Dietary Milk-Fat-Globule Membrane Affects Resistance to Diarrheagenic Escherichia coli in Healthy Adults in a Randomized, Placebo-Controlled, Double-Blind Study.

BACKGROUND: The milk-fat-globule membrane (MFGM) contains phospholipids and membrane glycoproteins that have been shown to affect pathogen colonization and gut barrier integrity. OBJECTIVE: In the present study, we determined whether commercial heat-treated MFGM can increase resistance to diarrheagenic Escherichia coli. METHODS: A randomized, placebo-controlled, double-blind, 4-wk parallel-intervention study was conducted in healthy adults. Participants were randomly assigned to a milk protein concentrate rich in MFGM [10 g Lacprodan PL-20 (Arla Foods Ingredients Group P/S), twice daily; n = 30; MFGM group) or a control [10 g Miprodan 30 (sodium caseinate), twice daily; n = 28]. After 2 wk, participants were orally challenged with live, attenuated diarrheagenic E. coli (10(10) colony-forming units). Primary outcomes were infection-induced diarrhea and fecal diarrheagenic E. coli excretion. Secondary outcomes were gastrointestinal symptoms [Gastrointestinal Symptom Rating Scale (GSRS)], stool frequency, and stool consistency (Bristol Stool Scale). RESULTS: Diarrheagenic E. coli resulted in increased fecal output, lower relative fecal dry weight, increased fecal E. coli numbers, and an increase in stool frequency and gastrointestinal complaints at day 1 after challenge. MFGM significantly decreased the E. coli-induced changes in reported stool frequency (1.1 ± 0.1 stools/d in the MFGM group; 1.6 ± 0.2 stools/d in the control group; P = 0.04) and gastrointestinal complaints at day 2 (1.1 ± 0.5 and 2.5 ± 0.6 GSRS scores in the MFGM and control groups, respectively; P = 0.05). MFGM did not affect fecal wet weight and E. coli excretion at day 2 after challenge. CONCLUSIONS: The attenuated diarrheagenic E. coli strain transiently induced mild symptoms of a food-borne infection, with complete recovery of reported clinical symptoms within 2 d. The present diarrheagenic E. coli challenge trial conducted in healthy adults indicates that a milk concentrate rich in natural, bioactive phospho- and sphingolipids from the MFGM may improve in vivo resistance to diarrheagenic E. coli. This trial was registered at clinicaltrials.gov as NCT01800396.

A randomized controlled trial investigating the neurocognitive effects of Lacprodan® PL-20, a phospholipid-rich milk protein concentrate, in elderly participants with age-associated memory impairment: the Phospholipid Intervention for Cognitive Ageing Reversal (PLICAR): study protocol for a randomized controlled trial.

BACKGROUND: Age-related cognitive decline (ARCD) is of major societal concern in an ageing population, with the development of dietary supplements providing a promising avenue for amelioration of associated deficits. Despite initial interest in the use of phospholipids (PLs) for ARCD, in recent years there has been a hiatus in such research. Because of safety concerns regarding PLs derived from bovine cortex, and the equivocal efficacy of soybean-derived PLs, there is an important need for the development of new PL alternatives. Phospholipids derived from milk proteins represent one potential candidate treatment. METHODS: In order to reduce the effects of age-associated memory impairment (AAMI) the Phospholipid Intervention for Cognitive Ageing Reversal (PLICAR) was developed to test the efficacy of a milk protein concentrate rich in natural, non-synthetic milk phospholipids (Lacprodan® PL-20). PLICAR is a randomized, double-blind, placebo-controlled parallel-groups study where 150 (N = 50/group) AAMI participants aged > 55 years will be randomized to receive a daily supplement of Lacprodan® PL-20 or one of two placebos (phospholipid-free milk protein concentrate or inert rice starch) over a 6-month (180-day) period. Participants will undergo testing at baseline, 90 days and 180 days. The primary outcome is a composite memory score from the Rey Auditory Verbal Learning Test. Secondary outcomes include cognitive (verbal learning, working memory, prospective and retrospective memory, processing speed and attention), mood (depression, anxiety, stress and visual analogue scales), cardiovascular (blood pressure, blood velocity and pulse wave pressure), gastrointestinal microbiota and biochemical measures (oxidative stress, inflammation, B vitamins and Homocysteine, glucoregulation and serum choline). Allelic differences in the Apolipoprotein E and (APOE) and Methylenetetrahydrofolate reductase (MTHFR) gene will be included for subgroup analysis. A subset (N = 60; 20/group)) will undergo neuroimaging using functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG) in order to further explore in vivo central mechanisms of action of Lacprodan® PL-20. This study will enable evaluation of the efficacy of milk-derived phospholipids for AAMI, and their mechanisms of action. TRIAL REGISTRATION: The trial is jointly funded by Arla Foods and Swinburne University of Technology, currently recruiting and is registered on the Australian New Zealand Clinical Trials Registry as ACTRN12613000347763.

Quantification of mannan-binding lectin.

Mannan-binding lectin (MBL) is attracting considerable interest due to its role in the immune defense. The high frequency of congenital MBL deficiency makes it feasible to evaluate clinical relevance through epidemiological investigations on fairly limited numbers of patients. MBL deficiency is determined by three mutant allotypes termed B, C and D in the coding region as well as mutations in the promoter region. It has been suggested that individuals, with deficiency-associated allotypes, may present significant amounts of low molecular weight MBL. We have compared the quantification of MBL by four commercially available assays with results obtained by our own in-house assays. Most assays are selectively sensitive for the wild type MBL (allotype A), but special combinations of antibodies also detect mutant forms of MBL. Thus a sandwich-type time-resolved immunoflourometric assay (TRIFMA), with a mouse monoclonal antibody (93C) as the catching and detecting antibody, shows B/B and D/D homozygous individuals to present signals corresponding to up to 500 ng MBL per ml (with plasma from an A/A individual as standard) as compared to less than 50 ng/ml and 200 ng/ml, respectively, when measured in other assays. In GPC at isotonic conditions the MBL in B/B and D/D individuals showed a Mr of 450 kDa. This MBL cannot bind to mannan. We further present a new method for quantifying the amount of MBL polypeptide chain. By applying plasma samples on SDS-PAGE at reducing conditions followed by Western blotting and quantification by chemiluminescense, this approach presents single polypeptide chains to the antibody independent of allotype differences in the collagen-like region. Titrations of recombinant MBL served as standard. In sera from homozygous mutants (O/O) the MBL concentrations estimated on Western blot were in the range of 100 to 500 ng/ml and correlated with that measured in the 93C-based TRIFMA.