03.20 ASCL1 Knockdown Suppresses Neuroblastoma Growth through Induction of Apoptosis

Posted on January 18, 2016

S. S. Erickson1, M. Balamurugan1, S. Kunnimalaiyaan1, T. C. Gamblin1, M. Kunnimalaiyaan1 1Medical College Of Wisconsin,Surgical Oncology,Milwaukee, WI, USA

Introduction: Neuroblastoma is a tumor of the developing sympathetic nervous system and one of the leading causes of childhood cancer deaths. Despite recent therapeutic advances, about 60 % of patients with high-risk neuroblastoma experience relapse. The carcinogenic mechanisms driving these cancers are poorly understood, so advances in this area are much needed. The transcription factor ASCL1 is expressed in the early stages of neurogenesis and plays an important role in neuronal fate determination. It has been shown to be highly expressed in neuroendocrine tumors, including neuroblastoma. Results from the overexpression (in non-endocrine airway epithelial cells of transgenic mice) and depletion (in lung cancer cells) of ASCL1 suggest an oncogenic role for this protein. Its downregulation may therefore be a potential treatment strategy for neuroblastoma. In this study, we examined the biological consequences of ASCL1 depletion in neuroblastoma cells.

Methods: NGP cells were transfected with a doxycycline-inducible shRNA sequence against ASCL1 to create an NGP-ASCL1 knockdown (KD) cell line. As a control, non-specific, no-target scrambled sequence containing plasmid was transfected. These cells were selected on puromycin media. Western analysis was used to study the effects of doxycycline treatment on levels of ASCL1, apoptotic proteins, and neuroendocrine tumor markers for up to eight days. The proliferation of both control and doxycycline-treated cells was measured by MTT assay.

Results: Doxycycline treatment induced RFP expression and decreased ASCL1 protein levels in NGP-ASCL1 KD cells. The amount of ASCL1 reduction was dependent on the amount of doxycycline added to the media. ASCL1 knockdown was associated with decreased cellular proliferation at the 4 day (21 % reduction, p=.005), 6 day (48 %, p=.001), and 8 day (61 %, p<.001) time points. Knockdown cells also exhibited lower levels of the neuroendocrine tumor marker CgA, the cell cycle driver cyclin D1, and the pro-survival proteins Mcl1 and survivin.

Conclusion: The controlled system we have developed for regulated expression of ASCL1 allows direct analysis of loss?of?function, with ASCL1 reduction being dependent on the amount of doxycycline added to the media. ASCL1 knockdown in NGP neuroblastoma cells inhibits their proliferative abilities, apparently through an apoptotic pathway. Our data lend further support to the idea that ASCL1 is an attractive molecular target in neuroblastoma.

03.21 PIM Kinase Inhibition Decreases Tumorigenicity of Neuroblastoma Cells

Posted on January 18, 2016

L. L. Stafman1, A. M. Waters1, E. F. Garner1, J. E. Stewart1, E. A. Beierle1 1University Of Alabama,Birmingham, AL, USA

Introduction:
Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid tumor of childhood. For those with advanced or metastatic disease, this tumor carries a dismal prognosis. Given the current limited treatment options, novel therapies are needed. Proviral insertion site in Moloney murine leukemia virus (PIM) proteins are a family of serine/threonine kinases which has been implicated in tumorigenesis in many solid tumors including glioblastoma, but not studied extensively in neuroblastoma. We hypothesized that neuroblastoma cells would express PIM and that treatment of these cells with AZD1208 (AZD), a PIM-specific inhibitor, would lead to decreased cellular proliferation, migration, and invasion.

Methods:
Four human neuroblastoma cell lines – SK-N-AS, SK-N-BE(2), SH-EP and WAC2 – were utilized. PIM protein expression was determined by immunoblotting of whole cell lysates. PIM inhibition was accomplished with AZD, a competitive inhibitor that binds to the unique ATP pocket in PIM proteins. Proliferation was measured using CellTiter96 assays. Migration and invasion assays were completed with Transwell plates. Data reported as mean ± SEM. Student’s t-test was used to compare data between groups, with p ≤ 0.05 considered significant.

Results:
PIM protein expression was detected in all 4 neuroblastoma cell lines with immunoblotting. Inhibition of PIM with AZD (5µM, 12 hours) significantly decreased cellular proliferation in all 4 cell lines compared to control (vehicle, 12 hours) (Figure 1). In addition, PIM inhibition (AZD, 1µM) significantly inhibited cellular migration in the SK-N-AS (5872 ± 240 vs. 5000 ± 200 cells, control vs. AZD, p=0.03), SK-N-BE(2) (6580 ± 256 vs. 4182 ± 200 cells, control vs. AZD, p=0.0003), SH-EP (17388 ± 451 vs. 15960 ± 487 cells, control vs. AZD, p=0.04) and WAC2 (18200 ± 576 vs. 1660 ± 372 cells, control vs. AZD, p=0.03). Finally, AZD-induced PIM abrogation also significantly inhibited cellular invasion in the SK-N-AS (16,763 ± 559 vs. 14,868 ± 385 cells, control vs. AZD, p=0.03), SK-N-BE(2) (8872 ± 161 vs. 4909 ± 57 cells, control vs. AZD, p<0.0001) SH-EP (13,947 ± 396 vs. 6668 ± 203 cells, control vs. AZD, p<0.0001), and WAC2 (6944 ± 377 vs. 5707 ± 268 cells, control vs. AZD, p=0.04) cell lines.

Conclusion:
All four neuroblastoma cell lines utilized expressed PIM. The PIM inhibitor AZD1208 had a significant impact upon these neuroblastoma cells, resulting in decreased proliferation, migration, and invasion. These results indicate that further exploration of the PIM kinase pathway in neuroblastoma should be undertaken.

03.16 Developmental Expression of Intestinal Alkaline Phosphatase Activity in Premature Infants

Posted on January 18, 2016

S. M. Koehler1, K. Fredrich1, M. Christensen1, T. Nghiem-Rao2, D. Gourlay1 2Children’s Hospital Of Wisconsin,Neonatology,Milwaukee, WI, USA 1Children’s Hospital Of Wisconsin,Pediatric Surgery,Milwaukee, WI, USA

Introduction: Intestinal alkaline phosphatase (IAP) is an enzyme secreted from the mucosa of the gut which exerts a number of beneficial effects on the gut, including the enhancement of barrier function, reducing excessive inflammation, maintaining commensal bacteria and detoxifies lipopolysaccharide. The premature gut has a permeable intestinal barrier and an immature microbiome which predispose the premature neonate to necrotizing enterocolitis (NEC). Minimal research has focused on the activity of IAP in the premature gut, thus we sought to determine normal IAP activity levels for premature infants.

Methods: This is an observational prospective trial involving 10 neonates, ranging in age from 26 to 37 weeks estimated gestational age, in the neonatal intensive care unit at Children’s Hospital of Wisconsin (CHW). Inclusion criteria included: capable of per rectum stooling, parents/guardians consent to the study, and born at or transferred to CHW within 72 hours of life. Patients were excluded if they had life threatening congenital anomalies, congenital intestinal obstruction, or colostomy/ileostomy due to causes other than NEC. If possible, the first stool was collected and then weekly thereafter. The stool was homogenized and the amount of protein per volume determined. An AP activity assay was performed using a p-Nitrophenyl phosphate (pNPP) colorimetric assay. Absorbance of all samples was measured in triplicate at 405nM. The AP activity was measured in the presence of IAP and tissue nonspecific alkaline phosphatase inhibitors. A standard curve of pNPP dilutions was used to quantify the amount of AP activity.

Results: AP activity in all stool samples of premature infants is almost completely inhibited by phenylalanine, an IAP specific inhibitor, but minimally inhibited by a tissue non-specific alkaline phosphatase inhibitor. This cohort of premature infants has a heterogeneous amount of AP activity. For the majority of patients, there is a substantial drop in the AP activity beginning at approximately day of life 4. This decline was followed by an increase in AP activity over the following 2-3 weeks which generally reached and just exceeded the AP activity in the first stool. Only one patient, who had stool samples at the appropriate time points, did not follow this trend of increasing AP activity after an initial drop and this patient was being treated with meropenem for bacteremia.

Conclusions: AP activity in the stool of premature infants is almost exclusively due to IAP as it is inhibited by phenylalanine. The activity of IAP, while initially higher, declines rapidly within days of birth and then rises slowly over the following 3 weeks. The relatively high initial level of AP activity could explain the decreased incidence of NEC in the early postnatal period. Larger studies will be necessary to determine if the heterogeneity of the baseline IAP levels has a physiologic significance.

03.17 Identification of Lactobacillus Strains in the Early Postnatal Rat Microbiota

Posted on January 18, 2016

M. Isani1, A. Grishin1, H. Ford1 1Children’s Hospital Los Angeles,Los Angeles, CA, USA

Introduction:
Necrotizing enterocolitis (NEC) affects premature infants and its etiology remains largely unknown. Colonization of the neonatal gastrointestinal (GI) tract with opportunistic pathogens, such as Coronobacter muytjensii, is thought to be a prime initiating event. Opportunistic pathogens compromise the gut barrier, leading to bacterial translocation, inflammation, and, ultimately, intestinal necrosis. Lactobacillus biotics have been shown in a number of studies to protect against NEC. However, results of Lactobacillus trials remain inconclusive due to the use of different species/strains and doses in different trials. Moreover, an important question of whether the lactobacilli used as probiotics are colonizing the intestine remains unanswered. We propose that an efficient probiotic strain should not only protect the intestinal epithelium, but should also be capable of colonizing the GI tract. To identify naturally occurring colonizing lactobacilli, we sought to isolate and characterize the strains of this genus in the intestines of 4 day old rats.

Methods:
Animal experiments were approved by CHLA IACUC. Neonates were obtained from timed pregnant Sprague Dawley rats purchased from Charles River or Harlan Labs. The neonates were kept in a temperature and humidity controlled incubator, and fed with formula for 4 days. Care was taken not to introduce extraneous bacteria during handling and feeding. To enumerate and isolate the lactobacilli, the content of the large intestine was serially diluted and plated on MRS agar. After 2 day incubation at 37oC, the emerging colonies were classified according to their appearance, and numbers in each class counted. Pure cultures were established for each class and maintained as frozen stocks. The lactobacilli were identified as Gram+ or Gram+/- non-spore-forming rods. The species identity was established by sequencing a variable region of the 16S rRNA gene and by biochemical tests.

Results:
Seven morphologically unique strains of lactobacilli were isolated from 138 rat pups belonging to 18 litters (Figure 1). Among the identified species, there were L. reuteri and L. murinus.

Conclusion:
Various lactobacilli are common first GI tract colonizers in neonatal rats. Availability of the pure cultures of the natural colonizing Lactobacillus strains will allow us to experimentally evaluate their ability to colonize the intestine and protect against NEC.

03.18 Alteration in VEGFR-2 Expression Contributes to Pathophysiology of Necrotizing Entercolitis (NEC)

Posted on January 18, 2016

J. A. Shepherd1, P. J. Matheson1, J. W. Smith1, R. N. Garrison1, C. D. Downard1 1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:

Necrotizing enterocolitis (NEC) alters ileal blood flow via dysregulation of mediators of vascular tone, but deranged intestinal angiogenesis might also contribute to impaired perfusion. Vascular endothelial growth factor receptor 2 (VEGFR-2) may have an important role in neonatal intestinal vascular development and control. We hypothesized that the gene and protein dysregulation that occurs during experimental NEC results in altered VEGFR-2 protein expression.

Methods:

Sprague-Dawley rats were randomly separated into NEC and CONTROL groups by litter. NEC groups were delivered by C-section 12 hrs prematurely, formula fed, exposed to intermittent cold and hypoxia, and given a single oral dose of lipopolysaccharide (n=95). CONTROLs were delivered vaginally and dam-fed. Ileum samples were obtained at 0, 12, 24, 48, 72 and 96 hours of life and Western blots were performed with antibodies against VEGFR-2 and β -actin for normalization by individual animal. Statistical analysis was performed using 2-way ANOVA and a priori P<0.05.

Results:

In NEC animals, ileal VEGFR-2 levels increase markedly from the 0 time point, peaking at 24 hours, before returning to baseline levels at 96 hours of life (see Figure 1). In the CONTROL animals, ileal VEGFR-2 concentration was significantly lower than in NEC animals until 96 hours of life.

Conclusion:

VEGFR-2 is the major signaling receptor for VEGFA, the vasoactive form of VEGF. In this experimental model of NEC, VEGFR-2 expression is increased in NEC compared to CONTROL animals, suggesting dysfunction in this intestinal microvascular angiogenesis pathway. When coupled with alterations in VEGFA levels in NEC, this could provide an explanation for vascular dysfunction and poor intestinal perfusion in NEC.

03.14 Genetic Pathway Analysis of Infants with Necrotizing Enterocolitis

Posted on January 18, 2016

C. C. Culbreath1, B. Halloran2, D. K. Crossman3, N. Ambalavanan2, T. Jilling2, C. A. Martin1 1University Of Alabama At Birmingham,Pediatric Surgery/Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Neonatology/Pediatrics,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Bioinformatics/Genomic Science,Birmingham, Alabama, USA

Introduction: Necrotizing enterocolitis (NEC) continues to be the leading gastrointestinal cause of demise in premature infants. Lack of representative animal models is a challenge in clarifying the pathogenesis of NEC. In depth molecular analysis of human tissue is hence required for understanding the mechanisms involved in NEC development.

Methods: Six premature infants that had a surgical resection for NEC and 6 age-matched ileal atresia controls were identified from the medical records. RNA was isolated from formalin fixed paraffin embedded tissue sections using the Rneasy FFPE kit from Qiagen. RNA quality was verified using an Agilent Bioanalyzer and by performing reverse transcription real time PCR analysis of housekeeping genes (GAPDH and 18S). Only RNA that exhibited good quality with both methods was used for microarray analysis. Gene expression analysis by microarray was then performed with the Illumina HT-12_V4 chip. A subsequent Ingenuity Pathway Analysis (IPA) was then done to identify top canonical pathways and regulators involved. All studies were done in accordance after approval by the University of Alabama at Birmingham Institutional Review Board.

Results:Greater than 47,000 genes were screened. A total of 47 genes were found to have a fold change > 2, and 37 genes had a fold change > 2 and p < 0.05. Pathway analysis determined top canonical pathways (Table 1), top upstream regulators, top diseases and biofunctions, top organ toxicity functions, and top regulator effector networks. The top upstream regulators were STAT3, Prolactin (PRL), IL1b, STAT1, and IFNg.

Conclusion:Paraffin sections are a reliable source of quality RNA for microarray analysis. Identification of relevant pathways and signal transducers by pathway analysis are being validated by protein expression to guide future studies in understanding mechanistic pathways resulting in necrotizing enterocolitis.

03.15 EGFR Signaling modulates HIF1α expression in intestinal epithelial and endothelial cells.

Posted on January 18, 2016

B. Aladegbami1, J. Guo1, B. W. Warner1 1Washington University,Pediatric Surgery,St. Louis, MO, USA

Introduction: Following massive small bowel resection (SBR), an important programmed compensatory process takes place in the remaining bowel termed adaptation. This adaptive response is characterized by increased enterocyte proliferation leading to taller villi, deeper crypts, and an expanded mucosal surface area. The magnitude of adaptation is regulated at least in part by intact epidermal growth factor receptor (EGFR) signaling. Recently, we have found that adaptation is also associated with diminished oxygen delivery, increased hypoxia-inducing factor (HIF1α) expression, and subsequent enhanced angiogenesis within the villi and submucosal layer in the remnant bowel. The aim of this study is to elucidate the potential role for EGFR signaling as a regulator of HIF1α expression in both enterocytes and endothelial cells.

Methods: Rat Intestinal Epithelial Cells (RIEC) and Human Intestinal Endothelial Cells (HIMEC) were serum-starved for 24 hours. The cells were then treated with either EGF (50ng/ml) alone, EGF plus EGFR inhibitors (Geftinib (ZD1839) or Analinoquinazolin (AG1478)) or the EGFR inhibitors alone for 1 hour. This was followed by a period of either normoxia or hypoxia (0.5 % Fi02) for another 4 hours. Cells were washed and harvested directly into 1X SDS sample loading buffer. A Western blot was then performed to assess the effects of EGFR signaling manipulation and expressions of HIF1α in these cell lines

Results: HIF1α expression could be detected only under hypoxic condition in both epithelial and endothelial cells. EGFR is activated at both normoxia and hypoxia conditions as demonstrated by EGFR phosphorylation in both cells. EGFR inhibitors effectively diminished EGFR and MAP-Kinase activity in both normoxia and hypoxia cells upon EGF stimulation. Interestingly HIF1α expression is also reduced in those cells with blunted EGFR activation.

Conclusion: EGFR signaling appears to play a role in the stimulation of HIF1α expression in both enterocytes and endothelial cells. These findings illuminate a novel mechanism for enhanced angiogenesis associated with resection-induced intestinal adaptation.

03.12 An Innovative Bioengineering Alternative To Open Fetal Repair Of Spina Bifida Defects

Posted on January 18, 2016

R. Marwan1, S. Williams1, J. Bardill2, D. Park2, N. Abd Aziz1, R. Marwan1 1University Of Colorado Denver,Pediatric Surgery,Aurora, CO, USA 2University Of Colorado Denver,Bioengineering,Aurora, CO, USA

Introduction: Neural tube defects (NTDs) are the leading cause of central nervous system malformation in humans & a devastating birth defect resulting in destruction of the developing spinal cord. Their sequelae are staggering & appear not only to have anatomical effects, but also functional, emotional & psychological morbidities including bladder & bowel incontinence, paralysis, musculoskeletal deformity, & shunt malfunctions & infections. Ultimately, its compound nature results in an immense financial burden amounting to $1,400,000. The morbidity & mortality associated with having a NTD is secondary not only to ongoing spinal cord injury as a result of prolonged mechanical trauma to the neural elements, but also chemical inflammation associated with exposure to amniotic fluid. Currently, pre-natal repair is performed relatively late, around 22-26 weeks of gestation & is associated with a 50% reduction in the need for ventriculoperitoneal shunting. However, it is associated with an increased risk to both mother & fetus including premature labor & prematurity among others. Reverse Thermal Gels are a group of biomimetic polymers that can undergo reversible phase transition from liquid to physical gel upon temperature change without the need for reactive species or outside energy sources making them suitable for biological use. We propose to test if an earlier repair using a bioengineering approach will result in better outcomes. This study focuses on the properties of our polymer

Methods: Surface ultrastructure of the polymer was studied using scanning electron microscopy of a 0.5cm3 mold of 3 different percent mass/volume (%m/v) freeze-dried polymer samples. Permeability to fluorescein and albumin was tested using 0.4mm pore polymer coated inserts in a 12 well plate. A micro-plate reader measured the absorbance of fluid in the bottom well. Degradation in both human & sheep amniotic fluid was studied using Gel Permeation Chromatography. Finally, proliferation of murine embryonic fibroblasts was measured using an MTT proliferation assay kit.

Results: Our polymer possesses a unique surface ultrastructure dictated by concentration and is tied to its biological properties. A 15% m/v sample has smaller pore sizes when compared to 10% & 13% samples. Permeability to fluorescein is predictable and depends on the pore sizes. At 1 day, 10% m/v polymer is more permeable to fluorescein than 13% & 15%. However, after 8 days, it is equivalent. There is minimal permeability to albumin. The polymer does not degrade in neither human nor sheep amniotic fluid for up to 30 days. Finally, compared to control media, murine embryonic fibroblasts possess a similar proliferation profile when subjected to polymer extract.

Conclusion: Our polymer is stable in amniotic fluid and possesses a unique ultrastructure and functional characteristics that supports its use as an innovative bioengineering approach to open fetal repair of NTDs.

03.13 Effects of an Artificial Placenta on Development and Brain Injury in Premature Lambs

Posted on January 18, 2016

N. L. Werner1, M. Coughlin1, C. J. Perez-Torres4, H. Parmar5, R. Shellhaas2, J. Barks6, J. R. Garbow3, G. B. Mychaliska1 1University Of Michigan,Pediatric Surgery,Ann Arbor, MI, USA 2University Of Michigan,Pediatric Neurology,Ann Arbor, MI, USA 3Washington University,Radiology,St. Louis, MO, USA 4Purdue University,Radiological Health Sciences,West Lafayette, IN, USA 5University Of Michigan,Radiology, Division Of Neuroradiology,Ann Arbor, MI, USA 6University Of Michigan,Pediatric Neonatolgy,Ann Arbor, MI, USA

Introduction: The effect of an extracorporeal artificial placenta (AP) on the developing brain is unknown. The aim of this study was to evaluate brain structure after AP support using magnetic resonance imaging (MRI).

Methods: Institutional guidelines for animal research were followed. AP lambs (n=5), with estimated gestational age (EGA) of 120±2 days (term=145 days), were cannulated for veno-venous ECMO on placental support and delivered. AP support with heparin was maintained for 7±0.5 days before euthanasia at EGA~127 days. Control(C) lambs, with an EGA of 120±2 days (n=3) or 127±2 days (n=2), were euthanized immediately after delivery. All brains were flushed with formalin for fixation and then characterized by MRI. A 25-direction diffusion sequence with an additional b=0 image (no diffusion weighting) was used to calculate fractional anisotropy (FA) and apparent diffusion coefficient (ADC) to qualitatively evaluate white-matter integrity. The b=0 image, plus a T1 sequence, were used to measure brain volume and hull, and to evaluate for hemorrhage.

Results: The AP brains had no evidence of hemorrhage or white-matter injury (Figure A, B). Brain volume appeared to increase with gestational age (C-120 day 1.77E4 mm3 v. C-127 day 1.79E4 mm3). AP brains were on average smaller than either control (1.55E4 mm3). The hull increased with gestational age (C-120 day 1.92E4 mm3 v. C-127 day 2.08E4 mm3). The volume/hull ratio, which is surrogate marker of cerebral folding, showed AP brains with a ratio between the 120 and 127 day controls (C-120 days 86% v. AP 89% v. C-127 days 92%). The ventricles of AP brains were mildly enlarged compared to controls (Figure C, D).

Conclusion: The artificial placenta provided long-term extracorporeal support with no evidence of brain hemorrhage or white-matter injury. The data suggest some ongoing brain development during AP support. Compared to controls, the AP brains were smaller and had larger ventricles; the clinical significance of these findings is unknown.

03.09 IGF-1 intraplacental gene transfer restores serum bioavailability of IGF-1 in growth restricted mice

Posted on January 18, 2016

S. M. Deeney1, K. N. Powers1, B. Dodson1, K. W. Liechty1, A. Marwan1, T. M. Crombleholme1 1Laboratory For Fetal And Regenerative Biology,Department Of Surgery, School Of Medicine, University Of Colorado Denver – Anschutz Medical Campus; Division Of Pediatric General Thoracic And Fetal Surgery, Colorado Children’s Hospital,Aurora, CO, USA

Introduction:

Intrauterine growth restriction (IUGR) is characterized by decreased serum bioavailability of IGF-1 both clinically and across a wide range of animal models. Our laboratory recently described a novel surgical mouse model of intrauterine growth restriction (IUGR) and rescued the IUGR phenotype using intraplacental adenovirus mediated gene transfer of human insulin-like growth factor-1 (Ad-IGF-1). Mouse birthweight, growth trajectory, glucose tolerance and blood pressure correct to control levels. The mechanism by which fetal reprogramming occurs has yet to be fully elucidated. We hypothesize that Ad-IGF-1 intraplacental gene transfer restores the bioavailability of serum levels of IGF-1 in IUGR pups.

Methods:
In time-mated e18 C57/BL6J dams, one of two naturally occurring mesenteric uterine artery branches were ligated in one pup (excluding the pups at the uterine ends), inducing IUGR (n=3). Control pups were those with dual arteries on the opposite uterine horn as well as sham-operated pups (n=5). A subset of IUGR pups (n=3) received 5 ul intraplacental injections of 1×10^8 pfu adenovirus expressing human IGF-1 after artery ligation. Serum samples were obtained by decapitation of the pups following delivery via hysterotomy at e20. Serum and placenta homogenate levels of mouse IGF-1 and IGFBP-3 were measured in duplicate by ELISA. Bioavailability of IGF-1 was defined as the molar ratio of IGF-1 to IGFBP-3. Statistical analysis was by Student’s T-test.

Results:
Serum levels of bioavailable IGF-1 were significantly reduced in IUGR pups compared to controls (P=0.01) and were restored with Ad-IGF-1 gene transfer (p>0.05). Placental levels of bioavailable IGF-1 were not different in all groups (p>0.05).

Conclusion:
Intraplacental Ad-IGF-1 gene transfer restores the serum levels of bioavailable IGF-1, while not changing these levels in the placenta itself. This is the first demonstration of how Ad-IGF-1 gene transfer restores pup serum bioavailable IGF-1 levels in IUGR mice, suggesting a possible mechanism by which IGF-1 induces fetal reprogramming of IUGR mice.

03.10 Ileal CCL3 and IL-12 Expression in Inflammatory Events Associated with Necrotizing Enterocolitis

Posted on January 18, 2016

K. Connolly1, P. J. Matheson2, J. A. Shepherd2, J. W. Smith2, R. N. Garrison2, C. D. Downard2 1University Of Louisville,School Of Medicine,Louisville, KY, USA 2University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction: Macrophages clear bacteria from the gut in the early stages of necrotizing enterocolitis (NEC), a potentially lethal inflammatory bowel disease of premature infants. Chemokine (C-C) ligand 3 (CCL3 or macrophage inflammatory protein-1a, MIP-1a) and Interleukin-12 (IL-12) are key chemokines/cytokines in this process. CCL3, a chemokine, recruits immune cells such as monocytes and neutrophils to the site of injury and induces the release of other pro-inflammatory cytokines including IL-1, IL-6, and tumor necrosis factor-a (TNF-a). IL-12, a pro-inflammatory cytokine, recruits and activates immune cells including T cells and NK cells.. We hypothesized that CCL3 and IL-12 expression in the ileum might be increased in the context of NEC-associated inflammation.

Methods: Sprague-Dawley rats were randomized to groups by litter. CONTROLS were delivered vaginally and dam-fed. NEC groups were delivered by C-section 12 hours prematurely, formula fed, exposed to intermittent cold and hypoxia, and given a single oral dose of lipopolysaccharide. Ileum samples were obtained at 0, 12, 24, 48, 72 and 96 hours of life and western blots were performed with antibodies against CCL3, IL-12, and b-actin for normalization by individual animal. Statistical analysis was performed using 2-way ANOVA and a priori P<0.05.

Results: Ileal CCL3 and IL-12 protein expression (see Figure) in NEC was below Control levels at 24 hours, but increased at 72 hours versus Controls (*P<0.05). The change in ileal protein expression occurred between the 24 and 48 hour time points for both mediators.

Conclusion: These data support our prior studies that demonstrated a similar pattern of CCL3 and IL-12 expression in the serum of NEC rats versus Control. Since macrophage function plays a key role in the early development of NEC, down-regulation of IL-12 in the setting of increased CCL3 might represent deranged or inhibited macrophage function in the NEC disease process.

03.11 Chronic Hypoxia Promotes Hypoxia-Inducible Factor-Independent Growth Response in Neuroblastoma

Posted on January 18, 2016

A. L. Alvarez1, B. T. Craig1, E. J. Rellinger1, J. Qiao1, D. H. Chung1 1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA

Introduction: Neuroblastoma is responsible for nearly 15% of all pediatric cancer-related deaths and remains a difficult pediatric solid tumor to cure. Acute tumor hypoxia in neuroblastoma is known to promote dedifferentiation and drug resistance in part by stabilizing hypoxia-inducible factors (HIF-1α and HIF-2α ) which function as key drivers of glycolysis, and has been well characterized. Chronic tumor cell hypoxia is also likely to exist in aggressive solid tumors such as neuroblastoma, yet its exact role in tumorigenicity remains poorly understood. We hypothesize that chronic hypoxia is a microenvironmental stress stimulus that plays a critical role in driving neuroblastoma progression. The aim of this study was to establish an in vitro model of chronic hypoxia to assess neuroblastoma cell growth.

Methods: Human neuroblastoma cell lines, BE(2)-C (MYCN-amplified) and SK-N-SH (MYCN-single copy), were cultured in 1% ambient oxygen for 1, 3, 7, or 10 d. Post-hypoxia cell viability was assessed over a time course (0 to 96 h) using CCK-8 assay. Soft agar colony formation after 2 weeks incubation in 0.4%/0.8% agarose gel was used to assess anchorage-independent growth. SDS-PAGE was used to assess protein expression. Two-tailed unpaired Student’s t-test was used for statistical analysis and a p value of <0.05 was considered significant.

Results: Ten days of 1% O2 increased the proliferative capacity of both cell lines compared to controls cultured at ambient O2 (p <0.01 for each individual comparison). Specifically, MYCN–single copy SK-N-SH cells demonstrated a two-fold increase in proliferation following 10 d exposure to 1% O2 compared to controls cultured in 21% O2. A more modest increase (1.5-fold) was observed in the MYCN-amplified BE(2)-C cells. Anchorage-independent growth was also observed in both BE(2)-C and SK-N-SH cells cultured for 10 d at 1% O2. The well-established mediators of the acute hypoxic response HIF-1α and HIF-2α were both upregulated by 1 d as expected, validating the hypoxia culture chamber. Interestingly, expression levels of both factors returned to basal levels by 10 d of continuous hypoxic exposure. Hexokinase-1 and lactate dehydrogenase-A, two downstream markers of glycolytic flux, were similarly decreased at 10 d of continuous exposure to 1% O2.

Conclusion: Neuroblastoma is a pediatric solid tumor with significant intra-tumoral heterogeneity featuring areas of necrosis and hemorrhage that may implicate chronic hypoxia as a key microenvironmental regulator of tumor progression. Our model shows that prolonged continuous exposure to subnormal oxygen levels induces a phenotype that enhances both cellular proliferation and anchorage independent growth. This behavioral change appears to be both a HIF- and glycolysis-independent phenomenon.

03.06 An Alternative DNA Repair Pathway Enhances Neuroblastoma Chemoresistance in Hypoxic Microenvironments

Posted on January 18, 2016

L. Gaston1, E. Corwin1, D. Bashllari1, B. Cummings1, A. Shiang1, X. Topalli1, V. Castle1, E. Newman1 1University Of Michigan,Pediatrics, Obstetrics And Gynecology, And Pediatric Surgery,Ann Arbor, MI, USA

Introduction: Neuroblastoma (Nb) is a neoplasm of neural crest origin that accounts for 8% of childhood malignancies and 15% of cancer deaths in this same population. This discrepancy may be attributed to high-risk patients presenting with widely disseminated disease, of which only 25% survive despite cytotoxic therapies. Metastatic Nb cells display increased chemoresistance and a hypoxic environment has been shown to dedifferentiate Nb cells to an immature neuroblastic state with rapid growth and cell survival advantage. Our preliminary data demonstrates that hypoxia induces expression of DNA Ligase III (lig3), a mediator of an alternate nonhomologus end-joining (altNHEJ) DNA repair program in Nb cells. Additional work from our laboratory has shown increased expression of this efficient yet error prone repair pathway in tumorigenic Nb cell lines and in tumors of patients with poor survival outcomes. Given this, we hypothesized that induction of altNHEJ confers a survival advantage to Nb cells in hypoxic microenvironments and to standard chemotherapeutic agents.

Methods: S-type Nb cells (SHEP) were divided into three treatment groups and cultured for 1) 72h at 21% O2, 2) 72h at 1% O2, and 3) 72h at 1% O2 followed by 24h reperfusion. Cells were fixed and analyzed by immunocytochemistry (ICC) for gH2AX and lig3 with DAPI mounting solution. Images were generated with fluorescent microscopy at 40x. Quantitative analysis was carried out with ImageJ software, expressed as mean number of foci/nucleus±SD. SHEP and IMR32 Nb cells were then cultured under the above conditions with doxorubicin, a standard cytotoxic utilized in Nb treatment (0, 1, 5, and 10µg/mL). Cell viability was determined at 72h by trypan blue exclusion. Lig3 expression was analyzed by ICC as described above. All experiments were repeated in triplicate and statistical analysis was performed with ANOVA.

Results: In hypoxic conditions, Nb cells acquired more DNA damage compared to normoxic controls (0.080±0.014 vs. 0.025±0.021 gH2AX positive foci/nucleus, p<.05) with decreased overall survival (38%±4 vs 78%± 12, p<.01). Hypoxic Nb cells had relative resistance to increasing doses of doxorubicin compared to normoxic controls at 1, 5, and 10µg/mL( p<.05). Lig3 expression increased in surviving hypoxic, doxorubicin-treated Nb cells in a dose-dependent manner (p<.0001).

Conclusion: Lig3, a major DNA repair factor in altNHEJ is activated in hypoxic conditions and confers Nb cell survival advantage to Doxorubicin. Lig3 or its interacting altNHEJ partners may prove promising therapeutic targets for Nb patients with high-risk disease.

03.07 The Artificial Placenta Promotes Type 2 Pneumocyte Maturation in Fetal Lambs

Posted on January 18, 2016

M. A. Coughlin1, N. L. Werner1, K. Marchetti1, L. Pierce1, T. Major1, G. B. Mychaliska1 1University Of Michigan,ECLS Laboratory,Ann Arbor, MI, USA

Introduction: Recreating the intrauterine environment with an extracorporeal artificial placenta (AP) to treat extreme prematurity is a promising strategy. The aim of this study was to assess lung development during AP support.

Methods: Institutional guidelines for animal research were followed. AP lambs of estimated gestational ages 116-120 days (term=145 days) were cannulated for VV-ECLS (internal jugular vein drainage and umbilical vein reinfusion) on placental support and delivered. They were intubated and the endotracheal tube filled with fluid and clamped (n=3) or held at a constant pressure of 8cmH2O (n=2). Tissue control (TC) animals were delivered and immediately euthanized. These were divided into early gestational age (115-122 days, n=4) and later gestational age (123-129 days, n=3). A bronchoalveolar lavage was obtained during necropsy on all animals and spun down to remove the solid cell component. The remaining fluid was used to obtain ELISA assays for surfactant related proteins B and C (SPB/SPC).

Results: The AP animals survived an average of 7 days. Levels of SPB in early vs. late tissue controls were 37.3±2 vs. 44.6±1.4 pg/ml (p=0.002). Levels of SPC in early vs. late tissue controls were 12.7±2 vs. 17±5.6 pg/ml (p=0.07). The average concentration of SPB in the AP lambs vs. later tissue controls was 41.1±5.5 vs. 44.6±1.4 pg/ml (p=0.24). The average concentration of SPC in the AP lambs vs. later tissue controls (Figure) was 22.4±1.8 vs. 17±5.6 pg/ml (p=0.02).

Conclusion: There was a significant difference seen in the levels of SPB and a trend toward increased levels of SPC with increasing ages of tissue controls. SPB levels are comparable between controls and AP animals. SPC levels were significantly higher in AP animals compared to controls. These data suggest that the artificial placenta promotes type 2 pneumocyte maturity.

03.08 Silencing of Intersectin 1 Decreases Neuroblastoma Tumor Growth in an Orthotopic Mouse Model

Posted on January 18, 2016

J. C. Harris1, A. Russo3, E. Herrero3, J. P. O’Bryan3, B. Chiu2 1Rush University Medical Center,Surgery,Chicago, IL, USA 2University Of Illinois At Chicago,Pediatric Surgery,Chicago, IL, USA 3University Of Illinois At Chicago,Pharmacology,Chicago, IL, USA

Introduction: Neuroblastoma is the most common solid extracranial tumor in childhood and accounts for 15% of all pediatric cancer deaths. Intersectin 1 (ITSN1) protein is involved in phosphoinositide 3-kinase (PI3K) signaling, which has been shown to stabilize the MYCN oncogenic transcription factor. Silencing ITSN1 in vitro significantly reduced the anchorage-independent growth of neuroblastoma cells. We hypothesize that silencing ITSN1 with shRNA in neuroblastoma cells leads to decreased tumor growth in an orthotopic mouse model.

Methods: We have previously created stable SK-N-AS neuroblastoma cell lines with empty vector (pSR) and vector that contains scrambled shRNA (pSCR), that served as control lines, or vector that contains shRNAs to ITSN1 (Sh#1 and Sh#2). Orthotopic neuroblastoma tumors of each cell line were established in three immunocompromised mice by injecting 1×106 cells directly into the adrenal gland. Tumor volume was monitored weekly with ultrasound, using tumor volume >1000mm3 as the end point. Harvested tumors were analyzed with anti-ITSN1 antibody on Western blot. Tumor growth was analyzed using Kaplan Meier curves and student’s T-test, with p<0.05 deemed statistically significant.

Results: Orthotopic tumors were successfully created in all cell lines. At day 25-post injection, tumor size for pSR was 1235.7±761.2mm3, pSCR was 1311.4±296.5mm3, Sh#1 was 574.4± 539.9mm3, Sh#2 was 439.5±95.3mm3. Sh#2 was significantly smaller than pSCR (p=0.027), and there was a trend toward significance that Sh#1 was smaller than pSCR (p=0.13). Overall survival was superior in Sh#2, 31±1.7 days, compared to pSCR, 25±0 days (p=0.025). Again, a trend towards overall survival in Sh#1, 31±7.2 days, compared to pSCR, 25±0 days (p= 0.11). There was no difference in tumor growth between pSR with the other groups, likely due to high variance within this group, overall survival of 29.67±14.1 days. Western blot analysis of the harvested tumor showed decreased ITSN1 expression in experimental groups Sh#1 and Sh#2 compared to the control groups pSR and pSCR.

Conclusion: Silencing ITSN1 expression in neuroblastoma cells leads to decreased tumor growth in vivo. We demonstrate that orthotopic animal models can serve as an authentic ‘read out’ of molecular manipulation of ITSN1 signaling in vitro. Thus, understanding the role of ITSN1 signaling pathways in tumor development may provide new insight into neuroblastoma tumorigenesis.

03.03 Sphingolipid Signaling in Enteric Glia: Implications for Intestinal Disorders of Inflammation

Posted on January 18, 2016

B. D. Bauman1, J. Meng1, S. Banerjee1, S. Roy1, W. Kennedy3, B. J. Segura1,2 2University of Minnesota,Pediatric Surgery,Minneapolis, MINNESOTA, USA 3University Of Minnesota,Neurology,Minneapolis, MINNESOTA, USA 1University Of Minnesota,General Surgery,Minneapolis, MINNESOTA, USA

Introduction:
Our studies presently underway are aimed at better understanding the underlying risks of pediatric intestinal disorders including inflammatory bowel disease and necrotizing enterocolitis (NEC) in premature infants. Our central hypothesis is that the relative prematurity of patients with NEC puts at them at greater risk of intestinal inflammation due to the underdeveloped nature of their enteric nervous system. Evidence from our lab and others suggests enteroglia may play a significant role in modulating intestinal inflammatory disorders through the maintenance of gut barrier function. Specifically, we have found that sphingosine-1-phosphate (S1P), a bioactive lipid, and FTY720, a known S1P agonist, mitigates intestinal inflmmation. To more fully understand the link between enteric glia, bioactive lipid signaling and NEC, we are now extending our studies into models of pediatric disease.

Methods:
Using in-vivo approaches involving DSS induced colitis in the mouse model, and in-vitro approaches involving the study of rat intestinal epithelial IEC-6 cells we investigated the putative signaling properties of the bioactive lipid sphingosine-1-phosphate (S1P)–known to cause calcium signaling in enteric glia, which bear its receptors based on our prior studies.

Results:

The S1P analog FTY720 (0.3 mg/kg daily by oral gavage) markedly attenuated the inflammatory response induced by 3.5% dextran sodium sulfate (DSS) by H&E analysis compared with controls in a mouse model of colitis at 3 and 5 days. Cultured rat intestinal epithelial cells (IECs) demonstrated enhanced tight junction formation (Occludin staining and transepithelial resistance) in response to enteric glial cell (EGC)-cultured media within 48 hours. By flow cytometry, Rat EGCs display traditional markers and the Toll like receptor 4 (TLR4). By QPCR, Rat EGCs show diminished expression of TLR4, TNFa, IL-1b, and iNOS response to S1P (1uM) treatment.

Conclusion:
Our data suggest that the sphingolipid analog FTY720 and S1P have effects upon inflammation in-vivo and in-vitro, respectively. This suggests that sphingolipids may modulate intestinal inflammation through a mechanism involving the enteric nervous system, potentially serving as a mode of preventive therapy in adults and children alike

03.04 The Feasibility of Spring Mediated Extra-Peritoneal Intestinal Lengthening

Posted on January 18, 2016

A. Scott1, J. D. Rouch1, N. Huynh1, E. Chiang1, S. L. Lee1, B. M. Wu2, J. C. Dunn1,2, S. Shekherdimian1 1University Of California – Los Angeles,Department Of Surgery, Division Of Pediatric Surgery,Los Angeles, CA, USA 2University Of California – Los Angeles,Department Of Bioengineering,Los Angeles, CA, USA

Introduction: Currently, models used for mechanical intestinal lengthening achieve lengthening within the intra-abdominal cavity. Here we show that spring-mediated lengthening can be achieved outside the intra-abdominal cavity through a stoma.

Methods: Using Sprague-Dawley rats, the defunctionalized limb of a Roux-en-y jejunojejunostomy was exteriorized from the peritoneal cavity through a defect created in the anterior abdominal wall (n=8). An encapsulated polycaprolactone spring was placed into the extra-abdominal segment. The stoma containing the spring was secured under the skin to protect it from desiccation and destruction. After 4 weeks, segments were retrieved for histological analyses. Animals who had blank capsules placed in the extra-peritoneal de-functionalized limb served as controls (n=3).

Results: Stomal segments were successfully lengthened from 1.0 cm to 2.5 ± 0.4 cm, whereas control segments were lengthened from 1.0 cm to 1.5 ± 0.1 cm (p<0.05). After lengthening, the exteriorized bowel remained viable and patent. Lengthened segments had increased smooth muscle thickness and crypt depth when compared to normal jejunal mucosa.

Conclusion: Spring-mediated mechanical lengthening is not limited to the abdominal cavity. Extra-peritoneal lengthening yields greater than a 2-fold increase in intestinal length. Lengthening through a stoma removes the need to enter the abdomen for subsequent re-lengthening of intestinal segments.

03.05 Escherichia coli 07:K1 CE10 Colonizes Neonatal Rats and Protects from Necrotizing Enterocolitis

Posted on January 18, 2016

J. D. Bowling1, J. Lim1, J. Golden1, A. Dossa1, B. Bell1, L. Chase1, J. Wang1, A. Grishin1, H. R. Ford1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction: Early post-natal microbiota is presumed to play a critical role in the pathogenesis of necrotizing enterocolitis (NEC), a severe gastrointestinal disease found often in premature infants. Whereas some colonizing bacteria may act as opportunistic pathogens, other bacteria might be innocuous and even protective. In our previous studies, the presence of E. coli CE10 in 4 day old rats negatively correlated with NEC. Here we tested the hypothesis that CE10 is capable of colonizing neonatal rats and protecting them from NEC.

Methods: E. coli CE10 was transformed with a plasmid (pJK_proK14_eGFR) conferring ampicillin resistance and expression of green fluorescent protein (GFP). Transformed bacteria were introduced to neonatal rats with the first feeding. Afterwards, the animals were subjected to 4 days of the NEC-inducing formula-feeding/hypoxia (FF/H) regimen. NEC was scored histologically. Stool and ileum samples were plated for total bacterial load and the numbers of E. coli C10. The level of apoptosis was analyzed using TUNEL staining and fluorescence microscopy.

Results: E. coli CE10 established itself as a first colonizer in animals that received it. FF/H alone caused NEC in 60% of neonatal rats. Introduction of E. coli CE10 reduced the incidence of NEC to 39% (p = 0.027). FF/H yielded a higher rate of apoptosis than E. coli CE10 via TUNEL staining. EC was scored histologically. Stool and ileum samples were plated for total bacterial load and the numbers of E. coli C10. The level of apoptosis was analyzed using TUNEL staining and fluorescence microscopy

Conclusion: E. coli 07:K1 CE10 is an efficient first colonizer in neonatal rats. CE10 significantly reduced the incidence of NEC. Bacteria similar to this strain can be used for the prophylaxis of NEC.

29.09 Deficiency in MFG-E8 Decreases Survival in Neonatal Sepsis by Exacerbating Intestinal Injury

Posted on January 18, 2016

L. W. Hansen1,2, W. Yang1,2, A. Khader1, J. M. Prince1,2, J. M. Nicastro1, G. F. Coppa1, P. Wang1,2 1Hofstra North Shore-LIJ School Of Medicine,Surgery,Manhasset, NY, USA 2The Feinstein Institute For Medical Research,Center For Translational Research,Manhasset, NY, USA

Introduction: Polymicrobial sepsis in neonates remains a major cause of morbidity and mortality. A breakdown in intestinal integrity contributes to the severity of this disease. Providing breast milk to premature infants can reduce the risk of necrotizing enterocolitis (NEC). Milk fat globule–EGF factor 8 (MFG-E8) is a secretory glycoprotein and is abundant in mammalian breast milk. MFG-E8 has anti-inflammatory activity and plays a major role in the maintenance of intestinal epithelial homeostasis. Thus, we hypothesize that MFG-E8 will reduce mortality in neonatal sepsis by protecting their intestinal integrity.

Methods: Newborn wild-type (WT) C57BL/6 and MFG-E8 knockout (KO) mice stayed with their mothers throughout the experiments. At age 5-7 days (3-4 g), the neonatal mice were made septic by intraperitoneal injection of cecal slurry (CS; 0.75 mg CS/g body weight) and monitored for a 7-day survival study. The CS was prepared from a mixture of cecal contents from 7 adult male WT mice (8-10 weeks old). To study intestinal injury, the neonatal mice were injected with 0.9 mg CS/g body weight (n=6-8 per group). At 10 h after injection, the small intestine was collected for analysis by ELISA, Western blot, myeloperoxidase activity, and endotoxin assay.

Results: The 7-day survival of septic WT neonates was 73%, while all the septic MFG-E8 KO neonates died within 2 days (Figure, P < 0.001). Protein levels of proinflammatory cytokines IL-6 (730.2 ± 105.9 pg/mg) and IL-1β (447.0 ± 45.6 pg/mg) in the intestine of septic MFG-E8 KO mice were increased 1.9- and 2.0-fold, respectively, compared to intestinal IL-6 (375.8 ± 50.9 pg/mg) and IL-1β (224.2 ± 47.9 pg/mg) in septic WT mice. The myeloperoxidase activity, a marker of neutrophil infiltration, in the intestine of septic MFG-E8 KO neonates was 3.8–fold higher than that in septic WT neonates (P < 0.05). There was a slight increase in serum endotoxin levels in septic MFG-E8 KO mice compared to septic WT mice (37.4 ± 9 vs. 25.8 ± 7 EU/ml). Additionally, septic MFG-E8 KO neonates had a reduced induction of phosphorylated AKT, which is an indication of activation of the cell survival pathway, compared to septic WT neonates.

Conclusion: Breast milk protein MFG-E8 can protect neonatal mice from polymicrobial sepsis by suppressing intestinal inflammation and promoting the cell survival pathway. Supplementation of MFG-E8 for critically ill infants may represent a viable therapeutic strategy.

29.10 Alum Pretreatment Enhances Phagocytosis and Improves Survival in Neonatal Polymicrobial Sepsis

Posted on January 18, 2016

B. Mathias1, J. Rincon1, A. L. Cuenca1, D. Nacionales2, L. L. Moldawer2, S. D. Larson1 1University Of Florida,Division Of Pediatric Surgery/Department Of Surgery,Gainesville, FL, USA 2University Of Florida,Department Of Surgery,Gainesville, FL, USA

Introduction: Each year, severe infection and sepsis in the neonatal period is responsible for over 3 million deaths worldwide. Contributing to this high mortality is the neonate’s functionally distinct innate immune response. In previous studies, we have demonstrated that adjuvant activation via the TLR4 receptor improves outcomes in neonatal sepsis. Unfortunately, TLR agonists (e.g. LPS, resiquimod) are contraindicated in clinical use due to sepsis-like symptoms they induce. Aluminum (alum) salts are currently used as adjuvants in pediatric vaccines to improve immune responses; however, the mechanisms by which alum mediate these responses are incompletely understood. Therefore, the purpose of our study was to determine if alum enhances immune effector cell recruitment and function in the setting of polymicrobial sepsis.

Methods: 5-7 day old (neonate) C57BL/6J (B6) mice received either no pretreatment (control) or subcutaneous (SQ) injection of aluminum hydroxide (alum; 100 µg) 24 h prior to sepsis. Neonates then underwent intraperitoneal (IP) administration of cecal slurry (CS; LD25-45) to induce intra-abdominal polymicrobial sepsis. Following injection of CS, mice were observed for 7 days to determine survival. Peritoneal wash was collected at 0, 2, 6 and 24 h following sepsis. Harvested cells were analyzed by flow cytometry for cell phenotype and phagocytosis activity (E.coli bioparticles).

Results: Subcutaneous alum pretreatment did not alter peritoneal macrophage or neutrophil recruitment following IP septic challenge. Despite a decrease in total peritoneal macrophages from baseline (0 h) in both groups, alum pretreatment significantly improved peritoneal macrophage phagocytosis at 6 h compared to controls (70.4±6.2 vs. 40.2±13.2; p<0.05). Alum pretreatment 24 h prior to septic challenge also demonstrated improved macrophage phagocytosis at baseline (68.9±19.2 vs. 17.8±26.6; p<0.05). Peritoneal neutrophil phagocytosis was unchanged at baseline and 6 h following septic challenge. However, neutrophil phagocytosis was a significantly increased at 18 h (88.2±9.9CS vs. 62.2±12.1; p<0.001) and 24 h (62.6±24.2 vs. 30.3±13; p<0.01) following sepsis in alum pretreated neonates compared to controls. Alum pretreated neonates had significantly improved survival compared to control animals when challenged with cecal slurry (p=0.023).

Conclusion: We demonstrate here that the vaccine adjunct alum modulates immune effector cell function and improves survival to polymicrobial sepsis in neonates. Alum pretreatment leads to improved phagocytosis in peritoneal macrophages and neutrophils likely influencing this survival advantage. These findings suggest that alum improves the neonate’s innate immune response and offers a potential novel clinical approach to preventing neonatal sepsis.

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