Jacob K. Olson

Heparin-binding epidermal growth factor–like growth factor overexpression in transgenic mice increases resistance to necrotizing enterocolitis

BACKGROUND Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency and the leading surgical cause of death in premature infants. We have shown that administration of exogenous heparin-binding epidermal growth factor-like growth factor (HB-EGF) in mice protects the intestines from experimental NEC. The aim of the current study was to evaluate the effect of gain-of-function of endogenous HB-EGF on susceptibility to NEC. METHODS Neonatal HB-EGF transgenic (TG) mice and their wild-type (WT) counterparts were exposed to experimental NEC. An additional group of HB-EGF TG pups were also exposed to NEC, but received the HB-EGF antagonist cross-reacting material 197 (CRM197) injected subcutaneously immediately after birth. To examine gut barrier function, HB-EGF TG and WT pups received intragastric fluorescein isothiocyanate-labeled dextran under basal and stressed conditions, and serum fluorescein isothiocyanate-labeled dextran levels were measured. RESULTS Wild-type mice had an incidence of NEC of 54.2%, whereas HB-EGF TG mice had a significantly decreased incidence of NEC of 22.7% (P = .03). Importantly, administration of CRM197 to HB-EGF TG pups significantly increased the incidence of NEC to 65% (P = .004). HB-EGF TG mice had significantly decreased intestinal permeability compared to WT mice both under basal and stressed conditions. CONCLUSIONS Our results provide evidence that overexpression of the HB-EGF gene decreases susceptibility to NEC and that administration of the HB-EGF antagonist CRM197 reverses this protective effect.

Exosomes secreted from bone marrow-derived mesenchymal stem cells protect the intestines from experimental necrotizing enterocolitis.

PURPOSE Treatment options for necrotizing enterocolitis (NEC) remain inadequate. Bone marrow-derived mesenchymal stem cells (BM-MSCs) can protect the intestines from NEC. Exosomes are nanoparticle-sized vesicles with important cell signaling capabilities. The objective of this study was to determine whether BM-MSC-derived exosomes can prevent NEC. METHODS Rat pups were either breast fed (Group 1) or subjected to experimental NEC and randomized to receive either no treatment (Group 2) or an intraperitoneal (IP) injection of PBS (Group 3), BM-MSC (Group 4), or BM-MSC-derived exosomes (Group 5). Histologic injury grade and intestinal permeability were determined. The effect of BM-MSC-derived exosomes on IEC-6 intestinal epithelial cells in an in vitro scrape model of wound healing was also determined. RESULTS Animals exposed to NEC that were either untreated or received PBS alone had an NEC incidence of 46% and 41%, respectively (p=0.61). Compared to untreated pups, the incidence of NEC was significantly lower in pups treated with either BM-MSC (9%, p=0.0003) or MB-MSC-derived exosomes (13%, p=0.0008). Similar results were found for intestinal permeability. Wound healing in IEC-6 cells was significantly increased by BM-MSC-derived exosomes. CONCLUSION BM-MSC-derived exosomes protect the intestines from NEC and may represent a novel, cell-free, preventative therapy for NEC in the future.

HEPARIN-BINDING EGF-LIKE GROWTH FACTOR PROMOTES INTESTINAL ANASTOMOTIC HEALING

BACKGROUND We have accumulated multiple lines of evidence supporting the ability of HB-EGF to protect the intestines from injury and to augment the healing of partial-thickness scald burns of the skin. The aim of the current study was to investigate the role of heparin-binding EGF-like growth factor (HB-EGF) in intestinal anastomotic wound healing. MATERIALS AND METHODS HB-EGF (-/-) knockout (KO) mice (n=42) and their HB-EGF (+/+) wild type (WT) counterparts (n=33), as well as HB-EGF transgenic (TG) mice (n=26) and their (WT) counterparts (n=27), underwent division and reanastomosis of the terminal ileum. In addition, WT mice (n=21) that received enteral HB-EGF (800 μg/kg) underwent the same operative procedure. Anastomotic bursting pressure was measured at 3 and 6 d postoperatively. Tissue sections were stained with hematoxylin and eosin to assess anastomotic healing, and Picrosirus red to assess collagen deposition. Immunohistochemistry using anti-von Willebrand factor antibodies was performed to assess angiogenesis. Complications and mortality were also recorded. RESULTS HB-EGF KO mice had significantly lower bursting pressures, lower healing scores, higher mortality, and higher complication rates postoperatively compared with WT mice. Collagen deposition and angiogenesis were significantly decreased in KO mice compared with WT mice. Conversely, HB-EGF TG mice had increased anastomotic bursting pressure, higher healing scores, lower mortality, lower complication rates, increased collagen deposition, and increased angiogenesis postoperatively compared with WT mice. WT mice that received HB-EGF had increased bursting pressures compared with non-HB-EGF treated mice. CONCLUSION Our results demonstrate that HB-EGF is an important factor involved in the healing of intestinal anastomoses.

Harvesting the benefits of biofilms: A novel probiotic delivery system for the prevention of necrotizing enterocolitis

BACKGROUND/PURPOSE Probiotics reduce the incidence of necrotizing enterocolitis (NEC) albeit only when administered at high frequency (at least daily). We have developed a novel probiotic delivery system in which probiotics are grown as a biofilm on microspheres, allowing enhanced efficacy with only a single treatment. METHODS Neonatal rats were subjected to experimental NEC. Pups received a single enteral dose of: (1) vehicle only, (2) unloaded microspheres, (3) MRS (broth)-loaded microspheres, (4) Lactobacillus reuteri, (5) L. reuteri grown on unloaded microspheres, or (6) L. reuteri grown on MRS-loaded microspheres. Intestinal injury was graded histologically and intestinal permeability determined by serum levels of enterally administered fluorescein isothiocyanate-labeled dextran. RESULTS 69% of untreated pups developed NEC, whereas 32% of pups treated with L. reuteri grown as a biofilm on unloaded microspheres (p=0.009) and 33% of pups treated with L. reuteri grown as a biofilm on MRS-loaded microspheres (p=0.005) developed NEC. No other group had a significant reduction in NEC. Furthermore, pups treated with L. reuteri grown as a biofilm had significantly reduced intestinal permeability. CONCLUSIONS A single dose of Lactobacillus biofilm grown on biocompatible microspheres significantly reduces NEC incidence and severity. This novel probiotic delivery system may be beneficial in the prevention of NEC in the future.

Stem cells and necrotizing enterocolitis: A direct comparison of the efficacy of multiple types of stem cells

PURPOSE Necrotizing enterocolitis (NEC) is a leading cause of gastrointestinal morbidity and mortality in premature infants. While studies have shown potential for stem cell (SC) therapy in experimental NEC, no study has compared different SC side-by-side. Our purpose was to determine whether one type of SC may more effectively treat NEC than others. METHODS Four SC were compared: (1) amniotic fluid-derived mesenchymal SC (AF-MSC); (2) amniotic fluid-derived neural SC (AF-NSC); (3) bone marrow-derived mesenchymal SC (BM-MSC); and (4) neonatal enteric neural SC (E-NSC). Using an established rat model of NEC, pups delivered prematurely received an intraperitoneal injection of SC. Control pups were injected with PBS. Additional controls were breast-fed by surrogates and not subjected to experimental NEC. Intestinal tissue was graded histologically. RESULTS NEC incidence was: PBS, 61.3%; breast-fed unstressed, 0%; AF-MSC, 19.1%; BM-MSC, 22.9%; AF-NSC, 18.9%; E-NSC 22.2%. All groups demonstrated statistical significance (p<0.05) compared to controls, and there was no difference between SC groups. CONCLUSION All four SC groups reduced the incidence and severity of experimental NEC equivalently. AF-MSC may be preferable because of availability of AF at delivery and ease of expansion, increasing potential for clinical translation. LEVEL OF EVIDENCE V (Animal study).

Heparin-binding EGF-like growth factor promotes neuronal nitric oxide synthase expression and protects the enteric nervous system after necrotizing enterocolitis

BackgroundNeonatal necrotizing enterocolitis (NEC) is associated with alterations of the enteric nervous system (ENS), with loss of neuronal nitric oxide synthase (nNOS)-expressing neurons in the intestine. The aim of this study was to investigate the roles of heparin-binding EGF-like growth factor (HB-EGF) in neural stem cell (NSC) differentiation, nNOS expression, and effects on ENS integrity during experimental NEC.MethodsThe effects of HB-EGF on NSC differentiation and nNOS production were determined using cultured enteric NSCs. Myenteric neuronal subpopulations were examined in HB-EGF knockout mice. Rat pups were exposed to experimental NEC, and the effects of HB-EGF treatment on nNOS production and intestinal neuronal apoptosis were determined.ResultsHB-EGF promotes NSC differentiation, with increased nNOS production in differentiated neurons and glial cells. Moreover, loss of nNOS-expressing neurons in the myenteric plexus and impaired neurite outgrowth were associated with absence of the HB-EGF gene. In addition, administration of HB-EGF preserves nNOS expression in the myenteric plexus and reduces enteric neuronal apoptosis during experimental NEC.ConclusionHB-EGF promotes the differentiation of enteric NSCs into neurons in a nitric oxide (NO)-dependent manner, and protects the ENS from NEC-induced injury, providing new insights into potential therapeutic strategies for the treatment of NEC in the future.

Single visit surgery for pediatric ambulatory surgical procedures: a satisfaction and cost analysis

BACKGROUND Single visit surgery (SVS) consists of same-day pre-operative assessment and operation with telephone post-operative follow-up. This reduces family time commitment to 1 hospital trip rather than 2-3. We began SVS for ambulatory patients with clear surgical indications in 2013. We sought to determine family satisfaction, cost savings to families, and institutional financial feasibility of SVS. METHODS SVS patients were compared to age/case matched conventional surgery (CS) patients. Satisfaction was assessed by post-operative telephone survey. Family costs were calculated as the sum of lost revenue (based on median income) and transportation costs (

An Enhanced Lactobacillus reuteri Biofilm Formulation that Increases Protection Against Experimental Necrotizing Enterocolitis

0.50/mile). RESULTS Satisfaction was high in both groups (98% for SVS vs. 93% for CS; p=0.27). 40% of CS families indicated that they would have preferred SVS, whereas no SVS families indicated preference for the CS option (p<0.001). Distance from the hospital did not correlate with satisfaction. Estimated cost savings for an SVS family was

Treatment of experimental necrotizing enterocolitis with stem cell-derived exosomes

188. Reimbursement, hospital and physician charges, and day-of-surgery cancellation rates were similar. CONCLUSIONS SVS provides substantial cost savings to families while maintaining patient satisfaction and equivalent institutional reimbursement. SVS is an effective approach to low-risk ambulatory surgical procedures that is less disruptive to families, facilitates access to pediatric surgical care, and reduces resource utilization. TYPE OF STUDY Cost Effectiveness Study. LEVEL OF EVIDENCE Level II.

Mice overexpressing the gene for heparin-binding epidermal growth factor-like growth factor (HB-EGF) have increased resistance to hemorrhagic shock and resuscitation

One significant drawback of current probiotic therapy for the prevention of necrotizing enterocolitis (NEC) is the need for at least daily administration because of poor probiotic persistence after enteral administration, increasing the risk of the probiotic bacteria causing bacteremia or sepsis if the intestines are already compromised. We previously showed that the effectiveness of Lactobacillus reuteri ( Lr) in preventing NEC is enhanced when Lr is grown as a biofilm on the surface of dextranomer microspheres (DM). Here we sought to test the efficacy of Lr administration by manipulating the Lr biofilm state with the addition of biofilm-promoting substances (sucrose and maltose) to DM or by mutating the Lr gtfW gene (encoding an enzyme central to biofilm production). Using an animal model of NEC, we determined that Lr adhered to sucrose- or maltose-loaded DM significantly reduced histologic injury, improved host survival, decreased intestinal permeability, reduced intestinal inflammation, and altered the gut microbiome compared with Lr adhered to unloaded DM. These effects were abolished when DM or GtfW were absent from the Lr inoculum. This demonstrates that a single dose of Lr in its biofilm state decreases NEC incidence. Importantly, preloading DM with sucrose or maltose further enhances Lr protection against NEC in a GtfW-dependent fashion, demonstrating the tunability of the approach and the potential to use other cargos to enhance future probiotic formulations. NEW & NOTEWORTHY Previous clinical trials of probiotics to prevent necrotizing enterocolitis have had variable results. In these studies, probiotics were delivered in their planktonic, free-living form. We have developed a novel probiotic delivery system in which Lactobacillus reuteri (Lr) is delivered in its biofilm state. In a model of experimental necrotizing enterocolitis, this formulation significantly reduces intestinal inflammation and permeability, improves survival, and preserves the natural gut microflora compared with the administration of Lr in its free-living form.