52 In Vitro Evidence of Cow's Milk Protein Sensitisation in Neonatal Necrotising Enterocolitis

Abstract

Background: Enteral feeding, particularly with formula feeds, has been implicated in the pathogenesis of necrotising enterocolitis (NEC). The direct role of milk antigens on mucosal and systemic inflammatory cascade in NEC has not been studied. The aim of the study is to characterise, in vitro, mucosal and systemic T-helper type 1/ type 2 (Th1/Th2) cytokine responses in NEC to mitogen and cow's milk protein (CMP) stimulation. Methods: 11 neonates Bell's stage 2–3 NEC [median post-conceptional age (range): 31(27–41) weeks] and 36 neonatal (non-inflamed) controls [preterm blood controls (n=21): 33(28–40) weeks; gut controls (n=15): 39(34–42) weeks] were studied. Mucosal specimens were obtained from surgical resection margins in 9/11 NEC and non-inflamed resections or biopsies in gut controls. Cytokine profile, interferon-gamma (IFNg), interleukin-4 (IL-4) and IL-5, was enumerated using the enzyme-linked immunospot (ELISPOT) assay, for peripheral blood (PBMC) and lamina propria mononuclear cells (LPMC), at rest and following stimulation with mitogens [PHA, ConA] and CMP [â-lactoglobulin (BLG), a-casein]. Results: PBMC: Compared to preterm blood controls, NEC infants had increased baseline secretion, vigorous responses to mitogens for all 3 cytokines (by 20–120 fold), a strong BLG response (IFNg > IL-4 > IL-5) and a smaller casein response (Table 1). LPMC: Gut controls had no detectable cytokine production at rest or with mitogen/antigen stimulation. NEC infants had only a small but significant increase in baseline cytokine secretion [median (25th–75th), IFNg: 3(0–15), p 0.003; IL-4: 2(1–9), p 0.002; IL-5: 1(0–4), p 0.13]. PHA and ConA produced an increase in mainly IFNg and IL-4 positive cells respectively [IFNg: 11(5–41), p< 0.05; IL-4: 13(4–20), p 0.01]. Only 3/9 NEC infants showed positive response to BLG (IFNg) and none to casein. Conclusion: Our study has demonstrated for the first time concomittant Th1/Th2 cytokine activation in NEC. We have also demonstrated significant CMP sensitisation primarily in the systemic compartment, without comparable mucosal activation, possibly suggesting a secondary event related to the alterations in mucosal barrier. These findings provide a novel mechanism for a potential direct contributory role of CMP in NEC inflammatory cascade. Clinical relevance with respect to e.g. post-NEC feeding regime management requires further investigation.