low molecular weight fractions of bimuno® exert immunostimulatory properties in murine macrophages

$: Low molecular weight fractions of BiMuno® exert immunostimulatory properties in murine macrophages$
J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 9 4 1 – 9 5 3

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Low molecular weight fractions of BiMuno� exertimmunostimulatory properties in murine macrophages

Laura E.J. Searlea,*, Gareth Jonesa, George Tzortzisb, Martin J. Woodwarda,c,Robert A. Rastallc, Glenn R. Gibsonc, Roberto M. La Ragionea,d

aDepartment of Bacteriology, Animal Health and Veterinary Laboratories Agency (AHVLA), Weybridge, Woodham Lane, New Haw,

Addlestone, Surrey KT15 3NB, UKbClasado Ltd., 5 Canon Harnett Court, Wolverton Mill, Milton Keynes MK12 5NF, UKcDepartment of Food and Nutritional Sciences, University of Reading, Whiteknights, Reading RG6 6UR, UKdDepartment of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK

A R T I C L E I N F O A B S T R A C T

Article history:

Received 18 March 2012

Received in revised form

24 June 2012

Accepted 15 July 2012

Available online 29 August 2012

Keywords:

Galactooligosaccharide (GOS)

Prebiotic

Immuno-modulation

Macrophages

Pro-inflammatory response

1756-4646/$ - see front matter Crown Copyrhttp://dx.doi.org/10.1016/j.jff.2012.07.002

* Corresponding author. Tel.: +44 01932 359E-mail address: [email protected]: GOS, galactooligosaccharide

Previous in vivo murine oral challenge studies have shown that the galactooligosaccharide

containing product, BiMuno�, reduced colonisation of S. Typhimurium. To gain further

insights into the mechanism of reduced colonisation, we wished to test the hypothesis that

the low molecular weight fractions of BiMuno� or a specific low molecular weight fraction

may have direct immuno-modulatory effects on murine macrophages. Cytokine responses

of murine macrophages in response to the commercially available product, BiMuno�, a

basal solution BiMuno� without GOS, purified low molecular weight fractions (referred to

as GOS), and the individual fractions of GOS (DP2, 3 and P4, with each fraction represent-

ing the increasing degree of complex polymerisation) were determined in vitro and ex vivo.

These studies demonstrated that BiMuno�, significantly stimulated both pro- and anti-

inflammatory cytokines in vitro (P 6 0.0394). Furthermore, the data indicate that the low

molecular weight fractions may be the primary stimulant of BiMuno� and specifically its

tri (DP3) and Ptetra-saccharide (DP P 4) fractions (P 6 0.0394).

Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Prebiotics have been defined as ‘‘a selectively fermented

ingredient that allows specific changes both in the composi-

tion and/or activity in the gastrointestinal microbiota that

confers benefits upon host well-being and health’’ (Gibson,

Probert, Van Loo, Rastall, & Roberfroid, 2004). Diets fortified

with prebiotic oligosaccharides, such as galactooligosaccha-

ride (GOS), increase numbers of bifidobacteria and lactobacilli

in the colon following a short feeding period (Gibson & Rober-

froid, 1995; Macfarlane, Steed, & Macfarlane, 2008; Tzortzis,

ight � 2012 Published by

478; fax: +44 01932 347(L.E.J. Searle).; AHVLA, Animal Health

Goulas, Gee, & Gibson, 2005a). Also, a growing body of evi-

dence suggests that prebiotics are associated with reducing

gastrointestinal colonisation by pathogens such as Salmonella

spp. (Agunos, Ibuki, Yokomizo, & Mine, 2007; Bailey, Blanken-

ship, & Cox, 1991; Searle et al., 2009; Spring, Wenk, Dawson, &

Newman, 2000). The administration of mannan-oligosaccha-

ride (MOS) in feed was associated with significant reductions

in numbers of S. Typhimurium recovered from the caeca of

chickens (Spring et al., 2000) and it has been demonstrated

that b 1–4 mannobiose reduced the invasion of S. Enteritidis

into the liver of chickens and reduced faecal shedding of

Elsevier Ltd. All rights reserved.

046.

and Veterinary Laboratories Agency; DP, degree of polymerisation

http://dx.doi.org/10.1016/j.jff.2012.07.002

mailto:[email protected]




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942 J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 9 4 1 – 9 5 3

the Salmonellae (Agunos et al., 2007). Our previous studies

demonstrated that BiMuno�, which contains the prebiotic

GOS, orally administered to mice was not only associated

with significantly reduced gastrointestinal colonisation and

systemic spread by S. Typhimurium but also alleviation of clin-

ical symptoms and pathology (Searle et al., 2009).

At present, the exact mechanisms of action of prebiotics

on reducing the carriage of pathogens remains largely un-

known. It is feasible that prebiotics reduce pathogen carriage

through microbiota dependent or independent immuno-

modulation (Macfarlane et al., 2008). The administration of

fructooligosaccharide (FOS), b 1–4 mannobiose, short chain

GOS/long chain FOS, cycloinulooligosaccharides or b-glucan

in feeds have been associated with increased production of

secretory IgA (sIgA) (Agunos et al., 2007; Benyacoub et al.,

2008; Hosono et al., 2003; Ishikawa & Nanjo, 2009; Lowry

et al., 2005; Scholtens et al., 2008). Furthermore, following

administration of FOS to BALB/c mice, sIgA was detected in

the Peyer’s patches (Hosono et al., 2003). Additionally, b-glu-

can, FOS and GOS have been associated indirectly with al-

tered host cytokine and cellular responses (Hosono et al.,

2003; Jung, Ha, Ha, & Han, 2004; Lowry et al., 2005; Vulevic,

Drakoularakou, Yaqoob, Tzortzis, & Gibson, 2008). To date,

no evidence is available addressing the question as to

whether or not GOS elicits host immune responses directly,

whereas published data illustrating the direct cytokine and

cellular effects of inulin, b-glucan and acidic human milk oli-

gosaccharides (HMO) on macrophages and T cells in vitro ex-

ists (Eiwegger et al., 2004; Kataoka, Muta, Yamazaki, &

Takeshige, 2002; Lee et al., 2001; Vos, M’Rabet, Stahl, Boehm,

& Garssen, 2007). We hypothesise that GOS stimulates the im-

mune system directly, through interacting with antigen pre-

senting cells such as macrophages. For example,

stimulation of antigen presenting cells to secrete pro-inflam-

matory cytokines may aid in pathogen clearance through

activation of macrophages to phagocytose the pathogen and

aid lymphocyte recruitment (Balaram, Kien, & Ismail 2009;

Michetti, Mahan, Slauch, Mekalanos, & Neutra 1992). In this

study, in vitro and ex vivo cytokine studies were utilised to test

this hypothesis; to determine whether low molecular weight

fractions of BiMuno�, and specifically which low molecular

weight fraction(s) of BiMuno�, have direct immuno-modula-

tory properties on murine macrophages.

2. Materials and methods

2.1. Test substances

2.1.1. BiMuno�

The GOS mixture (BiMuno�, Clasado Ltd, Milton Keynes, UK)

used in this study was produced from the activity of galac-

tosyltransferases from Bifidobacterium bifidum NCIMB 41171

using lactose as substrate (Tzortzis et al., 2005a; Tzortzis,

Goulas, & Gibson, 2005b). It has been shown to consist of

galactooligosaccharides in mainly the b 1–3, b 1–4, and b 1–6

linkages, as well as a disaccharide fraction of a 1–6 galactobi-

ose (Depeint, Tzortzis, & Vulevic, 2008; Tzortzis et al., 2005a).

Additionally, BiMuno� contains glucose, galactose and lactose

(which are involved in the manufacturing process) and the

stabilisers maltodextrin and gum arabic. In all assays, whole

BiMuno� was used at �5 mg ml�1. In all assays referring to fil-

tered BiMuno�, �5 mg ml�1 solutions were prepared and sub-

sequently filtered using a 0.22 lm filter (Sartorius stedim) to

remove bacterial debris, which is present due to the manufac-

turing process of the product. The syringe filters were made of

surfactant free cellulose acetate and function to remove

microorganisms, particles and precipitates larger than

0.2 lm from aqueous solutions.

2.1.2. BiMuno� without GOSBiMuno� without GOS was prepared to contain all of the sug-

ars in the commercially available BiMuno� product with the

exception of the low molecular weight fractions (referred to

as GOS). When diluted to 2.5 mg ml�1 BiMuno� without GOS

contained glucose (0.26 mg ml�1), galactose (0.25 mg ml�1),

lactose (1.3 mg ml�1), and the processing aids (maltodextrin

and gum arabic) (0.7 mg ml�1), representing 50% of the total

composition of BiMuno�. This was determined by isocratic

high performance liquid chromatography (HPLC) using an

Aminex HPX-87C Ca+2 resin-based column (Bio-Rad Laborato-

ries Ltd, UK) and high performance anion-exchange chroma-

tography coupled with pulsed amperometric detection

(HPAEC-PAD) using a pellicular anion-exchange resin based

column CarboPac PA-1 (Dionex Chromatography, Surrey, UK)

(Osman, Tzortzis, Rastall, & Charalampopoulos, 2010). In all

assays BiMuno� without GOS was used at �2.5 mg ml�1 as

these sugars make up 50% of BiMuno�. Solutions were filtered

prior to use in assays (0.22 lm filter, Sartorius stedim).

2.1.3. GOSThe various oligosaccharide fractions were purified from pre-

filtered BiMuno� by gel filtration on a Biogel P2 (Pharmacia)

column eluted at 3 ml min�1 with water (Tzortzis et al.,

2005a). This column allows the elution of compounds of be-

tween 100 and 1800 Da molecular weight. Individual fractions

were combined to create the low molecular weight complex

(referred to as GOS) equivalent to that found in BiMuno�

and this was used at a concentration of �2.5 mg ml�1 in all as-

says as GOS makes up 50% BiMuno�. In instances where the

individual fractions were used they were used at the following

concentrations; �1.1 mg ml�1 for DP2 (disaccharide),

�1.2 mg ml�1 for DP3 (trisaccharide) and �0.2 mg ml�1 for

DP P 4 (P tetrasaccharide). Solutions of GOS or the individual

fractions were filtered prior to use in assays (0.22 lm filter,

Sartorius stedim).

2.2. In vitro cytokine assays using murine macrophages

The BALB/c derived murine macrophages (RAW264.7), were

seeded at 2 · 105 cells ml�1 into T25 flasks (Nunc) and cul-

tured using standard procedures with Dulbecco’s Modified Ea-

gle’s Medium (DMEM) (Sigma–Aldrich) supplemented with

foetal calf serum (10%), 100x non-essential amino acids

(1%), 2 mM L-glutamine and gentamicin (50 lg ml�1) (Sigma–

Aldrich) for 48 h. Monolayers (that were P80% confluent)

were washed twice with Hanks balanced salts solution (HBSS)

(Sigma–Aldrich) to remove cell debris and residual gentami-

cin. Inocula (Table A.1) were prepared and delivered in indi-

vidual 10 ml volumes and were subsequently incubated at

37 �C, in the presence of 5% CO2, for the time points indicated

Table A.1 – Experimental designs for in vitro and ex vivo murine macrophage experiments.

Experimentalnumber

Experimentalname

Assaydetails

Testcondition

Timepoint (hours)

Cytokinestested

1 BiMuno�,

BiMuno� without GOS,

GOS

In vitro Negative 2,4,6 IFN-c, TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12p70

BiMuno� 2,4,6 IFN-c, TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12p70

Filtered BiMuno� 2,4,6 IFN-c, TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12p70

Filtered BiMuno�

without GOS

2,4,6 IFN-c, TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12p70

Filtered GOS 2,4,6 IFN-c, TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12p70

2 GOS fractions In vitro Negative 2,4,6 TNF-a

BiMuno� 2,4,6 TNF-a

Filtered GOS 2,4,6 TNF-a

Filtered DP2 2,4,6 TNF-a



3 BiMuno�, BiMuno� without GOS,

GOS and its fractions

Ex vivo Negative 6 TNF-a

BiMuno� 6 TNF-a

Filtered BiMuno� 6 TNF-a

Filtered BiMuno�

without GOS

6 TNF-a

Filtered GOS 6 TNF-a

Filtered DP2 6 TNF-a



J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 9 4 1 – 9 5 3 943

in Table A.1. Stimulation assays were conducted over a two-

six hour time point range as initial experiments showed that

six hours was sufficient to detect cytokine responses pro-

duced by murine macrophages. After the required time point

supernatants were harvested, filtered (0.22 lm filter, Sartorius

stedim) and stored at �80 �C. All assays were conducted in

duplicate on two consecutive days with lipopolysaccharide

(LPS, derived from E. coli O55:B5) (Sigma–Aldrich) (10 lg ml�1)

and concavalin A (ConA) (Sigma–Aldrich) (32 lg ml�1) serving

as positive controls. Samples were analysed using murine

specific cytokine plates (Meso-Scale Discovery (MSD)) as de-

scribed in subsequent sections.

2.3. Ex vivo cytokine assays on murine macrophages

2.3.1. Murine peritoneal lavageFifty, twelve-week-old female BALB/c (Charles, River) mice,

housed in accordance with UK animal welfare regulations,

were used to harvest peritoneal macrophages. Groups of five

mice were euthanized with CO2, positioned on their dorsal

recumbency and sprayed with 70% (v/v) ethanol. Then, 5 ml

of ice-cold DMEM (supplemented with foetal calf serum

(10%), 100· non-essential amino acids (1%), 2 mM L-glutamine

(Sigma–Aldrich) and Penicillin/Streptomycin (200U/200 lg,

respectively) (Invitrogen)) was injected into the peritoneal

cavity. The abdomen of the mice was then massaged for

two minutes prior to the abdominal cavity being opened asep-

tically. The peritoneal fluid was then harvested aseptically

using a Pastette, passed through a 40 lm cell strainer (BD Bio-

sciences), and checked for any bacterial contamination

microscopically using a Leica DFC320 light microscope (Leica

Microsystems). Those cell suspensions that were not contam-

inated were centrifuged at 550 · g for 10 min and the cellular

pellet resuspended in DMEM (as mentioned above). Cells were

seeded at 1–5 · 106 cells ml�1 in 24 well plates (Nunc) and

incubated at 37 �C, in the presence of 5% CO2, for 4 h to allow

the cells to adhere. Following this, cells were washed three

times with pre-warmed HBSS to remove any non-adherent

cells, the medium was replaced and the cells were main-

tained for 16 h at 37 �C, in the presence of 5% CO2. Following

a 16 h incubation, cells were washed twice with pre-warmed

HBSS to remove residual antibiotics, prior to delivery of the

inocula (Table A.1) in 1 ml volumes with LPS and ConA serv-

ing as positive controls. No regulated procedures were per-

formed, however all of the above studies were approved by

the local Animal Health and Veterinary Laboratories Agency

ethics committee.

2.4. Quantification of cytokine responses

Quantification of murine specific IFN-c, TNF-a, IL-2, IL-4, IL-6,

IL-10 and IL-12p70 (Th1 and Th2) cytokines in the superna-

tants of samples were conducted using the Meso-Scale Dis-

covery (MSD) 7-plex cytokine plate coupled to a Sector

Imager 6000 (MSD) reader as instructed by the manufacturer.

This sandwich ELISA technology allowed for the simulta-

neous quantification of 7 cytokines in the supernatants by

extrapolation against a known standard curve (0–

40,000 pg ml�1 mix containing the cytokines mentioned

above).

In essence, plates were incubated for 2 h at ambient tem-

perature with agitation (600 rpm) with samples or known

standards. Subsequently, plates were incubated (as men-

tioned above) with detection antibody (1 lg ml�1 anti-cyto-

kine antibody labelled with MSD SULFO-TAG + 0.7% bovine

gamma globulin) for 2 h and washed three times with

PBS + 0.05% (w/v) Tween. 2· read buffer (containing surfac-

tant) was added immediately prior to the plates being read


using the Sector Imager 6000 (MSD) which detects chemilumi-

nescence generated by the samples and quantifies the

amount of cytokine present by extrapolation against a stan-

dard curve.

The MSD TNF-a single-plex plate coupled to the Sector Im-

ager 6000 (MSD) was used, as instructed by the manufacturer,

to quantify murine specific TNF-a cytokines in the superna-

tants. The principle of the technique is that for the 7-plex kits,

however, in these instances only levels of TNF-a were quanti-

fied against a TNF-a standard curve (0–40,000 pg ml�1).

2.5. Analysis of potential GOS contamination by LPS bygel electrophoresis and cytokine assays

Samples of GOS were prepared (as described above) and then

separated by SDS gel electrophoresis according to the manu-

facturers instructions (NuPAGE� Novex 4–12% Bis–Tris Gel,

Invitrogen). Specifically, �25 mg ml�1 of GOS test samples

and 1 lg of LPS were centrifuged at 4000 · g through a 5 KDa

molecular weight cut off filter (Sartorius Stedim) and the

retentate and eluent collected. Polyacrylamide gels were then

loaded with GOS retentate (�25 mg ml�1), Novex Sharp Pro-

tein Standard (Invitrogen), GOS eluent (�2.5 mg ml�1), �1 mg

GOS, �0.1 mg GOS, 0.1 M PBS pH7.2, 1 lg LPS retentate, 1 lg

LPS eluent, LPS (10 lg and 50 ng). LPS was subsequently visu-

alised by silver staining, as previously described by Tsai and

Frasch (1982).

For cytokine assays T25 flasks of RAW264.7 cells were sub-

sequently incubated at 37 �C in the presence of 5% CO2 for 6 h

with the following inocula; media alone, or media containing

�25 mg GOS (�2.5 mg ml�1), 2.5 ng LPS (250 pg ml�1), 5 ng LPS

(500 pg ml�1), 50 ng LPS (5 ng ml�1), 125 ng LPS (12.5 ng ml�1),

250 ng LPS (25 ng ml�1), 500 ng LPS (50 ng ml�1), 5 lg LPS

(500 ng ml�1), 10 lg LPS (1 lg ml�1), 25 lg LPS (2.5 lg ml�1),

50 lg LPS (5 lg ml�1), 100 lg LPS (10 lg ml�1). Following incu-

bation the supernatants were harvested, filtered (0.22 lm fil-

ter, Sartorius stedim) and stored at �80 �C. Assays were

conducted in duplicate on two consecutive days. Samples

were analysed using murine specific cytokine plates (Meso-

Scale Discovery (MSD)) as described above.

2.6. Statistical analysis

Statistical analysis was carried out on complete data sets by

transforming the data onto the log2 scale and using Dunnetts

pair-wise statistical tests to compare differences induced by

all of the treatment regimes. All comparisons were based on

95% confidence intervals on the log2 scale and their associ-

ated P-value calculated (given to 4 decimal places).

3. Results

3.1. In vitro cytokine assays

3.1.1. Cytokine responses of RAW264.7 cells exposed toBiMuno�, BiMuno� without GOS and GOSRAW264.7 cells were incubated in the presence of BiMuno�,

filtered BiMuno�, filtered BiMuno� without GOS or filtered

GOS (equivalent to concentrations of 5 mg ml�1 BiMuno�)

with 2, 4 and 6 h incubation. BiMuno� alone, filtered BiMuno�

and filtered GOS induced significant increases in the produc-

tion of Th1 and Th2 cytokines when compared to negative

controls (Fig. B.1). Specifically, significant increases in TNF-a,

IL-2, IL-4, IL-6, IL-10 and IL-12p70 were detected in the super-

natants of RAW264.7 cells (P 6 0.0394) notably at 6 h after

exposure (Fig. B.1 and Table S.1). We note that although statis-

tically significant, only relatively marginal increases in IL-2,

IL-4 and IL-12p70 production were observed when compared

to TNF- a and IL-6 production. Filtered BiMuno� without

GOS did not induce significant changes in TNF-a, IL-6, IFN-c

or IL-12p70 cytokine production of RAW264.7 cells compared

to negative controls and, although significant, only marginal

increases in IL-2, IL-4 and IL-10 production (P 6 0.0426)

(Table S.1). Additionally, BiMuno�, filtered BiMuno� and fil-

tered GOS induced significant increases in TNF-a, IL-2, IL-6

and IL-10 production when compared to filtered BiMuno�

without GOS treated cells (P 6 0.0276) (Table S.1). Further-

more, BiMuno� and filtered GOS induced significant increases

in TNF-a, IL-2, IL-10 and IL-12p70 production when compared

to filtered BiMuno� treated cells (P 6 0.0286) (Table S.1). These

data suggest that the GOS component conveys immuno-stim-

ulatory properties which raised the question of whether this

could be attributed to a specific fraction of GOS.

3.1.2. Cytokine responses of RAW264.7 cells exposed to GOSfractionsRAW264.7 cells were incubated in the presence of filtered DP2,

filtered DP3 and filtered DP P 4 or BiMuno� and filtered GOS

as controls (equivalent to concentrations of 5 mg ml�1 BiMu-

no�), with 2, 4 and 6 h incubation. In this experiment, only

levels of TNF-a were quantified, partly due to large TNF-a re-

sponses detected in the experiments described above (1.3.1.1)

being indicative of immuno-stimulation and for cost reasons.

All fractions of GOS induced significant increases in TNF-a

production by RAW264.7 cells when compared to negative

controls (P 6 0.0019) notably at 4 and 6 h. DP3 and DP P 4 frac-

tions of GOS induced significant increases in TNF-a produc-

tion when compared to the DP2 (P 6 0.0213) notably at 4 and

6 h (Fig. C.1 and Table S.2). In the control experiments, BiMu-

no� alone and filtered GOS significantly increased TNF-a pro-

duction across the 2–6 h time points (P 6 0.0002) (Table S.2)

confirming the previous tests.

3.2. Ex vivo cytokine assays

Ex vivo harvested macrophages derived from BALB/c mice

were incubated in the presence of the test substances BiMu-

no�, filtered BiMuno�, filtered BiMuno� without GOS, filtered

GOS and the individual fractions that comprise GOS (DP2, 3

and P4) with 6 h incubation. With the exception of BiMuno�

without GOS all of the test substances significantly increased

TNF-a production when compared to negative controls

(P < 0.0001) (Fig. D.1 and Table S.3). Moreover, in line with

in vitro data using RAW264.7 macrophages, DP3 and DP P 4

significantly increased TNF-a production when compared

to DP2 (P 6 0.0063) (Fig. D.1 and Table S.3). BiMuno� alone, fil-

tered GOS, filtered DP3 and DP P 4 induced significant in-

creases in TNF-a production as compared to filtered

BiMuno� (P < 0.0001). Furthermore, BiMuno� alone, filtered

BiMuno�, filtered GOS, and all of the individual fractions of

Fig. B.1 – Cytokine responses of RAW264.7 cells exposed to BiMuno�, BiMuno� without GOS and GOS. Quantification of TNF-a

(A), IFN-c (B), IL-2 (C), IL-4 (D), IL-6 (E), IL-10 (F), IL-12p70 (G) cytokine production by RAW264.7 cells in response to BiMuno�,

filtered BiMuno� without GOS and filtered GOS. BiMuno�, filtered BiMuno� and filtered GOS induced significant increases in

Th1 and Th2 cytokine responses when compared to negative controls (P 6 0.0394) whereas filtered BiMuno� without GOS did

not induce such a cytokine response. Error bars, STDEV.


Table S.1 – Cytokine changes in RAW264.7 cells in response to BiMuno�, filtered BiMuno�, filtered BiMuno� without GOS or filtered GOS.

Cytokine Time (hours) Test condition a Test condition b Protein concentration (pg ml�1) P-Value 95% Confidence intervals

Test condition a Test condition b

TNF-a 2 Negative BiMuno� 9.46 2491.26 <0.0001 7.03, 9.17

4 Negative BiMuno� 5.37 23730.71 <0.0001 10.84, 13.58

6 Negative BiMuno� 2.72 32594.45 <0.0001 12.08, 15.76

2 Negative Filtered BiMuno� 9.46 457.14 <0.0001 4.60, 6.74



2 Negative Filtered GOS 9.46 2374.87 <0.0001 6.98, 9.12



2 Filtered BiMuno� without GOS BiMuno� 10.11 2491.26 <0.0001 6.91, 9.05



2 Filtered BiMuno� without GOS Filtered BiMuno� 10.11 457.14 <0.0001 4.47, 6.61



2 Filtered BiMuno� without GOS Filtered GOS 10.11 2374.87 <0.0001 �9.00, �6.85



2 Filtered BiMuno� BiMuno� 457.14 2491.26 0.0021 1.36,3.50



2 Filtered BiMuno� Filtered GOS 457.14 2374.87 0.0023 �3.45, �1.31



IL-2 2 Negative BiMuno� 0.02 1.23 0.0098 1.95, 7.81

2 Negative Filtered BiMuno� 0.02 0.51 0.0303 0.54, 6.39

2 Negative Filtered BiMuno� without GOS 0.02 0.40 0.0426 0.17, 6.02

2 Negative Filtered GOS 0.02 1.42 0.0088 2.11, 7.97

4 Filtered BiMuno� without GOS BiMuno� 0.06 6.20 0.0072 2.82, 8.13

6 Filtered BiMuno� without GOS BiMuno� 0.28 6.66 0.0103 1.94, 7.95

4 Filtered BiMuno� without GOS Filtered BiMuno� 0.06 1.34 0.0276 0.70, 6.00

4 Filtered BiMuno� without GOS Filtered GOS 0.06 4.33 0.0087 �7.77, �2.46

6 Filtered BiMuno� without GOS Filtered GOS 0.28 6.78 0.0009 �8.00, �1.99

6 Filtered BiMuno� BiMuno� 0.63 6.66 0.0286 0.62, 6.63

6 Filtered BiMuno� Filtered GOS 0.63 6.78 0.0274 �6.69, 0.67

IL-4 6 Negative BiMuno� 0.02 1.65 0.0098 2.29, 9.12

6 Negative Filtered BiMuno� 0.02 1.38 0.0144 1.67, 8.51

6 Negative Filtered BiMuno� without GOS 0.02 0.69 0.0252 0.87, 7.71

6 Negative Filtered GOS 0.02 0.81 0.0200 1.19, 8.03

94

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IL-6 2 Negative BiMuno� 7.63 150.61 0.0394 0.31, 8.332 Negative Filtered GOS 7.63 346.19 0.0177 1.41, 9.434 Negative BiMuno� 6.71 1544.18 0.0006 5.60, 11.386 Negative BiMuno� 8.77 3019.36 0.0003 6.27, 11.434 Negative Filtered BiMuno� 6.71 607.15 0.0014 4.24, 10.036 Negative Filtered BiMuno� 8.77 924.64 0.0009 4.55, 9.714 Negative Filtered GOS 6.71 3611.83 0.0004 6.77, 12.566 Negative Filtered GOS 8.77 8142.03 0.0002 7.59, 12.752 Filtered BiMuno� without GOS Filtered BiMuno� 8.54 43.16 0.0179 1.40, 9.422 Filtered BiMuno� without GOS Filtered GOS 8.54 346.19 0.0087 �3.11, 2.454 Filtered BiMuno� without GOS BiMuno� 7.54 1544.18 0.0009 5.03, 10.846 Filtered BiMuno� without GOS BiMuno� 13.89 3019.36 0.0005 5.28, 10.444 Filtered BiMuno� without GOS Filtered BiMuno� 7.54 607.15 0.0021 3.70, 9.486 Filtered BiMuno� without GOS Filtered BiMuno� 13.89 924.64 0.0017 3.57, 8.724 Filtered BiMuno� without GOS Filtered GOS 7.54 3611.83 0.0005 �12.01, �6.236 Filtered BiMuno� without GOS Filtered GOS 13.89 8142.03 0.0003 �11.77, �6.616 Filtered GOS BiMuno� 8142.03 3019.36 0.0290 �3.91, 1.25

IL-10 2 Negative BiMuno� 3.47 44.68 0.0015 2.33, 5.564 Negative BiMuno� 3.14 170.17 0.0035 3.52, 9.246 Negative BiMuno� 0.79 238.60 0.0001 5.84, 8.462 Negative Filtered BiMuno� 3.47 11.00 0.0316 0.24, 3.474 Negative Filtered BiMuno� 3.14 62.44 0.0087 2.07, 7.806 Negative Filtered BiMuno� 0.79 36.65 0.0007 3.22, 5.842 Negative Filtered GOS 3.47 28.36 0.0034 1.67, 4.904 Negative Filtered GOS 3.14 112.81 0.0050 2.91, 8.646 Negative Filtered GOS 0.79 221.10 0.0001 5.80, 8.426 Negative Filtered BiMuno� without GOS 0.79 6.57 0.0123 0.74, 3.362 Filtered BiMuno� without GOS BiMuno� 2.39 44.68 0.0011 2.61, 5.844 Filtered BiMuno� without GOS BiMuno� 0.13 170.17 0.0018 5.83, 12.856 Filtered BiMuno� without GOS BiMuno� 6.57 238.60 0.0002 4.03, 6.172 Filtered BiMuno� without GOS Filtered BiMuno� 2.39 11.00 0.0189 0.53, 3.764 Filtered BiMuno� without GOS Filtered BiMuno� 0.13 62.44 0.0033 4.39, 11.406 Filtered BiMuno� without GOS Filtered BiMuno� 6.57 36.65 0.0030 1.41, 3.552 Filtered BiMuno� without GOS Filtered GOS 2.39 28.36 0.0030 �5.19, �1.964 Filtered BiMuno� without GOS Filtered GOS 0.13 112.81 0.0023 �12.24, �5.236 Filtered BiMuno� without GOS Filtered GOS 6.57 221.10 0.0002 �6.14, �4.002 Filtered BiMuno� BiMuno� 11.00 44.68 0.0210 0.47, 3.706 Filtered BiMuno� BiMuno� 36.65 238.60 0.0025 1.55, 3.696 Filtered BiMuno� Filtered GOS 36.65 221.10 0.0026 03.65, �1.51

IL-12p70 4 Negative BiMuno� 0.10 12.70 0.0013 4.36, 7.594 Negative Filtered BiMuno� 0.10 8.69 0.0018 3.80, 7.034 Negative Filtered GOS 0.10 11.19 0.0015 4.12, 7.352 Filtered BiMuno� without GOS BiMuno� 2.07 8.76 0.0396 0.19, 5.016 Filtered BiMuno� without GOS BiMuno� 4.52 17.97 0.0364 0.21, 3.836 Filtered BiMuno� without GOS Filtered GOS 4.52 50.10 0.0070 �5.14, �1.526 Filtered BiMuno� BiMuno� 2.03 17.97 0.0077 1.42, 5.046 Filtered BiMuno� Filtered GOS 2.03 50.10 0.0022 �6.35, �2.73

aAt 6 h incubation LPS induced 38,117.03 pg ml-1 TNF-a,14.15 pg ml-1 IL-2, 1.82 pg ml-1 IL-4, 14,265.96 pg ml-1 IL-6, 269.62 pg ml-1 IL-10, 29.39 pg ml-1 IL-12p70.b At 6 h incubation ConA induced 23.22 pg ml-1 TNF-a, 0.78 pg ml-1 IL-2, 0.69 pg ml-1 IL-4, 10.65 pg ml-1 IL-6, 10.48 pg ml-1 IL-10, 7.10 pg ml-1 IL-12p70.

JO

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01

2)

94

1–

95

39

47

Fig. C.1 – Cytokine responses of RAW264.7 cells exposed to

GOS fractions. Quantification of TNF-a cytokine production

by RAW264.7 cells in response to the fractions of GOS.

BiMuno�, filtered GOS and the individual fractions induced

significant increases in TNF-a production when compared

to the negative control cells (P 6 0.0019). Additionally, levels

of TNF-a were significantly higher in cells incubated with

DP3 and DP P 4 fractions when compared to cells incubated

with the DP2 fraction (P 6 0.0213). Error bars, STDEV.


GOS significantly increased TNF-a production when com-

pared to filtered BiMuno� without GOS (P < 0.0001)

(Table S.3).

Table S.2 – TNF-a production from RAW264.7 cells in response

Cytokine Time (hours) Test condition a Test condition b Pr

Te

TNF-a 2 Negative BiMuno� 12

4 Negative BiMuno� 7.

6 Negative BiMuno� 7.

2 Negative Filtered GOS 12

4 Negative Filtered GOS 7.

6 Negative Filtered GOS 7.

2 Negative Filtered DP2 12

4 Negative Filtered DP2 7.








2 Filtered DP2 BiMuno� 28



2 Filtered DP2 Filtered GOS 28



2 Filtered DP2 Filtered DP3 28






aAt 6 h incubation LPS induced 39,792.33 pg ml�1 TNF-a and ConA induc

3.3. Analysis of GOS for LPS contamination

The following assays were conducted as a proof of principle,

to exclude the possibility that the strong TNF-a and IL-6 re-

sponses observed, which are stereotypical of LPS, were attrib-

uted to LPS. Varying concentrations of the low molecular

weight fractions (referred to as GOS) were analysed for LPS

contamination by silver staining of polyacrylamide gels and

LPS was not detected even at high concentrations (�1 mg

GOS). In all positive controls LPS (10 lg, 1 lg and 50 ng) was

detected (Fig. S.1). As GOS was purified by size exclusion chro-

matography using a Biogel P2 column (MW 100–1800 Da), it

should not contain LPS as the molecular weight of LPS is

>2 KDa (2–50 KDa range). It was concluded that, in the unli-

kely event that the low molecular weight fractions contained

trace amounts of LPS, it must be below the level of detection

by silver staining (<50 ng). We appreciate that LPS can exert

immuno-stimulatory effects at concentrations <50 ng ml�1

and a Limulus Amoebocyte Lysate (LAL) assay would be more

sensitive for the detection of endotoxin. However, GOS cross-

reacts with LAL and therefore gives false positive results and

thus was not used here. Confirmatory cytokine assays

showed that this theoretical maximal level of LPS contamina-

tion was not responsible for inducing the pronounced TNF-a

response of RAW264.7 cells when exposed to the low molecu-

lar weight fractions (Fig. S.1). Specifically, from the silver

stained gels of the electrophoresed GOS preparations it can

to the individual fractions that comprise GOS.

otein concentration (pg ml�1) P-Value 95% Confidenceintervals

st condition a Test condition b

.71 3607.77 <0.0001 6.54, 11.30

29 22757.53 <0.0001 9.74, 13.87

87 26191.11 <0.0001 10.41, 12.94

.71 2556.43 0.0002 5.94, 10.80

29 18032.71 <0.0001 9.38, 13.51

87 22018.47 <0.0001 10.24, 12.77

.71 287.52 0.0019 2.81, 7.66

29 2320.81 <0.0001 6.03, 10.16

87 1261.69 <0.0001 6.09, 8.62

.71 2430.05 0.0002 5.87, 10.73

29 18283.01 <0.0001 9.41, 13.54

87 25966.03 <0.0001 10.42, 12.94

.71 3791.73 0.0001 6.42, 11.27

29 18991.92 <0.0001 9.48, 13.61

87 29105.70 <0.0001 10.62, 13.18

7.52 3607.77 0.0104 1.22, 6.07

20.81 22757.53 0.0046 1.64, 5.77

61.69 26191.11 0.0002 3.06, 5.58

7.52 2556.43 0.0195 �5.56, �0.71

20.81 18032.71 0.0074 �5.41, �1.28

61.69 22018.47 0.0002 �5.49, �2.89

7.52 2430.05 0.0213 �5.49, �0.64

20.81 18283.01 0.0072 �5.44, �1.28

61.69 25966.03 0.0002 �5.59, �3.06

7.52 3791.73 0.0108 �6.04, �1.19

20.81 18991.92 0.0065 �5.52, �1.38

61.69 29105.70 0.0001 �5.38, �3.30

ed 54.48 pg ml�1 TNF-a.

Fig. D.1 – Cytokine responses of ex vivo harvested murine macrophages exposed to test substances. Quantification of TNF-a

cytokine production by ex vivo harvested macrophages (derived from BALB/c mice) in response to BiMuno�, filtered BiMuno�,

filtered BiMuno� without GOS, filtered GOS and the fractions of GOS. Significant increases in TNF-a were detected in the

supernatants of cells incubated with BiMuno�, filtered BiMuno�, filtered GOS and the individual fractions when compared to

the negative controls (P < 0.0001). Additionally, significant increases in TNF-a were detected in the supernatants of cells

incubated with DP3 and P4 when compared to DP2 (P 6 0.0063). Error bars, STDEV.


be concluded that the maximum amount of LPS contamina-

tion in �25 mg GOS could be 1.25 lg LPS and exposure of

RAW264.7 cells to this level of LPS induced 16,300 pg ml�1 of

TNF-a (±4072 pg ml�1), whereas GOS induced 28,970 pg ml�1

of TNF-a. Moreover, this level of TNF-a induced by GOS would

have to equate to 7.37 lg of LPS. It is acknowledged that LPS

from different sources exert different levels of cytokine re-

sponse. However, GOS should not contain LPS due to the nat-

ure of its production (being made from Gram positive and not

Gram negative bacteria) and moreover its stringent purifica-

tion during manufacture.

4. Discussion

In this study, in vitro and ex vivo studies were performed to

determine whether low molecular weight fractions of BiMu-

no�, and specifically which low molecular weight fraction(s),

have direct immuno-modulatory properties on murine mac-

rophages. Collectively, results from in vitro and ex vivo studies

showed that the low molecular weight fractions could exert

immuno-stimulatory effects on murine macrophages derived

from BALB/c mice and that the tri (DP3) and Ptetra-saccha-

ride (DP P 4) fractions, specifically, appear to be important

in stimulating cytokine responses. Previous studies by Searle

et al. (2009) demonstrated that BiMuno�, the entire commer-

cially available product, when administered orally was associ-

ated with a reduction in the colonisation, pathology and

clinical symptoms associated with murine salmonellosis. To

date, the exact mechanisms of action by which prebiotics re-

duce the carriage of pathogens remains largely unknown.

This study utilised murine macrophages, as a proof of princi-

ple, to test the hypothesis that the low molecular weight frac-

tions, derived from BiMuno�, may interact directly with

antigen presenting cells to evoke an immune response. We

showed that BiMuno� alone induced significant increases in

both pro- and anti-inflammatory cytokines in RAW264.7 cell.

Two murine specific cytokines, TNF-a and IL-6, were upregu-

lated by BiMuno� and it may be inferred important in Th1

mediated cellular responses for controlling pathogenic infec-

tions (Balaram et al., 2009; Eckmann & Kagnoff, 2001). It is

plausible that BiMuno� may stimulate antigen presenting

cells in the gut associated lymphoid tissue to aid Salmonella

clearance. As a result of this direct immuno-stimulatory ef-

fect, it seems reasonable to suggest that BiMuno� interacts di-

rectly with immune cells and/or their receptors as shown for

inulin and b-glucan activated RAW264.7 cells in vitro (Lee

et al., 2001; Lloyd, Viac, Werling, Remes, & Gatto 2007).

Questions arose from these observations as to which com-

ponent of BiMuno� is the active component and whether the

response by RAW264.7 cells was representative of macro-

phages in vivo. Data presented here indicate that the direct

immuno-stimulatory effect of BiMuno� is due to its low

molecular weight fraction component (referred to as GOS),

and more specifically the tri (DP3) and Ptetra-saccharide

(DP P 4) fractions (Tables S.2 and S.3). Ex vivo macrophages,

which would be in the natural resting state of the host, and

represent a more biologically relevant model, mounted com-

paratively large TNF-a responses to the low molecular weight

fractions and specifically the tri and Ptetra-saccharide frac-

tions (Table S.3). The direct immuno-modulatory properties

of GOS have not been documented previously and the data

produced in this study suggests a mechanism that may con-

tribute to the observations made by Vulevic et al. (2008) who

demonstrated that oral delivery of GOS was associated with

significant increases in human peripheral blood mononuclear

cell (PBMC) phagocytosis, natural killer (NK) cell activity and

altered cytokine profiles when compared to individuals fed

maltodextrin as a placebo (Vulevic et al., 2008).

Data presented here illustrate the complexity of the entire

commercially available product, BiMuno�, containing not

only low molecular weight fractions, simple monosaccha-

rides, disaccharides and complex stabilisers, but also

Table S.3 – TNF-a production from ex vivo harvested BALB/c murine macrophages in response to BiMuno�, filtered BiMuno�, filtered BiMuno� without GOS, filtered GOSand the individual fractions.

Cytokine Time (hours) Test condition a Test condition b Protein concentration (pg ml�1) P-Value 95% confidence intervals

Test condition a Test condition b

TNF-a 6 Negative BiMuno� 16.19 8176.53 <0.0001 8.33,9.71

6 Negative Filtered BiMuno� 16.19 126.98 <0.0001 2.27,3.65

6 Negative Filtered GOS 16.19 9284.09 <0.0001 8.51,9.89

6 Negative Filtered DP2 16.19 2192.58 <0.0001 6.43,7.81



6 Filtered DP2 BiMuno� 2192.58 8176.53 <0.0001 1.21,2.59

6 Filtered DP2 Filtered GOS 2192.58 9284.09 <0.0001 �2.77, �1.39

6 Filtered DP2 Filtered DP3 2192.58 4702.21 0.0063 �1.75, �0.38


6 BiMuno� Filtered BiMuno� 8176.52 126.98 <0.0001 5.37,6.75

6 BiMuno� Filtered BiMuno� without GOS 8176.52 10.01 <0.0001 8.99,10.37

6 BiMuno� Filtered DP3 8176.52 4702.21 <0.0001 0.14,1.53

6 Flitered BiMuno� without GOS Filtered BiMuno� 10.01 126.98 <0.0001 2.92,4.30

6 Flitered BiMuno� without GOS Filtered DP2 10.01 2192.58 <0.0001 �8.46, �7.09



6 Filtered BiMuno� Filtered DP3 126.98 4702.21 <0.0001 �5.92, �4.54

6 Filtered BiMuno� Filtered DP4 126.98 7738.58 <0.0001 �6.67, �5.29

6 Filtered BiMuno� Filtered GOS 126.98 9284.09 <0.0001 �6.94, �5.56


6 Filtered DP3 Filtered GOS 4702.21 9284.09 0.0082 �1.71, �0.33

aAt 6 h incubation LPS induced 7869.09 pg ml�1 TNF-a and ConA induced 7.93 pg ml�1 TNF-a.

95

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OU

RN

AL

OF

FU

NC

TI

ON

AL

FO

OD

S4

(2

01

2)

94

1–

95

3

Fig. S.1 – Detection of LPS by silver staining and cytokine assays. GOS and LPS samples electrophoresed and stained by silver

staining (A). Lane 1: GOS retentate (�25 mg ml�1) Lane 2: Novex sharp protein standard Lane 3: GOS eluent (�25 mg ml�1)

Lane 4: �1 mg GOS Lane 5: �0.1 mg GOS Lane 6: PBS negative control Lane 7: LPS retentate (1 lg) Lane 8: LPS eluent (1 lg) Lane

9: 10 lg LPS positive control Lane 10: 50 ng LPS positive control. Lane 10a: 50 ng LPS positive control when the gel was

overexposed. LPS was not detected in the negative control condition or any of the GOS preparations, whereas it was detected

in the positive controls. The limit of detection of the technique was 50 ng LPS and thus if present in �1 mg GOS, LPS is present

only in trace quantities (less than 50 ng). LPS calibration curve, and extrapolation in order to determine the theoretical LPS

content of GOS (B). Calibration curve of TNF-a production by RAW264.7 cells incubated with known concentrations of LPS

(ranging from 2.5 ng and 100 lg LPS) (as indicated by square symbols). From the calibration curve non linear regression data

analysis was conducted and showed that the maximum theoretical quantity of LPS (1.25 lg) in �25 mg GOS induced

16,300 pg ml�1 of TNF-a (±4072 pg ml�1) (as indicated by the circle symbol). In fact, �25 mg GOS induced 28,970 pg ml�1 of

TNF-a (as indicated by the triangle symbol). Furthermore, from this extrapolation it can be concluded that 7.37 lg LPS

contamination would need to be present in GOS to induce such a cytokine response.


bacterial cell debris. The complexity arises due to the manu-

facturing process that utilises whole cell B. bifidum (Tzortzis

et al., 2005a,b). Consequently, it was a prerequisite to deter-

mine whether the immuno-stimulatory effect of BiMuno�

was due to the bifidobacterial cell debris or sugars and thus

cytokine assays were conducted using filtered and unfiltered

BiMuno�. Filtering the mixture, using a 0.22 lm pore size,

served to eliminate cellular debris from the mixture. GOS

and BiMuno� without GOS did not contain bacteria, however,

were filtered for consistency with other treatments. We

showed that BiMuno�, filtered BiMuno� and filtered GOS sig-

nificantly increased levels of both pro- and anti-inflammatory

cytokines (TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12p70) in murine

macrophages when compared to negative controls

(P 6 0.0394), whereas sugars and stabilisers in the basal solu-

tion, BiMuno� without GOS, did not. In addition, an interest-

ing observation that filtering BiMuno� significantly reduced

its immuno-stimulatory effect (P 6 0.0286) was observed. This

indicates that bifidobacteria may contribute towards the

immunogenicity of BiMuno� or alternatively that the low

molecular weight fractions may be trapped in the filter along

with some of the other components of the product during the

filtering process. A key observation is that purified low molec-

ular weight fractions especially DP3 and DP4, which do not

contain bacteria, had direct immuno-stimulatory effects indi-

cates that the low molecular weight fractions derived from

BiMuno� may be, at least in part, responsible for the direct

immunogenic properties of the commercial product and

may prove efficacious at priming host immune responses.

However, we suggest that synthetic GOS be utilised in future

studies to definitively conclude whether GOS is the sole

immunogenic component of BiMuno�.

The immuno-stimulatory response of murine macro-

phages to the low molecular weight fractions (specifically

TNF-a and IL-6) resembles that induced by lipopolysaccharide

(LPS) (Beutler & Poltorak, 2001; Jiang, Akashi, Miyake, & Petty,

2000; Zughaier, Zimmer, Datta, Carlson, & Stephens, 2005).

Therefore, a necessary prerequisite of this study was to deter-

mine whether the low molecular weight fractions, derived

from BiMuno�, were contaminated with LPS and furthermore,

if it could account for the immuno-stimulatory properties of

the low molecular weight fractions even though it is unlikely

that they would contain any LPS, due to the manufacturing

and purification process. We demonstrated by silver staining

that GOS contained less than 50 ng LPS. Conducting a LAL as-

say, which is more sensitive at detecting endotoxin (as low as

5–10 pg ml�1), was not applicable in these experiments as

GOS interferes with the assay as do b-glucans (Morita, Tana-

ka, Nakamura, & Iwanaga, 1981). Whilst different endotoxins

have varying TNF-inducing activities, we demonstrated that

the maximum possible level of LPS contamination was not

responsible for the cytokine response induced by the low

molecular weight fractions. We argue that the low molecular

weight fractions should not contain any LPS and moreover no

viable Gram negative bacteria could be recovered from BiMu-

no� and we conclude that the pronounced TNF-a response

was due to the low molecular weight fractions not due to

hypothetically very low doses of LPS. As suggested previously,

synthetic GOS should be utilised in future studies to defini-

tively conclude that the immunogenicity of Bimuno� was

due to GOS and not hypothetical low molecular weight bacte-

rial components or trace amounts of LPS that could act syner-

gistically with the product. It may also be considered that

lipoteichoic acid (LTA) (Gram positive) could have contributed


to immune responses observed. However, as LTA is poorly

immunogenic compared to LPS, one can conclude that it

would be highly unlikely to evoke such pronounced

responses.

Heightened TNF-a and IL-6 production by macrophages in

response to low molecular weight fractions may promote

antigen presenting cell recruitment thus may enhance the

phagocytosis of pathogens and aid pathogen clearance. How-

ever, the results presented here only represent the single GOS

mixture tested and thus variations in efficacy may be ob-

served with different GOS mixtures.

Acknowledgments

The authors acknowledge CLASADO Ltd. for supplying the

test substance BiMuno�, purified GOS and its individual frac-

tions and for funding the studies. We acknowledge Dr. Daryan

Kaveh, Dr. Shelley Rhodes and Dr. Philip Hogarth for their

guidance in the peritoneal lavage technique and cytokine as-

says and members of the Animal Service Unit (ASU) for their

technical support. We acknowledge the Cell and Tissue Cul-

ture Section at AHVLA (Weybridge) for preparing RAW264.7

cells and Dr. Nick Coldham and Mr. Phillip Humphryes for

their guidance in the detection of LPS.

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