A randomized, placebo-controlled, double blind volunteer study into the effect of milk ingredients on gastroenteritis caused by an attenuated E. coli.

Food-borne infections
Food-borne infections occur frequently. The WHO reported in 2007 that in
industrialized countries, the percentage of the population suffering from
food-borne diseases each year is up to 30%. This is probably an
underestimation, since recent data from a Dutch study indicates that the
incidence of infectious intestinal disease is 964 per 1000 person years 3.
Food-borne infections are also frequently encountered by travelers to tropical
countries, with incidences up to 80%. After some days of diarrhea, stomach
pain, nausea or vomiting, most infections are self-limited and cured. However,
such ordinary infections can be life-threatening in people with reduced
resistance (e.g. young children, elderly, or persons taking immune-suppressive
drugs). Treatment of food-borne infections with antibiotics is usually
non-effective. Moreover, many bacterial pathogens become resistant to these
drugs. Therefore, it is important to search for alternative means to prevent or
treat these infections.

Milk ingredients
Enhancement of human resistance to food-borne infections is an attractive
option. Milk ingredients can contribute to enhanced human resistance to
infectious disease. By strengthening the gut barrier, milk constituents may
prevent translocation. In addition, the immune response towards an infection is
important in clearing pathogens. Milk constituents have also been shown to
modify immune responses. Finally, the microbiota composition of the GI tract
can influence the course of infections, by production of antimicrobial agents
in the large intestine, competition on nutrients for bacterial growth and
mucosal adhesion sites, and by modifying the immune response. Several milk
constituents have been shown to modify the gut microbiota composition. All
together, these activities can result in diminished pathogenic load and
diarrhea. Until now, studies into activities of individual milk components are
mainly limited to in vitro studies and animal infection models. Human evidence
is scarce, more difficult to obtain, but highly needed. Because of evolutionary
reasons, it can be expected that milk constituents acts synergistically in
protecting against gastrointestinal infections.
A broad range of other antimicrobial agents are present in the bovine milk fat
globule membrane. Pathogen decoy activity is the predominantly mentioned
bioactivity. Constituents of the bovine milk fat globular membrane appear to be
good inhibitors of rotavirus adhesion in vitro, but in-vivo evidence is scarce,
except for MUC1 and lactadherin 4. Since norovirus and several bacterial
pathogens can bind sialic acid 5 and sugar moieties 6, bovine milk fat globule
constituents containing sialic acid and sugar groups, such as milk
gangliosides, mucin and other glycosylated proteins, theoretically may act as
decoy, thus preventing adherence of these pathogens to the intestinal
epithelium. However, this needs to be confirmed. Except for sphingolipids, the
effect of the milk fat membrane constituents is less well studied for bacterial
pathogens than for viral pathogens.
Sweet buttermilk powder, which is rich in milk fat globule membrane components,
protected rats against Listeria-infection when compared to skim milk 7. This
effect can be explained by the bactericidal effect of digestion products of
sphingolipids, i.e. lysosphingolipids and sphingosine, or by the presence of
milk fat globule membrane constituents that act as pathogen decoy, such as
gangliosides, MUC1 and lactadherin.
So far, evidence from the milk fat globule membrane is mainly obtained from
in-vitro data, whereas data from animal models is limited and scattered over
different pathogens. Human evidence is scarce.

We want to determine whether Lacprodan® PL-20 can effectively protect against
enterotoxigenic E.coli induced travellers* diarrhea in humans. In the present
double-blind, placebo-controlled, randomized parallel study, the effect of oral
milk protein concentrate vs placebo will be studied on the resistance of humans
to enterotoxigenic Escherichia coli infection (ETEC). Lacprodan® PL-20 is a
milk protein concentrate rich in phospholipids and a source of phosphatidyl
serine and sphingomyelin. The main hypothesis is that Lacprodan® PL-20 will
improve human resistance to ETEC as measured by decreased fecal excretion of
ETEC with time and less ETEC-induced daily fecal output.

Study design
Subjects, recruited from the Wageningen/Ede area, will participate in a
randomized, double-blind, placebo-controlled, parallel intervention study of 4
weeks after receipt of signed informed consent. Subjects consume either
Lacprodan® PL-20 or placebo. Subjects will be instructed to maintain their
habitual diet and usual pattern of physical activity.

Dietary restrictions and replacing soy products
Subjects will be instructed to maintain their habitual diet, except for their
dairy intake and intake of products with high amounts of prebiotic fibers and
probiotics. Dairy has a high calcium content and contributes significantly to
total daily calcium intake. Calcium can significantly reduce the
gastro-intestinal symptoms induced by the ETEC strain1. To standardize and
decrease dietary calcium intake of the subjects, low-calcium soy products will
be provided to the subjects for the entire study. The low-calcium soy products
will be purchased from Provamel Nederland BV. The soy products will be provided
as single portions and can be stored at room temperature. Subjects will be
instructed to consume the low calcium soy products at breakfast and dinner. The
subjects are not allowed to consume other dairy products during the study.

Lacprodan® PL-20
Lacprodan® PL-20 is a milk protein concentrate rich in phospholipids and a
source of phosphatidyl serine and sphingomyelin. The placebo Miprodan® 30 is a
sodium caseinate and is a powder of identical appearance. The placebo contains
an identical amount of calcium but does not contain the bioactive components.
Subjects are requested to mix the Lacprodan® PL-20 or the Miprodan® 30 powder
twice daily in their soy drinks, once at breakfast and once at dinner, and
during the entire study. Lacprodan® PL-20 will be supplied as a powder in
sachets. The sachets have to be stored under cool and dry conditions to
prevent deterioration due to humidity and high temperatures. The shelf life is
a minimum of 12 months if kept under the prescribed storage conditions.

Oral ETEC Vaccine
After an adaptation period of 2 weeks to the intervention products, subjects
will be infected with a single oral dose of attenuated ETEC strain E1392-75-2A
at a dose of 1010 CFU (Bovee-Oudenhoven et al., 2003). Oral infection will
occur between 11.00 h and 12.00 h AM. Before taking ETEC, subjects are not
allowed to eat for 4 hrs and not to drink for 2 hrs. Thereafter, and under
supervision of the project team, they will get a NaHCO3 solution (100 ml 2%
NaHCO3) to neutralize the gastric acid. After 5 minutes, they get a fruit juice
(100 ml) containing the ETEC strain at the above-mentioned dose. Subjects go
home, but are not allowed to drink and eat for 1 hour.

Diaries and biological samples
Before and after infection, the subjects are asked to fill in a 2x24 hrs
nutrition diary and report and estimate amounts of all foods and drinks eaten
(online dairy; LimeSurvey2012). Bowel habits (defecation frequency) and
frequency and severity of gastrointestinal symptoms (flatulence, bloating,
abdominal pains and cramps) are self-recorded daily in an online diary
(LimeSurvey2012), using Visual Analogue Scales (VAS; range 0-5 from none to
severe) wherever appropriate.
Blood samples (10 ml) will be taken by qualified staff of a local hospital on 1
time point before and on 2 time points (day 3 and 14) after ETEC infection.
Before (on 2 separate days) and after ETEC infection (on 5 separate days), 24
hrs fecal samples will be collected. All materials and information needed for
proper collection of the fecal samples (stool collection kit) will be supplied
by NIZO food research and delivered to the subjects. Feces will be frozen
immediately after defecation. Subjects will be asked to store feces in
mini-freezers, supplied by NIZO food research. Every 2-3 days, the frozen feces
will be transported to the lab, weighed, homogenized, and analyzed for ETEC by
QPCR. Homogenized fecal sub-samples will be frozen and stored (at -20 oC) for
later analyses. Diarrhea will be quantified by analyses of fecal wet and dry
weight. Results will be compared with self-reported information on stool
consistency (Bristol stool scale).
In addition, before and after infection (on 2 separate days) 2 mL saliva will
be collected.

The MIRAGE study is a parallel, double-blind, placebo-controlled 4-weeks
intervention with a milk and whey protein concentrate in healthy volunteers. In
this study, the effect of an intervention with Lacprodan® PL-20 vs placebo
(Miprodan® 30) on several infection markers in response to an ETEC challenge is
investigated. The infection markers of interest, the primary and secondary
study outcomes, are mentioned below. Participants will be randomly assigned to
the milk and whey protein concentrate or placebo group (n=30 per group).
Subjects will be instructed to maintain their usual pattern of physical
activity and their habitual food intake, but to standardize their dietary
calcium intake. After an adaptation period of 2 weeks, subjects will be orally
infected with a live, but attenuated, ETEC vaccine (strain E1392-75-2A;
collection NIZO food research; dose will be 1010 CFU). Before and after
infection, an online diary will be kept to record all food and drinks
consumption (2x2 days) to assess the habitual dietary intake. The diary will
also be used for daily recording of bowel habits and frequency and severity of
gastrointestinal complaints. The following biological samples will be
collected: 4x10 ml venous blood, a single fecal bolus (for screening) and 7x24
hrs feces. Blood is sampled for immune response analyses and the fecal samples
are collected to quantify several infection- and immune system markers and to
verify dietary calcium intake. Saliva is sampled three times before and after
infection to quantify immune system markers.

Benefits for subjects to participate in the MIRAGE study
There are no direct benefits for the subjects from participation to the MIRAGE
study. The single oral administration of the ETEC vaccine strain to the
subjects offers no protection against E. coli infections in the future.
Previous studies with this vaccine strain have shown that single oral
administration leads to a rise of specific serum antibody titers, but the
quality and quantity of the effect is considered inadequate for significant
protection against subsequent infections. Only after repeated vaccinations
protection would be induced against a very specific (and thus small) group of
bacterial pathogens. Although there are not direct benefits for the study
subjects, a positive study outcome can offer advantages for population groups
in the future. When Lacprodan® PL-20 does improve resistance to ETEC infection,
it will be possible to decrease intestinal infection incidence by providing
relatively simple dietary advices, e.g. to travelers to tropical countries.

Safety information on ETEC strain
ETEC strain E1392-75-2A (supplier: Acambis, Cambridge, UK) is a spontaneous
mutant unable to produce toxins. The strain obtained is 100% pure. Because of
its streptomycin-resistance it can be discriminated from other E. coli species
that are part of the endogenous microbiotia and excreted in feces. ETEC
E1392-75-2A is sensitive to ciproxin, which is a commonly used antibiotic for
treatment of E. coli infections in humans. Vaccination experiments with this
ETEC strain in humans are published by e.g. Tacket et al. (1997)2. In their
study, after oral administration of 1010 CFU, 15% of the vaccinated persons
suffered from self-limited, mild diarrhea with spontaneous recovery after 1-3
days. The most recent human intervention study at NIZO food research with this
strain performed in 2010 showed that 79% of infected volunteers suffered from a
mild and transient diarrhea for 1-3 days when orally dosed with log1010 colony
forming units. Besides this, 74% of the volunteers experienced a mild abdominal
pain, 63% reported bloating and 26% reported fever. Other symptoms, e.g. nausea
and vomiting, were not reported and complications are not expected.

Safety information on Lacprodan® PL-20
Lacprodan® PL-20 is a milk cream powder reduced in triglycerid fat. This
product is an approved food ingredient under the term *fat-reduced cream
powder*. The product is manufactured, packaged and labelled according to the
relevant EU-regulations for food and food ingredients, and/or FAO/ WHO Codex
Alimentarius, when relevant. This includes that the milk/milk constituents used
as raw material origins from healthy cows. The milk used in the production is
included in monitoring programmes for undesirable substances, as required by
regulations or HACCP-based risk assessment. The production plants are approved
by the competent authorities and included in the EU-register of approved food
establishments.