ill trauma patients will display increased blood lymphocyte HVEM expression and that such alteration should be predictive of secondary infectious events.

 

Methods: Critically ill trauma patients prospectively enrolled from the Trauma ICU were compared with healthy controls. Leukocytes were isolated from whole blood, stained for CD3 (lymphocytes) and HVEM by flow cytometry. Charts were reviewed for injuries sustained, APACHE II score, and hospital course including secondary infections.

 

Results: Trauma patients (N=31) compared with healthy controls (N=10) were slightly older (46.7 +/-2.4 vs 36.8+/- 2.1 years; p=0.03) but matched for male sex (74% vs 60%; p=0.4). Trauma patients had higher presenting White Cell Count (13.9 +/- 1.3 vs 5.6 +/-0.5 x106/ml; p=0.002), lower percentage of CD3+ lymphocytes (7.5% +/-0.8 vs 22.5% +/-0.9; p<0.001), but significantly greater expression of HVEM+/CD3+ lymphocytes (89.6% +/-1.46 vs 67.3% +/-1.7; p<0.001). The most common source of infection was ventilator associated pneumonia. Among trauma patients, those who developed a secondary infection during the hospitalization had a higher APACHE II score (20.6 +/- 1.6 vs 13.6 +/-1.4; p=0.03) and markedly lower HVEM expression on CD3+ lymphocytes (75% +/-2.6 vs 93% +/-0.7; p<0.01).

 

Conclusion: In keeping with prior observations on critically ill septic patients, we demonstrate that HVEM expression is increased on CD3+cells after trauma. However, patients who develop an infection during their hospital stay have a lower percentage HVEM+ CD3+ cells circulating. This suggests that HVEM signaling in lymphocytes plays a role in maintaining host defense to infection in critically injured trauma patients. Given the expanding availability of check-point protein mediators, HVEM expression may play the roles of a marker of risk for infection as well as a potential therapeutic target to modulate the immune response to trauma, offering a means for preventing trauma related complications.
 

27.04 Topical Neck Cooling Prolongs Survival of Rats with Septic Shock

Posted on January 31, 2019

E. J. Charles1, A. Y. Zhang1, D. Wu1, R. G. Sawyer2, Z. Yang1  1University Of Virginia,Surgery,Charlottesville, VA, USA 2Western Michigan University,Surgery,Kalamazoo, MI, USA

Introduction: Septic shock often results in end-organ damage and high mortality. We have previously demonstrated that systemic hypothermia prolongs survival of rats with septic shock by inhibiting inflammatory responses. In the present study, we hypothesized that topical neck cooling would inhibit systemic inflammation and thus prolong survival of rats with septic shock.

Methods: Septic shock was induced by cecal ligation and incision (CLI) in Sprague Dawley rats. One hour after CLI, rats were randomized to no further treatment (control, N=12) or topical neck cooling for two hours by wrapping an ice-filled Penrose drain around the neck (N=10). An additional group of rats (N=5) underwent bilateral cervical vagotomy just prior to CLI, followed by topical neck cooling. Primary endpoint was survival duration (hours). Parallel groups of rats (N=4-5 per group) were euthanized three hours after CLI for evaluation of inflammatory markers.

Results: Topical neck cooling prolonged survival after CLI, with a median [IQR] of 10.4 [7.7-11.4] hours for neck cooling rats compared with 6.3 [5.1-7] hours for control (p=0.001). Kaplan-Meier survival analysis is shown in the figure (hazard ratio 0.14, 95% confidence interval 0.05-0.44). Median survival for vagotomy plus neck cooling group was significantly less than both other groups (2.8 [2.4-3] hours). Neck skin temperature was significantly lower in the neck cooling group than control group (16.7±1.4 vs. 30.5±0.6?C, p<0.0001), but the rectal core temperatures were maintained similar between control and neck cooling groups for up to 3 hours (36±0.2 vs. 36.4±0.3?C, p=0.3). Neck cooling rats awoke from anesthesia 71 minutes earlier than control (109±9 vs. 180±7 minutes, p=0.0004). None of the vagotomy plus neck cooling rats awoke prior to death. Significantly more splenic contraction (decreased weight) and fewer circulating leukocytes were found in control rats. Splenic tissue IL-1β and TNF-α protein and mRNA levels were significantly higher in control and vagotomy plus neck cooling groups compared to neck cooling alone (p<0.05). Plasma IL-1β  and TNF-α  levels were significantly higher in control and vagotomy plus neck cooling animals compared to neck cooling alone (p<0.05).

Conclusion: Topical neck cooling significantly prolonged survival of rats with septic shock and was associated with decreased splenic and systemic pro-inflammatory mediator expression, an effect abrogated by vagotomy. Neck cooling may be effective by activating the vagus nerve, leading to a reduced systemic inflammatory response. Topical cooling to target the vagus nerve is a non-invasive treatment that may provide a clinical benefit for septic patients.

27.03 Rapid Transfuser Impact on Whole Blood Platelet Count, Platelet Function, and Hemostatic Potential

Posted on January 31, 2019

M. Zaza1, Y. Wang1, M. George1, C. E. Wade1, J. C. Cardenas1, B. A. Cotton1  1McGovern Medical School at UTHealth,Acute Care Surgery,Houston, TX, USA

Introduction:  Rapid infusion devices are often employed to deliver large volumes of warmed fluids and blood products. Typically, RBCs and plasma are delivered through the rapid infuser device, while platelets and cryoprecipitate are given through a separate intravenous line, as neither platelets nor cryoprecipitate are approved for use with such devices. The restriction on their use is due to concern with component injury and adherence to device filters and circuits. Many centers have recently adopted whole blood (WB) for emergency use. Given that WB contains platelets and fibrinogen, the purpose of this study was to evaluate the impact of rapid infusers on WB platelet count, platelet function, and overall clot strength.

Methods:  Five units of WB (three days or less from donation to testing) were obtained from our regional blood center provider. A baseline samples was obtained from each unit from a non-filtered infusion line (BASELINE). Following this, samples were obtained on WB from four different infusion models: filtered blood tubing (FILTER), filtered blood tubing on a pressure bag at 300 mmHg (PRESSURE), and through a primed Belmont FMS 2000 run at 70 mL/hr (BELMONT70) and 100ml/hr (BELMONT100). Platelet count was assessed by complete blood count (HemaVet 950), while platelet function (AA, ADP, Thrombin, Ristocetin, P-selectin, and Annexin-v) was assessed with Multiplate. Overall hemostatic potential was assessed by thrombelastography (R-value, k-time, angle, MA, lysis) while thrombin generation (lag time, ETP, peak, ttpeak) was assessed with calibrated automated thrombogram (CAT). All testing was performed using STATA 12.1. Means were assessed with ANOVA (Bonferroni correction) while medians were assessed with Kruskal-Wallis. 

Results: There was a statistically significant decrease in platelet count from baseline when samples were obtained from the BELMONT70 and BELMONT100 (TABLE). However, there were no differences detected in platelet function between infusion models; all p>0.20. While there were no differences in any thrombelastography parameters between baseline and infusion models (all p>0.20), there were significant differences in all thrombin generation parameters by CAT (TABLE). * = p<0.05

Conclusion: The use of rapid infusers with WB appears appropriate despite containing platelets and fibrinogen, which, as concentrates, are not approved for use with these devices. While platelet count is significantly decreased when WB is subjected to a rapid infuser device, platelet function and overall clot strength are preserved. Moreover, when compared to standard transfusion tubing or pressure bag, thrombin generation is accelerated and thrombin potential increased when WB is administered through a rapid infuser device. 

 

27.02 Salvage of the Zone of Stasis with Stem Cell Conditioned Media is Mediated by Reduction in Apoptosis

Posted on January 31, 2019

G. Garg1,2, J. S. Vazquez1,2, J. H. Chen1,2, B. C. Carney2,4, M. V. Cruz1,2, T. E. Travis1,2,3, L. T. Moffatt2,4, J. W. Shupp1,2,3,4  1MedStar Washington Hospital Center,The Burn Center,Washington, DC, USA 2MedStar Health Research Institute,Firefighters’ Burn And Surgical Research Laboratory,Washington, DC, USA 3Georgetown University School of Medicine,Department Of Surgery,Washington, DC, USA 4Georgetown University School of Medicine,Department Of Biochemistry,Washington, DC, USA

Introduction: Burn progression, in which initially uninjured skin adjacent to deep partial or full thickness burns (the zones of stasis) becomes necrotic, occurs over the first 24-72 hours after injury. Many factors likely contribute to burn wound progression, such as inflammation, oxidative stress, and disruption of cell cycle regulation. Media used to grow mesenchymal stem cells (MSCs) is rich in cytokines and cell mediators that promote healing and can therefore potentially impact multiple pathways. The present study aimed to use MSC-conditioned media in an easily applied topical gel to treat the zone of stasis in an attempt to prevent burn progression, salvaging healthy tissue.

Methods:  A rat comb burn model was used to generate serial burns with viable interspaces. Burn areas including interspaces were treated with two applications of conditioned media (n=6) or vehicle (n=6). The applications were at 30 minutes and 24 hours after injury. Digital photographs and Laser Doppler Imaging (LDI), of burns and interspaces were obtained daily for seven days from the burn area on the left flank. Percentages of interspace conversion were calculated from digital photos using ImageJ software, and perfusion units obtained from regions of interest in LDI analysis. Biopsies were obtained from the burn and interspaces on the right side and then paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). Immunofluorescent staining was used to quantify caspase-3 expression in cells in the papillary dermis.

Results: The percentage of converted interspace area was significantly reduced in the treated rats compared to control each post-burn day (1.25% vs. 27.2% on day 1, 16.2% vs. 47.3% on day 2; p < 0.05). Interspace perfusion was significantly increased in the treated rats compared to sham rats (p<0.05). H&E stained sections demonstrated that the treatment group had increased epidermal thickness, though not statistically significant. Further, Immunofluorescent staining demonstrated an 88.4% decrease in caspase-3 expression in the treated rats compared to sham rats (p<0.05).

Conclusion: MSC-derived conditioned media is a novel topical therapy to prevent burn progression. This may be mediated by an inhibition of apoptosis. Further study is warranted to evaluate the molecular pathways associated with this positive effect in order to identify a potential therapeutic target. This therapy may serve to reduce the areas of necrotic tissue requiring excision and grafting, therefore minimizing the associated complications of surgery including bleeding, infection, and length of stay.

27.01 Differential Effects of Bacteria on Endothelium and Epithelium in the Ex Vivo Perfused Human Lung

Posted on January 31, 2019

J. T. Ross1, N. Nesseler2, E. Minus3, J. E. Gotts4, M. A. Matthay4  1University Of California – San Francisco,Department Of Surgery,San Francisco, CA, USA 2University Hospital of Rennes,Intensive Care Unit, Department Of Anesthesia And Critical Care,Rennes, BRITTANY, France 3Amherst College,Amherst, MA, USA 4University Of California – San Francisco,Departments Of Medicine And Anesthesia, Cardiovascular Research Institute,San Francisco, CA, USA

Introduction: ARDS is an important complication of sepsis, but develops less frequently in non-pulmonary than in pulmonary sepsis. Patients with non-pulmonary sepsis often develop pulmonary edema, but rarely progress to ARDS. We hypothesized that there may be a differential susceptibility of the lung endothelium and alveolar epithelium to bacterial-induced injury. The goal of these studies was to measure endothelial and epithelial injury in the ex vivo lung when exposed to S. pneumonia instilled into the air spaces or injected into the perfusate.

Methods: Human lungs not suitable for transplantation were received, and the right or left lung was selected based on gross appearance. The pulmonary artery and main bronchus were cannulated. A 3 Fr catheter was passed via the bronchus into the distal airspaces. The lung was perfused with DME-H21 and 5% bovine serum albumin at 37°C with a mean pulmonary arterial pressure of 10 mmHg (Figure 1). Perfusate drained passively from the pulmonary veins and collected in a reservoir for recirculation. Lungs were inflated with room air at a continuous positive airway pressure of 8 cmH2O. 100ml of fresh human blood was added to the perfusate. In a subset of lungs, 1010 cfu S. pneumonia were added to either the perfusate or distal airspaces. Alveolar fluid clearance (AFC) was measured at 0 and 5 hours by instilling 100ml of 5% albumin into the distal airspaces and comparing protein concentrations at 5 and 35 minutes. Endothelial and epithelial permeability were evaluated by measuring weight gain of the lung over 6 hours and the accumulation of IgM from the perfusate into the airspaces.

Results: Of the 48 lungs perfused, 77% had a normal starting AFC. Lungs in the control group had minimal weight gain (mean 321 ± 212g, 68 ± 28% of start weight), preserved AFC (normal AFC at 5h in 91%) and minimal accumulation of IgM in the airspaces (mean 0.004g, IQR 0.0002-0.004g). Addition of S. pneumoniae into the distal airspaces was associated with significant weight gain compared to controls (mean 127 vs 68%, p = 0.022), significant decrease in AFC (p<0.0001) and a trend towards accumulation of IgM in the airspaces. S. pneumoniae in the perfusate was associated with significant weight gain (mean 155 vs 68%, p = 0.012) but preserved AFC and minimal IgM accumulation.

Conclusion: The results indicate that the human lung endothelium is susceptible to injury from S. pneumonia in either the vascular compartment or the airspaces. In contrast, the alveolar epithelium is susceptible to injury from S. pneumoniae in the airspaces, but resistant to high doses of S. pneumoniae in the vascular compartment. This pattern suggests one explanation for the lower incidence of ARDS after non-pulmonary sepsis.

 

26.10 CD146+ Adipose-Derived Stromal Cells (ASCs) Have Enhanced Regenerative Effects

Posted on January 31, 2019

M. R. Borrelli1, C. Blackshear1, S. Vistnes1, R. Patel1, M. Longaker1, D. C. Wan1  1Stanford University,Palo Alto, CA, USA

Introduction: Adipose-derived stromal cells (ASCs) are cells isolated from the stromal vascular compartment of adipose tissue with extensive proliferative capacity and multilineage differentiation abilities. These characteristics make them strong candidate cells in regenerative medicine. Recent work indicates that ASCs are a heterogeneous mix of cells, and are comprised of multiple stem and progenitor subpopulations. Further elucidation of the exact identity and regenerative capacities of each of the ASC subpopulations can help to develop therapies able to target specific regenerative processes.

Methods:  We previously identified a subpopulation of ASCs positive for the surface marker CD146. Flow cytometry was used to isolate CD146+ ASCs from human lipoaspirate samples (n=3). Gene expression analysis was compared between CD146+ and CD146- ASC populations, and unsorted fat cells, for a number of proangiogenic (ANGPT1, VEGF, FGF) and adipogenic (PPARg, LPL, FABP4) factors. To explore in vivo regenerative potential, freshly isolated CD146+ ASCs were also injected into the subcutaneous tissue overlying the scalp of recipient immunodeficient mice. Graft retention was followed by CT scanning every two weeks for a total of 12 weeks, at which point mice were sacrificed and the fat grafts were explanted and procssed for histology and confocal microscopy.

Results: CD146+ ASCs showed a significant upregulation of VEGF and LPL expression compared to unsorted fat cells and CD146- cells (*p > 0.05, ***p > 0.005) (Figure 1.A).  CD146+ and unsorted fat cells had significantly higher graft retention volumes compared to CD146- ASCs and fat alone. Transplantation of fat alone resulted in the poorest graft retention (Figure 1.B). Fat grafts isolated from mice transplanted with CD146+ ASCs showed the greatest CD31 immunofluorescent staining, and the greatest graft vascularization in confocal microscopy.

Conclusion: We have identified a subpopulation of ASCs positive for the surface marker CD146 with enhanced angiogenic effects. These effects are likely mediated by increased expression of proangiogenic factors such as VEGF. This work suggests that enriching lipoaspirates with CD146+ ASCs may enhance fat graft vascularization and retention in the clinical setting.

 

26.09 PIM3 Kinase Promotes Tumor Metastasis in Hepatoblastoma Cells

Posted on January 31, 2019

R. Marayati1, L. L. Stafman1, A. P. Williams1, J. L. Easlick2, H. R. Markert1, J. M. Aye3, J. E. Stewart1, E. A. Beierle1  1University Of Alabama at Birmingham,Division Of Pediatric Surgery, Department Of Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Division Of Transplantation, Department Of Surgery,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Division Of Pediatric Hematology Oncology, Department Of Pediatrics,Birmingham, Alabama, USA

Introduction: Hepatoblastoma (HB) is the most common primary liver tumor in children. Despite an increasing incidence and major advances in care for other pediatric solid tumors, therapy for HB has remained virtually unchanged for the last 20 years. Over half of patients initially present with metastatic or advanced disease and the prognosis for these children remains dismal. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human HB cells and function to promote tumorigenesis. CRISPR-Cas9 gene editing technology is a powerful approach for loss of function studies. We aimed to use CRISPR-Cas9 mediated PIM3 knockout (KO) to confirm and verify the role of PIM3 kinase in promoting cell proliferation and survival and further characterize the effects of PIM3 KO on migration and invasion of HB in vitro as distinct early steps of the metastatic cascade.

Methods: CRISPR-Cas9 gene editing technology was used to introduce inactivating deletions in the PIM3 gene and achieve stable PIM3 KO in the long-term passaged human HB cell line, HuH6. PIM3 gene editing was confirmed by PCR and Sanger sequencing and resulting knockout of protein expression by immunoblotting. Cell proliferation and viability were measured using the CellTiter 96® and alamarBlue® colorimetric assays, respectively. Migration and invasion were evaluated using modified Boyden chambers with 8 µm micropore Transwell inserts coated with collagen and MatrigelTM (BD Biosciences) respectively. Student’s t-test was used with mean ± standard error of the mean reported and p≤0.05 significant.

Results: Immunoblotting confirmed expression and stable KO of PIM3 in the HuH6 human HB cell line. PIM3 KO HuH6 cells exhibited a significant decrease in proliferation (18.4%, p=0.024) and viability (27.1%, p<0.001) compared to control HuH6 cells. Migration was significantly decreased with PIM3 KO (983 ± 132 vs 375 ± 46 cells, control vs PIM3 KO, p<0.001). Additionally, PIM3 KO resulted in significantly decreased invasion through Matrigel (153 ± 46 vs 63 ± 30 cells, control vs PIM3 KO, p=0.002).

Conclusion: Stable CRISPR-Cas9 PIM3 KO leads to decreased cell proliferation, viability, migration, and invasion of human HB cells. These findings suggest that PIM3 promotes the metastatic phenotype in HB in vitro and that targeting PIM3 may provide a novel therapeutic strategy for children with metastatic or relapsed disease.
 

26.08 Hepatic Progenitor Cells are Generated from a GMP-Compliant Human Induced Pluripotent Stem Cell Line

Posted on January 31, 2019

A. I. Squillaro1,2, A. M. Fode2, L. A. Nucho2, S. M. Zuber1,2, D. F. Chang2, C. R. Schlieve1,2, T. C. Grikscheit1,2,3  1Children’s Hospital Los Angeles,Division Of Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Developmental Biology And Regenerative Medicine At The Saban Research Institute,Los Angeles, CA, USA 3University Of Southern California,Keck Medical School,Los Angeles, CA, USA

Introduction:  Donor scarcity remains an obstacle for patients requiring orthotopic liver transplantation, although in some inborn errors of metabolism, replacement of an entire organ may not be necessary to prevent the buildup of toxic metabolites. Liver cell transplantation has been proposed for these patients; however, primary liver cells have limited expansion in vitro and still require donor tissue. Human induced pluripotent stem cells (hiPSC) offer a renewable source for cellular therapies that, unlike human embryonic stem cells (hESC), are derived from postnatal tissues. We initially derived hepatic progenitor cells from hESC after 17 days of directed differentiation. However, in order to plan to meet regulatory standards, we next hypothesized that hepatic progenitor cells (HPC) might be be generated from a fully characterized GMP hiPSC line, LiPSC-GR1.1, in a variation of our tested differentiation protocol. 

Methods: LiPSC-GR 1.1 hiPSC were cultured in suspension for 17 days and differentiated into hepatospheres containing HPC. These hepatospheres were then analyzed by H&E and immunofluorescence staining for hepatic nuclear 4α (HNF4α), α- fetoprotein (AFP), and cytokeratin 19 (CK19), a marker for cholangiocytes. Immunofluorescent staining for proliferating cell nuclear antigen (PCNA) evaluated the presence of proliferation. 

Results: H&E revealed formed hepatospheres. Immunofluorescence staining for hepatocyte markers HNF4α, an important regulator in metabolism, and AFP confirmed that hepatic progenitor cells were formed after 17 days of directed differentiation in vitro (Figure 1A). Cholangiocytes were detected in the HPC by the presence of CK19. Active proliferation of cholangiocytes were identified by PCNA co-staining (Figure 1B). 

Conclusion: A fully characterized, GMP-compliant hiPSC line can produce hepatic progenitor cells after 17 days in vitro. The day 17 cells form hepatospheres that contain hepatocytes and cholangiocytes which are actively proliferating. Further investigation of hepatic progenitor cells from this source may provide a functional cellular therapy that salvages patients from congenital metabolic diseases. 

26.07 The dysbiotic EdnrBNCC-/- microbiome enhances epithelial tight junction development in pseudo germ-free mice

Posted on January 31, 2019

K. P. Kuruvilla1,3, J. F. Pierre2, A. Gosain1,3  1University of Tennessee Health Science Center,Department Of Surgery,Memphis, TN, USA 2University of Tennessee Health Science Center,Department Of Pediatrics,Memphis, TN, USA 3Children’s Foundation Research Institute, Le Bonheur Children’s Hospital,Division Of Pediatric Surgery,Memphis, TN, USA

Introduction: Hirschsprung disease (HSCR) results from incomplete development of the enteric nervous system (ENS). Hirschsprung-Associated Enterocolitis (HAEC) is the most significant and life-threatening complication of HSCR and the occurrence of HAEC even after surgical resection of bowel lacking an ENS suggests that pathophysiology of HAEC is multifactorial. We have previously shown that EdnrBNCC-/- mouse model of HSCR exhibit colonic dysbiosis prior to development of HAEC. We have also observed that tight junctions (TJ), which are critical determinants of intestinal epithelial barrier integrity, mature earlier in EdnrBNCC-/- mice (vs. wildtype) but then are downregulated just prior to HAEC onset. In this study, we sought to determine the impact of the dysbiotic EdnrBNCC-/- microbiome in regulating the expression of TJ using pseudo germ-free (PGF) mice. We hypothesized that the dysbiotic EdnrBNCC-/- microbiome would trigger downregulation of TJs and promote HAEC.

Methods: Wild-type PGF pups were generated by treatment of pregnant dams with broad spectrum antibiotics and antifungals and maintained PGF until weaning. At postnatal day P21, PGF animals received oral gavage of either EdnrBNCC-/- microbiota for fecal microbiota transplant (FMT) or sterile phosphate buffered saline (PBS) as control. Mice were separately housed and followed for 1 week (P28). Prior studies have shown that P21 coincides with the onset of HAEC in EdnrBNCC-/- mice and P28 is the mean time of death from HAEC.

Results: 16S pyrosequencing and total copy number confirmed the PGF status of neonatal pups. PGF pups showed reduction in body weight gain as compared to non-PGF WT pups beginning about P10. However, upon weaning and gavage with either FMT or PBS, both groups started gaining body weight. In the seven days following gavage, neither FMT nor PBS-treated mice showed signs of HAEC based on clinical severity scoring (hunched posture, ruffled hair coat, lethargy and weight loss). Immunohistochemistry revealed increased expression of the TJ proteins occludin (Ocln) and zona occludens-1 (ZO-1) in the ileum and colon of the FMT group as compared to PBS. There were no differences in the expression of claudin-3 (Cldn3) or E-cadherin (adherens junction protein).

Conclusions: Increased expression of TJ proteins (Ocln and ZO-1) in FMT mice indicates that the dysbiotic EdnrBNCC-/- microbiome aids in strengthening the epithelial barrier of these intestinal tissues. This result is consistent with our prior, in vivo, observations in EdnrBNCC-/- mice prior to HAEC and suggests that EdnrBNCC-/- microbiome alone is not sufficient to cause HAEC. The specific components of the EdnrBNCC-/- microbiome contributing to TJ maturation and the mechanistic pathways are under active investigation.

 

26.06 Neonatal Mice Have Aberrant Splenic/Bone Marrow Dendritic Cell Responses to Sepsis Compared to Adults

Posted on January 31, 2019

R. B. Hawkins1, J. C. Rincon1, S. L. Raymond1, S. D. Larson1  1University Of Florida,Division Of Pediatric Surgery,Gainesville, FL, USA

Introduction: Neonatal sepsis is a leading cause of morbidity and mortality, with over 1 million annual deaths worldwide. Neonates are at higher risk of sepsis and poor outcomes due to quantitative and functional impairments in immunity. Previous work from our laboratory has demonstrated that neonates rely on innate immunity during sepsis. However, functional aspects of innate immunity including neutrophil motility, neutrophil extracellular trap production, cytokine production, and antigen presentation are impaired in neonates. The purpose of this study was to better understand the quantitative and functional differences in neonatal dendritic cells at baseline and in response to sepsis compared to adults. We hypothesized that neonates have decreased dendritic cell populations at baseline and that antigen-presenting capabilities in response to sepsis are impaired.

Methods:  Neonatal and adult C57BL/6 mice received an intraperitoneal injection of cecal slurry (CS) at 1.1 mg/g body weight to induce sepsis (LD40-70). Mice were euthanized 18 hours following CS. Splenocytes were harvested and myeloid (GR1+CD11b+) and dendritic cell activation (MHCII, CD80, CD86) markers were analyzed with flow cytometry. For in vitro studies, bone marrow-derived cells (BMDCs) were isolated from naïve neonatal and adult B6 mice and differentiated for 7 days with 20 ng/mL GM-CSF. Cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 24 hours, and dendritic cell activation markers were analyzed using flow cytometry.

Results: Splenic GR1+CD11b+ populations were similar at baseline between neonatal and adult mice. However, 18 hours following CS, neonates had significant depletion of GR1+CD11+ cells (p<0.01) while adults had no significant change (Figure 1). Similarly, following in vitro stimulation with LPS, neonatal BMDCs experienced no expansion of the GR1+CD11b+ lineage, while adult BMDCs had significant myeloid expansion (p<0.001). In vitro LPS stimulation induced increased expression of MHCII with concurrent diminishment of CD86 and CD80 in adult but not neonatal BMDCs (p<0.001). 

Conclusion: The adult response to sepsis involves early expansion of myeloid cells that differentiate into macrophages or neutrophils to fight infection. Neonatal mice have aberrant downregulation of splenic and bone marrow dendritic cells in response to infectious stimuli in both in vivo and in vitro experiments. Taken together, these results suggest that neonatal myeloid cells have reduced antigen presentation function, which may partially explain the diminished neonatal ability to mount an early, effective immune response during sepsis.

 

26.05 Intestinal Permeability Changes Following Small Bowel Resection

Posted on January 31, 2019

C. C. Courtney1, K. M. Seiler1, E. J. Onufer1, K. G. McDonald2, R. D. Newberry2, B. W. Warner1  1Washington University,Division Of Pediatric Surgery,St. Louis, MO, USA 2Washington University,Department Of Internal Medicine,St. Louis, MO, USA

Introduction:  The intestinal epithelial barrier selectively permits the absorption of nutrients while providing a defense against antigens and intraluminal toxins. It achieves this barrier function through both structural complexes and specialized delivery systems. Our lab has discovered injury to the liver associated with massive small bowel resection (SBR) independent of TPN, with associated translocation of bacteria to the liver, spleen, and lymph nodes. This suggests that there are alterations in the permeability of the intestine following SBR. The purpose of this study was to determine the most plausible mechanism by which the intestinal barrier is altered by looking at both paracellular and transcellular pathways within the intestine.

Methods:  Male B6 mice underwent 50% proximal SBR or sham operation (transection with reanastomosis alone). Paracellular permeability was measured by fluorescein isothiocyanate-conguated (FITC)-dextran absorption and tight junction protein networks. FITC-dextran was injected into the distal bowel and serum uptake was quantified using a fluorimeter. Intestinal tissue was preserved in a glutaraldehyde solution and embedded for electron microscopy (EM). Single cell RNA sequencing and mass spectrometry were used to evaluate occludin and claudin-3/4 expression. The number of Goblet cell associated passages (GAPs) were quantified using intravital two-photon (2P) imaging as these structures facilitate the transcellular delivery of luminal antigens to the underlying immune cells. 

Results: FITC-dextran uptake into the serum following massive SBR was increased compared to sham mice (1.83 ± 0.15 ng/µL vs. 1.34 ± 0.22 ng/µL). EM of tight junction complexes were more obscured with irregular arrangements in resected mice compared to sham operated mice (graphic 1). Single seq analysis after SBR showed a 21% decrease in occludin expression and a 30% decrease in claudin 2 and 3 expression compared to sham mice. Compared with unoperated mice, the number of GAPS appeared similarly increased after both SBR fand sham operation in both proximal and distal small intestine.

Conclusion: Massive SBR is associated with increased intestinal barrier permeability as revealed by increased FITC dextran absorption into the serum. This appears to be in part due to structural changes at the tight junction level and reduced expression of tight junction proteins occludin, claudin-2/3. The expression of GAPs was not statistically different between SBRs and shams suggesting that the alterations in permeability are primarily driven by changes at the paracellular level. These findings suggest potential targets to restoring the intestinal barrier, thus preventing intestinal resected-associated liver disease.

 

26.04 Prenatal Probiotic Administration Protects the Neonatal Intestine from Necrotizing Enterocolitis

Posted on January 31, 2019

M. M. Elbahrawy1, C. E. Pisano1, R. D. Shelby1, J. B. Navarro2, S. D. Goodman2, G. E. Besner1  1Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital,,Department Of Pediatric Surgery,Nationwide Children’s Hospital,,Columbus, OH, USA 2Center for Microbial Pathogenesis, The Research Institute at Nationwide Children’s Hospital,Columbus, OH, USA

Introduction: We have previously shown that enteral administration of the probiotic Lactobacillus reuteri (Lr) in its biofilm state to premature rat pups exposed to experimental necrotizing enterocolitis (NEC) protects the intestines from injury. In the current study, we investigated whether administration of the probiotic biofilm enterally to the pregnant mother can protect the intestines of the progeny from NEC.

Methods:  For administration in its planktonic (free-living) state, Lr was pelleted and resuspended in sterile 0.9% saline. For administration in its biofilm state, dextranomer microspheres were loaded with maltose (DM-maltose) by overnight incubation to reach equilibrium, followed by Lr incubation with the loaded microspheres at room temperature for 30 minutes to induce biofilm formation. For 7 days prior to delivery, dams received a daily gavage dose of: (1) 0.2 ml of sterile water, (2) 0.2 ml of Lr (2×108 CFU), or (3) 0.2 ml of Lr (2×108 CFU) + DM-maltose (2 mg). Pups were delivered prematurely via Cesarean section on day 21 of gestation and subjected to experimental NEC via repeated exposure to hypoxia/hypothermia/hypercaloric feeds. Additional pups from untreated dams were delivered by C-section, placed with surrogate dams, unstressed, and breast fed. After 4 days, all pups were sacrificed and intestinal samples were collected. Intestinal histologic injury was graded blindly by two independent investigators.

Results: None of the unstressed breast-fed control pups developed NEC, whereas 63.2% of pups delivered from untreated dams and exposed to experimental NEC developed histologic injury. Compared to the progeny from untreated dams, 27.3% of pups from dams treated with Lr (p=0.030), and only 10.5% of pups from dams treated with Lr + DM-maltose developed NEC (p=0.0009) (see Figure). Furthermore, progeny of dams who were treated with Lr + DM-maltose and exposed to experimental NEC had significantly decreased mortality (20.5%) compared to pups from untreated dams (61.9%) (p < 0.001).

Conclusion: Prenatal Lactobacillus reuteri in its biofilm state administered enterally to pregnant rats is most effective in reducing NEC incidence and mortality in offspring exposed to experimental NEC and may represent a novel method of reducing clinical NEC in the future.

26.03 Osteosarcoma Tumor Excision Increases Vascular Endothelial Growth Factor Receptor in the Lungs

Posted on January 31, 2019

M. P. Kallis1,3, C. Tzanavaris1,3, S. Wang1,3, C. Maloney1,3, M. Symons1,3, B. M. Steinberg1,3, S. Z. Soffer2,3  1The Elmezzi Graduate School of Molecular Medicine,Northwell Health,Manhasset, NY, USA 2Cohen Children’s Medical Center, Northwell Health,Division Of Pediatric Surgery,New Hyde Park, NY, USA 3The Feinstein Institute for Medical Research,Center For Oncology And Cell Biology,Manhasset, NY, USA

Introduction:

Excision of the primary tumor is the mainstay of treatment for osteosarcoma (OS), but may result in a significant increase in pulmonary metastasis. Previous studies in our lab have shown that gefitinib decreases surgically accelerated pulmonary metastasis in an animal model of OS via its effect on tumor associated macrophages. Angiogenesis is essential for tumor growth and metastasis, but also increases following systemic inflammation and surgery. The purpose of this study is to determine whether vascular endothelial growth factor receptor (VEGFR), used as a marker of a pro-angiogenic tumor environment, specifically increases in response to removal of the primary tumor or is a general response to surgical stress.

Methods:

K7M2 OS cells were injected into the tibia of syngeneic BALB/c mice. One week post implantation, mice were randomly assigned to 4 groups: control, amputation of primary tumor, amputation of contralateral limb, amputation of primary tumor plus perioperative gefitinib. Mice were sacrificed 3 weeks later, and lungs were harvested to assess pulmonary metastatic nodules. Lung sections (10 high-power fields per mouse) were analyzed by immunohistochemistry for the presence of VEGFR, quantified by ImageJ photo processing software.

Results:

Mice that underwent amputation of the primary tumor had increased pulmonary metastases compared to tumor bearing controls (17 vs 9.5, p<0.05). Histological lung sections from these two groups were analyzed for VEGFR positivity. Lung sections from mice that underwent amputation of tumor showed an increase in the number of VEGF+ cells (105.6 vs 74.8 per field, p<0.05). Surgical stress, represented by amputation of the contralateral limb, had no effect on metastasis with metastatic nodules akin to controls (7 vs 9.5, p<0.05). Lung sections from the contralateral amputation group showed a marked reduction in VEGFR+ cells (58.4 per field, p<0.05). Gefitinib suppressed both the increase of gross metastatic nodules and the increase of VEGFR+ cells induced by amputation of the primary tumor (6 vs 17, p<0.05; 74 vs 105.6 per field, p<0.05).

Conclusions:

Post-surgical metastatic enhancement reflects distinct microenvironmental changes that occur upon removal of the primary tumor. Among these is a pro-angiogenic tumor environment. These changes are independent of intrinsic surgical stress. Gefitinib prevents this pro-angiogenic switch, further supporting the use of gefitinib as a clinically important anti-metastatic strategy.

 

26.02 Ablation of Prominin-1 Hepatic Cells After Bile Duct Ligation Mitigates Pro-Fibrobrogenic Expression

Posted on January 31, 2019

M. R. Fenlon1, N. Malkoff1, J. Xu1, E. Mahdi1, A. Glazier1, C. Lee1, R. Oweis1, K. Asahina2, K. S. Wang1  1Children’s Hospital of Los Angeles,Surgery,Los Angeles, CA, USA 2Keck School of Medicine,Pathology,Los Angeles, CA, USA

Introduction:
Biliary Atresia (BA) is the most common cause of end-stage liver failure in children. The expansion of intrahepatic ductular reactions (DR), comprised of reactive cholangiocytes, has been strongly linked to liver fibrosis, and correlates with progression toward cirrhosis post-Kasai portoenterostomy. We previously demonstrated the expansion of Prominin-1 (Prom1)-expressing hepatic progenitor cells (HPCs) within DR and evolving fibrosis in BA. Curiously, while null mutation of Prom1 leads to decreased DR and fibrosis in a murine model of BA induced by Rhesus rotavirus, targeted ablation of preexisting Prom1+ HPCs in adult mice undergoing bile duct ligation (BDL) does not. We therefore hypothesized that de-novo Prom1 expression following HPC activation, is responsible for fibroblastic activation in cholestatic liver injury.

Methods:
Adult C57BL6 mice (8-10 weeks) were bred to heterozygosity for Prom1-Cre/ERT2 and ROSA-lsl-Diphtheria Toxin A (DTA). BDL vs sham laparotomy (day 0) was performed to induce cholestatic liver injury. DTA-mediated targeted ablation of all cells expressing Prom1 was accomplished by tamoxifen (TAM)-dependent Cre recombination. Intraperitoneal TAM vs corn oil control (Oil) was administered either day -7 (Pre-BDL) to target preexisting Prom1-expressing HPCs or day +2 (Post-BDL) to target all cells expressing Prom1, including those with de novo expression. At day +10, whole liver RNA was collected for gene expression analysis with quantitative PCR. ANOVA with post-hoc Tukey test was used for statistical analysis (α <0.05 significance).

Results:

With all experimental conditions, BDL-injured mice developed the characteristic weight loss, extra-hepatic biliary dilation, ascites and jaundice. Neither pre- nor post-Sham Prom1-expressing cell ablation significantly altered gene expression compared to Oil Sham. Pre-BDL Prom1-expressing cell ablation increased fibroblastic αSMA and Vimentin expression compared to Pre-Sham (30.5±30.6 vs 0.3±0.2, p<0.01, 10.4±5.3 vs 1.3±0.7 [WK1] [FM2] p<0.001, mean±SD, N=8) whereas Post-BDL ablation (compared to post-Sham) did not (3.8±2.3 vs 2.8±1.3, 1.1±0.4 vs 1.0±0.7, 2.9±0.9 vs 1.5±0.5, all p>0.05, N=9). Moreover, Post-BDL expression of Ck19, αSMA, and Vimentin was significantly lower compared to Pre-BDL groups (3.8±2.3 v 29.6±27.0, p<0.01, 1.1±0.4 v 30.5±30.6, p<0.001, 2.6±0.9 v 10.4±5.3 p<0.001, N=9).

Conclusion:
Post-injury Prom1-expressing cell ablation mitigates the characteristic fibroblastic upregulation of cholestatic liver injury, whereas pre-injury ablation does not. This suggests that a de novo population of Prom1-expressing cells, but not preexisting Prom1-HPCs, are involved in cholestatic liver fibrosis. Further characterization of this cellular population may identify actionable pathways in the treatment of BA.

26.01 C23, a Peptide Derived from CIRP, Suppresses Inflammation and Reduces Lung Injury in Neonatal Sepsis

Posted on January 31, 2019

N. Denning1,3,4, W. Yang1,3,4, L. Hansen3, J. M. Prince1,2,3, P. Wang1,3,4  1The Feinstein Institute for Medical Research at Northwell Health,Center For Immunology And Inflammation,Manhasset, NY, USA 2Cohen Children’s Medical Center at Northwell Health,Division of Pediatric Surgery,New Hyde Park, NY, USA 3Zucker School of Medicine at Hofstra/Northwell,Department Of Surgery,Manhasset, NY, USA 4Feinstein Institute for Medical Research,Elmezzi School Of Molecular Medicine,Manhasset, NY, USA

Introduction: Neonatal sepsis remains a leading cause of infant mortality. Cold inducible RNA binding protein (CIRP) is an inflammatory mediator that induces TNF-α  production in macrophages. C23 is a CIRP-derived oligopeptide that can prevent CIRP from binding to its receptor. We hypothesized that treatment with C23 would reduce systemic inflammation and protect the lungs in neonatal sepsis. 

Methods: Sepsis was induced in C56BL/6 mouse pups (5-7 days) by intraperitoneal injection of adult cecal slurry (.525 mg/g body weight, LD100).  One hour later pups received retro-orbital injection of C23 (8 mg/kg) or equivalent volume of vehicle (0.9% NS).  Ten hours after sepsis induction blood and tissues were collected for analysis.  

Results: C23 treatment resulted in a 58% and 69% reduction in serum levels of pro-inflammatory cytokines IL-6 and IL-1β , respectively.  C23 reduced markers of organ injury. AST was reduced by 40% in C23-treated pups compared to vehicle-treated septic pups, from 168.30 ± 21.08 to 99.89 ± 40.07 (p<0.05). LDH was decreased by 45%, from 168.3 ± 46.76 in vehicle animals to 92.12 ± 56.52 in C23-treated mice (p<0.05). In the lungs, C23 treatment reduced expression of cytokines IL-6 and IL-1β  by 78% and 74%. In addition, the mRNA level of neutrophil chemoattractant KC was reduced by 84%. (Table). These results corresponded to a reduction in histologic lung injury score.  Vehicle- treated pups scored 0.49 ± 0.19, while C23 treatment reduced scores to 0.29 ± 0.12 (p<0.05; Max = 1). Apoptosis in the lungs, measured by TUNEL assay, was also decreased by 53% with C23 treatment (p<0.05). 

Conclusion: Inhibition of CIRP with C23 treatment is protective in septic neonatal mice as demonstrated by reduced inflammatory markers systemically and in the lung. Therefore, C23 has promising therapeutic potential in treatment of neonatal sepsis. 
 

25.10 Bmi1 Loss Partially Reverses Kras-driven Changes in Gene Regulatory Networks in Pancreatic Neoplasia

Posted on January 31, 2019

J. K. Thompson1, J. Blumberg5, O. Alkhalili5, H. Crawford2,4, M. Pasca Di Magliano1,3, F. O. Bednar1  1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Internal Medicine,Ann Arbor, MI, USA 3University Of Michigan,Cell And Developmental Biology,Ann Arbor, MI, USA 4University Of Michigan,Molecular And Integrative Physiology,Ann Arbor, MI, USA 5University Of Michigan,Literature, Science, And Arts,Ann Arbor, MI, USA

Introduction: KRAS is the primary oncogenic driver in human pancreatic ductal adenocarcinoma (PDA). Pancreatic acinar cells are most susceptible to transformation by oncogenic Kras in mouse models of PDA. The earliest stage of transformation consists of conversion of the acinar cells to a duct-like progenitor phenotype in a process called acinar-ductal metaplasia (ADM). Transcription factor (TF) gene regulatory networks control ADM progression and pancreatic tissue repair. We have shown that deletion of Bmi1, a component of the Polycomb Repressor Complex 1 (PRC1), disrupts oncogenic Kras-driven ADM in mice. We hypothesize that loss of Bmi1 inhibits the Kras-driven changes in TF networks to abolish pancreatic neoplasia.

Methods: Mice with a pancreatic acinar cell-specific, tamoxifen inducible Cre recombinase (Ela-CreER) were bred with strains containing the oncogenic Kras G12D allele, the conditional knockout Bmi1 allele (Bmi1 flox) and the fluorescent protein tdTomato in the Rosa26 locus. We activated the Cre recombinase with five daily gavages of tamoxifen (4mg/day) and induced acute pancreatitis with intraperitoneal caerulein injections (x8/day for two consecutive days). We isolated acinar cell progeny from primary pancreata with fluorescence-activated cell sorting (FACS) using the tdTomato lineage marker one week after acute pancreatitis. Total cell RNA was isolated from the sorted cells using the RNeasy Micro Kit (QIAGEN) and complementary DNA (cDNA) was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). We utilized a panel of 26 TaqMan qPCR probes specific for pancreatic developmental TFs and three acinar cell and duct markers (amylase, elastase, keratin 19 – CK19) to characterize the TF networks within the isolated cells.

Results: Tamoxifen gavage induced a high level of tdTomato expression in the pancreata from Ela-CreER expressing mice. RT-qPCR analysis after FACS identified Kras-driven loss of expression of most acinar cell-specific TFs one week after pancreatitis induction consistent with active ADM. Genetic deletion of Bmi1 partially restored amylase and elastase expression at the RNA level. Bmi1 loss also led to recovery of the acinar cell compartment from ADM. These findings correlated with increased expression of Mist1 and Nr5a2, key acinar fate-specific TFs. We did not observe recovery of other acinar TFs, including Pdx1 and Ptf1a/p48, one week after acute pancreatitis in cells with oncogenic Kras and deleted Bmi1.

Conclusion: Oncogenic Kras-driven ADM is controlled by changes in master TF gene regulatory networks. Bmi1 deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis.

 

25.09 The role of intestinal alkaline phosphatase in common bile duct ligation induced liver fibrosis

Posted on January 31, 2019

Y. Liu1, T. Liu1, V. Robin1, A. Fatemeh1, C. Paul1, F. Matthew1, R. Hodin1, R. Hodin1  1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA

Introduction:
Biliary cirrhosis can result from many conditions including sclerosing cholangitis, malignancy, or benign biliary obstruction.  Mice undergoing common bile duct ligation (CBDL) have been shown to have impaired intestinal barrier function, bacteria overgrowth, and liver fibrosis.  Interestingly, the process of liver fibrosis is ameliorated with depletion of intestinal bacteria.  Intestinal alkaline phosphatase (IAP) is an enzyme located on the brush border of intestinal epithelial cells and released into the gut lumen and has been shown to both detoxify LPS and maintain the intestinal barrier through upregulating tight junctions. We hypothesized that IAP may protect against liver fibrosis in a CBDL model. 

Methods:
Adult (WT and IAP KO) mice were subjected to either sham or bile duct ligation (+/- IAP in their drinking water) and sacrificed for tissue harvest after 1 and 3 weeks. 

Results:
Compared to sham controls, stool IAP activity, AKP3, and AKP6 mRNA expression were significantly decreased after CBDL both at 1 week (sham vs. CBDL: 114.56 vs. 27.50; 1 vs. 0.36; 1 vs. 0.64) and 3 weeks (sham vs. CBDL: 114.56 vs. 32.97, 1 vs. 0.58; 1 vs. 0.69). Additionally, ZO-1, ZO-2 and Occludin mRNA expression were decreased (sham to CBDL 1 week: 1 vs. 0.71, 1 vs. 0.58, 1 vs. 0.65; 3 weeks: 1 vs. 0.64, 1 vs. 0.54, 1 vs. 0.60), Gut barrier function became impaired after CBDL as determined by FITC-Dextran assay (sham to CBDL 1 week: 156.12 vs. 70.92 pg/ml). IAP treatment maintained tight junction protein gene expression in the CBDL mice (sham vs. CBDL 3 weeks: 1 vs. 1.22, 1 vs. 0.92, 1 vs. 1.07). In regard to the effect of IAP on CBDL induced liver fibrosis, we found that liver ACAT, Collagen I, TIMP, and TGF-b mRNA expression were increased in IAP KO compared to WT mice (4.87 vs. 9.67; 47.74 vs. 122.69; 49.55 vs. 70.12; 2.40 vs. 4.37).  Furthermore, enteral IAP supplementation decreased expression of these genes in both WT and IAP KO mice 3 weeks after CBDL(WT mice: 2.97, 28.48, 28.67, 1.79; KO mice: 2.89, 28.40, 35.27, 2.89).

Conclusion:
Bile duct ligation may lead to intestinal barrier dysfunction by decreasing IAP levels, and further increasing the liver fibrosis process. Oral IAP may represent a novel therapy against liver fibrosis. 
 

25.08 Depletion of the Gut Microbiome Decreases Pancreatic Cancer Metastases

Posted on January 31, 2019

S. Kurtom1,2, V. Sethi1, A. Ferrantella1, B. Giri1, H. K. Jacob1, S. Ramakrishnan1, A. Saluja1, V. Dudeja1  1University Of Miami,Surgery,Miami, FL, USA 2Virginia Commonwealth University,Surgery,Richmond, VA, USA

Introduction:

The gut is comprised of trillions of bacteria that play a crucial role in intestinal homeostasis. A growing body of evidence shows that the gut’s microbiome plays a role in modulating the immune system. Our study aimed to evaluate microbiome-driven immunological mechanisms, specifically toll-like receptor (TLR) activation, in metastatic murine pancreatic ductal adenocarcinoma (PDAC).  TLR2 and TLR4 are important mediators of the inflammatory response in cancer. We hypothesize that gut microbiome depletion decreases liver metastases, via abrogation of the TLR-induced inflammatory response.

Methods:
C57BL/6J mice received either oral saline or a gut sterilizing cocktail of poorly absorbable broad spectrum antibiotics (Vancomycin, Ampicillin, Amphotericin B, Metronidazole, and Neomycin). These mice then received intrasplenic injection of pancreatic cancer cells derived from KPC (Kras LSL.G12D/+; p53 R172H/+;Pdx::Cre) and PKT (Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox) mice. To evaluate the role of TLR activation, intrasplenic KPC injection was subsequently repeated in TLR2-/- or TLR4-/- mice. Mice were sacrificed and the tumors were immunophenotyped and immunostained for multiple antigens.

Results:
Gut microbiome depletion decreased hepatic metastases in KPC injected wild type and TLR4-/- mice and PKT injected WT mice. This tumor inhibitory effect of gut microbial depletion disappeared in the TLR2-/- KPC group. Antibiotic-treated mice had a decrease in intrametastatic IL-17+ CD4+ cells.

Conclusion:
Gut microbiota promote liver metastases in pancreatic cancer, potentially via modulating IL-17+ CD4 cells mediated through the TLR2 pathway. 
 

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