Background A major cause of enteric infection, Gram-negative pathogenic bacteria activate mucosal inflammation through lipopolysaccharide (LPS) binding to intestinal toll-like receptor 4 (TLR4). Breast feeding lowers risk of disease, and human milk modulates inflammation. Objective This study tested whether human milk oligosaccharides (HMOSs) influence pathogenic Escherichia coli-induced interleukin (IL)-8 release by intestinal epithelial cells (IECs), identified specific proinflammatory signalling molecules modulated by HMOSs, specified the active HMOS and determined its mechanism of action. Methods Models of inflammation were IECs invaded by type 1 pili enterotoxigenic E. coli (ETEC) in vitro: T84 modelled mature, and H4 modelled immature IECs. LPS-induced signalling molecules co-varying with IL-8 release in the presence or absence of HMOSs were identified. Knockdown and overexpression verified signalling mediators. The oligosaccharide responsible for altered signalling was identified. Results HMOSs attenuated LPS-dependent induction of IL-8 caused by ETEC, uropathogenic E. coli, and adherent-invasive E. coli (AIEC) infection, and suppressed CD14 transcription and translation. CD14 knockdown recapitulated HMOS-induced attenuation. Overexpression of CD14 increased the inflammatory response to ETEC and sensitivity to inhibition by HMOSs. 2′-fucosyllactose (2′-FL), at milk concentrations, displayed equivalent ability as total HMOSs to suppress CD14 expression, and protected AIEC-infected mice. Conclusions HMOSs and 2′-FL directly inhibit LPS-mediated inflammation during ETEC invasion of T84 and H4 IECs through attenuation of CD14 induction. CD14 expression mediates LPS-TLR4 stimulation of portions of the ‘macrophage migration inhibitory factors’ inflammatory pathway via suppressors of cytokine signalling 2/signal transducer and activator of transcription 3/NF-κB. HMOS direct inhibition of inflammation supports its functioning as an innate immune system whereby the mother protects her vulnerable neonate through her milk. 2′-FL, a principal HMOS, quenches inflammatory signalling.

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid‐state NMR spectroscopy with 100–111 kHz magic‐angle spinning (MAS). The excellent resolution in the Cα‐Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα–Hα detection block was developed and applied for the sequence‐specific backbone and aliphatic side‐chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

The immature neonatal intestinal immune system hyperreacts to newly colonizing unfamiliar bacteria. The hypothesis that human milk oligosaccharides from colostrum (cHMOSs) can directly modulate the signaling pathways of the immature mucosa was tested. Modulation of cytokine immune signaling by HMOSs was measured ex vivo in intact immature (fetal) human intestinal mucosa. From the genes whose transcription was modulated by cHMOSs, Ingenuity Pathway Analysis identified networks controlling immune cell communication, intestinal mucosal immune system differentiation, and homeostasis. cHMOSs attenuate pathogen-associated molecular pattern-stimulated acute phase inflammatory cytokine protein levels (interleukin-8 (IL-8), IL-6, monocyte chemoattractant protein-1/2 and IL-1β), while elevating cytokines involved in tissue repair and homeostasis. In all, 3′-, 4-, and 6′-galactosyllactoses of cHMOSs account for specific immunomodulation of polyinosinic:polycytodylic acid-induced IL-8 levels. cHMOSs attenuate mucosal responses to surface inflammatory stimuli during early development, while enhancing signals that support maturation of the intestinal mucosal immune system.

Background: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation.

Escherichia coli is, to date, the most used microorganism for the production of recombinant proteins and biotechnologically relevant metabolites. High density cell cultures allow efficient biomass and protein yields. However, their main limitation is the accumulation of acetate as a by-product of unbalanced carbon metabolism. Increased concentrations of acetate can inhibit cellular growth and recombinant protein production, and many efforts have been made to overcome this problem. On the other hand, it is known that E. coli is able to grow on acetate as the sole carbon source, although this mechanism has never been employed for the production of recombinant proteins. By optimization of the fermentation parameters, we have been able to develop a new acetate containing medium for the production of a recombinant protein in E. coli BL21(DE3). The medium is based on a buffering phosphate system supplemented with 0.5% yeast extract for essential nutrients and sodium acetate as additional carbon source, and it is compatible with lactose induction. We tested these culture conditions for the production of MNEI, a single chain derivative of the sweet plant protein monellin, with potential for food and beverage industries. We noticed that careful oxygenation and pH control were needed for efficient protein production. The expression method was also coupled to a faster and more efficient purification technique, which allowed us to obtain MNEI with a purity higher than 99%. The method introduced represents a new strategy for the production of MNEI in E. coli BL21(DE3) with a simple and convenient process, and offers a new perspective on the capabilities of this microorganism as a biotechnological tool. The conditions employed are potentially scalable to industrial processes and require only low-priced reagents, thus dramatically lowering production costs on both laboratory and industrial scale. The yield of recombinant MNEI in these conditions was the highest to date from E. coli cultures, reaching on average ~180 mg/L of culture, versus typical LB/IPTG yields of about 30 mg/L.