79.18 Biliary Excretion of Diclofenac Metabolites Leads to Increased Rates of Anastomotic Leakage in Rats

Posted on January 26, 2017

S. Yauw1, R. Lomme1, P. Van Den Broek2, R. Greupink2, F. Russel2, H. Van Goor1  1Radboudumc,Department Of Surgery,Nijmegen, GELDERLAND, Netherlands 2Radboudumc,Department Of Pharmacology And Toxicology,Nijmegen, GELDERLAND, Netherlands

Introduction:
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been associated with anastomotic leakage in humans and animals, but underlying mechanisms are unknown. Some studies suggest an increase of bile toxicity by specific NSAIDs, causing small intestinal damage. In this experiment we determined the relevance of biliary diclofenac excretion in anastomotic leakage.

Methods:
Randomized controlled blinded experiment assessing anastomotic leakage, leak severity and anastomotic strength in 138 male Wistar rats, using bile duct and duodenal catheterization techniques and 72 rats as bile donors. It was studied if administration of ‘diclofenac bile’ induces leakage in control rats and, in turn, if diversion or replacement of bile reduces leakage in diclofenac (oral or intramuscular) treated rats. Diclofenac biliary metabolites and plasma levels were determined with HLPC and LCMS.

Results:
Leak rate was 28% after administration of ‘diclofenac bile’ compared to 6% (p=0.089) after control bile. Following oral diclofenac administration 76% leaked compared to 47% (p=0.127) when bile was replaced with control bile. After intramuscular administration 67% leaked and 50% if the rat’s own bile was diverted and returned, compared to 25% (p=0.060) when bile was drained or 20% (p=0.117) when replaced with control bile, respectively. Grading according to an Anastomotic Complication Score revealed signs of leakage were significantly more severe in ‘diclofenac bile’ groups; (p=0.006 versus control bile in control groups; p=0.016 versus control bile in oral diclofenac groups; p=0.025 versus bile drainage in intramuscular diclofenac groups; p=0.283 versus control bile in intramuscular diclofenac groups. Anastomotic strength results grossly supported macroscopic findings. Bile analysis showed diclofenac metabolite levels peak within two hours after administration, with diclofenac-acyl-glucuronide and unchanged diclofenac as main metabolites. Plasma levels of diclofenac did not change following exchange of ‘diclofenac bile’ by ‘control bile‘, or vice versa, and could not explain the different leak rates.

Conclusion:
Altered bile composition aggravates ileal anastomotic leakage in rats treated with diclofenac, which may result from biliary excretion of diclofenac metabolites. This is a newly identified pathway in the pathophysiology of anastomotic leakage and its exact mechanism and clinical relevance should be clarified by further research.
 

79.17 Beta-glucuronidase Inhibitor Reduces Diclofenac Induced Anastomotic Leakage

Posted on January 26, 2017

S. Yauw1, M. Arron1, R. Lomme1, P. Van Den Broek2, R. Greupink2, A. Bhatt3, M. Redinbo3, H. Van Goor1  1Radboudumc,Department Of Surgery,Nijmegen, GELDERLAND, Netherlands 2Radboudumc,Department Of Pharmacology And Toxicology,Nijmegen, GELDERLAND, Netherlands 3University Of North Carolina At Chapel Hill,Department Of Biochemistry And Microbiology,Chapel Hill, NORTH CAROLINA, USA

Introduction:
Use of diclofenac in colorectal surgery has been associated with increased rates of anastomotic leakage. Previous experiments suggest drug metabolites in bile play a role in this process. Cleavage of the diclofenac-acyl-glucuronide metabolite by bacterial glucuronidase in the gut releases a harmful aglycone, which causes small intestinal mucosal damage in mice. Administration of a beta-glucuronidase inhibitor (Inh1) prevents this. It was studied if inhibition of glucuronidase prevents the development of diclofenac-induced anastomotic leakage in rats.

Methods:
In one hundred and eight male Wistar rats an anastomosis of the ileum was constructed. Animals were allocated to six groups, three with diclofenac and three without diclofenac. For the diclofenac groups, group Dic received diclofenac only and the groups Dic-Inh1-low and Dic-Inh1-high received diclofenac and beta-glucuronidase inhibitor in either a low (800ug/kg/d) or high dose (4000ug/kg/d) orally. For the non-diclofenac groups, animals received either low dose beta-glucuronidase inhibitor (group Inh1-low), vehicle (methylcellulose; group Veh) or no solution (group Neg). All solutions were given from the day of surgery until sacrifice on day three. Plasma levels of diclofenac were determined to rule out a systemic effect. Outcomes were anastomotic leakage, leak severity score, bursting pressure and breaking strength.

Results:
Anastomotic leak rates were 67% in group Dic, 33% in group Dic-Inh1-low (p=0.094) and 50% in group Dic-Inh1-high (p=0.500). Leak severity was reduced in group Dic-Inh1-low (p=0.029), but not in the high dose group (p=0.293). In non-diclofenac cohort rates were 0% in group Neg, 17% in group Inh1-low (0.230), 17% in group Veh (p=0.227). Bursting pressure and breaking strength were not significantly different. Plasma levels of diclofenac were not changed by Inh-1.

Conclusion:
Beta-glucuronidase inhibitor reduces anastomotic leakage when given in low doses, suggesting a toxic effect of diclofenac metabolite on anastomotic healing. The absent effect in the high dose might be due to an intrinsic toxicity of the inhibitor or clumping of the substance. These findings improve our understanding of the pathogenesis of anastomotic leakage.
 

79.16 Mechanical Properties and Healing in Burn Wounds

Posted on January 26, 2017

D. Koganti1, S. Tannouri1, C. Lotto1, E. Macarak2, J. Uitto2, J. Rosenbloom2  1Thomas Jefferson University,Dermatology/Surgery,Philadelphia, PA, USA 2Thomas Jefferson University,Dermatology,Philadelphia, PA, USA

Introduction:  Burn wounds lead to severe disability due to contractures from abnormal healing. The mechanical properties of burns may be a consequence of aberrant scarring. We propose a protocol using a murine model for the mechanical testing of burn wounds. We then investigate the effects of topical therapies on burns using this model.

Methods:  C57BL/6 mice underwent deep thermal injury by a heated brass plate on their dorsum. Burned and unburned skin was harvested at nine days, two weeks, and five weeks post-burn injury. We created a reproducible, standardized protocol using a custom 3-D stamp and tensometer to determine the mechanical properties of the skin. The effects of topical treatments (povidine-iodine, mupirocin, silver sulphadiazine) on the unburned and burned skin were then investigated.

Results: Tensile strength of burned skin was less than unburned skin at nine days (0.86 N/m2 vs. 3.21 N/m2, Student’s t- test, p=0.008), two weeks (0.31 N/m2 vs. 3.26 N/m2, p=0.004), and five weeks (0.69 N/m2 vs. 4.44 N/m2, p= 0.000006).  At two weeks, burns treated with povidine-iodine (0.31 N/m2 vs 0.12 N/m2, p=0.03) and mupirocin (0.31 N/m2 vs. 0.10 N/m2, p=0.02) had less tensile strength than untreated burns. At five weeks, silver sulphadiazine weakened the burn vs. burned untreated skin (0.32 N/m2 vs 0.69 N/m2, p=0.01). Also, at five weeks, unburned skin with any of the agents (povidine-iodine: 2.03 N/m2 vs 4.44 N/m2, p=0.0002; mupirocin: 1.56 N/m2 vs 4.44 N/m2, p=0.0001; silver sulphadiazine: 1.50 N/m2 vs 4.44 N/m2, p=0.00009) was found to be weaker than unburned, untreated skin.

Conclusion: The tensile strength of a burn is decreased compared to unburned skin in a murine model, reflecting that the process of wound healing after burns causes weaker regenerated skin.  Some commonly used topical therapies seem to lead to weaker healed skin than untreated burns. These agents also decrease the tensile strength of unburned skin and warrant further investigation. Using this protocol, tensile strength may be used as a parameter to evaluate the effect of therapies on healing of burn wounds.

 

79.15 Long non-coding RNA GAS5 Regulates Proinflammatory Macrophage Polarization

Posted on January 26, 2017

C. E. Liechty1, J. Hu1, C. Zgheib1, K. W. Liechty1, J. Xu1  1Laboratory For Fetal And Regenerative Biology, Children’s Hospital Colorado And The University Of Colorado Anschutz Medical Campus,Department Of Surgery,Aurora, CO, USA

Introduction:

Macrophages have the ability to dynamically transition between M1 (pro-

inflammatory) and M2 (anti-inflammatory) phenotypes in response to signals from the

surrounding microenvironment. Prolonged M1 macrophage polarization is thought to play a

central role in the chronic inflammation and pathogenesis of diabetic wounds. Recent studies

reveal that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological

processes. However, the role of lncRNAs in macrophage polarization or in diabetic wounds

remains undefined. We have previously shown that lncRNA GAS5 (Growth Arrest-Specific 5) is

up-regulated in diabetic wounds. We hypothesize that increased GAS5 expression contributes

to prolonged M1 macrophage polarization in diabetic wounds.

Methods:

To test our hypothesis, we incubated the murine macrophage cell line RAW264.7

with media containing 5 mM glucose (low glucose), or 25 mM glucose (high glucose) for 4, 8,

and 24 hours and examined GAS5 gene expression by Real-time PCR. In addition, we

examined macrophage polarization and STAT1 gene expression in response to GAS5

overexpression using plasmid transfection.

Results:

High glucose conditions significantly induced macrophage GAS5 gene expression in a

time dependent manner. Plasmid mediated over-expression of GAS5 resulted in significantly

increased gene expression of M1 macrophage markers (iNOS, TNFa, and IL1-Beta), while

demonstrating no change M2 macrophage markers (Arg1, and Mrc1). Mechanistically, GAS5

overexpression promoted M1 macrophage through significantly induced STAT1 expression, the

crucial transcriptional factor for M1 activation.

Conclusion:

These findings provide the first evidence that the lncRNA GAS5 is involved in the

regulation of macrophage polarization. Furthermore, these results suggest a potential role of

lncRNA GAS5 in the pathogenesis of the diabetic wound healing impairment and may represent

a novel therapeutic target to correct the diabetic wound healing impairment.

79.14 Phenotypic and Genotypic Changes in Metastatic Neuroblastoma

Posted on January 26, 2017

W. E. Barry1, J. R. Jackson1, G. Asuelime1, Z. Wan2, R. C. Seeger2, E. S. Kim1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Pediatrics,Los Angeles, CA, USA

Introduction:  Neuroblastoma is the most common solid tumor in children and despite current multimodal therapies, survival is poor.  While massive primary tumors are a clinical challenge for these children, the main cause of death is recurrent, incurable, metastatic disease following surgical resection.  It is critical to further characterize the biologic differences between the primary tumor and the metastatic cells which appear to respond differently to treatment and are the major cause of mortality in these patients. We hypothesize that cells derived from liver and bone marrow metastases in a murine model will be phenotypically and genotypically different when compared to their parental cell of origin. 

Methods:  Multiple metastatic cell lines were created de novo from the bone marrow and liver of NSG mice utilizing two human neuroblastoma cell lines (CHLA 255, SH-SY5Y). A metastatic neuroblastoma mouse model of minimal residual disease was used whereby the parental cell line was injected into the renal capsule, followed by primary tumor resection 7 days later. The metastases were allowed to grow until euthanasia criteria were met. Metastatic neuroblastoma cells from the liver and bone marrow were harvested and cultured. In vitro studies comparing the parental cell line to the metastatic cell lines were performed. Response to increasing doses of chemotherapy (etoposide) was analyzed using a DIMSCAN cytotoxicity assay. Gene expression was characterized using a low-density metastatic pathway PCR microarray. The PCR-array was performed using the parental and metastatic cell lines from the SH-SY5Y cell line. 

Results: Metastatic cell lines derived from the liver and bone marrow had a significantly lower response to increasing doses of etoposide (p<0.05) compared to the parental cell line, which followed a dose response curve. PCR array revealed numerous genes which were upregulated in the liver and bone marrow cell lines compared to the parental (Figure). Numerous neuroblastoma associated genes appeared to be upregulated including APC, CDH1, CXCL12, FGFR4, MET and NR4A3. These genes play critical roles in cell adhesion, migration, proliferation and survival. Many of the other upregulated genes were also vital to cell migration and adherence including CDH6 and MMP10. 

Conclusion: Metastatic neuroblastoma cells appear to have significantly different gene expression as well as response to standard chemotherapy when compared to the parental cell line. These preliminary data provides the background for further in vitro and in vivo studies to elucidate the effects of treatments particularly on genes specific to metastatic disease in our novel murine model of minimal residual disease.

79.13 Stem Cell Expansion in Spring Mediated Distraction Enterogenesis in a Murine Model

Posted on January 26, 2017

N. Huynh1, J. D. Rouch1, G. Dubrovsky1, A. Scott1, M. G. Martin2, S. Shekherdimian1, J. Dunn1  1University Of California – Los Angeles,Division Of Pediatric Surgery, Department Of Surgery, David Geffen School Of Medicine,Los Angeles, CA, USA 2University Of California – Los Angeles,Division Of Gastroenterology And Nutrition, Department Of Pediatrics, David Geffen School Of Medicine,Los Angeles, CA, USA

Introduction:
Distraction enterogenesis (DE) has been explored as a novel treatment in patients with short bowel syndrome (SBS). Self-expanding springs have been shown to lengthen intestines in animal models. Evidence of epithelial cellular proliferation in lengthened segments is a marker for successful intestinal growth. In this study, we evaluate stem cell growth in intestinal crypts after spring-mediated intestinal lengthening in a murine model.

Methods:
A 10 mm compressed nitinol spring was placed in a 5 mm isolated intestinal segment in mice. A non-compressed nitinol spring placed in a 5 mm isolated intestinal segment served as a control. After 1 to 2 weeks, intestinal segments were examined for size, histological analysis, and stem cell proliferation in intestinal crypts using immunofluorescence for OLFM4 and PCR for mRNA expression of Lgr5.

Results:
Intestinal segments lengthened from 5 to 9.3 ± 1.0 mm, whereas control segments lengthened from 5 to 6.0 ± 0.5 mm (p<0.01). OLFM4 immunostaining showed increased stem cell compartment growth in lengthened and control segments in comparison to normal intestine (Figure), 50.0 ± 9.4 µm and 63.1 ± 15.2 µm? versus 26.4 ± 9.6 µm (p<0.01), respectively. Lgr5 expression was increased in lengthened and control segments in comparison to normal intestine by 9-fold and 10-fold, respectively. Histologically, lengthened and control segments had increased smooth muscle thickness and crypt depth in comparison to normal small intestine.

Conclusion:
Spring-mediated DE in a mouse model led to a significant increase in intestinal length and stem cell expansion in intestinal crypts. Although control segments did not significantly lengthen, the obstruction from intestinal isolation led to an increase in stem cell proliferation. This model can be used to study factors that optimize DE and further suggests that spring-mediated intestinal lengthening is a promising treatment for SBS.
 

79.12 Protective Role of Intestinal HSP70 in Barrier Maintenance and Milk Induction of HSP70.2

Posted on January 26, 2017

R. M. Rentea1, Y. Guo2, X. Zhu3, M. W. Musch3, D. M. Gourlay4, J. L. Liedel5  1Children’s Mercy Hospital- University Of Missouri Kansas City,Pediatric Surgery,Kansas City, MO, USA 2University Of Chicago,Pediatrics, Section Of Neonatology,Chicago, IL, USA 3University Of Chicago,Medicine,Chicago, IL, USA 4Children’s Hospital Of Wisconsin,Pediatric Surgery,Milwaukee, WI, USA 5Albert Einstein College Of Medicine,Pediatrics And Critical Care,Bronx, NY, USA

Introduction:  Necrotizing Enterocolitis (NEC) is a gastrointestinal disease of complex etiology that effects 1 in 10 premature infants. We previously demonstrated that formula feeding inhibits ileal expression of Heat Shock Protein -70 (Hsp70), a critical stress protein within the intestine and that may serve to lower the injury threshold for subsequent stressors. Barrier function for the premature intestine is critical. It is unknown whether decreased neonatal intestinal Hsp70 increases permeability. We sought to determine whether reduced Hsp70 protein expression increases neonatal intestinal permeability. 

Methods:  Young adult mouse colon cells (YAMC) were utilized to evaluate barrier function (translocation of fluorescent dextran) as well as intestine from Hsp70-/- pups (KO). Sections of intestine were analyzed by Western blot, immunohistochemistry, and real time PCR. Statistical data were analyzed by paired students t-test and expressed as a mean +/- SEM with p<0.05 considered significant.

Results: YAMC cells were sub-lethally heated or treated with expressed milk (EM) to induce Hsp70. Immunostaining demonstrates co-localized Hsp70 and tight junction protein Zona Occludens-1 (ZO-1), suggesting physical interaction to protect tight junction function. The permeability of YAMC monolayers increases following oxidant injury and is partially blocked by Hsp70 induction either by prior heat stress or EM (fluorescent dextran-70 flux oxidant injury 12 µg/mL vs. heat shock 1.5 µg/mL p<0.007 or EM 3.0 µg/mL p<0.01). Consistent with previous results in the rat, Hsp70 was detected in the ileum of all mother fed mouse pups on day 3.  RT-PCR analysis demonstrated that the Hsp70 isoforms, 70.1 and 70.3 predominate in WT pup, however, Hsp70.2 predominates in the KO pups via a 3 fold change (p<0.05).   While Hsp70 is present in WT milk, it is not present in KO EM (450 ng/mL vs. 0.10 p<0.001).

Conclusion: Hsp70 associates with ZO-1 to maintain epithelial barrier function. Both induction of Hsp70 and exposure to EM prevent stress-induced increased permeability. Hsp70.2 is present in both WT and KO neonatal intestine suggesting a crucial role in epithelial integrity.  Induction of the Hsp70.2 isoform appears to be mediated by mother’s milk.  These results suggest that mothers milk feeding modulates Hsp70.2 expression and could attenuate injury leading to NEC. 
 

79.10 Chemokine Ligand 5 (CXCL5) Regulates Intestinal Triglyceride Absorption After Small Bowel Resection

Posted on January 26, 2017

B. Aladegbami1, Y. Xie2, J. Guo1, C. Erwin1, N. Davidson2, B. Warner1  1Washington University,Pediatric Surgery,St. Louis, MO, USA 2Washington University,Gastroenterology,St. Louis, MO, USA

Introduction: Following massive small bowel resection (SBR), an important adaptive process occurs that is characterized by increased enterocyte proliferation, increased vessel density and an expanded mucosal surface area. CXCL5 is a proangiogenic chemokine and CXCL5 Knock out mice (CXCL5-KO) undergo normal structural adaptation but have impaired angiogenesis and a perturbed profile of intestinal absorption. The goal of this study was to further characterize the role of CXCL5 in intestinal triglyceride absorption and to understand its effect on body composition and metabolism.

Methods: CXCL5 knockout mice and C57BL/6 wild type (WT) mice were subjected to 50% proximal small bowel resection (N= 10 and 7, respectively).  On post-operative day 14 (POD14) Pluronic F127 was injected via tail vein and mice were then gavaged enterally with mixture of 20 % intralipid, corn oil and radioactive 3[H]-triolein in order to measure triglyceride (TG) absorption. Serum was collected at 1, 2 and 3 hours’ time point and remnant intestine was sectioned into 9 equal segments for analysis. Indirect calorimetry was used to measure metabolic activity and body composition determined by MRI.

Result:  CXCL5-KO mice showed a decreased level of radiation (TG absorption) in the serum at the 1 (CXCL5-KO: 5482.5±1407.1; WT: 15481±4351.6, p =0.03) and 2 (CXCL5-KO: 8964.7±1118.6; WT: 16413±1126.8 p =0.01) hours’ time point.  Total TG increased 57 fold in WT at 3 hours, but only 37 fold in CXCL5-KO (p=0.015).  In addition, CXCL5-KO mice showed a non-significant but higher level of radiolabeled TG in the intestine when compared to WT (Area under the curve CXCL5-KO: 0.35; WT: 0.11, p =0.07 (NS)). These findings are all consistent with a delayed TG absorption profile and a compensatory increase in production by the Liver.

  CXCL5-KO mice had no difference in respiratory exchange ratio, locomotion, and body composition when compared to WT both pre- and postoperatively. However there was a consistent decrease in respiratory exchange ratio, locomotion and lean body mass in all post-surgical mice when compared to their pre-operative levels.

Conclusion: Our results suggest a potential function of CXCL5 and/or angiogenesis in the regulation intestinal triglyceride absorption. However, this effect does not translate to a difference in whole body metabolism and body composition in the early postoperative period. 

79.09 Necrotizing Enterocolitis Is Associated With Changes In Intestinal Epithelial Claudin-2 Expression

Posted on January 26, 2017

G. J. Ares1,2, D. R. Wood1, C. Y. Yuan1, C. J. Hunter1  1Northwestern University,Pediatrics,Chicago, IL, USA 2University Of Illinois At Chicago,General Surgery,Chicago, IL, USA

Introduction:
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in neonates, affecting 5-10% of patients in the neonatal intensive care unit (NICU). NEC is mostly seen in premature infants, but its pathophysiology remains unknown. Tight junction (TJ) proteins are paracellular complexes in intestinal villi important in maintenance of protective intestinal barrier. Claudin-2 (C2) is an intercellular pore forming TJ protein that regulates permeability. We hypothesize that Claudin-2 expression levels are upregulated in human NEC, as well as in an experimental model of bacteria-induced NEC.

Methods:
Caco-2 cells were exposed to LPS in an in vitro model of NEC in a 10 day time course experiment with samples taken on days 0, 1, 3, 5, 7, and 10 along with untreated controls per day. An in vivo model of rat pups subjected to hypoxia twice per day and fed clean formula (control) vs bacteria + formula (NEC). Cells were fragmented into cytosol, membrane, nucleus, and cytoskeleton compartments. C2 was analyzed by immunofluorescence mean fluorescent intensity (MFI), western blot, and quantitative PCR in Caco-2 cells, rat intestinal segments, and human intestines from patients with NEC vs humans without NEC requiring bowel resection. Western blots were standardized to loading controls.  Data was analyzed with student’s T-test.

Results:
Staining showed increased expression of C2 in humans with NEC vs control (MFI=710±24 vs 1530±98 (p<0.0005), respectively) and in rats with experimental NEC vs control (MFI= 514±22 vs 706±21 (p<0.0005), respectively). There appears to be a change in localization of C2 from cytosol to membrane in IF for Caco-2 cells. Western blot and qPCR confirmed 2-fold increase in C2 expression in the cellular NEC model vs control over time, with an increased proportion of C2 in the membrane compartment. In rats, western blot showed increased C2 expression in NEC vs control, and a greater than 2-fold increase on qPCR (p<0.001). Figure 1 shows western blot for C2 in Caco-2 cells exposed to LPS (L) vs control group (C) and the measured immunoblot density of LPS group relative to Control per day.

Conclusion:
In conclusion, human cells and experimental cellular and rat models both showed increased expression of Claudin-2 TJ protein in the intestinal epithelium with NEC. The change in expression of this pore forming protein may play an important role in the breakdown of intestinal barrier integrity in NEC. Changes seen in localization may elucidate the mechanism by which this breakdown occurs. Further research in TJ proteins could help define the pathophysiology of NEC and provide targets for therapy.

79.08 Localization of muscarinic receptors to the crypt stem cell compartment of the murine small intestine

Posted on January 26, 2017

C. J. Greig1, S. J. Armenia1,2, V. R. Klump3, R. A. Cowles1  1Yale School Of Medicine,Department Of Surgery,New Haven, CT, USA 2New Jersey Medical School,Newark, NJ, USA 3Yale School Of Medicine,Department Of Dermatology,New Haven, CT, USA

Introduction:

Maintenance of the highly plastic intestinal epithelium relies upon a small population of stem cells localized to intestinal crypts. These specialized stem cells are responsible for both self-renewal and generation of all mature intestinal epithelial cell types, but the mechanisms by which these processes are controlled remain incompletely understood. Recent evidence suggests that muscarinic acetylcholine (ACh) signaling is involved in epithelial barrier function, epithelial proliferation and inhibition of apoptosis. Furthermore, all muscarinic ACh receptor (mAChR) subtypes have been documented throughout the gastrointestinal tract but the precise location of these receptors within the functional crypt-villus unit is unclear. Thus identification of ACh receptors present in the region of the stem cell niche would allow for design of therapies targeting the muscarinic cholinergic signaling system. We hypothesized that the intestinal crypt base would express only specific mAChRs that drive proliferation in this critical area of the mucosa.

Methods:

With institutional approval, proximal and distal intestinal segments from the jejunum and ileum were procured from wild type C57Bl/6 mice aged 12-14 weeks. Collected segments from each region of the bowel were divided and either formalin fixed and used to create paraffin sections or used for RNA extraction in an RNase-free environment per standard protocols. Paraffin sections on specialized membrane slides were used in capturing cells at the base of the intestinal crypt, performed on a LCM microscope at 100X magnification into RNAlater solution to allow for subsequent RNA extraction without significant degradation. Care was taken to avoid any subepithelial tissue and include only the bottom half of the crypt, where the intestinal stem cells are thought to reside. Using primers targeting the five subtypes of mAChRs (M1-M5), RT-PCR was performed using RNA from full-thickness intestinal and LCM crypt cell (LCM-CC) samples to determine the presence or absence of each subtype.

Results:

In full-thickness intestinal samples, mAChR subtypes M1-M4 were found in all regions of the jejunum and ileum examined. The M5 mAChR subtype was present in the proximal jejunum, but was absent from all other regions of the bowel. For LCM-CC samples, only the M1 mAChR subtype was present and was found in crypt cells from all regions of the intestine examined.

Conclusion:

All subtypes of muscarinic acetylcholine receptors were present throughout the small intestine with the exception of the M5 mAChR, which appears to be localized to the proximal jejunum. However, when cells from the crypt base were collected by laser capture microdissection and analyzed, only the M1 subtype was present by RT-PCR. Given the known location of the stem cell niche to this area, the cholinergic system, possibly via the M1 muscarinic acetylcholine receptor, is likely to be a key mediator of intestinal homeostasis.

79.07 Intestinal Inflammation in Neonates Undergoing Intestinal Resection

Posted on January 26, 2017

S. M. Koehler1, S. Welak2, K. Fredrich1, M. Christensen1, M. Knezevich1, D. Gourlay1  1Children’s Hospital Of Wisconsin,Pediatric Surgery,Milwaukee, WISCONSIN, USA 2Children’s Hospital Of Wisconsin,Neonatology,Milwaukee, WISCONSIN, USA

Introduction: Necrotizing enterocolitis (NEC) is the most common inflammatory intestinal disease in premature neonates and a significant cause of infant mortality. The purpose of this study is to compare levels of intestinal inflammation in neonates with NEC to those with congenital bowel malformations.  We hypothesize that NEC will have high levels of inflammation at the involved portion of the bowel, but areas remote from the disease will be uninflamed similar to intestine resected for congenital malformations. 

Methods: This is an IRB approved prospective study involving discarded tissue from neonates undergoing intestinal resection for NEC, history of NEC, or congenital anomalies. Formalin-fixed sections were utilized to access for histological grade of injury.  Levels mRNA expression of interleukin 6 (IL6), interleukin 1 beta (IL1B), toll-like receptor 4 (TLR4), tissue necrosis factor alpha (TNFα), tissue nonspecific alkaline phosphatase (TNAP), (intestinal alkaline phosphatase (IAP) and NADPH oxidase 1 (NOX1) were evaluated via quantified PCR.  Statistical analysis was performed using a Student’s t-test and a p-value of <0.05 was considered significant. 

Results: Fifteen samples were collected from eight patients at nine operations. Four samples were considered diseased and were from patients with acute NEC near the site of perforation. The remaining samples were considered healthy.  Three were from patients with acute NEC at a site remote from the perforation, three from patients with a history of NEC undergoing stoma reversal, and five from patients with congenital bowel malformations. When comparing mRNA expression of inflammatory and anti-inflammatory markers between intestinal samples from active NEC with the remaining samples, there were significantly higher levels of TNAP (12205 vs. 1270, p=0.009), IL6 (86654 v 24807, p=0.044) and IL1B (133568 vs 14009, p=0.047). Interestingly, some “healthy” samples were highly inflamed and contained ulcerated mucosa. When these tissue samples were compared to those samples with less evidence of histological damage, regardless of diagnosis, there were significantly higher levels of TNAP (10538 vs 1010, p=0.019), IL6 (81377 vs 21260, p=0.036) and TLR4 (12265 vs 4795, p=0.008).

Conclusions: This preliminary data confirms that there are high levels of inflammation in active NEC despite histological grade.  The level of inflammation is similar to samples with mucosal ulceration.  While both groups showed elevation in TNAP and IL6, they did vary in expression of TLR4 and IL1B.  However, the receptor for IL1B shares a signaling domain with toll-like receptors thus this may simply represent an alternative signaling pathway.  Furthermore, while TNAP is not typically considered an inflammatory enzyme, its expression is increased during periods of intestinal inflammation.  Further studies will be necessary to confirm these findings and further evaluate the relative activity of TNAP and IAP. 

 

79.06 Altered Development of Tight Junctions in a Mouse Model of Hirschsprung’s-Associated Enterocolitis

Posted on January 26, 2017

K. P. Kuruvilla1,3, P. K. Shukla2, R. Rao2, F. J. Cailleux1,3, A. Gosain1,3  1University Of Tennessee Health Science Center,Surgery,Memphis, TN, USA 2University Of Tennessee Health Science Center,Physiology,Memphis, TN, USA 3Le Bonheur Children’s Hospital,Children’s Foundation Research Institute,Memphis, TN, USA

Introduction: Hirschsprung’s-associated enterocolitis (HAEC), a common and potentially life-threatening complication of Hirschsprung’s disease, has a multifactorial pathophysiology that includes alterations in the intestinal barrier, impaired gut mucosal immunity and dysbiosis of the intestinal microbiota. Tight junctions form an intercellular barrier between epithelial cells and function to regulate permeability. Failure of tight junctions has been implicated in multiple forms of intestinal inflammation (e.g. inflammatory bowel disease, necrotizing enterocolitis). We sought to determine the role of tight junction proteins in the development of HAEC.

Methods: Following Institutional Animal Care and Use Committee approval, proximal colon (PC) and distal colon (DC) segments were harvested from post-natal day (P) 18 and 22 Wnt1-Cre/EdnrB -/- mice, which model HAEC (EdnrB-null), and control mice (n=4/group/time point). Quantitative real-time PCR (qPCR) was used to compare gene expression of Occludin, ZO-1, Claudin-1, -2 and -3 between EdnrB-nulls and controls. Confocal microscopy was used to analyze tight junction protein (Occludin and ZO-1) expression and localization over time and between groups. Delta-delta threshold cycle methodology was used to compare qPCR results between groups, with >2-fold changes taken as significant. ImageJ was used for comparison of immunohistochemistry data.

Results: At P18, control animals showed robust staining for ZO-1 and Occludin in the DC, indicating well-formed tight junctions, while 2/4 EdnrB-nulls showed minimal staining but robust expression of tight junction genes (Occludin, Claudin-1, Claudin-2), indicating ongoing development. However, in the PC at P18, EdnrB-nulls had well-developed Occludin and ZO-1 by microscopy while staining was minimal in controls. By P22, both EdnrB-nulls and controls had robust staining for Occludin and ZO-1 in the DC, but in the PC, controls continued to demonstrate decreased tight junction staining versus EdnrB-nulls. Gene expression of Occludin, Claudin-1 and Claudin-2 was down-regulated in EdnrB-nulls versus controls at P22.

Conclusion:  Tight junction gene expression and protein localization to the epithelial barrier showed both temporal and regional variability between EdnrB-null animals and controls. EdnrB animals demonstrated earlier development of PC tight junctions and later development of DC tight junctions as compared to controls. Decreased tight junction gene expression at P22 precedes the known onset of HAEC in this model. The mechanisms and functional consequences of temporal and regional variability in tight junctions and implications for the development of HAEC are the subject of ongoing work.

79.05 Metabolic Dysfunction and Caloric Deficiencies are Associated with Neonatal Necrotizing Enterocolitis

Posted on January 26, 2017

T. J. Sinclair1, D. Zhang1,2, B. X. Ling1, H. J. Cohen4, K. G. Sylvester1,3  1Stanford University School Of Medicine,Department Of Surgery,Stanford, CA, USA 2University Of Science And Technology Beijing,School Of Computer And Communication Engineering,Beijing, HAIDIAN, China 3Lucile Packard Children’s Hospital Stanford,Center For Fetal And Maternal Health,Stanford, CA, USA 4Stanford University School Of Medicine,Department Of Pediatrics,Stanford, CA, USA

Introduction:
We hypothesize that prematurity predisposes to metabolic vulnerability, and in combination with clinical exposures such as nutrition, leads to the development of acquired diseases of prematurity including necrotizing enterocolitis (NEC). Routine newborn screening (NBS), that includes measurements of amino acids (AA) and fatty acid metabolism associated acylcarnitines (AC) can be re-purposed to elucidate the metabolic impact of prematurity and clinical care on disease development.

Methods:
We performed a retrospective longitudinal cohort study by querying a multicenter, longitudinal database that included 995 preterm infants (<32 weeks gestation) and 72 cases of NEC. Patient samples were obtained on day of life 1, 7, 28, and 42. Metabolite data from each time point included 72 AA and AC measures. Nutritional data (total calories, enteral feed, and total parenteral nutrition) were collected and average at each of the same time points. Cases of NEC were separated into sub-cohorts based on age of diagnosis: day of life 8-28, 29-42, and > 42. Control subjects were alive at the end of the study and had no history of bowel disease. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for all the NEC cases using the Day 1 analyte level, as well as for each sub-cohort using the analyte levels at each time point prior to the age of NEC diagnosis. Corrections were made for birth weight, gestational age, and multiple hypothesis testing with a false discovery rate of 5%. Loess curves were plotted over time for the significant analytes in each analysis and for the nutritional data. 

Results:
Day 1 analyte levels of phenylalanine (OR 1.6, CI 1.4-2.2), citrulline/arginine ratio (OR 0.4, CI 0.2-0.8), and arginine (OR 1.4, CI 1.1-1.9) were significantly associated with NEC and these differences persisted over time. In the sub-cohort analyses, the number of metabolites that were significantly associated with NEC increased with proximity to the day of diagnosis and included a combination glycine, citrulline, essential amino acids methionine and phenylalanine, and predominantly short chain AC’s. Subjects with NEC were administered significant less enteral feeds and more TPN components over time, but also received significantly lower weight-adjusted total calories (p=2.9×10-4).

Conclusion:
Premature infants that develop NEC demonstrate progressive metabolic dysfunction both at birth and over time compared to controls on serial laboratory sampling obtained prior to diagnosis. The significant analytes involved specific biochemical pathways including homocysteine metabolism, nitric oxide synthesis, the urea cycle, and short chain fatty acid metabolism. The progressive metabolite abnormalities observed in infants that acquire NEC may reflect differences in metabolic function, nutritional content, and caloric deficiencies. 
 

79.04 Decreased Angiogenesis in Slit3Knockout Mice with CDH Results in Reduced Pulmonary Arterial Branching

Posted on January 26, 2017

M. Shah1, M. R. Phillips1, T. Rao1, L. J. Edwards1, Y. Z. Lee1, S. E. McLean1  1University Of North Carolina At Chapel Hill,Chapel Hill, NC, USA

Introduction:
Congenital diaphragmatic hernia (CDH) is a structural birth defect associated with pulmonary hypoplasia and pulmonary arterial hypertension (PAH). Impaired angiogenesis is thought to play a role in the pathophysiology of PAH in CDH. We hypothesize that decreased angiogenesis in Slit3 knockout (KO) mice with CDH and PAH results in attenuated pulmonary arterial (PA) branching patterns.

Methods:
Slit3 wild-type (WT) and KO mice were harvested at 2-weeks, 4-weeks, and 2-3 months (adult) of age. The pulmonary arterial system was perfused with Microfil casting agent and scanned with MicroCT (Scanco). Vascular tree analysis was performed using AMIRA software, based on size and degree of vessel branching (rank) from the main pulmonary artery. Lungs were harvested for RNA isolation. Real-time PCR was performed for angiogenesis markers. Lungs were also lysed in RIPA buffer for protein isolation for use in Western Blot analysis.

Results:
There was a 2-fold decrease in FGF1 expression in KO vs. WT mice at 4-weeks, and a 2-fold decrease in HIF1A and eNOS expression in adult KO vs. WT mice. There was a 28% decrease in eNOS protein levels in KO vs. WT mice based on Western Blot. There was a trend towards fewer number of vessels in 2-week, 4-week, and adult KO vs. WT mice, with a more pronounced difference in adult mice (5404 vs. 7247, p=0.32, 8989 vs. 14229, p=0.12, 11293 vs. 21292 vessels, p=0.06, respectively). Number of ranks were similar in KO vs. WT mice at 2-weeks (61-70 vs. 51-60 ranks), with a trend towards decreased number of ranks in 4-week and adult KO vs. WT mice (61-70 vs. 81-90, 61-70 vs. 91-100 ranks, respectively). There were also fewer vessels per rank in KO vs. WT mice at 2-weeks, 4-weeks, and adult (1185 vs. 1902, 2186 vs. 3675, 2660 vs. 5311, respectively for ranks 31-40, p<0.05). 

Conclusion:
Slit3 KO mice with CDH and PAH have decreased angiogenesis over time with decreased pulmonary arterial branching patterns. Further elucidation of the mechanisms of decreased angiogenesis is needed in order to further understand the mechanisms leading to PAH in CDH.

79.03 Mitochondrial DNA Promotes a Pro-Inflammatory Profile in Intestinal Epithelial Cells

Posted on January 26, 2017

V. Siow1,2, E. A. Novak1, G. J. Vincent1, K. Cunningham1,2, K. P. Mollen1  1Children’s Hospital Of Pittsburgh,Pediatric General And Thoracic Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh,General Surgery,Pittsburgh, PA, USA

Introduction:  The role of mitochondrial dysfunction in the pathogenesis of inflammatory bowel disease (IBD) is just beginning to be understood. Our lab and others have shown evidence of mitochondrial stress and a disruption in mitochondrial function within the intestinal epithelium of humans with IBD as well as mice undergoing experimental colitis. We now hypothesize that mitochondrial stress leads to a release of the damage-associated molecular pattern (DAMP) mitochondrial DNA (mtDNA), which in turn contributes to immune activation and the potentiation of inflammation.

Methods: Serum samples were obtained from patients with severe active IBD and from control subjects according to University and IRB protocol. MtDNA content was determined using qRT-PCR. Mice were subjected to 3% Dextran Sodium Sulfate (DSS) for 7 days. The mitochondria within the intestinal epithelium of control mice vs. DSS-subjected mice were analyzed via electron microscopy. Total DNA was isolated from murine intestinal crypts in control and DSS-subjected mice. This DNA was then utilized to determine the levels of mtDNA relative to nuclear DNA via qPCR and the ΔΔCt method. Intestinal epithelial HT29 cells were treated with mtDNA at 37°C. RNA was then isolated, cDNA synthesized, and qPCR setup to determine the expression of cytokines in control vs. mtDNA-treated cells.

 

Results: For the first time, we show evidence of mtDNA release in the bloodstream of humans with severe active IBD. Mice undergoing experimental colitis exhibit dramatically altered mitochondrial structure as well as a decrease in overall mtDNA within the intestinal epithelium as compared to control mice.  Although total mtDNA content was decreased, there was evidence of free mtDNA within the cytosol of intestinal epithelial cells of mice subjected to experimental colitis. Additionally, HT29 cells treated with mtDNA exhibited an increase in the expression of pro-inflammatory cytokines, including TNF-α as well as TLR9 expression.

Conclusion: Here we investigate the role of mtDNA in intestinal inflammation. Our previous studies demonstrate a role for mitochondrial dysfunction in the pathogenesis of colitis.  However, it remains unclear how this dysfunction leads to inflammatory change. We now demonstrate that the amount of circulating mtDNA in the blood of patients with IBD is increased as compared to healthy control patients, suggesting that a release of mtDNA from dysfunctional mitochondria occurs during disease. We also show that intestinal epithelial cells treated with mtDNA demonstrate an increase in expression of pro-inflammatory cytokines. Our studies suggest that mtDNA may contribute to local and systemic inflammation in patients with IBD. Strategies aimed at limiting mitochondrial dysfunction or sequestering cell-free mtDNA may lead to new therapeutic approaches to combat disease.

 

79.02 The IEC-6 Cell Line as a Model of Muscarinic Signaling Pathways in the Murine Intestinal Epithelium

Posted on January 26, 2017

S. J. Armenia1,2, C. J. Greig1, R. A. Cowles1  1Yale University School Of Medicine,Department Of Surgery,New Haven, CT, USA 2New Jersey Medical School,Newark, NJ, USA

Introduction:  Growth of the intestinal epithelium is critical during intestinal adaptation and after intestinal mucosal injury. While several peptide hormones have been shown to stimulate intestinal mucosal growth, the role of enteric neurotransmitters, such as acetylcholine (ACh), in mucosal homeostasis remains poorly defined. Previous work documenting serotonergic regulation of enterocyte proliferation implicated muscarinic ACh receptors (mAChRs) in the signaling mechanism. The distribution of the five mAChR subtypes on dividing enterocytes has not been reported but may assist in the development of targeted therapies aimed at stimulating intestinal mucosal growth. In order to establish an in vitro model for study of cholinergic signaling in the intestinal mucosa, we aimed to document the presence of mAChRs in cultured enterocytes. We hypothesized that while mAChRs would be widely distributed in the small bowel, the IEC-6 cell line would express enterocyte-specific muscarinic receptors allowing these cells to respond to muscarinic receptor agonists in a well-controlled model system. 

Methods:  The IEC-6 small intestine epithelial cell line was cultured under standard conditions and, with institutional approval, small intestinal segments were obtained from male wild type C57Bl/6 mice between the ages of 12-14 weeks. RNA was extracted from IEC-6 cells and whole intestinal segments in an RNase-free environment per standard protocols. RT-PCR was performed using targeted primers to document the presence or absence of each of the five mAChRs. PCR products were separated by gel electrophoresis and visualized using ethidium bromide staining.  To assess the responses of epithelial cells to agonists, cultured IEC-6 cells were treated with serial dilutions of bethanechol, a muscarinic receptor agonist, for 48 hours. Cellular viability and proliferation were assessed using an MTT assay. Statistical analysis was performed using Student’s t-test, with significance assumed when p<0.05.

Results: In whole intestinal segments, the presence of RNA from all five mAChRs was demonstrated by RT-PCR. In cultured IEC-6 cells, RNA for two of the five mAChR subtypes (M1 and M4) was amplified by RT-PCR. Treatment of IEC-6 cells with bethanechol stimulated cellular proliferation in a dose-dependent manner. 

Conclusion: Muscarinic ACh receptors are widely distributed in the small intestine. While all mAChR subtypes can be documented in whole small intestinal segments, only M1 and M4 mAChRs appear to be expressed in IEC-6 cells. The ability of bethanechol to stimulate IEC-6 cell proliferation suggests that ACh may play an important role in enterocyte proliferation in-vivo. The specific mAChR responsible for this action and the subsequent intracellular signaling pathways warrant further study of this model. 

 

79.01 FAK Inhibition Abrogates Neuroblastoma Cancer Stem Cell Phenotype in Patient-Derived Xenografts

Posted on January 26, 2017

L. Stafman1, E. Garner1, A. Hjelmeland1, J. Stewart1, S. Mruthyrunjayappa1, K. Yoon1, S. Cramer1, E. Beierle1  1University Of Alabama,Birmingham, Alabama, USA

Introduction:

Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Focal adhesion kinase (FAK) is a tyrosine kinase affecting proliferation, adhesion, and migration, and is prominent in aggressive forms of NB. FAK inhibition in immortalized human NB cell lines has been shown to decrease NB tumorigenicity. Immortalized cell lines do not always recapitulate the human condition, so we have adopted the use of patient-derived xenografts (PDXs), which are tumors implanted into immunosuppressed mice from human patients.  Cancer stem cells are a subpopulation of cells with stem cell-like properties. NB cancer stem cells can be identified by expression of cell surface proteins, including CD133, and the ability to grow as spheres in vitro. We hypothesized that FAK inhibition would decrease tumorigenicity and cancer stem cell maintenance in human NB PDXs.

Methods:
Cells from two human NB PDXs (COA3, COA6) were treated with the FAK inhibitors 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) or PF-573,228 (PF). FAK expression and phosphorylation was determined with immunoblotting. Viability and proliferation were assessed with alamarBlue and CellTiter 96 assays, respectively. Expression of the cell surface protein and stem cell marker, CD133, was determined using flow cytometry after 24 hours of treatment with PF or Y15. The ability of the NB PDXs to grow as spheres was assessed using an in vitro limiting dilution analysis. Student’s t-test, extreme limiting dilution analysis, and χ2 statistics were used. Data reported as mean ± SEM with p<0.05 significant.

Results:
FAK expression and phosphorylation was inhibited in both COA3 and COA6 cells following treatment with PF or Y15.  FAK inhibition with the small molecules (PF or Y15) significantly decreased viability and proliferation in a dose dependent fashion in both NB PDXs. Additionally, PF or Y15 treatment yielded a significant decrease in the expression of the cancer stem cell marker, CD133 (18.5% ± 0.2 untreated cells vs. 11% ± 0.3 and 5.3% ± 0.1, PF or Y15, respectively, p< 0.02). FAK inhibition with PF or Y15 also significantly decreased sphere formation in both human NB PDXs in culture (Figure), indicating a decrease in cell “stemness”. 

Conclusion:
Inhibition of FAK with small molecule inhibitors decreased viability, proliferation, and cancer stem cell maintenance in human NB PDXs. Multiple clinical trials examining the safety and efficacy of FAK inhibitors are currently ongoing, although none have yet been undertaken in children or patients with NB. Our data indicate that FAK inhibitors warrant further exploration as a novel therapy for NB.
 

78.20 Flow Based Human Dendritic Cell Dengue Virus Neutralization Assay

Posted on January 26, 2017

S. L. Keith1, N. Graham2, E. Sendra2, B. Kirkpatrick2, J. E. Boyson1  2University Of Vermont College Of Medicine / Fletcher Allen Health Care,Department Of Medicine,Burlington, VT, USA 1University Of Vermont College Of Medicine / Fletcher Allen Health Care,Department Of General Surgery,Burlington, VT, USA

Introduction:  Over 40% of the world’s population lives in dengue virus (DENV) endemic areas.  Therefore, the development of a safe and efficacious dengue virus vaccine is a global health priority. Althought the first dengue vaccine was recently licensed in 3 countries, it has only a 30% efficacy against some DENV serotypes in DENV-endemic areas. Interestingly, this low efficacy was not predicted during pre-clinical testing. The reasons underlying these discrepant results are unclear. One of the primary correlates of vaccine efficacy is the ability to elicit neutralizing antibodies. The standard assay in the field is a plaque reduction neutralizing titer 50% (PRNT50) assay that assesses the ability of a vaccinee’s serum to neutralize virus infection of Vero cells, which do not have the same attachment factors and receptors as human cells. This raises the question of whether the currently used PRNT50 assay is the best method for neutralizing antibody determination.

Methods:  The purpose of this study was to develop a neutralization assay using primary human cells and to determine whether this assay would yield comparable results to the standard PRNT50 assay. Human CD14+ monocytes were enriched from peripheral blood mononuclear cells. Monocytes were then differentiated into immature dendritic cells (immDC) using GM-CSF and IL-4 for use in a neutralization assay.  Antisera with known PRNT50 values for DENV1 were obtained from volunteers vaccinated with the NIH DENV TV005 vaccine (UVM Vaccine Testing Center) and incubated with DENV1 prior to adding to human immDCs.  Cells were then harvested and DENV infection was assessed using flow cytometry.  An inhibition curve (IC50) was calculated and compared to the PRNT50 value.

Results: Preliminary data indicate patient to patient variability in the level of DENV infection of monocytes. A comparison of Vero cell PRNT50 and human immDC IC50 values using DENV serotypes 1 and 4 suggests no significant difference in neutralizing antibody titers.

Conclusion: Our data indicate that the standard PRNT50 assay is suitable for testing DENV vaccinine response, at least for serotypes 1 and 4. Serotypes 2 and 3 have yet to be tested. This assay may be important in evaluating DENV candidate vaccines in the future.?

 

78.19 HDAC6 Inhibition Increases Macrophage and Innate Immune Cell Proportions in Spleen of Septic Mouse

Posted on January 26, 2017

T. Zhao1, B. Liu1, B. Pan1, X. Cheng1, E. Chen1, Y. Li1, H. B. Alam1  1University Of Michigan Hospital,General Surgery/ Surgery/Medical School,Ann Arbor, MI, USA

Introduction: We have previously demonstrated that Tubastatin A (Tub A), a selective inhibitor of histone deacetylase 6 (HDAC6), improved survival, and increased phagocytosis of splenocytes in a mouse model of lethal cecal ligation and puncture (CLP) induced septic shock. The present study was designed to test the hypothesis that inhibition of HDAC6 with Tub A may affect the spleen cell population.

Methods: C57BL/6J mice were subjected to CLP, and 1 h later given an intraperitoneal injection of either Tub A (70 mg/kg) dissolved in dimethyl sulfoxide (DMSO), or DMSO only (n=7/group). Sham-operated animals were treated in an identical fashion, without CLP. Forty-eight hours later, spleen was harvested and homogenized into single cell suspension. Erythrocytes were lysed, and single-cell suspension was made for analysis. The splenocytes were washed, blocked with anti-mouse CD16/32, stained with anti-mouse B220 PE-Cy7, CD3 APC-eFluor® 780, CD11b FITC, Gr-1 PerCP-Cy5.5 and F4/80 Antigen APC, and subjected to flow cytometry. Data was acquired on an LSRII Flow Cytometer (BD Biosciences) and analyzed with FlowJo (FlowJo, LLC). Analysis of variance (ANOVA) with the Bonferroni post-hoc test was used for multiple comparisons among 3 groups.

Results: In spleen at 48 h after CLP, it was found that there were no statistical differences in percentages of neutrophils (CD11b+ Gr-1+) and T lymphocytes (CD3+) among three groups. The percentages of innate immune cells and macrophages were not statistically different between sham and vehicle-treated CLP groups. However, there were statistical differences in percentages of innate immune cells (CD11b+; p=0.0444), macrophages (CD11b+ F4/80+; p=0.0019), and B lymphocytes (B220+; p=0.0125) among three groups. Treatment with Tub A significantly increased percentages of innate immune cells (10.8±0.4 vs.6.2±1.3%, p<0.05) and macrophages (7.0±1.1 vs. 2.9±0.3%, p<0.01; Figure 1) as compared to vehicle-treated CLP group. Animals from the vehicle-treated CLP group showed decreased percentage of B lymphocytes compared to sham group (59.1±2.9 vs. 44.3±2.6%, p<0.05).

Conclusion: Selective inhibition of HDAC6 significantly increased macrophage and innate immune cell proportions in spleen in the lethal septic model. These results may explain the beneficial effects of Tub A treatment in a septic model as previously reported.

 

78.18 Alterations In The Ceramide-Sphingosine Pathway During Active Urinary Tract Infections.

Posted on January 26, 2017

G. E. Martin1, R. Boudreau1, C. Couch1, M. Edwards1, G. Erich1,2, P. Jernigan1, A. Mahdy1, A. Seitz1  1University Of Cincinnati,College Of Medicine, Department Of Surgery,Cincinnati, OH, USA 2University Of Duisburg-Essen,Department Of Molecular Biology,Essen, NORTH RHINE-WESTPHALIA, Germany

Introduction: Recurrent or “chronic” urinary tract infections (UTIs) constitute a large health burden among women in the United States, with more than one quarter of young women developing recurrent UTIs despite adequate treatment of the initial organism. Pathogenesis of recurrent UTIs is poorly understood and requires a better understanding of the urogenital tract’s innate immunity. Previous research by our group has shown that sphingosine plays an important role in the innate immunity of the lung by prevention of bacterial invasion. Other groups have shown a similar role in the skin, oropharynx and gingiva.  Given sphingosine’s role in the skin and respiratory tract, we hypothesized that it is also an important part of innate immunity in the bladder, and uroepithelial cells obtained from human patients with UTIs would have decreased levels of sphingosine and increased levels of ceramide relative to uninfected patients. 

Methods: Samples were obtained from patients with confirmed recurrent UTIs (infected group) and those without recurrent UTIs (control group). Uroepithelial cells were isolated from these samples and stained with anti-sphingosine, anti-ceramide, and anti-acid ceramidase antibodies. Images were obtained by scanning laser confocal microscopy and analyzed using the image analysis software Imaris. Cumulative fluorescence per cell was quantified and averages obtained for infected and uninfected patients.   Cells were then stained for ceramide and acid ceramidase and also underwent analysis.

Results: Decreased sphingosine staining was noted in uroepithelial cells from the infected group with subsequent quantitative fluorescent analysis of these samples showing a 41% decrease in the fluorescence present in the infected urine samples.  Uroepithelial cells from the infected group showed an increase in the intensity of ceramide staining and decreased acid ceramidase staining compared to the non-infected group.

Conclusion: This work provides evidence that the ceramide-sphingosine lipid-rheostat is altered during an active human urinary tract infection.  We have shown that uroepithelial cells have decreased concentrations of sphingosine and acid ceramidase and increased concentrations of ceramide upon active infection. This pathway could provide a target for novel therapies for recurrent UTIs and potentially decrease the burden of UTIs associated with antibiotic-resistant bacteria.

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