Interrogation of Milk-Driven Changes to the Proteome of Intestinal Epithelial Cells by Integrated Proteomics and Glycomics.

Bovine colostrum is a rich source of bioactive components which are important in the development of the intestine, in stimulating gut structure and function and in preparing the gut surface for subsequent colonization of microbes. What is not clear, however, is how colostrum may affect the repertoire of receptors and membrane proteins of the intestinal surface and the post-translational modifications associated with them. In the present work, we aimed to characterize the surface receptor and glycan profile of human HT-29 intestinal cells after exposure to a bovine colostrum fraction (BCF) by means of proteomic and glycomic analyses. Integration of label-free quantitative proteomic analysis and lectin array profiles confirmed that BCF exposure results in changes in the levels of glycoproteins present at the cell surface and also changes to their glycosylation pattern. This study contributes to our understanding of how milk components may regulate intestinal cells and prime them for bacterial interaction.

Immunomodulatory effects of natural polysaccharides assessed in human whole blood culture and THP-1 cells show greater sensitivity of whole blood culture.

Immunomodulatory drugs are available to maintain immune homeostasis but some have undesirable side effects. Six oligo- and poly-saccharides were assessed for their pro- and anti-inflammatory responses in two in vitro model systems, the monocytic THP-1 cell line and human whole blood cultures (HWBC). The compounds were first characterised for their molecular mass and physical properties. Following incubation with lipopolysaccharide (LPS) or the compounds, cytokine and chemokine secretion was assayed in both models and intracellular TNF-α was measured by flow cytometry in HWBC cell sub-populations. LPS, inulin, galacturonan, heteroglycan and fucoidan demonstrated pro-inflammatory properties and intracellular TNF-α expression was increased in the monocytes of HWBC. Mannan and xyloglucan did not elicit any significant responses. Inulin induced maximum cytokine secretion and heteroglycan induced maximum chemokine secretion in HWBC. This study emphasises the potential of inulin and heteroglycan as potential immunomodulatory therapeutics and that HWBC had a greater and more varied response in comparison to THP-1 cells.

Comparative study of the skin penetration of protein transduction domains and a conjugated peptide.

PURPOSE: We examined the ability of a protein transduction domain (PTD), YARA, to penetrate in the skin and carry a conjugated peptide, P20. The results with YARA were compared to those of a well-known PTD (TAT) and a control, nontransducing peptide (YKAc). The combined action of PTDs and lipid penetration enhancers was also tested. METHODS: YARA, TAT, YKAc, P20, YARA-P20, and TAT-P20 were synthesized by Fmoc chemistry. Porcine ear skin mounted in a Franz diffusion cell was used to assess the topical and transdermal delivery of fluorescently tagged peptides in the presence or absence of lipid penetration enhancers (monoolein or oleic acid). The peptide concentrations in the skin (topical delivery) and receptor phase (transdermal delivery) were assessed by spectrofluorimetry. Fluorescence microscopy was used to visualize the peptides in different skin layers. RESULTS: YARA and TAT, but not YKAc, penetrated abundantly in the skin and permeated modestly across this tissue. Monoolein and oleic acid did not enhance the topical and transdermal delivery of TAT or YARA but increased the topical delivery of YKAc. Importantly, YARA and TAT carried a conjugated peptide, P20, into the skin, but the transdermal delivery was very small. Fluorescence microscopy confirmed that free and conjugated PTDs reached viable layers of the skin. CONCLUSIONS: YARA and TAT penetrate in the porcine ear skin in vitro and carry a conjugated model peptide, P20, with them. Thus, the use of PTDs can be a useful strategy to increase topical delivery of peptides for treatment of cutaneous diseases.