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Necrotizing Enterocolitis Is Induced by Bacterial Fermentation of Formula: Role of Innate Immunity In a Novel Porcine Model

Saturday, October 20, 2012: 9:51 AM
Versailles Ballroom (Hilton Riverside)
Shreyas K. Roy, MD, CM1, Qinghe Meng1, Benjamin Sadowtiz1, Gary Nieman1, Tamer Ahmed1, David Clark, MD2, Michael Ratner, MD3 and Robert N. Cooney, MD3, (1)Surgery, SUNY Upstate Medical University, Syracuse, NY, (2)Pediatrics, Children's Hospital Albany, Albany, NY, (3)SUNY Upstate Medical University, Syracuse, NY

Purpose: Although Necrotizing Enterocolitis (NEC) is the leading gastrointestinal cause of neonatal mortality, a unifying mechanism incorporating all clinical associations is lacking. We Hypothesize that NEC occurs secondary to bacterial fermentation of infant formula. In this study, we specifically hypothesized acidic byproducts of bacterial fermentation cause intestinal inflammation. We evaluated intestinal epithelial cell expression of TLR-2, TLR-4, NF- kB, IL-1b, and TNF-a to elucidate the role of innate immunity in the mediation of bacterial fermentation-induced NEC.

Methods: Piglets (n=9) were anesthetized, surgically instrumented, and divided into 3 groups: Bacteria + Formula (n = 3): Formula was incubated with non-toxigenic E. Coli until pH reached 5.5, confirming fermentation, and fed to animals via post-pyloric gastrostomy tube. Bacteria Only (n = 3):  Formula incubated with E. Coli was centrifuged to produce a bacterial pellet which was resuspended in normal saline (pH 5.5) and fed to animals. Formula Only (n = 3): Formula was fed to animals. After 6-h intestine was removed and washed with cold PBS. Intestinal mucosal scrapings were frozen and stored at -700C. Real-time PCR was performed to quantify TLR-2, TLR-4, NF- kB, IL-1b, and TNF-a mRNA with b-actin as control.  Electrophoretic mobility shift assay for NF-kB activation was performed along with Western Blot analysis for relative abundance of NF-kB and TLR-4. Data are expressed as fold-induction, analyzed with ANOVA (p<0.05).

Results: Bacteria + Formula group developed classic features of NEC: intestinal inflammation, necrosis, pneumatosis intestinalis, and portal venous gas. Bacteria Only and Formula Only groups displayed normal intestinal histology. Bacteria + Formula group had the greatest increase in mRNA expression of TLR-2, IL-1, TNF-a, and NF-kB (2.1, 31, 3.5 and 1.7 folds respectively), and along with the Bacteria Only group (1.6, 29, 3 and 1.5 folds respectively), was significantly elevated compared to Formula Only. Bacteria + Formula group had decreased TLR-4 mRNA and protein levels vs. other groups. EMSA showed NF-kB binding activity was increased in Bacteria + Formula group. NF-kB protein (p 65 subunit) was increased in the Bacteria + Formula and Bacteria Only (1.9 and 1.67 folds) vs. Formula Only groups.

Conclusions: Confirming our hypothesis, this novel animal model of NEC produced the clinical, gross pathologic and histological NEC phenotype in the Bacteria + Formula group and not in animals fed bacteria or formula alone. The mRNA expression of TLR-2 and NF-kB were equivalently elevated in Bacteria + Formula and Bacteria Only vs. Formula Only group, suggesting innate immune signaling alone is insufficient for development of NEC. IL-1b was significantly elevated in Bacteria + Formula vs. other groups corroborating gross and histological findings. These results represent a novel, clinically relevant porcine model to assist in elucidating the alternations in the innate immune system in NEC pathogenesis.