87.19 Square Knots vs. One Handed Sliding Crossed Knots in #1 Polydioxanone (PDS) Suture

Posted on January 31, 2019

B. Slatnick1, E. Day1, S. Asghar1, V. Y. Dombrovskiy1, T. Davidov1  1Rutgers, Robert Wood Johnson Medical School,General Surgery,New Brunswick, NJ, USA

Introduction:

Surgeon preference dominates choice on number of knots and type of suture material for surgical closures.  For abdominal wall closures, #1 Polydioxanone suture (PDS) is recommended due its high tensile strength and absorbable nature. While “square knots” on monofilament suture are anecdotally known to be stronger than sliding knots, this is not well quantified. Furthermore, the optimal number and type of knots with PDS suture for abdominal wall closure is poorly defined. Our previous study shows that addition of one-handed sliding crossed knots with PDS suture decreases knot failure rate in a linear fashion. However, even with 12 knots there was a 10% failure rate. This study seeks to determine the mechanical properties of square knots (or two handed knots) on PDS and define the optimal number of knots required to prevent knot failure.

Methods:

Square crossed knots were tied with 4, 6, or 8 throws using #1 PDS Suture. A single surgeon hand tied a total of 90 samples in randomized order: 30 samples for each group over 2 sessions to prevent fatigue. Using a tensilometer, knots were tested to the point of knot failure—defined as the knot untying before the suture breaks. The peak force in Newtons (N) was recorded at knot failure. We then compared knot failure rates and peak forces between this model and our previous study. Chi squared analysis, t-test, and general linear modeling was used for intergroup comparisons.

Results:

Square knots had decreased knot failure rates compared to one handed sliding crossed knots with the same number of throws. Square knots with 4, 6, and 8 throws had knot failure rates of 90, 0 and 0% respectively. There was no difference in knot failure rates between 4 one handed sliding crossed knots and 4 square knots (P= 0.45); however, with 6 or 8 square knots there were no failures compared to 70% and 43% for one handed sliding crossed knots respectively. At 4 knots, 52 N were required on average for knot failure in the one-handed sliding crossed group vs. 70 N for square knots.

Conclusion:

Square knots are superior to one-handed sliding crossed knots using #1 PDS. Square knots with 6 throws are the optimal number to prevent knot failure.

87.18 Endovascular Occlusion Model for Retrohepatic Inferior Vena Cava Injuries in Pediatric Trauma

Posted on January 31, 2019

L. Carrillo1, M. Skibber1, A. Kumar1, C. Cox1  1McGovern Medical School at UTHealth,Department Of Pediatric Surgery,Houston, TX, USA

Introduction: The Resuscitative Endovascular Balloon Occlusion (REBOA) device has been adapted for Inferior Vena Cava (IVC) use as an adjunct to control massive hemorrhage in both animal models and human cases. While rare, IVC injuries in unstable pediatric patients have been associated with a 67% mortality rate.

Methods:  A simulated venous system was designed with modeled IVC components and a 1L/min outflow. These IVC segments were derived from measurements of 300 pediatric trauma computer tomography (CT) scans and reflect post and prehepatic diameters for the five largest Broselow categories. A REBOA device was inserted into the system with the balloon placed in the IVC model. Pressure monitors were placed distally and on a tank replicating the capacitance of the venous system. A Flow meter was placed distally to the segment and balloon. Flow and pressure readings were recorded as the REBOA device was inflated at .2cc/second.

Results: With the low flow of the venous system approximately 80-90% of the inflation volume occurs with the first 10% of occlusion. This reduction in flow corresponded with an increased pressure difference, reflecting the increased volume in the venous system.

Conclusion: Pediatric IVC injuries with significant hemorrhage may be amenable to endovascular occlusion as an adjunct to resuscitation and operative management

 

87.17 Effects of Immunologic Breast Milk Components on Intestinal Epithelial Cell Viability and Protection

Posted on January 31, 2019

J. Ayivor2, B. Sims1, K. Brawner1, C. Martin1  1University Of Alabama at Birmingham,Department Of Pediatric Surgery,Birmingham, Alabama, USA 2Oakwood University,Department Of Biological Sciences,Huntsville, AL, USA

Introduction:  Necrotizing Enterocolitis, (NEC) is the leading cause of intestinal morbidity and mortality in premature infants, characterized by epithelial cell injury and sepsis.  Breast milk has been shown to decrease the occurrence of NEC; the exact mechanisms that facilitate this protective process are not clear. Human Milk Oligosaccharides, (HMOs) are complex non-digestible pre-biotic sugars that have been shown to stimulate protective immune responses and beneficial growth of intestinal flora. Exosomes are cell derived proteins found in breast milk known to regulate intracellular signaling, inflammation, and immune responses. The objective of this study was to assess the impact of exosomes and human milk oligosaccharides compared to milk ultrafiltrate on the protection of intestinal epithelial cells. Rat intestinal epithelial cells (IEC-6) were used for experimentation.

Methods:  Upon reaching confluence, the IEC-6 were pre-treated for 2 hours with 0.1 µg/ml, 1µg/ml or 10 µg/ml of exosomes, 1µg/ml, 10 µg/ml HMOs, or a 5 µg/ml dose of breast milk ultra-filtrate. Following pretreatment, cells were injured with H2O2 for 2 hours. Cell viability was assessed through trypan blue staining.

Results: An Ordinary ANOVA was used to determine significance.  Exosomes and HMOs were found to be protective against cell injury with the p value <0.001. 

Conclusion: Ultrafiltrate was not protective against cell injury.  It was determined that breast milk is immunologically active and protective against epithelial cell injury. Exosomes and HMOs improved cell viability; breast milk ultrafiltrate did not. This suggests that isolated and concentrated breast milk components may have an added therapeutic benefit. Future studies will further clarify these mechanisms of protection.

 

87.16 Toll-like Receptor Expression in Lung, Liver, and Ileum in a Rat Model of Necrotizing Enterocolitis

Posted on January 31, 2019

J. E. Schucht1,3, B. M. Ebenschweiger1, P. J. Matheson1,2,3, J. W. Smith1,2,3, C. D. Downard1,2  1University of Louisville,Department Of Surgery,Louisville, KENTUCKY, USA 2Robley Rex Veterans Affairs Medical Center,Research And Development,Louisville, KY, USA 3University of Louivsville,Department Of Physiology And Biophysicsj,Louisville, KENTUCKY, USA

Introductions: Necrotizing enterocolitis (NEC) is a surgical emergency in premature infants, and the etiology is not fully understood.  It is known that NEC etiology is associated with formula feeding, hypothermia, hypoxia, and maternal smoking. In NEC, altered cytokine and growth factor expression, and impaired intestinal blood flow lead to intestinal necrosis.  Toll-like receptors (TLRs) are gateway molecules to the innate immune system, and TLRs are activated by various pathogen-associated molecular patterns (PAMPs).  We hypothesized that TLR expressions will be enhanced in a rat model of NEC compared to controls. 

Methods: Sprague- Dawley rats were randomized by litter to undergo NEC induction via a known experimental NEC protocol and control group. NEC pups were delivered via C-section and underwent a 48 hour protocol. Control pups were delivered vaginally and dam-fed. At 48 hours, lung, liver, and ileum samples were collected from both groups. ELISA was performed on tissue homogenates for TLR2, TLR3, TLR4, TLR7, and TLR9 according to manufacturers’ directions. Statistical analysis was performed via 2-way analysis of variance (ANOVA) and post-hoc Tukey-Kramer HSD test.

Methods: Thirty-four (34) rat pups (n=15 in Control and n=19 in NEC) each had lung, liver, and ileum samples taken for a total of 102 samples. In lung tissue, TLR4 and TLR7 levels were elevated in the NEC group vs. Controls (P < 0.05), while TLR3 showed a trend toward elevation in NEC (P = 0.09). TLR2 and TLR9 levels in the lung were not statistically different. In liver, TLR2, TLR3, TLR7, and TLR9 were increased in NEC vs. Control (P < 0.05). In the ileum, TLR7 was statistically elevated in NEC vs. Control.

Conclusions: Overall, the NEC group showed a general trend of elevation of TLR protein expression at 48 hrs after the start of the NEC model compared to time matched Controls in lung and liver, however in ileum only TLR7 was significantly increased in NEC groups at 48 hrs.  In spite of prior work suggesting that TLR2 and TLR4 play key roles in NEC development, we did not find increased TLR2 and TLR4 in this study.  Examination of other time points in this NEC protocol are warranted to examine the TLR expression patterns over time in these organs. 

87.15 In vivo rat evaluation of novel PLA/PCL polymeric patch for potential spina bifida coverage

Posted on January 31, 2019

M. Oria1, R. Tatu2, C. Lin2, J. Peiro1  1Cincinnati Children’s Hospital Medical Center,Pediatric Surgery,Cincinnati, OH, USA 2University Of Cincinnati,Department Of Biomedical Engineering,Cincinnati, OH, USA

Introduction:
Current therapeutic strategies for spina bifida repair showed a limited number of options in the market and any of them have all the requirements as the perfect patch. In fact, sometimes the surgical procedures become really challenge using different patches to fully cover the spina bifida lesion. For this purpose, the blend films of poly (lactic acid) and poly (ε-caprolactone) was designed and validated in vitro to cover all these requirements but was never tested in vivo.

Methods:
Blend films of poly (L-lactic acid) (PLA) and poly (ε-caprolactone) (PCL) were fabricated by solvent casting the blend consisted 83% PLA and 17% PCL in chloroform as solvent. The blend solution poured in Teflon mold and dried for 24h to attain films. A total of 26 adult Sprague-Dawley rats weighing 200-250 gwere used in this project. 2 sets of experiments were performed A) Subcutaneous implantation of the patch: 1 cm x 1 cm blend patch was surgically implanted under the skin the back of the animals during 2 weeks till tissue and patch were harvested for histological analysis and B) Dural implantation of the patch: A rat laminectomy model was used to determine the effects of PLA-PLC as a dural substitute. Once a week after surgery and during for 4 weeks an electrophysiological test was assessed using six needle subcutaneous electrodes to assess the motor function in the rats. For the recording of the evoked potentials to assess the function of the motor tract we recorded motor evoked potentials (MEP) and compound motor action potential (CMAP) as a control. After 4 weeks tissue was harvested for histological analysis. 

Results:
The blend patch was analyzed in terms of rejection from the animal, inflammation reaction and functional interaction with spinal cord tissue in rat animal models. No evidence of adverse or inflammatory reactions was observed in both models of subcutaneous implantation, neither as a dural substitute. No signs of astrogliosis in the neural tissue was observed and no functional alteration of the motor evoked potentials after 4 weeks of implantation as dural substitute in the rat spinal cord.

Conclusion:
The results of the present study suggest that PLA-PLCpatch are the ideal form factor for a minimally-invasive application such as fetoscopy and will serve as our SB repair patch. Blended patches when are in contact with the spinal cord as a dural substitute will not induce any adverse effect such as scar formation or tethering cord and functions of the spinal cord. These findings encourage further investigation of this patch with the possibility for future design of cellular or/and pharmacological treatment bipolar combination.
 

87.14 Modeling High-Risk Hepatoblastoma

Posted on January 31, 2019

A. M. Ibarra1,2, S. E. Woodfield1,2, R. H. Patel1,2, S. F. Sarabia1,2, K. B. Ghaghada1,2, D. Lopez-Terrada1,2, S. A. Vasudevan1,2  1Baylor College Of Medicine,Surgery/Pediatrics,Houston, TX, USA 2Texas Children’s Hospital,Surgery/Pediatrics,Houston, TX, USA

Introduction:
Hepatoblastoma (HB) is the most common pediatric liver malignancy with most patients under the age of five. Patients have an overall survival (OS) rate of 80% at five years after diagnosis; however patients with high-risk, metastatic disease have a five-year OS of only about 40%. There are currently no cell line xenograft models of aggressive, high-risk HB available for preclinical testing.

Methods:
Two million HepT1 cells were orthotopically injected into the livers of immunocompromised mice (NSG) to generate intrahepatic tumors. Growth of tumors in vivo was monitored with ELISA for levels of serum human α-fetoprotein (AFP), MRI, and bioluminescent imaging (BLI) as the HepT1 cell line was transduced with luciferin and emitted a signal with intraperitoneal injection of luciferase. We also analyzed cell line DNA for the described mutations, CTNNB1 (aa5-80 (exon 3)) and NFE2L2 (c.89T>C). 

Results:
We injected 9 mice with HepT1 cells that were confirmed to have the CTNNB1 and the aggressive NFE2L2 mutations. Two out of 9 mice (22%) grew tumor within the first 2 weeks after implantation, and after 4 weeks, we had 100% take of HepT1 tumors in animals. Serum human AFP increased in the animals with the growth of tumors. Most importantly, we saw clear presence of vena caval tumor thrombus, extrahepatic disease including periportal lymph nodes, and lung metastasis in 75% of animals harboring HepT1 tumors.  

Conclusion:

Building on our previous work developing orthotopic xenograft mouse models of HB with the widely available HepG2 and Huh-6 cell lines (Woodfield et al., 2017), we developed a more aggressive orthotopic xenograft mouse model of HB with intrahepatic injection of HepT1 cells. The tumors generated with this cell line were found to invade major blood vessels and metastasize, unlike other previously described cell line models of HB. This model will facilitate pre-clinical studies of aggressive HB. 

87.13 Tgf-β Suppression with Wnt Activation Increases Intestinal Stem Cell Proliferation in Vitro

Posted on January 31, 2019

S. M. Zuber1, G. Levin1, C. R. Schlieve1, A. I. Squillaro1, K. L. Fowler1, L. A. Nucho1, T. C. Grikscheit1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:
Intestine organoid units (OU), multicellular epithelial and mesenchymal cell clusters, include stem and progenitor cells and can be cultured long-term in a reduced-factor medium with subsequent generation of tissue-engineered small intestine (TESI) after implantation in vivo. Application of Y-27632 (Rho-Kinase Inhibitor), A-83-01 (Tgf-β Inhibitor), and CHIR99021 (GSK3 Inhibitor) (YAC) small molecules to primary hepatocytes is known to convert terminally differentiated cells to bipotent liver progenitors. Tgf-β suppression and Wnt activation also induce intestinal stem cell proliferation. We therefore hypothesized that addition of YAC inhibitors might increase the proliferation and size of cultured OU to increase TESI yields for future human therapies.

Methods:
Small intestine from 2-week old C57/BL6 mice was harvested to generate OU that were maintained on reduced-factor Matrigel for 5 days (n=6). OU were cultured in DMEM supplemented with 10% fetal bovine serum, non-essential amino acids, and antibiotics in either the presence or absence of YAC small molecules refreshed every two days. Eight random fields in each well were imaged to assess the number and average area of OU (ImageJ software) and evaluated by ANOVA (GraphPad Prism 7). OU were collected on Day 5 either for whole mount or in agarose embedded in paraffin, then sectioned at 8 µm thickness. OU were co-stained for E-cadherin (epithelial marker) and Ki-67 (proliferation marker) for immunofluorescence. Paraffin-embedded sections were imaged with fluorescence microscopy and whole mount fixed OU were imaged with confocal microscopy. For each condition, OU containing a lumen were randomly imaged. The ratio of Ki-67 positive cells and total cells stained with E-Cadherin was measured and evaluated by paired t-test.  

Results:
On day 5, OU treated with YAC were significantly larger than OU cultured in control media (49928 µm2 vs 9529 µm2, p=0.0036) (Figure 1A and 1B). While there was initially a significant increase in number of OU in each well with YAC application after day 1 (170.6 vs 264.5, p<0.0001), there was no significant difference in OU number on Day 5 (p=0.07). There was an increase in intestinal cell proliferation as seen by the significantly higher ratio of Ki-67 positive cells over total cells stained with E-cadherin in our YAC group (0.488 vs 0.124, p=0.0248) (Figure 1C).

Conclusion:

While there is not an increase in the overall number of OU, the addition of YAC small molecules in culture increases both the size and proliferation index of intestine organoid units. Ongoing investigation will determine if this approach increases the yield of resulting tissue-engineered small intestine growth.

87.12 Identification of suitable reference gene for qPCR analysis in pediatric inflammatory bowel disease

Posted on January 31, 2019

C. Buonpane1,2, G. Ares1,2,4, B. Benyamen1, C. Yuan3, D. Wood3, C. J. Hunter1,2,3  1Ann & Robert H. Lurie Children’s Hospital,Pediatric Surgery,Chicago, IL, USA 2Feinberg School Of Medicine – Northwestern University,Surgery,Chicago, IL, USA 3Northwestern University,Pediatrics,Chicago, IL, USA 4University Of Illinois At Chicago,Surgery,Chicago, IL, USA

Introduction: Pediatric inflammatory bowel disease (IBD) accounts for 10-15% of IBD cases and is associated with considerable morbidity for patients. Dysregulated microRNAs (miRNA), small non coding RNA molecules that modulate gene expression, have been the target of research in IBD diagnosis, surveillance and therapy. Proper selection of reference genes, which are a prerequisite for accurate measurement of miRNA expression, are currently lacking. We hypothesize that different reference genes should be used for various tissue and disease types. This study aims to identify suitable reference genes for the study of pediatric intestinal tissue in IBD. 

Methods: Five candidate reference genes (miR-16, 193a, 27a, 103a, 191) were selected based on their previous use as standards in colorectal cancer. Expression levels of the reference genes were analyzed by RT-qPCR in 28 pediatric intestinal samples (13 IBD, 15 control). Criteria used for designation of suitable reference genes: (1) ubiquitous- present in all tissue samples with quantification cycle (Cq) value 15-35 (2) uniform expression- no differential expression between control and disease samples (p>0.05) (3) stability- stability value (SV) <0.5 by NormFinder. The effects of normalizers on the expression of target miRNA-874 was also assessed.

Results:

Patients included had a mean age of 10 ± 5 years, 72% were male, 29% had Crohn’s Disease, 18% had Ulcerative Colitis and 53% were controls. For the study of Crohn’s disease in small bowel samples, we found four miRNAs that met criteria (miR-16, miR-193a, miR-27a, miR-191). The best combination was miR-27a/191 (Cq 19.11-28.87, p=0.052, SV=0.147). None of the candidate reference genes met criteria for use in the study of colonic tissue in Crohn’s Disease. For ulcerative colitis, miR-16, miR-27a and miR-191 met criteria. The best combination was miR16/27a (Cq 18.53-22.45, p=0.556, SV=0.178).

The effects of reference gene selection on target miR-874 expression were significant (see Figure). MiR-874 expression was decreased in Crohn’s disease small bowel compared to controls (miR-103a as reference, p<0.0001; miR-27a/191 as reference, p=0.002). With miR-193a as reference, miR-874 expression was significantly higher in ulcerative colitis samples than controls (p=0.03); however, with miR-16/27a as reference, miR-874 expression was not different between disease and control samples (p=0.5).

Conclusion:Our results demonstrate that reference gene choice for qPCR analysis has a significant effect on study results and that proper data normalization is imperative.  We recommend miR-27a/191 for the study of small bowel in Crohn’s Disease and miR-16/27a for the analysis of ulcerative colitis samples.
 

87.11 Underdeveloped Intestinal Epithelial Mitochondria Contributes To Metabolic Injury In Newborn Gut

Posted on January 31, 2019

K. Wang1,2, Z. Sun1, G. Tao1, P. Lin1, J. Dunn1, K. Sylvester1  1Stanford University,Department Of Surgery, School Of Medicine,Palo Alto, CA, USA 2First Hospital of China Medical University,Gastrointestinal Surgery,Shenyang, LIAONING, China

Introduction:

Necrotizing Enterocolitis (NEC) occurs in preterm newborns following gut colonization and the introduction of enteral feeding. Newborn enteral feeding produces multiple short-chain fatty acids (SCFAs) that are absorbed (propionate, acetate) for systemic utilization or metabolized (butyrate) in the gut. Luminal production of SCFAs is essential for normal gut mucosal function, however we have previously noted a developmental stage sensitivity to butyric acid (BA). We hypothesized that enteric utilization of BA would be coupled to mitochondrial oxidative capacity and oxidative stress buffering according to developmental stage of the host.

Methods:

Human intestinal epithelial stem cells (hIESCs) and their differentiated enterocyte progeny (withdrawal of Wnt agonists) were subject to a titrated dose of NaBu (sodium butyrate) for a determination of cell sensitivity. Following treatment with 10mM NaBu for 24h, LDH release assay was used to detect cellular death and Western blot analyses of cellular lysates for metabolic driver proteins. An in vivo model of SCFA induced metabolic challenge was developed through  oral (300mM, 20μl/g body weight twice a day for 3 days) administration to 14d and 28d day mice to represent preterm and term newborns respectively. After 72 hours, mouse stool was assayed for enterocyte specific keratin proteins, markers of intestinal epithelial injury. Mitochondria from 14d and 28d mice were isolated for a determination of mitochondrial Oxidative capacity the oxygen consumption and mitochondrial membrane permeability transition (MPT) detection.

Results:

Following treatment with 10mM NaBu for 24h, hIESCs and differentiated enterocytes from a preterm human newborn demonstrated a significant increase in sensitivity compared to a term human newborn by LDH release assay (0.20±0.03 vs 0.08±0.07,p=0.07, hIESCs; 0.34±0.14 vs 0.05±0.03,p=0.03, differentiated cells). AKT and PGC-1α were significantly downregulated by NaBu in both groups. After 72 hours of NaBu exposure, Keratin protein content in the 14d mouse stool significantly increased compared to the untreated group, suggesting a significant increase in intestinal epithelial cell injury. In functional studies, ADP-induced oxygen consumption was found to be significantly lower in the isolated mitochondria from the gut of 14d mice compared to 28d mice (39.8±12.3 vs 143.84±11.3 pmol/s*ml, p=0.015). MPT assay indicated that mitochondrial swelling and membrane permeability were significantly greater in 14d mice compared to 28d mice under identical conditions.

Conclusion:

Premature developmental stage of enterocytes in both human and mouse is associated with increased sensitivity to NaBu exposure. Mitochondrial capacity for oxidative phosphorylation and redox buffering appears to be deficient in less mature enterocytes. These observations are consistent with age dependent metabolic injury pattern that may be applicable to human NEC.?

87.10 Use of Mesenchymal Stem Cells and Nanosilk to Bioengineer a Patch for Wound Healing and Fetal Interventions

Posted on January 31, 2019

S. A. Hilton1, L. C. Dewberry1, C. Zgheib1, M. M. Hodges1, E. J. Burtch2, J. Jacot2, P. Rozance3, S. Seal4, K. W. Liechty1  1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2University Of Colorado Denver,Department Of Bioengineering,Aurora, CO, USA 3University Of Colorado Denver,Department Of Pediatrics,Aurora, CO, USA 4University of Central Florida,Department of Material Science Engineering, AMPAC And NSTC Center,Orlando, FL, USA

Introduction

Spina bifida encompasses a range of caudal neural tube defects affecting 1 in every 2000 live births in the United States, the most severe being myelomeningocele where the spinal cord herniates out of the back. Amniotic fluid stem cells are an easily obtained source of autologous cells capable of differentiation into all three germ lines. Silk fibroin (SF) is a biocompatible and biodegradable polymer, which can be tailored to different nanostructures for biomedical application. We have tested multiple formulations of nanosilk including a liquid and matte formulation. We have previously shown that the nanosilk liquid improves biomechanical properties of human skin and both liquid and matte formulations and can be used for drug delivery. We hypothesized, we could isolate and differentiate ovine amniotic fluid stem cells and use these cells to populate a nanosilk matte that could be applied to a variety of wound healing applications including fetal repair of myelomeningocele. 

 

Methods

Nanosilk (matte formulation) was created from silk fibroin isolated from the cocoons of Bombyx mori silk worms, cut into small pieces, and dissolved in solution to obtain the viscosity needed for electrospinning nanofibers. Ovine amniotic fluid mesenchymal stem cells (oAF MSCs) were isolated from mid-gestation amniotic fluid collected during fetal intervention. oAF MSCs were cultured with the nanosilk for 7 days.

Nine female 12 week old mice that are bred homozygous diabetic (Db/Db) were used. Db/Db mice were used as this is an established murine model of abnormal diabetic wound healing. A single 8mm wound was made on the dorsal neck skin of each mouse with a punch biopsy. Wounds were treated with one time administration of phosphate buffered saline control or Nanosilk only and Nanosilk + oAF MSCs at the time of wounding.

 

Results

oAF MSC cells were isolated from 7 ewes within 7-21 days after amniotic fluid harvest. oAF MSCs were confirmed with IHC markers (positive for CD29, CD90, CD44, and negative for CD45) and showed the ability to differentiate into multiple cell lineages including adipocytes, osteocytes, and neural cells (Figure 1). Diabetic mice treated with nanofiber had improved wound healing (day 16 compared to day 18). Nanofiber cultured with ovine amniotic stems cells had similar wound healing to the nanofiber. The nanofiber did not incorporate into the wound and acted as a biologic dressing that was shed as the wound healed (Figure 2).

 

Conclusions 

oAF MSCs can be quickly isolated and cultured for incorporation into nanofiber silk patch that can be applied to wounds and possibly for fetal repair of myelomeningocele. The nanofiber silk patch is able to hold the MSCs stable in culture for several weeks and presents a possible delivery method for these cells. No adverse effects on wound healing are seen when the nanosilk or nanosilk + oAF MSCs are used and may have improved wound healing. 

87.09 Varying Degrees of Plication to Optimize Spring-Mediated Intestinal Lengthening

Posted on January 31, 2019

G. Dubrovsky1, A. Thomas2, S. Shekherdimian1, J. Dunn2  1University Of California – Los Angeles,Los Angeles, CA, USA 2Stanford University,Palo Alto, CA, USA

Introduction:
Intestinal lengthening with endoluminal springs has been well studied in animal models, and has been shown to lengthen a segment of intestine by as much as 3-fold. Springs are secured inside the intestine by surgically narrowing the intestine around the spring. This method of intestinal lengthening can be performed within a functional segment of bowel, and does not interfere with normal digestion or peristalsis. However, the amount of intestinal narrowing or plication necessary for lengthening has not been studied. The goal of this study is to determine if lengthening can be optimized by changing the amount of intestinal plication performed to secure an endoluminal spring.

Methods:
Juvenile mini-Yucatan pigs underwent surgical implantation of a nitinol spring within a segment of intestine via enterotomy that was closed. Plication or narrowing of the intestine around the spring with sutures was performed to help secure the spring within the intestine. There were three total groups of pigs; in the first group, the diameter of the intestine was reduced by 50%, in the second group it was reduced by 30%, and in the third group it was reduced by only 10% (FIGURE). Pigs were kept on a liquid diet post-operatively. 3 weeks after the original operation the pigs were euthanized and intestinal segments were examined for longitudinal lengthening and for histological changes.

Results:
All pigs tolerated the surgeries without complications. There were no cases of obstruction or perforation and all springs remained in place at the site of original implantation. While 50% plication resulted in 3-fold lengthening, 30% plication resulted in a 1.8-fold increase in length, and 10% plication resulted in a 1.3-fold increase in length. All lengthened segments showed significant increases in both the thickness of the muscularis propria as well as the depth of crypts as compared to normal segments of intestine.

Conclusion:
Intestinal plication is effective in securing springs within a segment of intestine to achieve intestinal lengthening, and the degree of plication effects the amount of lengthening that can be achieved. 50% reduction in the diameter of the bowel allows for maximal intestinal lengthening, while still avoiding bowel obstruction. These results will be important in guiding potential future therapies in the treatment of short bowel syndrome.
 

87.08 Broad Spectrum Antibiotics Reduce Adaptation and Liver Steatosis in a Zebrafish Short Bowel Model

Posted on January 31, 2019

K. Gee1, M. Isani1, K. Maselli1, T. Grikscheit1  1Children’s Hospital Los Angeles,Surgery,Los Angeles, CA, USA

Introduction: Intestinal failure associated liver disease (IFALD) is associated with short bowel syndrome (SBS). The pathogenesis may include bile acid dysmetabolism linked to changes in the microbiome. We previously showed that prolonged antibiotic administration with intestinal resection durably alters the intestinal microbiome in neonates.  We hypothesized that administration of broad spectrum antibiotics might reduce intestinal adaptation and affect liver steatosis and components of IFALD in a SBS zebrafish model.

Method: Adult male zebrafish comprised five groups: starvation(n=25), sham laparotomy(n=29), SBS(n=29), sham+ antibiotics(n=37), and SBS+ antibiotics(n=29). Sham fish had a laparotomy only. SBS surgery consisted of laparotomy with distal intestinal ligation and proximal stoma creation. Antibiotic groups received 100μg/ml ampicillin, 5μg/ml kanamycin, and 250 ng/ml amphotericin B every 3 days. Weekly weights were recorded until harvest at 2 weeks. Villus height, epithelial perimeter, intestinal circumference, and liver steatosis droplets per high power field were measured on H&E slides. BrdU injection prior to harvest followed by immunofluorescent detection assessed proliferating cells. cyp7a1, fxr, and tnfa expression were determined by RT-PCR. 

Results: SBS+ antibiotics fish lost a significant percentage of initial weight compared to sham+ antibiotics at 14 days (95% ± 3% v 73.3% ± 2.7%, p<0.0001). There was a decrease in villus height (279.2μm ± 50.8μm v 175μm ± 31.8μm, p=0.0016) and epithelial perimeter (589.8μm ± 155.9μm v 374.1μm ± 79μm, p=0.0005) between SBS and SBS+ antibiotics fish. There was no significant change in intestinal circumference between SBS groups. Additionally, SBS fish had significantly more steatosis in their liver compared to all other cohorts (p<0.0001). BrdU+ cells/villi were increased in SBS fish compared to sham (8.9 ± 3.7 v 2 ± 0.7, p=0.02) as well as SBS + antibiotics fish vs sham+ antibiotics (9.59 ± 2.86 v 1.95 ± 1.8, p=0.02). On RT-PCR analysis, there was increased gene expression of hepatic cyp7a1 (p=0.01) and decreased gene expression of intestinal fxr in SBS fish when compared to SBS+ antibiotic fish (p=0.03). SBS fish had increased expression of intestinal tnfa when compared to sham fish (p=0.04).

Conclusion: Administration of broad spectrum antibiotics to SBS zebrafish significantly reduces villus height and epithelial perimeter, classic indicators of successful intestinal adaptation, when compared to SBS fish that were not exposed to antibiotics. There was increased expression of hepatic cyp7a1 and decreased expression of intestinal fxr accompanied by liver steatosis when compared to SBS + antibiotics fish. While avoidance of IFALD is critical in patients with SBS, additional research to manipulate the FXR pathway without dysregulation of the microbiome is needed.

 

87.07 Role of Luminal Signaling in Parenteral Nutrition Associated Injury

Posted on January 31, 2019

A. Munoz Abraham1,3, A. Price2, K. Blomenkamp2, C. Manithody2, H. Osei1,3, P. Rajalakshmi2, V. Kakrala2, C. Denton2,3, J. Krebs2, J. Friend2, W. Phillips2, M. Westrich2, J. Greenspon1,3, G. A. Villalona1,3, A. Jain2,3  1Saint Louis University School Of Medicine,Pediatric Surgery,St. Louis, MO, USA 2Saint Louis University School Of Medicine,Pediatrics,St. Louis, MO, USA 3Cardinal Glennon Children’s Hospital,St. Louis, MO, USA

Background:

Total Parenteral Nutrition (TPN) provides all nutritional needs intravenously. It remains an essential lifesaving therapy, however despite widespread use; enthusiasm is tempered due to significant side effects and a lack of understanding into mechanisms leading to TPN injury. Using a novel ambulatory TPN piglet surgical model, developed in our lab to recapitulate long term human TPN delivery we tested our hypothesis that an altered luminal signaling is a major driver for TPN injury.

 

Methods:

30 animals were randomly allocated to receive TPN (n=22) or enteral nutrition (EN, n=8) for approximately 3 weeks. A subgroup of TPN animals (n=14) underwent bowel resection. Gut, liver and serum were collected. Samples underwent histology, serum biochemistry, ELISA and western blot assays. Pair wise t-test for normally distributed data otherwise pairwise Mann Whitney U test were conducted. All tests were 2 tailed using a significance level of 0.05.

 

Results:

We successfully placed surgical catheters and achieved bowel resection in animals. TPN resulted in cholestatic liver injury with significant bilirubin elevation and hepatomegaly, p=0.046. Stellate interlobular fibrosis was noted with TPN. TPN caused an increase in hepatic inflammation noted on CD3 immunohistochemistry (p=0.005). Interestingly, no significant differences in serum ALT were noted. TPN resulted in marked gut atrophy and reduction in muscularis mucosa compared to EN animals demonstrated grossly and histologically. The mean, (±SE) for gut density (g/cm) and villous / crypt (V/C) ratio was 6.34±1.67, 2.67±0.18 for EN vs 2.96±0.34, 2.06±0.16 for TPN (p=0.002, p=0.03 respectively). While the key luminal signaling regulators, gut FXR, TGR5, EGF and MAPKinase were significantly downregulated with TPN (p<0.05), CyP7A1 was several folds higher in TPN animals (p<0.05). Additionally, gut resection (SBS animals) impaired improvement in cholestasis or hepatic fibrosis despite luminal agonists (CDCA) secondary to impaired gut-systemic signaling. As expected, in SBS activation of gut FXR and apical sodium dependent BA transporter was noted with CDCA vs TPN (qPCR p=0.02, p=0.04; respectively). CDCA, however preserved gut mass (gm/cm, p=0.04) demonstrated grossly and histologically.

Conclusion:

Our study reports novel finding of an increased CD3 staining, interlobular stellate hepatic fibrosis and muscularis mucosa reduction associated with TPN therapy. We also noted significant alteration is key hepatobiliary receptors driving luminal signaling and its impairment with gut resection, despite preservation of gut growth with luminal FXR activators. Our study using a novel surgical piglet model presents a paradigm shift to our understanding of TPN injury, as secondary to altered luminal-systemic cross talk.

87.06 Dual Target Inhibition of RET and the RAS-MAPK Pathway with RXDX-105 as a Novel Therapeutic in Neuroblastoma

Posted on January 31, 2019

S. Flynn1, J. Lesperance2, N. Phanhthilath2, M. Paul2, P. Zage2  1University Of California – San Diego,Surgery,San Diego, CA, USA 2University Of California – San Diego,Pediatrics,San Diego, CA, USA

Introduction:

Novel therapies are needed for high risk neuroblastoma, where outcomes remain poor despite aggressive multimodal therapy. The RET tyrosine kinase is highly expressed in neural crest and neuroblastoma cells and is felt to play a role in their proliferation and survival. BRAF mutations are uncommon, but have been described in neuroblastoma tumors, and Eleveld et al. demonstrated high rates of RAS-MAPK mutations in relapsed neuroblastoma. RXDX-105 is a small molecule multikinase inhibitor with potent activity against RET and BRAF, currently under Phase I/Ib clinical trials in adult solid tumors. Given the suggested role of RET and the RAS-MAPK pathway in neuroblastoma survival, proliferation, and relapse, we investigated dual inhibition of these pathways with RXDX-105 as a potential new therapeutic for neuroblastoma.

Methods:
The effect of RXDX-105 on cell proliferation and viability was evaluated using Incucyte ZoomTM continuous live cell imaging and alamarBlue® assays. Caspase 3/7 cleavage assays were used to asses for apoptosis. Pathway inhibition effects on cell cycle were measured using flow cytometry with propidium iodide staining. Inhibition of RET and the RAS-MAPK pathways were measured with Western blot analysis of treated and untreated cells. The efficacy of RXDX-105 against neuroblastoma tumors in vivo was evaluated with an orthotropic xenograft model in immunocompromised mice treated with oral RXDX-105. Tumors were assessed with in vivo luminescence and final tumor weight.

Results:
IC50 values for the tested cell lines ranged between 5mcM and 14mcM following 72 hours of RXDX-105 exposure. Drug treated cells showed an increase in caspase 3/7 cleavage as well as G1 cell cycle arrest. RXDX-105 treatment potently inhibited RET phosphorylation as well as phosphorylation of downstream proteins of BRAF, including MEK and ERK. Daily oral treatment with RXDX-105 in vivo resulted in reduced tumor growth.

Conclusion:
Inhibition of RET and BRAF/RAS-MAPK pathway inhibits neuroblastoma cell growth and viability through combination of induction of apoptosis and cell cycle arrest. Inhibition of these pathways with RXDX-105 shows promise as a novel therapeutic in preclinical xenograft models.
 

87.05 A Bioadhesive Reverse Thermal Gel for Minimally Invasive In Utero Patching of Myelomeningocele

Posted on January 31, 2019

J. R. Bardill1,2, D. Park1, A. I. Marwan1,2  1University Of Colorado Denver,Bioengineering,Aurora, CO, USA 2University of Colorado Denver Anschutz Medical Campus,Pediatric Surgery,Aurora, CO, USA

Introduction:

Myelomeningoceles (MMC) are one of the most common congenital birth defects of the spinal cord. Prenatal repair of these defects became the standard of care, however, open approaches present significant risks to mother and fetus.  Our laboratory has developed a novel Reverse Thermal Gel (RTG) to deliver a minimally invasive patch for MMC coverage. Preliminary studies demonstrated long-term in-vitro gel stability and minimally invasive application in a mouse MMC model with partial defect coverage.

In preparation to transition to the ovine model, an RTG patch with surface adhesion in the amniotic environment and cellular scaffolding properties to integrate with host will be needed.  Here, we demonstrate the chemical synthesis and characterization of a bioadhesive RTG, in-vitro cellular scaffolding properties, and the biological applicability in a mouse MMC defect model.

Methods:

A. Synthesis of Bioadhesive RTG:  Dopamine was conjugated to the RTG polymer backbone and verified using proton nuclear magnetic resonance (H NMR) spectroscopy and infrared (IR) spectroscopy.  Mechanical properties were tested using rheology. B. In-vitro RTG cellular scaffold:  Mouse skin fibroblasts and human epidermal keratinocytes (HEKA) were mixed (separately) with RTG-media solution, followed by gelling at 37°C to encapsulate the cells within RTG.   Fibroblasts were stained for vimentin and alpha smooth muscle actin (α-SMA), and HEKAs were stained with keratin 14 (k14) antibodies.  C. Adhesive RTG injection into mouse MMC defect embryos:  In-vivo applicability was tested in Grhl3 mice to assess MMC defect coverage by the adhesive RTG.  D. Inflammatory response:  Defect tissue was immunostained for CD68 and F4/80 macrophages. 

Results:

H NMR and IR verified aromatic and hydroxyl peaks from dopamine are present in the polymer backbone.  The elastic strength of the adhesive RTG increased 500-1000 Pascal’s compared to the regular RTG.  Dermal fibroblasts expressed vimentin and α-SMA markers.  HEKAs in RTG culture expressed k14 markers. The adhesive RTG was applied to mouse MMC defects by needle injection and improved defect coverage, with an average of 72% defect coverage at harvest.  CD68 and F4/80 macrophage positive cells were not significantly different when comparing adhesive RTG and saline injections in defect embryos.

Conclusion:

We synthesized and characterized a bioadhesive RTG with increased elastic strength.  This adhesive RTG has in-vitro fibroblast and keratinocyte cellular scaffolding properties cultured in an RTG matrix.  Finally, the adhesive RTG can be injected in a minimally invasive manner to improve mouse MMC defect coverage compared to the non-adhesive RTG with minimal macrophage response.  Overall, this work has shown the adhesive RTG is a promising candidate for future application in the ovine model. 

 

 

87.04 The Role of Enterococcus faecalis in the Pathogenesis of Experimental Enterocolitis

Posted on January 31, 2019

P. T. Delaplain1, J. Wang1, A. Grishin1, H. R. Ford1,2  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2University Of Miami,Miller School Of Medicine,Miami, FL, USA

Introduction:  The exact relationship between intestinal bacterial populations and the development of necrotizing enterocolitis (NEC) is currently unknown. We hypothesize that strains of bacteria colonizing the neonatal gut could be either innocuous/protective, or opportunistically pathogenic. We investigated the ability of two different strains of E. faecalis, one of the common first intestinal colonizers, to induce or prevent intestinal inflammation in neonatal rats.  

Methods:  Strains of E. faecalis previously isolated from 4-day-old rats were examined for a variety of characteristics and two strains, 8 and BB70, were chosen for in vivo experiments. Newborn rats from timed pregnant mothers were formula-fed and subjected to hypoxia every 8 hours to induce NEC.  Neonates were inoculated with or without 108 cfu of E. faecalis 8 or BB70 during the first feed. During the second feed, the animals were challenged with, or without 106 cfu of Cronobacter muytjensii 51329, a known NEC pathogen. All animals were sacrificed on day 4. Intestinal contents were serially diluted and plated on blood agar, BHI-azide agar, and MRS agar to quantify total bacteria, E. faecalis, and lactic bacteria. Isolates from BHI-azide were further examined using diagnostic media to identify E. faecalis strains. NEC was scored histologically. Results were compared using χ2 test.

Results: Strain 8 and Strain BB70 were found in the intestines of animals inoculated with these strains at average relative abundance of 28.2% and 49.5%, respectively. These strains were not found in control animals. Strain 8 increased NEC pathology, whereas strain BB70 decreased it (Figure 1).

Conclusion: E. faecalis strains 8 and BB70 appear to act as opportunistic pathogen and protective commensal, respectively. Our results show that pathogenic and protective properties of E. faecalis may be strain-specific. E. faecalis strains similar to BB70 may be useful in preventing clinical NEC.

87.03 Natural History of the Heterogeneous Pulmonary Response Following Tracheal Occlusion

Posted on January 31, 2019

R. Marwan1, S. Al-Juboori1, E. Dobrinskikh2, J. Haines3, R. Marwan1  1University Of Colorado Denver,Pediatric Surgery,Aurora, CO, USA 2University Of Colorado Denver,Medicine,Aurora, CO, USA 3University Of Colorado Denver,Biochemistry And Molecular Genetics,Aurora, CO, USA

Introduction:  Infants with severe congenital diaphragmatic hernia (CDH) offered experimental tracheal occlusion (TO) have inconsistent clinical outcomes. Understanding such clinical variability is crucial to advancing neonatal care but the reasons for such deviations are currently unknown. Our laboratory has proposed a novel concept of heterogeneous topological zones formation in fetal lungs following TO. However, the temporal pattern of airspace morphometry and metabolic landscape changes following TO is not fully elucidated. Our objective is to explore the natural history of heterogeneous changes in fetal lungs following TO.

Methods:  We evaluated fetal lungs after 1 and 4 days following TO; global and local metabolic changes, as well as morphometric indices were examined using mass spectrometry-based metabolomics, Fluorescence Lifetime Imaging Microscopy (FLIM) and Tissue Airspace Ratio (TAR), respectively.

Results: 1 day (D1) after TO, there is an appearance of two distinct morphometric areas varying between small (high TAR values) and large (small TAR values) airspaces, with an overall shift toward smaller airspaces compared to controls. In contrast, by day 4 (D4) TO lungs have a higher frequency of large airspaces (small TAR values) compared to control. D4 TO lungs global metabolomics data demonstrate a significant decrease in glucose and hexose phosphate, and an increase in lactate production, compared to D1 TO lungs. Moreover, there is a suppression of the tricarboxylic acid cycle (TCA) with a significant decrease in citrate and fumarate at D4. These changes are accompanied by significant increase in spermidine and spermine at D4, which are important for cellular growth and proliferation. Locally on FLIM, D1 TO lungs demonstrate two types of heterogenous zones. Namely, zones which have free/bound NADH ratio similar to control lungs, and zones with decreased free/bound NADH ratio- indicating increased OXPHOS in these regions. Similar to the histological findings, FLIM on D4 TO lungs demonstrates appearance of zones with enlarged airspaces, which have metabolic shift towards glycolysis, accompanied by decreased FLIM-surfactant signal only in those areas.

Conclusion: In normal fetal lungs, we report a novel unique natural history of heterogeneous changes; TO leads to heterogeneous pulmonary response. Initially, there is formation of zones with small airspaces, followed by airspace enlargement over time.  While FLIM of D1 TO lungs reveals zones with increased OXPHOS, FLIM in D4 TO lungs demonstrate a shift toward glycolysis in the enlarged airspaces with decreased surfactant production. We speculate that the “best responders” to tracheal occlusion should have bigger lungs with small airspaces and normal surfactant production.
 

87.02 PIM Kinases Play a Role in Hepatoblastoma Resistance to Cisplatin

Posted on January 31, 2019

L. Stafman1, A. P. Williams1, J. Aye1, J. Stewart1, E. A. Beierle1  1University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction:  

Despite increasing incidence, treatment for hepatoblastoma has not changed significantly over the past 20 years. Treatment strategies primarily rely on cisplatin, but chemoresistance remains a significant challenge. Stem cell-like cancer cells (SCLCCs) are a subset of cancer cells thought to play a role in chemoresistance. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia (PIM) kinases, specifically PIM3, play a role in hepatoblastoma and decrease the SCLCC phenotype, and that the combination of PIM inhibition with cisplatin acts synergistically to decrease hepatoblastoma cell proliferation. We therefore sought to develop a cisplatin resistance model and evaluate the effect of cisplatin and PIM inhibition in combination on the SCLCC phenotype and the effect of PIM inhibition on cisplatin-resistant hepatoblastoma cells.

Methods:

Cisplatin-resistant human hepatoblastoma cells were developed through serial passage of the human hepatoblastoma HuH6 cells in athymic nude mice and treatment of the mice with cisplatin (2 mg/kg/day three consecutive days weekly). PIM inhibition was achieved using the small molecule pan-PIM inhibitor, AZD1208. Cell viability and proliferation were assessed using the alamarBlue® and CellTiter 96® colorimetric assays. Combination indices were calculated and isobolograms constructed using the method of Chou and Talalay. CD133 expression was determined using flow cytometry and an extreme limiting dilution analysis was used to determine sphere frequency. PIM3 expression was assessed by Western blotting.

Results:

Cell viability and proliferation in the presence of cisplatin was increased in the cisplatin-resistant cells compared to cisplatin-naïve cells, confirming the cisplatin resistance model. Cisplatin-resistant hepatoblastoma cells both expressed more CD133 and formed tumorspheres more readily than cisplatin-naïve cells, indicating an increase in the SCLCC phenotype. Cisplatin-resistant hepatoblastoma cells exhibited higher PIM3 expression. Finally, PIM inhibition in combination with cisplatin resulted in combination indices of 0.25 and 0.35, indicating that PIM inhibition sensitized even cisplatin-resistant cells to cisplatin (Figure).

 

Conclusion:

Together, these findings provide evidence that PIM inhibition may be promising in combination with the standard chemotherapeutic cisplatin in hepatoblastoma to target SCLCCs and prevent chemoresistance to cisplatin.

87.01 The Antimicrobial Effects of Lactobacillus reuteri in Protection Against Necrotizing Enterocolitis

Posted on January 31, 2019

R. D. Shelby1, L. Mashburn-Warren2, J. B. Navarro2, N. Tengberg1, J. Allen2, M. T. Bailey2, S. D. Goodman2, G. E. Besner1  1Nationwide Children’s Hospital, The Research Institute, Center for Perinatal Research,Pediatric Surgery,Columbus, OH, USA 2Nationwide Children’s Hospital, The Research Institute,Center For Microbial Pathogenesis,Columbus, OH, USA

Introduction: Despite decades of research, necrotizing enterocolitis (NEC) remains the most common gastrointestinal (GI) surgical emergency in the premature infant. While the pathogenesis of NEC remains unclear, alterations in the intestinal microbiome likely play a mechanistic role. Our laboratory has demonstrated the efficacy of Lactobacillus reuteri(Lr) in protecting the intestines from experimental NEC when the probiotic is administered in its biofilm state. When grown as a biofilm on maltose-loaded dextranomer microspheres (DM), the Lr-DM-maltose product significantly restores the intestinal microbiome, and decreases the incidence and severity of NEC after a single dose. Lr exhibits both anti-inflammatory and anti-microbial properties – the latter attributed to the production of reuterin, an anti-microbial compound that inhibits harmful gut bacterial growth. In the current study, we developed a reuterin-deficient mutant formulation of Lr (Lr-gldC) to examine the importance of the antimicrobial effects of Lr in protecting the intestines from NEC. 

Methods: Using a validated rodent model of NEC, premature rat pups were delivered via C-section and subjected to repeated episodes of hypercaloric formula feeding, hypoxia, and hypothermia for 96 hours. Rat pups were randomized to one of several treatment groups prior to initiation of formula feeding and stress: 1) NEC + sterile water (vehicle control, N=51), 2) NEC + planktonic (free-living) Lr (N=45), 3) NEC + Lr on maltose loaded microspheres (Lr + DM-maltose; N=56), 4) NEC + Lr-gldC (mutant form of Lr) (N=12); or 5) NEC + Lr-gldC + DM-maltose (N=42). Control pups were unstressed and breast fed (N=13).Rat pups were sacrificed when they exhibited signs and symptoms of NEC, or after 96 hours. Intestinal tissue was collected at sacrifice for blinded histopathological analysis.

Results: As expected, compared to no treatment, administration of a single dose of Lr in its biofilm state (Lr+ DM-maltose) significantly decreased the incidence of NEC (67% vs. 18%, p< 0.0001), whereas administration of Lr in its planktonic state had no significant effect. In contrast, compared to administration of Lr+ DM-maltose, administration of Lr-gldC + DM-maltose resulted in significant (albeit not total) loss of beneficial effects (18% vs.41%, p=0.008). 

Conclusion: In conclusion, the ability of Lr to produce reuterin contributes to, but is not solely responsible for, the ability of the probiotic to reduce both the severity and incidence of NEC. Other factors such as the anti-inflammatory properties of Lr may also play a role, and will be investigated in future studies. These results may contribute to our understanding of the etiology of NEC, and will be important as we translate this technology to the bedside.

 

86.20 Cartilage Oligomeric Matrix Protein Expression Correlates with Disease Stage In Colon Cancer.

Posted on January 31, 2019

N. P. Omesiete1, J. Jandova1, H. C. Jecius1, V. Nfonsam1  1University of Arizona,Surgery,Tucson, AZ, USA

Introduction:
Colon cancer (CC) is an important contributor to cancer morbidity and mortality. In our previous study we demonstrated that Cartilage Oligomeric Matrix Protein (COMP) was significantly overexpressed in Young patients with CC. The aim of this study was to establish the correlation between COMP expression levels, the stage of disease and Carcinoembryonic Antigen (CEA) Level.

Methods:
FFPE samples of CC and matching noninvolved tissues from 12 CC patients (stage I: n=3; stage II: n=4; stage III: n=4 and stage IV: n=1) were obtained from pathology archives. The samples were deparaffinized and the CC tissues were microdissected. RNA was isolated, and used for expression profiling of 770 cancer-related genes. COMP levels in serum (n=4) were measured using ELISA assay. Immunohistochemistry was performed using anti-human COMP antibody. CEA levels were checked for all the patient samples.

Results:

Gene expression profiling of 770 cancer related genes revealed increasing mRNA expression levels of COMP with increasing disease stage. Comparison between Stage I and Stage II tumors revealed higher COMP levels in Stage II tumors and TLR2, IL8, RIN1, IRAK3 and CACNA2D2 as top 5 highest correlated genes in pairwise expression association of COMP. Comparison between Stage II T4 and Stage III tumors did not show significant change in expression of COMP. However, when Stage I and Stage III tumors were compared, there was almost seven times higher COMP expression in Stage III tumors than in Stage I. VEGFC, MA3K8, SFRP1 and PRKACA, genes involved in various cellular processes such as proliferation, invasion, differentiation and angiogenesis were the highest co-expressed genes with COMP. Analysis of serum from four CC patients revealed positive correlation between COMP levels and stage of disease and CEA levels. The higher the COMP levels higher the stage of disease and CEA levels. These results were confirmed by immunohistochemistry.

Conclusion:
Increasing COMP expression and protein levels in CC tumors are associated with higher disease stage. COMP can be detected in serum of CC patients and shows strong positive correlation with stage of disease and CEA levels. COMP is a potential biomarker for aggressive CC and more studies should be done to establish this.

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