2.14 Generation of Synthetic Intestinal Bioscaffold with Similarities to Native Mammalian Small Intestine

Posted on January 18, 2018

M. R. Ladd1, C. Costello2, B. Johnson1, C. Gosztyla1, A. Werts1, L. Martin1, W. Fulton1, P. Lu1, H. Jia1, E. Banfield1, J. Sung1, S. Wang1, T. Prindle1, Y. Yamaguchi1, C. Sodhi1, J. C. March2, D. J. Hackam1  1Johns Hopkins University School Of Medicine,General Surgery,Baltimore, MD, USA 2Cornell University,Ithaca, NY, USA

Introduction: Short bowel syndrome is a devastating disease with limited treatment options. The development of an artificial intestine offers a potential solution, however, the ability to develop functional small intestine has been limited in part due to scaffolds with inadequate mechanical properties to promote intact intestinal tissue formation. The goal of this study is to develop and evaluate intestinal-like scaffolds that mimic the mechanical properties of native small intestine and thus to cross a technical hurdle in the generation of an artificial intestine.

Methods:  Intestinal-like scaffolds were fabricated from poly(glycerolsebacate) using a serial fabrication technique with laser indentation into agarose gel to create microvilli that mimic the height and width of native intestinal villi. The pore size and crosslinker were varied from the villus portion to the basal portion to further approximate the native bowel. We evaluated the tensile properties of these synthetic scaffolds (n=10, in triplicate) and compared them to porcine intestine from 3-week-old piglets (n=9, in triplicate). The in vitro degradation of the scaffolds in media and media plus digestive enzymes (to mimic the intestinal environment) was characterized by mass loss (n=3 per condition) and scanning electron microscopy. Scaffolds were coated with matrigel and seeded with murine intestinal stem cell cultures (n=3) harvested to evaluate for cell attachment. Young's modulus, a measure of stiffness, was calculated as the slope of the linear portion of each stress-strain curve.

Results: Our novel scaffolds demonstrated projections which approximated villi seen in intestine. The Young’s modulus of the scaffolds was 5.6 MPa vs. 1.03 MPa for small intestine. The ultimate tensile strength and maximum load of the scaffolds were 1.45 MPa and 3.2 N compared to 1.11 MPa and 1.3 N for the small intestine. Strain at failure was higher in the intestine (175% vs. 77%). In vitro degradation studies demonstrated 42% mass loss at 5 weeks yet the villus structures were still present, consistent with that seen in the native mammalian state. When seeded with murine intestinal stem cells, the scaffolds demonstrated good cell attachment by confocal microscopy and scanning electron microscopy (SEM).

Conclusion: We have successfully developed synthetic scaffolds with mechanical properties that approximate those of native piglet small intestine and allow for attachment of stem cells suggesting they may be suitable for tissue engineered small intestine.  

 

2.13 The Effects of Gestational Psychological Stress on Neonatal Mouse Intestinal Development

Posted on January 18, 2018

J. Shah1,2, S. B. Deas2,3, J. H. Neilson2,3, C. Ren4, T. Jilling4, K. M. Brawner2, C. A. Martin2  1University Of Alabama at Birmingham,Department Of Clinical & Diagnostic Sciences,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Department Of Medicine,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Department Of Pediatrics,Birmingham, Alabama, USA

Introduction: Psychological stress during pregnancy has been shown to cause subsequent harm to the fetus and newborn.  Many studies focus on neurodevelopmental outcomes, but little is known about the effect of gestational stress on intestinal immunity and development. We have shown that psychological stress disrupts immune development, but its effect on intestinal development is not known. The purpose of this study was to determine the effect of psychological stress during pregnancy on the newborn’s intestinal architecture and growth.

Methods:  8-week-old C57BL/6 littermates underwent timed breeding. Pregnant dams were subjected to one hour of daily psychological stress by using a well-established restraint model during days E7-E14 of the gestational period. The distal ileum of 2-week-old offspring of stressed mothers and non-stressed controls was harvested for histological analysis. Slides were blinded and Axiovision Rel. 4.7 software was used to measure villus height and crypt depth. To determine the effect of excess stress hormones on intestinal proliferation, an explant model was used. 2 mm biopsies were taken from wild type non-stressed mice and treated with 100 mM of corticosterone for 24 hours. RNA was isolated and RT-PCR was performed to determine the effect of corticosterone on the intestinal stem cell marker Leucine-rich-repeat-containing G-protein-coupled Receptor 5 (LGR5) and growth factors Epidermal Growth Factor Receptor (EGFR) and Insulin Growth Factor-1 (IGF-1). A non-parametric T test was used to determine any significant differences between the groups. Results were expressed as the mean SEM.

Results: 7 mice were included in each group. The villus height was 126.4  5.6 m for control and 100.4  4.5 m for stress mice, p value <0.05. The crypt depth was 63.9  1.2 m for control and 53.7  2.3 m for the stressed group, p value <0.05. RT-PCR revealed that explants exposed to corticosterone had a 2.1-fold increase in LGR5 compared to controls, p value = 0.04. There was no significant difference in the IGF-1 and EGFR expression between control and treatment groups.

Conclusion: Here we establish that neonatal mice with mothers that were subjected to psychological stress during pregnancy have significantly shorter villi and crypts compared to controls.  In addition, pups from stressed mothers had higher expression levels of the intestinal stem cell marker LGR5, which may suggest a compensatory response to stress.  Future studies will further clarify the temporal relationship between intrauterine stress and intestinal development as well as the mechanisms of how excess stress hormones affect neonatal intestinal development. These findings will aid in determining the effect of gestational psychological stress on intestinal development and stem cell proliferation

2.12 Double Plication for Spring-Mediated In-Continuity Intestinal Lengthening in a Porcine Model

Posted on January 18, 2018

G. Dubrovsky1, N. Huynh2, A. Thomas2, S. Shekherdimian1, J. C. Dunn1,2  1University Of California – Los Angeles,Division Of Pediatric Surgery, Department Of Surgery, David Geffen School Of Medicine,Los Angeles, CA, USA 2Stanford University,Division Of Pediatric Surgery, Department Of Surgery, Stanford University School Of Medicine,Palo Alto, CA, USA

Introduction:
Spring-mediated distraction enterogenesis has been shown to increase the length of an intestinal segment in rat and pig models by as much as 3-fold, and therefore has potential as a new therapy for patients with short bowel syndrome. However, this method for intestinal growth has required the spring to be confined within a segment of intestine that has two closed ends and that has been defuctionalized. The goal of this study is to use suture plication to confine a spring within a functional intestinal segment while maintaining the luminal patency of the intestine and allowing for the normal flow of GI contents.

Methods:
Juvenile mini-Yucatan pigs underwent placement of a nitinol spring within a functional segment of jejunum in continuity. A 20 French catheter was passed temporarily, and sutures were used to plicate and narrow the intestinal lumen to the diameter of the catheter around the encapsulated spring on both sides (FIGURE). Pigs were maintained on a liquid diet post-operatively. The intestine was re-examined 3 weeks after spring placement for lengthening and for histological changes.

Results:
All pigs tolerated a liquid diet with appropriate weight gain. In the absence of plication, springs passed through the intestine within a week. Double plication allowed the spring to stay in place within the jejunum for 3 weeks. Compared to uncompressed springs that showed no change in the length of plicated segments, compressed springs caused a nearly 1.5-fold increase in the length of plicated segments. Histology showed a significant increase in both the thickness of the muscularis propria from 230 µm to 450 µm and in the crypt depth from 210 µm to 420 µm in lengthened segments compared to normal segments of intestine.

Conclusion:
Intestinal plication is an effective method to confine endoluminal springs. The confined springs are able to lengthen intestine while allowing for normal GI function and growth of animals, making this a clinically relevant model. We also see histological changes consistent with intestinal growth at the cellular level. This approach may be useful to lengthen intestine in patients with short bowel syndrome.
 

2.11 FAK Inhibition Decreases Stemness and Tumorigenicity of Tumor Initiating Cells in Neuroblastoma

Posted on January 18, 2018

L. Stafman1, E. Garner1, J. Aye1, A. Williams1, J. Stewart1, K. Yoon1, K. Whelan1, E. Beierle1  1University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction:
Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and despite aggressive therapies, over 50% of patients with high risk tumors will suffer disease relapse. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase present in aggressive NB. Tumor initiating cells (TICs) are cancer cells with stem cell-like properties thought to be responsible for tumor recurrence and chemoresistance. We previously demonstrated that FAK inhibition decreased CD133 expression and tumorsphere formation, both markers of TICs, in NB patient-derived xenografts (PDXs). We hypothesized that targeting FAK would decrease stemness and tumorigenicity in NB TIC population.

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). Expression of TIC markers, Sox2, Oct4, and Nanog, were assessed by immunoblotting. Cells were separated by CD133 surface expression using microbeads to obtain TIC (CD133-enriched) and non-TIC (CD133-depleted) populations. Tumorsphere formation assays were performed. Invasion was evaluated using Transwell inserts. Cell cycle analyses were performed by flow cytometry. Student’s t-test, extreme limiting dilution analysis, and χ 2 statistics were used with mean ± standard error of the mean reported and p ≤  0.05 significant.

Results:
FAK inhibition with PF or Y15 significantly decreased expression of the TIC markers Sox, Oct4, and Nanog in NB PDXs. Additionally, treatment with PF or Y15 decreased tumorsphere formation in both TICs and non-TICs (p<0.01). Invasion was significantly decreased with FAK inhibition in both TIC (98% ± 2% decrease with 5 μ M PF, 86% ± 11% decrease with 5 μM Y15) and non-TIC (96% ± 1% decrease with 5 μM PF, 62% ± 9% decrease with 5 μM Y15) populations. FAK inhibition with both small molecules resulted in significantly decreased invasion in both the TIC and non-TIC cell populations. FAK inhibition also led to cell cycle arrest with significant increases in G1 and decreases in S phase of the cell cycle in both TICs and non-TICS (Figure).

Conclusion:
Inhibition of FAK with small molecule inhibitors decreased expression of TIC markers in human NB PDXs. NB TICs (CD133-enriched cells) were not immune to FAK inhibition and exhibited decreased tumorsphere formation, invasion, and cell cycle arrest similar to their non-TIC (CD133-depleted) counterparts. These findings suggest that FAK supports the NB TIC phenotype and targeting FAK in this cell population may be an important therapeutic strategy to decrease NB chemoresistance and relapse. 
 

2.10 FAK Inhibition Decreases Tumorigenicity in a PDX Model of Primary and Metastatic Wilms Tumor

Posted on January 18, 2018

J. Aye1, S. Mruthyunjayappa1, L. Stafman1, E. Garner1, J. Stewart1, E. Mroczek-Musulman1, K. Yoon1, K. Whelan1, E. Beierle1  1University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction:  

Approximately 12% of patients with Wilms tumor (WT) will have metastatic disease at diagnosis and often have a grave prognosis.  We established a patient-derived xenograft (PDX) of metastatic WT, including a liver metastasis (COA 42), along with its matched isogenic primary renal tumor (COA 25).  Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in the tumorigenesis of pediatric renal tumors.  To date, the role of FAK in metastatic WT has not been investigated.  We hypothesized FAK inhibition would decrease tumorigenicity in this PDX model.

Methods:

Two human WT samples, primary renal tumor, COA 25, and hepatic metastasis, COA 42, were implanted in athymic nude mice.  H&E staining of the patient’s renal primary and liver metastasis and subsequent PDXs, COA 25 and COA 42, was performed.  FAK expression and phosphorylation was detected with immunohistochemical staining (IHC) of patient samples and by immunoblotting of PDX protein lysate.  COA 25 and COA 42 cells were treated with small molecule FAK inhibitors, PF-573228 (PF) and 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), for 24 hours. Cell viability and proliferation were assessed with alamarBlue and CellTiter 96 assays, respectively.  Cell cycle analysis was performed by flow cytometry.  Results were compared with student’s t-test and p≤0.05 was considered significant.

Results:

H&E staining confirmed COA 25 and COA 42 recapitulated the patient’s primary and metastatic tumor, respectively, and IHC confirmed FAK expression and phosphorylation in these tissues.  Immunoblotting revealed FAK was present and phosphorylated in both PDXs.  FAK inhibition with PF (10 μM) significantly decreased cell viability by 63% and proliferation by 47% in the COA 25 cells (renal primary) compared to untreated cells.  Y15-induced FAK inhibition (10 μM) of COA 25 cells also significantly decreased cell survival by 61% and proliferation by 36%.  Treatment of the COA 42 cells (hepatic metastasis) with PF and Y15 (10 μM) also significantly reduced cell survival by 83% and 68%, respectively, and proliferation by 63% and 72%, respectively.  Flow cytometry revealed G1 cell cycle arrest for both COA 25 and COA 42 treated with PF and Y15 (10 μM) compared to untreated cells (Figure).

Conclusion:

FAK protein was expressed and phosphorylated in primary renal and metastatic WT PDXs.  FAK inhibition with two small molecules led to decreased tumor cell viability and proliferation along with cell cycle arrest in both the primary renal tumor and liver metastasis.  These findings suggest that further exploration of FAK as a therapeutic target for metastatic WT should be undertaken.  

 

2.09 Lipocalin-2 Increases Weight Loss, Intestinal Permeability and Mortality in a Short Bowel Model

Posted on January 18, 2018

S. M. Alaish1, D. R. Shores2, A. Zhang1, M. Wang1, H. Jia1, W. Fulton1, C. Sodhi1, D. J. Hackam1  1Johns Hopkins University School Of Medicine,Pediatric Surgery/Surgery,Baltimore, MD, USA 2Johns Hopkins University School Of Medicine,Pediatric Gastroenterology/Pediatrics,Baltimore, MD, USA

Introduction: Short bowel syndrome (SBS) causes an intestinal dysbiosis and increased intestinal permeability which can lead to sepsis and even death. As an iron-sequestering protein in the antibacterial innate immune response, Lipocalin-2 (LCN2) prevents bacterial iron uptake and controls bacterial growth. In a mouse model, we have previously shown that LCN2 mRNA and protein increase in the intestine following 75% small bowel resection.  We now hypothesize that LCN2 may serve as a potential defense mechanism in the intestine against the dysbiosis that occurs in SBS and tested this by evaluating the outcome of LCN2-/- mice exposed to a model of SBS.

Methods: Under an ACUC-approved protocol, we performed a 75% small bowel resection (SBR) on both C57BL/6J (n=6) and LCN2-/- (C57BL/6J background) (n=6) mice, which mimics the extensive resection seen in SBS patients. Sham-operated C57Bl/6J (n=6) and LCN2 -/- (n=6) mice served as controls. Mice were weighed daily. On postoperative day 6, the mice were injected with BrdU.  24 hours later, the mice were gavage fed FITC-Dextran, serum was collected, intestinal permeability was assayed, and the mice then underwent euthanasia. Intestinal tissue was collected and processed for staining.  Statistical analysis was performed using ANOVA with p<0.05 considered significant.

Results:LCN2 -/- mice exposed to SBR lost significantly more weight than shams (p<0.05) but significantly less than wild-type SBR mice (p<0.05). Similarly, intestinal permeability was increased in LCN2 -/- SBR mice as compared to shams (p<0.05) but less than wild-type SBR mice (p<0.05), while enterocyte proliferation was similar for the LCN2-/- SBR mice and the wild-type SBR mice. Strikingly, survival was significantly greater in the LCN2-/- SBR mice as compared to the wild-type SBR mice (87.5% vs. 56.1%, p<0.05), suggesting that in disagreement with our original hypothesis, the presence of LCN2 exerts pro-weight loss and pro-permeability effects that contribute to increased mortality, and which are reversed in LCN2 -/- mice.

Conclusion:Contrary to our hypothesis, LCN2 exerts surprising, deleterious effects on gut permeability and weight loss in mice, contributing to increased mortality, in a mouse model of short bowel resection.  Such effects may reflect the pro-inflammatory role of LCN2 which has been observed in other systems, and suggest that LCN2 inhibition may offer a novel therapeutic approach for gut protection in children with SBS.

 

2.08 Fetal Lung Transcriptome Patterns in an Ex Vivo Compression Model of Diaphragmatic Hernia

Posted on January 18, 2018

Z. D. Fox1,2, G. Jiang2, K. A. Walker2, K. Ho3, A. P. Liu3, S. M. Kunisaki1,2  1University Of Michigan Medical School,Ann Arbor, MI, USA 2Michigan Medicine,Pediatric Surgery Section, Department Of Surgery,Ann Arbor, MICHIGAN, USA 3University Of Michigan,Mechanical Engineering,Ann Arbor, MICHIGAN, USA

Introduction: The molecular mechanisms associated with potentially lethal pulmonary hypoplasia and pulmonary hypertension in congenital diaphragmatic hernia (CDH) remain poorly understood. Periostin (POSTN) has been shown to be an important stress response molecule during the saccular stage of lung development and is a critical regulator of alveolar septation in association with α-smooth muscle actin (SMA) myofibroblasts. This study employed an ex vivo model to determine whether mechanical compression affects POSTN and other pulmonary transcripts during fetal CDH lung development.

Methods: Sprague-Dawley dams were gavaged with nitrofen (100 mg) at E9.5 gestation to induce fetal CDH pulmonary hypoplasia. Whole fetal rat lungs from nitrofen-exposed and control (vehicle only) dams at E15.5 were explanted and cultured ex vivo in customized chambers under static mechanical compression (0.2 or 0.4 kPa, n=8/group) for 12 hrs to mimic physiologic compression forces that occur in CDH in vivo. Lungs were evaluated for mesenchymal (POSTN, SMA, TGF-β) and epithelial (SP-C) expression by qPCR and immunohistochemistry (IHC). Statistical comparisons normalized to lungs at 0 kPa were made by analysis of variance with significance set at p<.05.

Results: Control lungs exposed to 0.2 and 0.4 kPa showed significant increases in POSTN expression (1.79±.10; 2.12±.39, respectively; both p<.001). In contrast, compressed nitrofen-exposed lungs revealed significant decreases in POSTN expression (0.4 kPa: 0.67±0.15, p<.001). IHC confirmed  increased presence of POSTN in control lungs, but not in nitrofen-exposed lungs (Figure). TGF-β was significantly increased in nitrofen-exposed lungs (1.39±.12, p=.045) and control lungs under increased compression (0.2 kPa: 1.33±.08, p=.036). Compression alone did not alter SMA expression in control lungs, but nitrofen-exposed lungs revealed significantly increased SMA at both 0.2 and 0.4 kPa (2.04±.15; 2.22±.11; both p<.001, respectively). Control lungs exposed to 0.4 kPa showed significant increases in SP-C (1.20±.20, p<.001). Conversely, nitrofen-exposed lungs had a significant reduction in SP-C expression at 0.2 and 0.4 kPa (0.53±.04, p<.01; 0.69±.23, p<.001; respectively).

Conclusion: Collectively these data suggest that mechanical compression induces a distinct transcriptome pattern within the nitrofen CDH fetal lung characterized by downregulation of POSTN and SP-C and upregulation of TGF-β and SMA. This ex vivo compression system may serve as a novel functional platform to better understand the impact of mechanical stress on the complex genetic control of matricellular dynamics during CDH lung development.

2.07 The In Vitro and In Vivo Effects of Granulocyte Colony Stimulating Factor on Metastatic Neuroblastoma

Posted on January 18, 2018

W. E. Barry1, G. Asuelime1, L. Wang2, E. S. Kim1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Pathology,Los Angeles, CA, USA

Introduction: We have previously described a small subpopulation of neuroblastoma (NB) cancer stem cells (CSCs) that express the granulocyte colony stimulating factor (G-CSF) receptor. In previous studies, the ligand G-CSF leads to increased primary tumor growth and increased metastasis in mice, but the impact of G-CSF on established metastatic disease is not fully understood. Despite these concerns, G-CSF is commonly administered to children with high-risk metastatic NB.  We therefore hypothesize that G-CSF will lead to a more aggressive phenotype in vitro and decreased survival in a murine NB metastatic model of minimal residual disease (MRD).

Methods: Metastatic cell lines from the liver and bone marrow of NSG mice were derived after orthotopic implantation of the human parental NB cell lines (CHLA-255 and SH-SY5Y).  DIMSCAN cell viability, colony formation and Matrigel invasion assays were performed using these metastatic cell lines with and without the addition of G-CSF. In vivo, 1 million cells (SH-SY5Y) were injected into the renal capsule of 14 NSG mice. The primary tumor was resected on post-injection day 7 and the mice were randomly assigned to either receive treatment of G-CSF (n=7) or no treatment (n=7). Mice receiving G-CSF were given 250ug/kg/day intraperitoneal for 14 days.  Metastatic disease was assessed with bioluminescent imaging and confirmed with histopathology.  Survival was analyzed using log-rank analysis, metastasis with Fisher’s exact test, and in vitro studies with Student’s t-test. 

Results: In vitro, increasing doses of G-CSF had no significant impact on cell viability by DIMSCAN assay, nor did G-CSF increase the invasive capacity of metastatic NB cells by matrigel invasion assay. Using a colony formation assay, the addition of G-CSF was found to increase colony counts in parental neuroblastoma cells (SH-SY5Y) and bone marrow metastatic cells compared to control (p≤0.01). In vivo, the burden of metastatic disease at week 4 by bioluminescent imaging, as well as the post-necropsy burden of liver disease were increased in mice treated with G-CSF, however this did not reach statistical significance (p=0.2 for both). Overall survival was significantly decreased in the mice treated with G-CSF (Figure).

Conclusions: In vitro, G-CSF does not appear to impact cell viability or invasiveness of metastatic NB cells but may have a modest effect on anchorage-independent growth, a hallmark of carcinogenesis. In vivo, G-CSF leads to decreased survival in a metastatic neuroblastoma mouse model of minimal residual disease. Further studies are necessary to validate and fully elucidate the potential etiologies of these findings to determine the safety of its use in patients with high-risk NB.

2.06 An Organotypic Spinal Cord Slice Culture Model of Fetal Myelomeningocele Hydrogel Patch Repair

Posted on January 18, 2018

K. A. Walker1, G. Jiang1, J. Di Bernardo1, K. O’Shea3, L. D. Shea4, S. M. Kunisaki1  4University Of Michigan,Biomedical Engineering,Ann Arbor, MI, USA 1University Of Michigan (Michigan Medicine),Surgery, Pediatric Surgery Section,Ann Arbor, MI, USA 3University Of Michigan (Michigan Medicine),Cell And Developmental Biology,Ann Arbor, MI, USA

Introduction:  Fetoscopic repair is an emerging surgical option to help preserve neural function in prenatally diagnosed myelomeningocele (MMC). However, the exact in utero repair strategy that would facilitate maximal regeneration of damaged neural tissue remains unknown. Our laboratory has recently developed a hydrogel-based surgical patch to facilitate tissue coverage during fetoscopic MMC repair. The purpose of this study was to determine whether an organotypic slice culture (OSC) model of fetal MMC could be established to study the effects of patch repair on underlying spinal cord development.

Methods:  Sprague-Dawley pregnant dams were gavaged with retinoic acid (60 mg/kg) at E10 gestation to induce fetal MMC. E21 rat pup spinal cords were longitudinally sectioned (400 microns) and cultured ex vivo in the presence of fibrin hydrogels (40 mg/mL) loaded with 6×105 rat dorsal root ganglion cells (group 1, n=10). Sections exposed to acellular fibrin hydrogels were used as controls (group 2, n=10). Neurotrophin expression, cell proliferation, apoptosis, and neural phenotype were subsequently analyzed at E21+7 and E21+14 by phase microscopy, qPCR, ELISA, and immunohistochemistry (IHC). The optical density (OD) of IHC sections was quantified using ImageJ. Statistical comparisons were by the unpaired t-test with significance set at p<0.05.

Results: Fetal OSCs were successfully cultured for up to 14 days while maintaining morphological integrity and demonstrating progressive neurite extension. qPCR showed upregulation of several neurotrophic genes, including neurotrophin-4/5 (NT4/5), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 compared to native control tissues. ELISA demonstrated an increase in NT4/5 over time whereas BDNF and CNTF decreased over time. IHC (Figure) showed no significant differences between group 1 and group 2 in terms of proliferation and apoptosis based on Ki67 and Cas3 expression, respectively. The neural progenitor marker, Nestin, was significantly increased in group 1 compared to group 2 at E21+7 (mean OD: 0.60±0.14 vs. 0.33±0.16, p<0.03), but these levels were similar at E21+14 (mean OD: 0.50±0.06 vs. 0.53±0.07, p=0.38). At both time points, there were no significant differences with respect to astrocyte (GFAP) and neuronal (beta-III tubulin, NeuN, and MAP2) differentiation with the damaged spinal cord. 

Conclusion: For the first time, this study demonstrates a novel ex vivo OSC model that can be sustained for a sufficient period of time to study the impact of patch repair on MMC neural tissue. Further work utilizing this organotypic approach to evaluate the effects of biomaterials, cells, and growth factors on MMC spinal cord development is warranted.

 

2.05 Alum Pretreatment Improves Survival from Murine Neonatal Sepsis and Alters Levels of IL-10 and pAKT

Posted on January 18, 2018

R. B. Hawkins1, J. C. Rincon1, S. L. Raymond1, R. Ungaro1, J. L. Wynn1, L. L. Moldawer1, S. D. Larson1  1University Of Florida College Of Medicine,Department Of Surgery,Gainesville, FLORIDA, USA

Introduction: Sepsis is a leading cause of morbidity and mortality in the neonatal population. Neonates have attenuated immunity compared to adult counterparts, but this difference is poorly understood. Interleukin-10 (IL-10) has emerged as an important anti-inflammatory cytokine that is associated with impaired neutrophil function and increased mortality from neonatal sepsis. Aluminum hydroxide (alum) is often used as an adjuvant in pediatric vaccines, but its use as an immune stimulant alone has not been well described. We have previously demonstrated that alum pretreatment improves survival from polymicrobial sepsis in neonatal mice. We sought to better understand the mechanism of alum’s survival benefit through its effects on IL-10 and phosphorylated protein kinase B (pAKT).

Methods:  Wild-type neonatal mice aged 5-7 days were used for experiments. Control mice received a sham injection while alum-pretreated mice received a subcutaneous injection of 50 µg alum 24 hours prior to induction of sepsis or sacrifice. 24 hours later, some mice were sacrificed and blood samples were obtained to evaluate for baseline cytokine changes associated with alum pretreatment. Sepsis was induced using the cecal slurry method. Mice were sacrificed at 6, 18, and 24 hours after induction of sepsis and blood and spleen samples were obtained. IL-10 cytokine analysis was performed using a customized mouse cytokine magnetic bead Milliplex® panel. pAKT levels were determined from splenic tissue at 6 and 24 hours following induction of sepsis using a Western blot assay.

Results: Neonatal mice who received alum pretreatment had a significant decrease in circulating IL-10 levels compared to non-treated counterparts at baseline. After induction of sepsis, both alum-pretreated and non-treated groups had significant elevation in IL-10 levels. There was no significant difference in circulating IL-10 levels between non-treated and alum-pretreated mice at 6, 18, and 24 hours after induction of sepsis. pAKT levels were significantly elevated at 6 and 24 hours after induction of sepsis. Alum pretreatment caused significantly less elevation of pAKT at 6 and 24 hours following induction of sepsis.

Conclusion: IL-10 is an important anti-inflammatory cytokine that may contribute to a diminished innate immune response in neonates. Alum pretreatment is associated with decreased circulating IL-10 levels at baseline, which may be partially responsible for alum’s survival benefit in the cecal slurry model. The PI3K-AKT pathway is involved in modulation of circulating IL-10 levels, and our data suggest that alum pretreatment limits production of pAKT following sepsis. These data suggest a possible therapeutic role for alum or other immunomodulators to affect the PI3K-AKT and IL-10 pathways to improve survival from neonatal sepsis.
 

2.04 Hydrogen Sulfide Donor GYY4137 Protects The Intestines Following Injury via eNOS Dependent Pathways

Posted on January 18, 2018

N. A. Drucker1, A. R. Jensen1, M. J. Ferkowicz1, T. A. Markel1  1Indiana University School Of Medicine,Pediatric Surgery,Indianapolis, IN, USA

Introduction: Intestinal ischemia in adults and necrotizing enterocolitis (NEC) in premature infants are both devastating intestinal conditions with poor outcomes.  GYY4137 is a long-acting hydrogen sulfide (H2S) donor, which may be protective against intestinal injury in these conditions.  We hypothesized that administration of GYY4137 would improve mesenteric perfusion, reduce intestinal injury, and reduce the inflammatory response in intestinal I/R injury and NEC, and that these benefits would be mediated through endothelial nitric oxide-dependent pathways.

Methods:  Adult male C57Bl/6 wild type (WT) and eNOS knockout (eNOSKO) mice underwent superior mesenteric artery occlusion for 60 minutes.  Prior to abdominal closure, 50mg/kg GYY4137 or PBS vehicle was administered intraperitoneally.  In separate groups, NEC was induced in WT and eNOSKO pups via maternal separation, formula feeding, enteral lipopolysaccharide, and intermittent hypoxic and hypothermic stress.  Pups received daily intraperitoneal injections of 50mg/kg GYY4137 in 10µL vehicle.  Laser Doppler Imaging was used to assess mesenteric perfusion in the adult animals at baseline and 24 hours post-ischemia.  Pups were assessed at baseline and P9.  After euthanasia, the terminal ileum of each animal was fixed, paraffin embedded, sectioned, and stained with H&E.  Sections were blindly graded using published injury scores.  Intestinal tissue was homogenized and cytokines measured by ELISA.  Data were compared using Mann-Whitney, and p-values <0.05 were significant.

Results: After I/R injury (A) and NEC (B), GYY4137 improved perfusion in WT mice compared to vehicle, but this effect was lost in the eNOSKO animals.  Histologic injury followed a similar pattern with reduced intestinal injury in WT mice treated with GYY4137, and no significant improvement in the eNOSKO group.  Cytokine expression after GYY4137 administration was altered by the ablation of eNOS in both I/R injury (C) and NEC (D).  Significant differences were noted in IL-6, IP-10, and VEGF in the NEC pups, but only in IP-10 in the adult mice after I/R.  

Conclusion: GYY4137, a long-acting donor of H2S, has potential as a therapeutic compound for intestinal ischemia.  It improves mesenteric perfusion and intestinal injury after ischemia and NEC, and these benefits appear to be mediated through endothelial nitric oxide dependent pathways.  

 

2.03 IL6 Is An Important Paracrine Factor Of USC Mediated Intestinal Protection Following Ischemia

Posted on January 18, 2018

B. S. Morocho1, N. A. Drucker1, J. P. Te Winkel1, M. J. Ferkowicz1, T. A. Markel1  1Indiana University School Of Medicine,Pediatric Surgery,Indianapolis, IN, USA

Introduction: Human umbilical mesenchymal stromal cells (USC) improve survival and mesenteric perfusion following intestinal ischemia.  USCs likely act via the paracrine release of specific key mediators, of which IL-6 may serve an important role. We hypothesized that IL-6 would play a key role in stem cell mediated intestinal protection following ischemia and reperfusion (IR) injury.

Methods: Eight to ten week old C57BL/6J male mice were used: 1) IR + Vehicle, 2) IR + USC, 3) IR + Negative Control siRNA treated USC and 4) IR + IL-6 siRNA treated USC. Mice were anesthetized using isoflurane and a midline laparotomy was performed. Initial perfusion of the small intestine was imaged using laser Doppler imaging. The superior mesenteric artery was then occluded for 60 minute with an atraumatic vascular clamp.  After ischemia, the clamp was removed and intestines allowed to recover.  Immediately prior to closure, mice were injected intraperitoneally with either 250 ul phosphate buffered saline vehicle, or with USC transfected with siRNA as above.  siRNA efficacy was confirmed by RT-PCR.  After 24 hours of recovery, perfusion was reassessed, mice were euthanized, and the intestines were harvested for histological analysis. Tissue blocks were H&E stained and sectioned.  Mucosal injury was assessed and graded by an established injury grading scale.  Perfusion was expressed as percentage of baseline (mean+/-SEM) and analyzed by student’s t test.  Histology was presented as injury score (median and IQR) and analyzed by Mann-Whitney.  P<0.05 was significant. 

Results: IL-6 siRNA effectively knocked down USC IL-6 production.  USCs, as well as USCs transfected with a negative siRNA control significantly increased perfusion (A) and improved mucosal injury scores (B) compared to vehicle following injury (Perfusion: Vehicle 42.49+/-9.28%, USC 78.3+/-8.83, siRNA Negative Control: 77.8+/-4.37,p<0.05; Histology: Vehicle: 3(2.75), USC: 1(0.75), siRNA Negative control: 1(1),p<0.05. USCs transfected with IL-6 siRNA yielded significantly lower mesenteric perfusion and worse mucosal injury scores compared to negative control (Perfusion: siRNA Negative control: 77.8+/-4.37, IL-6 siRNA: 28.4+/-5.84,p<0.05; Histology: siRNA Negative control:1(1), IL-6 siRNA: 3.5(2.25),p<0.05). 

Conclusion: USC mediated intestinal protection was negatively impacted by the decrease in IL-6 production from USCs. IL-6 likely acts in an anti-inflammatory manner, possibly through the membrane bound IL-6 receptor. Further studies are required to delineate the downstream effects of IL-6 during stem cell mediated intestinal protection. 

 

2.02 Macrophage-promoted Invasion of Osteosarcoma Requires Receptor-interacting Protein Kinase 2 (RIPK2)

Posted on January 18, 2018

C. Maloney1,2, M. P. Kallis1,2, M. C. Edelman4, M. Symons3, B. M. Steinberg1,3, S. Z. Soffer2,3  1Feinstein Institute Of Medical Research,Elmezzi School Of Molecular Medicine,Manhasset, NY, USA 2Hofstra Northwell School Of Medicine,Surgery,Manhasset, NY, USA 3Feinstein Institute Of Medical Research,Karches Center For Oncology And Cell Biology,Manhasset, NY, USA 4Hoftstra Northwell School Of Medicine,Pathology And Laboratory Medicine,Manhasset, NY, USA

Introduction: We have previously shown that macrophages promote osteosarcoma (OS) invasion in vitro which is inhibited by gefitinib, an epidermal growth factor receptor (EGFR) inhibitor (p<0.001). In vivo, gefitinib reduces both the incidence of gross lung metastasis and the metastatic outgrowth of pulmonary micro-metastases. However, EGFR is neither present on macrophages nor on OS cells, suggesting a non-EGFR mechanism for these findings. An alternative target of gefitinib, RIP2K, is expressed in antigen-presenting cells and plays a central role in NOD-mediated innate immune responses and NF-κB activation. The role of RIPK2 in tumor-associated macrophages has not been studied. We investigated the role of RIP2K in macrophage-promoted OS invasion in vitro and the effect of gefitinib on pulmonary macrophage phenotype in vivo

Methods: In vitro, mouse OS cells (K7M2) were incubated ± conditioned media from wild type or RIPK2 knock-out mouse bone marrow-derived macrophages (BMDMs) ± the RIP2K inhibitor OD36 (5.3nM).  Invasive capacity was assessed utilizing 3D-invasion assays. In vivo, K7M2 cells were implanted into the tibia of BALB/c mice (n=6). Mice were treated with either control or gefitinib-impregnated chow beginning 1 week post-implantation. Non-tumor-bearing wild type BALB/c mice were used as controls. Four weeks post-implantation, lungs were harvested and homogenized into a single-cell suspension. The cells were washed, stained with antibodies targeting the macrophage markers CD45, CD11b and F4/80, and MHC II, which is upregulated on pro-inflammatory macrophages, and subjected to flow cytometry. Data was acquired on an LSRFortessa Flow Cytometer (BD Biosciences) and analyzed with FlowJo (FlowJo, LLC).   

 

Results: In vitro, conditioned medium from wild-type BMDMs significantly promoted OS invasion (<0.001) while conditioned medium from RIPK2 knockout macrophages did not.  Conditioned medium from macrophages pretreated with the RIPK2 inhibitor also inhibited macrophage-promoted invasion (p<0.01).  Addition of the inhibitor to tumor cells in the absence of macrophages had no effect on OS invasion (p=0.876). In vivo, macrophages in the lungs of tumor-bearing mice showed decreased MHCII expression, a pro-inflammatory anti-tumor marker, when compared to non-tumor bearing control mice.  Conversely, macrophages from gefitinib-treated tumor-bearing mice displayed an increase in MHCII expression compared to untreated tumor-bearing mice, suggesting that gefitinib suppressed or reversed the polarization of the macrophages. 

Conclusions: Macrophage-promoted invasion of osteosarcoma requires RIPK2 activity. Gefitinib promotes a pro-inflammatory phenotype in pulmonary macrophages and prevents outgrowth of pulmonary metastases in vivo.  Upfront treatment with gefitinib may limit metastatic progression of OS by modulating macrophages via RIP2K inhibition.

 

 

2.01 PROMININ-1 Hepatic Progenitor Cell Ablation Increases Hepatic Fibrosis in Bile Duct Ligation

Posted on January 18, 2018

M. R. Fenlon1,2, J. Xu1, J. Zagory1, K. Asahina2, K. Wang1  1Children’s Hospital Los Angeles,Surgery,Los Angeles, CA, USA 2University Of Southern California,Los Angeles, CA, USA

Introduction:
In Biliary Atresia (BA), post-Kasai hepatoenterostomy survival with native liver inversely correlates with extent of liver fibrosis. We previously demonstrated an expansion of Prom1+ Hepatic Progenitor Cells (HPCs) within areas of developing ductular reactions in the Rhesus Rotavirus (RRV) model of experimental BA (Mavila, Hepatology 2014). Here, the degree of Prom1+ cell expansion correlated with the extent of fibrosis. We also previous demonstrated differential regenerative capacity of Prom1+ HPCs in neonatal mice compared to adult (Zhu, Cell, 2016). Moreover, null mutation of Prom1 leads to decreased ductular reaction and periportal fibrosis in RRV-induced BA (Zagory, unpublished data). Herein, we hypothesized that selective Prom1+ cell ablation is associated with decreases ductular reaction and liver fibrosis in an adult model of cholestasis caused by bile duct ligation (BDL).

Methods:
C57BL6 mice were bred to heterozygosity for both Prom1-Cre and inducible-Diphtheria Toxin Receptor (iDTR). 8-10 week old transgenic mice underwent BDL. Two weeks prior to BDL, tamoxifen was administered to induce cre-recombination in Prom1+ HPCs, resulting in targeted expression of iDTR. 1 week prior to BDL, Diphtheria Toxin (DT) or saline was injected to induce selective ablation of Prom1+ HPCs expressing iDTR. At 5 days post-BDL, whole liver tissue was collected and analyzed for histology and gene expression.

Results:
The extent of ductular reaction following BDL ± DT, as demonstrated by biliary epithelial marker CYTOKERATIN-19 (CK19) immunofluorescence, was comparable. Compared to sham, BDL resulted in increased Sirius red staining indicating increased deposition of excess extracellular matrix, a surrogate marker for liver injury (n = 13). Sirius red staining was greater post-BDL in DT treated mice compared to saline. Quantitative PCR analysis of hepatic fibrosis markers demonstrated trends towards increased fibrosis in BDL mice after DT ablation compared to saline group: α-Smooth Muscle Actin 4.19 vs 5.73, p=0.34; Vimentin 1.77 vs 6.31, p=0.15 (median fold expression of NS BDL and DT BDL groups).

Conclusion:
Contrary to our expected result, selective ablation of Prom1-expressing HPC leads to increased fibrosis following BDL in adult mice. This may be the consequence of differential regenerative capacity of Prom1+ HPC in adults compared to neonates. Further studies are essential to elucidate the functional role of Prom1+ HPC in infants with BA.
 

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