An enhanced Lactobacillus reuteri biofilm formulation that increases protection against experimental necrotizing enterocolitis.

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.

Evaluating the efficacy of different types of stem cells in preserving gut barrier function in necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. Increased intestinal permeability is central to NEC development. We have shown that stem cells (SCs) can reduce the incidence and severity of NEC. Our current goal was to investigate the efficacy of four different types of SC in preservation of gut barrier function during NEC. MATERIALS AND METHODS: We compared (1) amniotic fluid-derived mesenchymal SC, (2) bone marrow-derived mesenchymal SC, (3) amniotic fluid-derived neural SC, and (4) enteric neural SC. Premature rat pups received an intraperitoneal injection of 2 × 106 SC or phosphate-buffered saline only and were then subjected to experimental NEC. Control pups were breastfed and not subjected to NEC. After 48 h, animals received a single enteral dose of fluorescein isothiocyanate -labeled dextran (FD70), were sacrificed 4 h later, and serum FD70 concentrations determined. RESULTS: Compared to breastfed, unstressed pups with intact gut barrier function and normal intestinal permeability (serum FD70 concentration 2.22 ± 0.271 µg/mL), untreated pups exposed to NEC had impaired barrier function with significantly increased permeability (18.6 ± 4.25 µg/mL, P = 0.047). Pups exposed to NEC but treated with SC had significantly reduced intestinal permeability: Amniotic fluid-derived mesenchymal SC (9.45 ± 1.36 µg/mL, P = 0.017), bone marrow-derived mesenchymal SC (6.73 ± 2.74 µg/mL, P = 0.049), amniotic fluid-derived neural SC (8.052 ± 1.31 µg/mL, P = 0.0496), and enteric neural SC (6.60 ± 1.46 µg/mL, P = 0.033). CONCLUSIONS: SCs improve gut barrier function in experimental NEC. Although all four types of SC reduce permeability equivalently, SC derived from amniotic fluid may be preferable due to availability at delivery and ease of culture, potentially enhancing clinical translation.

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.

Development of a Standardized Scoring System to Assess a Murine Model of Clostridium difficile Colitis.

Background: Clostridium difficile infection is the most common cause of antimicrobial-associated diarrhea. Our aim was to introduce a novel and efficient clinical sickness score (CSS), and to define a detailed histologic injury score (HIS) in a murine model of C. difficile colitis. Methods: Mice received an antibiotic cocktail (kanamycin, gentamicin, colistin, metronidazole, and vancomycin) for 96 h. After 48 h, mice received an intraperitoneal injection of clindamycin, followed by oral C. difficile (1.5 × 107 CFU). Signs of sickness were scored using a novel CSS (range 0-12) with scores ≥6 consistent with C. difficile colitis. Intestinal tissue was analyzed utilizing an adapted HIS (range 0-9) with scores ≥4 consistent with C. difficile colitis. Stool was analyzed for C. difficile, and survival evaluated. Results: No control mice showed signs of sickness, whereas 23% of mice receiving antibiotics alone and 65% of mice exposed to antibiotics and subsequently C. difficile demonstrated signs of sickness (p = 0.0134). No control mice had histologic injury, whereas 8% of mice receiving antibiotics alone and 75% of mice exposed to antibiotics followed by C. difficile had evidence of histologic injury (p = 0.0001). Mice exposed to C. difficile lost more weight, although not significant (p = 0.070). Mice that received C. difficile had decreased survival compared to control mice and mice receiving antibiotics only (p = 0.03). Conclusions: We have developed a novel clinical scoring system, and detailed histological grading system, that enables the objective evaluation of a murine C. difficile colitis model. This model allows the study of this disease in a host that demonstrates clinical and histologic signs comparable to human C. difficile infection. This will allow for improved study of therapeutics for this disease in the future.

Treatment of experimental necrotizing enterocolitis with stem cell-derived exosomes.

PURPOSE: Necrotizing enterocolitis (NEC) remains a devastating disease in premature infants. We previously showed that four stem cell (SC) types equivalently improve experimental NEC. Exosomes are intercellular nanovesicles containing RNA, miRNA, DNA, and protein. Because SC therapy faces challenges, our aim was to determine if the beneficial effects of SC are achievable with cell-free exosomes. METHODS: Exosomes from four SC types were compared: (1) amniotic fluid-derived mesenchymal SC (AF-MSC); (2) bone marrow-derived MSC (BM-MSC); (3) amniotic fluid-derived neural SC (AF-NSC); and (4) neonatal enteric NSC (E-NSC). Rat pups exposed to NEC received a varying concentration of a single type of exosome with control pups receiving PBS only. Intestinal damage was graded histologically. RESULTS: The incidence of NEC was 0% in unstressed pups and 60.7% in control pups subjected to NEC. Exosomes (4.0×108) reduced NEC incidence to: AF-MSC 25.0%; BM-MSC 23.1%; AF-NSC 11.1%; E-NSC 27.3%. When administered at a concentration of at least 4.0×108, all groups demonstrated a significant reduction in NEC compared to untreated pups. At this minimum concentration, there was no difference in treatment efficacy between exosomes and the SC from which they were derived. CONCLUSION: Stem cell-derived exosomes reduce the incidence and severity of experimental NEC as effectively as the stem cells from which they are derived, supporting the potential for novel cell-free exosome therapy for NEC. TYPE OF STUDY: Basic science.

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).

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 ($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 $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.

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.

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.

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

BACKGROUND: The aim of the current study was to determine whether overexpression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) could protect the intestines from injury after hemorrhagic shock and resuscitation in mice. METHODS: Hemorrhagic shock and resuscitation was induced in HB-EGF transgenic and wild type mice. Cross-reacting material 197 (5 mg/kg) was administered to a subset of HB-EGF transgenic mice to block the overexpressed HB-EGF. Intestinal histologic injury scores, intestinal epithelial cell apoptosis indices, and gut barrier function were determined. The Student t test and 1-way analysis of variance were employed to compare the differences between groups. RESULTS: All mice subjected to hemorrhagic shock and resuscitation had significantly increased intestinal histologic injury scores, apoptosis indices, and intestinal permeability compared with sham-operated mice. Compared with wild type mice, HB-EGF transgenic mice had significantly decreased histologic injury (mean injury grade 2.79 ± 0.84 vs 3.88 ± 1.43, P = .02), apoptosis indices (mean apoptosis index 8.77 ± 5.23 vs 17.91 ± 13.23, P = .03), and mucosal permeability (FITC-dextran 4 clearance 13.06 ± 5.67 vs 20.03 ± 7.81 nL/min/ m(2), P = .02) at 3 hours of reperfusion. HB-EGF transgenic mice subjected to hemorrhagic shock and resuscitation and treated with cross-reacting material 197 had a significantly increased histologic injury (mean injury grade 3.63 ± 1.00 vs 2.79 ± 0.84, P = .04) and mucosal permeability (FITC-dextran 4 clearance 22.87 ± 9.69 vs 13.06 ± 5.67 nL/min/cm2, P = .01) at 3 hours of reperfusion compared with non-cross-reacting material 197 treated transgenic mice, with no significant changes in apoptosis indices. Cross-reacting material 197 did not reverse the decreased apoptosis observed in HB-EGF transgenic mice subjected to hemorrhagic shock and resuscitation, which suggests that mechanisms in addition to decreased apoptosis may be responsible for the intestinal cytoprotective effects of endogenous HB-EGF overexpression. CONCLUSION: Overexpression of HB-EGF increases resistance to hemorrhagic shock and resuscitation in mice.

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.