Synbiotic therapy (Bifidobacterium longum/Synergy 1) initiates resolution of inflammation in patients with active ulcerative colitis: a randomised controlled pilot trial.
ABSTRACT Ulcerative colitis (UC) is an acute and chronic inflammatory disease of the large bowel with unknown aetiology. The immune response against normal commensal microorganisms is believed to drive inflammatory processes associated with UC. Therefore, modulation of bacterial communities on the gut mucosa, through the use of probiotics and prebiotics, may be used to modify the disease state.
A synbiotic was developed for use in UC patients combining a probiotic, Bifidobacterium longum, isolated from healthy rectal epithelium, and a prebiotic (Synergy 1), a preferential inulin-oligofructose growth substrate for the probiotic strain. Treatment was employed in a double blinded randomised controlled trial using 18 patients with active UC for a period of one month. Clinical status was scored and rectal biopsies were collected before and after treatment, and transcription levels of epithelium related immune markers were measured.
Sigmoidoscopy scores (scale 0-6) were reduced in the test group (start 4.5 (1.4), end 3.1 (2.5)) compared with placebo (start 2.6 (2.1), end 3.2 (2.2)) (p=0.06). mRNA levels for human beta defensins 2, 3, and 4, which are strongly upregulated in active UC, were significantly reduced in the test group after treatment (p=0.016, 0.038, and 0.008, respectively). Tumour necrosis factor alpha and interleukin 1alpha, which are inflammatory cytokines that drive inflammation and induce defensin expression, were also significantly reduced after treatment (p=0.018 and 0.023, respectively). Biopsies in the test group had reduced inflammation and regeneration of epithelial tissue.
Short term synbiotic treatment of active UC resulted in improvement of the full clinical appearance of chronic inflammation in patients receiving this therapy.
- SourceAvailable from: Giovanni Cammarota[Show abstract] [Hide abstract]
ABSTRACT: Over the past recent years, a great number of studies have been directed toward the evaluation of the human host-gut microbiota interaction, with the goal to progress the understanding of the etiology of several complex diseases. Alterations in the intestinal microbiota associated with inflammatory bowel disease are well supported by literature data and have been widely accepted by the research community. The concomitant implementation of high-throughput sequencing techniques to analyze and characterize the composition of the intestinal microbiota has reinforced the view that inflammatory bowel disease results from altered interactions between gut microbes and the mucosal immune system and has raised the possibility that some form of modulation of the intestinal microbiota may constitute a potential therapeutic basis for the disease. The aim of this review is to describe the changes of gut microbiota in inflammatory bowel disease, focusing the attention on its involvement in the pathogenesis of the disease, and to review and discuss the therapeutic potential to modify the intestinal microbial population with antibiotics, probiotics, prebiotics, synbiotics and fecal microbiota transplantation.Pharmacology [?] Therapeutics 01/2015; · 7.75 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is a chronic inflammation of the small intestine and colon caused by a dysregulated immune response to host intestinal microbiota in genetically susceptible subjects. A number of fermented dairy products contain lactic acid bacteria (LAB) and bifidobacteria, some of which have been characterized as probiotics that can modify the gut microbiota and may be beneficial for the treatment and the prevention of IBD. The objective of this review was to carry out a systematic search of LAB and bifidobacteria probiotics and IBD, using the PubMed and Scopus databases, defined by a specific equation using MeSH terms and limited to human clinical trials. The use of probiotics and/or synbiotics has positive effects in the treatment and maintenance of UC, whereas in CD clear effectiveness has only been shown for synbiotics. Furthermore, in other associated IBD pathologies, such as pouchitis and cholangitis, LAB and bifidobacteria probiotics can provide a benefit through the improvement of clinical symptoms. However, more studies are needed to understand their mechanisms of action and in this way to understand the effect of probiotics prior to their use as coadjuvants in the therapy and prevention of IBD conditions.BioMed Research International 01/2014; · 2.71 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Probiotics are microorganisms that are ingested either in combination or as a single organism in an effort to normalize intestinal microbiota and potentially improve intestinal barrier function. Recent evidence has suggested that inflammatory bowel disease (IBD) may result from an inappropriate immunologic response to intestinal bacteria and a disruption in the balance of the gastrointestinal microbiota in genetically susceptible individuals. Prebiotics, synbiotics, and probiotics have all been studied with growing interest as adjuncts to standard therapies for IBD. In general, probiotics have been shown to be well-tolerated with few side effects, making them a potential attractive treatment option in the management of IBD. To perform a systematic review of randomized controlled trials on the use of probiotics, prebiotics, and synbiotics in IBD. In our systematic review we found 14 studies in patients with Crohn's disease (CD), 21 studies in patients with ulcerative colitis (UC), and five studies in patients with pouchitis. These were randomized controlled trials using probiotics, prebiotics, and/or synbiotics. In patients with CD, multiple studies comparing probiotics and placebo showed no significant difference in clinical outcomes. Adding a probiotic to conventional treatment improved the overall induction of remission rates among patients with UC. There was also a similar benefit in maintaining remission in UC. Probiotics have also shown some efficacy in the treatment of pouchitis after antibiotic-induced remission. To date, there is insufficient data to recommend probiotics for use in CD. There is evidence to support the use of probiotics for induction and maintenance of remission in UC and pouchitis. Future quality studies are needed to confirm whether probiotics, prebiotics, and synbiotics have a definite role in induction or maintenance of remission in CD, UC, and pouchitis. Similar to probiotics, fecal microbiota transplantation provides an alternate modality of therapy to treat IBD by influencing the intestinal flora.Clinical and experimental gastroenterology. 01/2014; 7:473-87.
INFLAMMATORY BOWEL DISEASE
Synbiotic therapy (Bifidobacterium longum/Synergy 1)
initiates resolution of inflammation in patients with active
ulcerative colitis: a randomised controlled pilot trial
E Furrie, S Macfarlane, A Kennedy, J H Cummings, S V Walsh, D A O’Neil, G T Macfarlane
See end of article for
Dr E Furrie, Microbiology
and Gut Biology Group,
University of Dundee,
Medical School, Dundee
DD1 9SY, UK;
Revised version received
13 May 2004
Accepted for publication
22 June 2004
Gut 2005;54:242–249. doi: 10.1136/gut.2004.044834
Background and aims: Ulcerative colitis (UC) is an acute and chronic inflammatory disease of the large
bowel with unknown aetiology. The immune response against normal commensal microorganisms is
believed to drive inflammatory processes associated with UC. Therefore, modulation of bacterial
communities on the gut mucosa, through the use of probiotics and prebiotics, may be used to modify the
Methods: A synbiotic was developed for use in UC patients combining a probiotic, Bifidobacterium
longum, isolated from healthy rectal epithelium, and a prebiotic (Synergy 1), a preferential inulin-
oligofructose growth substrate for the probiotic strain. Treatment was employed in a double blinded
randomised controlled trial using 18 patients with active UC for a period of one month. Clinical status was
scored and rectal biopsies were collected before and after treatment, and transcription levels of epithelium
related immune markers were measured.
Results: Sigmoidoscopy scores (scale 0–6) were reduced in the test group (start 4.5 (1.4), end 3.1 (2.5))
compared with placebo (start 2.6 (2.1), end 3.2 (2.2)) (p=0.06). mRNA levels for human beta defensins
2, 3, and 4, which are strongly upregulated in active UC, were significantly reduced in the test group after
treatment (p=0.016, 0.038, and 0.008, respectively). Tumour necrosis factor a and interleukin 1a, which
are inflammatory cytokines that drive inflammation and induce defensin expression, were also significantly
reduced after treatment (p=0.018 and 0.023, respectively). Biopsies in the test group had reduced
inflammation and regeneration of epithelial tissue.
Conclusions: Short term synbiotic treatment of active UC resulted in improvement of the full clinical
appearance of chronic inflammation in patients receiving this therapy.
immune response towards the commensal gut microbiota
plays a key role in the development and maintenance of this
condition.1–5For example, knockout or transgenic mice with
genetic susceptibilities to IBD only acquire characteristic
lesions when their colon is populated with normal commen-
sal bacteria while germ free animals do not manifest an
inflammatory response.6 7Evidence from human studies has
also suggested that mucosal bacterial populations in UC may
be altered towards a more proinflammatory phenotype.2 8–11
Manipulation of the mucosal microbiota to reduce the
inflammatory potential of colonising bacteria is therefore an
attractive therapy for UC. One option is to use antibiotics to
remove species involved in inducing the inflammatory
response. However, antibiotic therapy has had limited success
in UC, possibly due to the fact that treatment needs to be
customised for individual patients.12–16An alternative is to use
probiotic bacteria that interact with the host epithelium to
resolve inflammation. Probiotics have been defined as live
microbial feed supplements that beneficially affect the host
by improving its intestinal microbial balance.17The most
widely used probiotics in humans are bifidobacteria and
lactobacilli but other organisms such as Escherichia coli and
the yeast Saccharomyces boulardii have been reported to have
some beneficial effects in maintaining remission in inflam-
matory bowel disease (IBD).18–20
Probiotic therapy can be potentially improved through
combination with a prebiotic (usually a non-digestible
oligosaccharide that is not absorbed in the upper gut) that
promotes growth of the probiotic in the large bowel. This
lcerative colitis (UC) is a relapsing inflammatory
Evidence from animal models suggests that an altered
combination is known as a synbiotic. A further advantage of
using a synbiotic is that the prebiotic component would
promote the growth of indigenous organisms in the gut with
probiotic properties.21Few clinical trials have been performed
with probiotics to treat or maintain remission in IBD and the
resulting success has been variable.22–25The best known
product, VSL#3, uses a mixture of eight different bacterial
strains that have been reported to prevent the onset of
Assessments of inflammation in UC have traditionally been
done by the gastroenterologist on examination of the bowel
at endoscopy and by a pathologist who examines microscopic
inflammatory responses in biopsies. However, new sensitive
methods of measuring changes in gene expression in
inflamed tissue have been developed. Proinflammatory
cytokines have been measured before in probiotic therapy
in ex vivo colonic biopsy specimens from Crohn’s patients
where decreased expression of tumour necrosis factor a
(TNF-a) occurred,28but these cytokines can be produced by a
variety of infiltrating cells and give little indication of the
immune responsiveness of the epithelium.29 30A newly
described group of antimicrobial peptides known as human
beta defensins (hBD) are expressed uniquely by epithelial
cells. Six hBD (hBD1–6) are currently recognised. hBD1 is
constitutively expressed and its promoter has no functioning
Abbreviations: BHI, bowel habit index; CAI, clinical activity index; CRP,
C reactive protein; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; HS, histology score; hBD, human beta defensin; IBD,
inflammatory bowel disease; IL-1a, interleukin 1a; IL-10, interleukin 10;
PCR, polymerase chain reaction; SS, sigmoidoscopy score; TNF-a,
tumour necrosis factor a; UC, ulcerative colitis; WC, Wilkins-Chalgren
response elements; therefore, the level of expression of hBD1
mRNA directly allows absolute determination of the number
of epithelial cells in each biopsy.31–33hBD2 and 3 have been
shown to be significantly upregulated in UC but not in
Crohn’s disease34–36and are consequently excellent target
genes for assessing inflammatory responses in UC epithelia.
In this pilot study, we have developed a synbiotic for
treating UC patients and, to our knowledge, this is the first
report of synbiotic therapy in the treatment of UC. To
investigate the effectiveness of the synbiotic, we used
traditional methods of assessing inflammation, in conjunc-
tion with new techniques that directly measure the inflam-
matory state of the gut epithelium in a quantitative way.
The principal aim of this work was to induce clinical
improvement in UC patients receiving synbiotic therapy in
comparison with the placebo group. The second objective was
to assess the effects of synbiotic feeding on the mucosa, with
reference to inflammatory mediators induced in active colitis.
Gastroenterology Outpatients Clinic, Ninewells Hospital,
were asked to give written consent to take part in this
investigation. Eighteen patients accepted the invitation.
Eligible patients were aged 24–67 years who had not received
antibiotics in the last three months and were not taking
healthy control biopsies were obtained from other patients
attending the clinic who had been shown by sigmoidoscopy
and histology to have no evidence of inflammatory bowel
disease. These studies were approved by the Tayside
Committee on Medical Research Ethics, Dundee (study
patients withactiveUCattending the
Eighteen study numbers were assigned and randomised
using a table of random digits.37Nine patient numbers were
assigned to the test group and nine to the placebo group. The
18 patients were randomly assigned to either group, and
given a study number (SO1 to 18). This assignment was not
divulged to the clinician, patient, or inhouse researcher who
carried out the experimental measurements. A description of
patient involvement is given in table 1. Two patients did not
attend the first study visit because they had taken antibiotics
after recruitment, and therefore 16 patients entered the
study. Their characteristics are given in table 2. Patients were
recruited on the basis of active inflammation; time lag
between recruitment and initiation of the study was up to
two months. In one case (table 2) a placebo patient had
entered remission after recruitment but before commence-
ment of the study and had a sigmoidoscopy score of 0 and a
clinical activity index of 1. Recruits were maintained on the
therapy they were receiving at initiation of the trial which
was not altered during the study. Each patient was assessed
in the IBD research clinic using the clinical activity index38
and sigmoidoscopic appearance scored,39as described in
table 3. They were also requested to keep a daily bowel habit
diary, previously validated by Cummings and colleagues,40in
which details of the amount and consistency of each stool
were recorded, together with the presence of blood or mucus,
and whether the subject experienced abdominal pain. Rectal
biopsies were also taken for histology scoring by the
Department of Pathology, Ninewells Hospital (no inflamma-
tion=0, mild inflammation=1, moderate inflammation=2,
and severe inflammation=3). Venous bloods were taken for
measurement of C reactive protein (CRP; Biochemical
Medicine, Ninewells Hospital, UK). Further biopsies were
used for inhouse assessment of mucosal inflammatory
Test patients were given 261011
Bifidobacterium longum in a gelatin capsule and a sachet
containing 6 g of prebiotic fructo-oligosaccharide/inulin mix
(Synergy 1; Orafti, Tienen, Belgium), twice daily for four
weeks. Placebos were given in an identical capsule, contain-
ing potato starch, and sachets of 6 g of powdered maltodex-
trose (Orafti) to simulate the prebiotic. The synbiotic/placebo
was taken after breakfast and following the evening meal to
minimise the inhibitory effects of gastric acid on the
probiotic. At the end of one month, each patient was
reassessed in the clinic and scored in the same way as the
pretreatment visit. Biopsies were taken for histology and
measurements of inflammatory markers, while CRP levels
were measured as before.
freeze dried viable
Production of the probiotic
Nineteen different bifidobacterial isolates were assessed for
suitability as a probiotic strain (10 were isolated from healthy
colonic mucosae, five were obtained from the faeces of
healthy donors, and four were culture collection strains).
They comprised six strains of B adolescentis, two B bifidum, two
B infantis (DSM 86184, ATCC 15617), two B longum, and one
Description of patient involvement during the feeding study
Synbiotic (9 starting patients) Placebo (9 starting patients)
One removed (antibiotics)
No adverse responses
One removed (antibiotics)
Two withdrew due to deterioration of condition
Remaining 6 patients completed study, 5 with full
Post-treatment Remaining 8 patients completed
the study, 7 with full biopsy retrieval
Clinical details of patients on commencement of
Duration of disease (y)
Initial clinical activity index
Initial sigmoidoscopy score
Initial CRP (mg/l)
Values are mean (range) or number.
*Disease extent unknown for one placebo patient.
?Ulcerative colitis related drugs only.
CRP, C reactive protein.
Synbiotic therapy improves active ulcerative colitis 243
each of B angulatum, B breve, B catenulatum, B dentium, B lactis
(DSM 10140), B pseudocatenulatum, and B suis (ATCC 17533).
The organisms were assessed for aerotolerance, acid toler-
ance, bile salt resistance, adhesion to epithelial cells, and
their abilities to utilise oligofructose as an energy source.
Their abilities to survive freeze drying and long term storage
were also determined. The organism which performed best in
all cases was a B longum strain isolated from the healthy
rectal mucosa. Its identity was confirmed by partial sequen-
cing (500 of 1500 bp) of the variable region of the 16S rRNA
gene. Further analysis of the ability of the selected organism
to alter the profile of cytokine expresssion in a model
epithelial cell line (HT29) was investigated. Proinflammatory
cytokine interleukin 1a (IL-1a) was measured by both real
time polymerase chain reaction (PCR) for mRNA levels and
cytokine ELISA for secreted proteins, either alone or in
coculture with the selected B longum, B bifidum, and a
commensal Escherichia coli. The selected probiotic was found
to significantly reduce IL-1a produced by HT-29 cells with
respect to mRNA levels (p,0.05) and protein (p,0.05),
unlike the other two organisms which increased expression
of this proinflammatory cytokine. The organism was subse-
quently grown overnight in batch culture under anaerobic
conditions at 37˚C in Wilkins-Chalgren (WC) broth. Bacteria
were harvested by centrifugation (14 000 g, 30 minutes) and
washed twice in anaerobic phosphate buffered saline. The
pellet was frozen overnight at 280˚C and then lyophilised
Holbrook, New York, USA). The resulting powder was
weighed, and in order to determine cell viability and check
purity, serial dilutions were made and plated onto WC agar
plates. These were incubated aerobically and anaerobically to
maximise contaminant detection. All quantitation and purity
tests were checked by two independent microbiologists
during production and packaging of the probiotic.
RNA and cDNA preparation
Biopsies were macerated using liquid nitrogen snap freezing
and mechanical grinding. RNA was purified using the RNA
easy kit (Qiagen, Hilden, Germany) with an initial clean-up
stage using a Qiashredder column (Qiagen) and an additional
step of DNA digestion to ensure no genomic DNA contam-
ination. Samples were reverse transcribed using the AMV RT
kit (Promega, Madison, Wisconsin, USA) according to the
manufacturers instructions and aliquoted before storage at
Preparation of standards for quantitation of DNA
Standard amounts of DNA corresponding to the target
sequences are needed to carry out real time PCR. This was
achieved by making purified plasmid DNA containing the
target sequences. Briefly, cDNA from normal healthy colon,
or mononuclear blood cells, was amplified using the specific
PCR primer pair (see table 4). Product of correct size and
sequence was purified using the Qiaquick PCR purification
kit (Qiagen) and ligated into a vector using the pGEM-T easy
vector systemI (Promega).
(Promega) were transformed with each ligated vector, and
after overnight incubation positive colonies were chosen.
From each selected colony the plasmid was purified using the
Wizard plus SV miniprep system (Promega). Concentration
of the plasmid preparation was determined by agarose gel
Biolabs, Beverly, Massachusetts, USA). Samples were diluted
to 1010molecules/ml, aliquoted, and stored (280˚C).
JM109 competentE coli
standards (New England
Real time quantitative PCR
The appropriate plasmid preparation was diluted to give a
standard curve of 106–101molecules/ml for all assays, except
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which
had a standard curve of 108–101molecules/ml. Real time PCR
Baron and colleagues39)
System used to score inflammation in the colon at sigmoidoscopy (modified from
On touching mucosa
Blood in lumen
Some patchy changes
All vessels obscured
Severe active disease
levels in rectal biopsies
Real time polymerase chain reaction primers used to quantitate mRNA/rRNA
Gene targetPrimer pair 59 to 39
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) GGAAGGTGAAGGTCGGAGTC
Human beta defensin 1 (hBD1)
Human beta defensin 2 (hBD2)
Human beta defensin 3 (hBD3)
Human beta defensin 4 (hBD4)
Interleukin 1a (IL-1a)
Tumour necrosis factor 1a (TNF-a)
Interleukin 10 (IL-10)
Bifidobacterial genus specific
244 Furrie, Macfarlane, Kennedy, et al
was carried out using an iCycler and the iQ SYBR Green
Supermix (BioRad, Hercules, California, USA). Test samples
were added in triplicate at 2 ml/well in a 20 ml total reaction
Tissue specimens were fixed in formalin and embedded in
paraffin. Sections (4 mm thick) were cut using a microtome
(Leica RM 2135) and mounted onto clean glass slides which
had been coated with poly-L-lysine (Superfrost plus; VWR
International Ltd, Poole, Dorset, UK) to improve tissue
adherence. Four serial sections were stained with haematoxy-
lin and eosin, using standard methods, to visualise tissue
Significant differences between the pre-synbiotic and post-
synbiotic therapy groups and between the post-synbiotic and
post-placebo groups were assessed for hBD and cytokine
results using the Mann-Whitney test for non-parametric
Significant difference in clinical outcome was assessed using
the difference in sigmoidoscopy score (SS) in the synbiotic
group over four weeks compared with the difference in SS in
the placebo group using a two sample t test with equal
variances. Significance was given for p values less than 0.05.
Inflammatory markers in healthy and UC tissues
All results shown for inducible hBD (2–4) were normalised
for epithelial cell numbers, as determined by levels of hBD1,
the constitutive epithelial cell housekeeping gene. Figure 1
shows that there were significant increases in mRNA
expression for all inducible defensins measured (hBD2
p,0.0001, hBD3 p=0.0010, hBD4 p,0.0001) in UC rectal
biopsies (n=18) compared with normal healthy rectal
biopsies (n=12). Analysis of the proinflammatory cytokines
TNF-a and IL-1a were also significantly increased in UC
compared with normal controls (p=0.0150 and p=0.0089,
respectively) while there was no significant difference in the
immunomodulatory cytokine interleukin 10 (IL-10).
Effect of synbiotic on inflammatory markers in the gut
Figure 2 shows the results from the two trial groups,
before and after the four week feeding period. Significant
differences in expression of inducible hBD occurred in the
synbiotic patients (hBD2 p=0.0156, hBD3 p=0.0379, hBD4
p=0.0078). No significant differences between the post-
synbiotic group and the post-placebo were observed for hBD
??????? ????????? ??
????????? ?? ???? ???????? ???? ?????????? ??? ???? ??????
hBD3 (p=0.0010), hBD4 (p,0.0001), tumour necrosis factor a (TNF-a)
(p=0.0150), interleukin 1a (IL-1a) (p=0.0089)?, and interleukin 10 (IL-
10) mRNA in ulcerative colitis (UC) rectal biopsies (n=18) versus normal
rectal biopsies (n=12). Bars represent mean (SD) and results were
normalised for epithelial cell numbers through expression levels of hBD1
for inducible hBD (2–4), and for total cells through glyceraldehyde-3-
phosphate dehydrogenase levels for cytokines.
Comparison of human beta defensin (hBD)2 (p,0.0001),
????????? ?? ???? ???????? ???? ?????????? ??? ???? ??????
necrosis factor a (TNF-a), interleukin 1a (IL-1a), interleukin 10 (IL-10))
mRNA concentrations in mucosal tissue before and after four weeks’
consumption of synbiotic or placebo. Bars represent means (SD). All
results shown for inducible hBD (2–4) were normalised for epithelial cell
numbers, as determined by levels of hBD1, the constitutive epithelial cell
housekeeping gene, and cytokine levels were normalised for total cells
per biopsy using glyceraldehyde-3-phosphate dehydrogenase.
Significance between the pre- and post-synbiotic groups were hBD2
(*p=0.0156), hBD3 (*p=0.0379), hBD4 (*p=0.0078), TNF-a
(*p=0.0175), IL-1a (*p=0.0379), and IL-10 (NS). Significance between
the post-synbiotic and post-placebo groups were hBD2 (NS), hBD3 (NS),
hBD4 (NS), TNF-a (?p=0.0177), IL-1a (?p=0.0051), and IL-10 (NS).
Human beta defensin (hBD) 2–4 and cytokine (tumour
Synbiotic therapy improves active ulcerative colitis245
due to the small number of placebos who completed the trial.
Similar results were obtained for the proinflammatory
cytokines TNF-a and IL-1a, with significant reductions in
expression in the post-feeding synbiotic patients compared
with the pre-synbiotic group (p=0.0175 and p=0.0379,
respectively). On comparison of the levels of inflammatory
cytokines for the post-synbiotic group versus the post-placebo
group, we found a significant reduction for both TNF-a and
IL-1 a levels (p=0.0177 and p=0.0051, respectively). The
most marked reduction was seen with IL-1a, which returned
to levels found in normal healthy tissue (see fig 1). No
significant difference was seen in IL-10 between the placebo
and synbiotic groups.
Presence of bifidobacteria on the mucosa
Levels of bifidobacterial specific total rRNA were determined
using real time PCR in mucosal biopsies pre- and post-
treatment. The synbiotic group had a starting mean of
1406 molecules of bifidobacterial rRNA per biopsy, which
rose to a mean of 58 699 at the end of the study, a 42-fold
increase. The placebo group had a mean starting level of 3527
bifidobacterial rRNA copies per biopsy increasing to 16 285
over the same four week interval, a 4.6-fold increase. Samples
were normalised for number of epithelial cells by hBD1
Clinical outcome for patients was assessed by sigmoidoscopy
score (SS), clinical activity index (CAI), and bowel habit
index (BHI). Results are expressed as the difference in values
recorded on day 1 and day 28 of the trial for SS and CAI, and
as the percentage change over the four week period for BHI.
SS (maximum change possible=6) was decreased by a mean
of 1.3 points over the four week period in the synbiotic group
(n=8); five patients improved, two remained the same, and
one increased from 4 to 5. The placebo group had an increase
of 0.58 over the same period (n=6); three had increased
scores and three had decreased scores. SS was reduced
markedly in the synbiotic group after treatment (start 4.5
(1.4), end 3.1 (2.5)) compared with the placebo group (n=6;
start 2.6 (2.1), end 3.2 (2.2)). The difference in pre- and post-
feeding between the synbiotic and placebo groups was
borderline significant (p=0.06). CAI (maximum score 19)
was reduced in five patients in the synbiotic group with three
showing an increase in score (start 5.6 (3.7), end 5.3 (3.4)).
The placebo group had three patients with improving scores,
one which remained the same and two with increased CAI
(start 4.9 (3.2), finish 3.1 (2.5)). Mean BHI increased in the
placebo group by 70.4% compared with a reduction of 20.4%
in the synbiotic group.
Correlation of hBD levels with sigmoidoscopy scores
Analysis of the relationship between SS and levels of
individual hBD showed that there was no correlation
between hBD1 with SS, before or after treatment, in either
the test or placebo group (results not shown). However, in
both synbiotic and placebo groups pretreatment, there was a
positive correlation between SS and mRNA for hBD2–4
whereby in more active disease higher levels of inducible hBD
were detected (fig 3A). At the end of the feeding study,
comparison of SS and hBD showed that this positive
correlation remained only in the placebo group whereas
hBD2 and hBD4 manifested a loss of correlation with SS
score, and hBD3 had an inverse relationship in the synbiotic
Correlation of hBD levels with histology scores (HS)
A positive correlation was evident in the relationship between
HS and hBD2–4 in both synbiotic and placebo groups, before
??????? ? ??????
????????? ?? ????
????????? ?? ????
0–3) with inducible hBD mRNA synthesis (B) in synbiotic patients and placebos. Lines represent exponential best fits for synbiotic (broken lines) and
placebo (solid lines) data sets, respectively.
Comparison of sigmoidoscopy scores (scale 0–6) with inducible human beta defensin (hBD) mRNA synthesis (A) and histology scores (scale
246Furrie, Macfarlane, Kennedy, et al
the feeding study (fig 3B). After treatment, the placebos
retained a positive correlation for all three inducible beta
defensins, as occurred with SS. In the synbiotic group, a
positive correlation for hBD2 and 4 remained when compar-
ing HS with mRNA concentrations but hBD3 levels related
poorly with HS in this group (fig 3B).
C reactive protein (CRP) levels in blood
Only five of the eight synbiotic patients had elevated CRP
(mean 6.0 (SD 6.5)), and only one of six patients in the
placebo group who completed the study had elevated CRP
(mean 1.6 (SD 3.6)). After treatment, the synbiotic group
showed a reduction in CRP (mean 1.8 (SD 3.9)) after four
weeks. None of the placebo patients had raised circulating
levels of CRP.
Histopathology of rectal biopsies
Histology scoring produced results similar to SS; four
individuals showed a reduction in score and two remained
the same, with a mean reduction in HS in the synbiotic group
over the four week period (start 1.7 (1.4), end 1.1 (1.2))
compared with the mean increase in the placebo group (start
0.9 (0.9), end 1.9 (1.1)), with three showing an increase in
score and two remaining the same. Two representative paired
biopsies are shown in fig 4: A (SS=6, HS=3) and C
(SS=3.5, HS=1.5) were from a synbiotic patient, pre- and
post-feeding, respectively, while B (SS=1.5, HS=1.5) and D
(SS=5.0, HS=2.5) were from a placebo treated patient, at
the beginning and end of the study. Comparison of fig 4A and
C shows resolution of acute inflammatory activity following
synbiotic consumption. The crypt abscesses in fig 4A have
disappeared and the epithelium shows a more regenerative
appearance in fig 4C. Small crypt abscesses are evident in the
rectal biopsy from the placebo patient (fig 4B). This
inflammation has been exacerbated in fig 4D, with a larger
abscess visible in the middle of the section, with crypt
rupture, and a general increase in the numbers of infiltrating
Consumption of synbiotic twice daily over four weeks
significantly reduced mucosal inflammatory markers in
active UC. This was concurrent with a reduction in colitis at
the macroscopic and microscopic level. However, although
lowered SS were observed in the synbiotic group, they were
not as marked as the reductions in HS and inflammatory
markers. This may indicate that changes in inflammatory
mediators at the molecular level could precede gross clinical
changes scored through sigmoidoscopy by several weeks. This
contention is supported by the relationship seen in HS and
not SS after synbiotic feeding, with both hBD2 and 4 showing
that although local inflammation was resolving in these
patients, more general changes in colonic appearance, such as
vessel pattern, take longer to return to normal. Alternatively,
this therapy may be resolving the local inflammation through
direct contact with the epithelium but is insufficient in all
cases of UC (three of eight patients receiving synbiotic
therapy had no improvement in SS) in reducing the systemic
chronic inflammatory condition. It is possible that these
patients may improve their clinical scores if this treatment is
given in conjunction with other therapies or through
selection of a different probiotic organism. Unfortunately,
no significant difference in SS or CAI between the synbiotic
and placebo groups was found. This may be due to the small
number of placebos who completed the study. Two placebos
requested withdrawal because of exacerbation of symptoms,
and refused to undergo sigmoidoscopy or provide a final
biopsy due to discomfort. There were no withdrawals from
the synbiotic group after commencement of the study and no
reports of adverse reactions.
Markers of active disease in biopsies from UC patients
include proinflammatory cytokines such as TNF-a, IL-8, IL-
1a, and IL-1b.29These molecules are upregulated in active UC
but they are not specific for the epithelium as the large
numbers of infiltrating leucocytes in the mucosa contribute
greatly to their formation.28–30When using a probiotic or
synbiotic, it is important to be able to assess directly its
effects on the epithelial barrier as well as the underlying
compared with a placebo patient at the start (B) and the end (D) of the study.
Representative histopathology of rectal mucosa from a patient with ulcerative colitis pre-synbiotic therapy (A) and post-treatment (C),
Synbiotic therapy improves active ulcerative colitis247
immune system as the epithelium is the first point of host
contact for the organisms. Human beta defensins are good
indicators of mucosal immunity because they are uniquely
expressed by epithelial cells.31 32Two types of expression
occur: hBD1 is a non-inducible constitutively expressed
molecule while hBD2–4 are upregulated by bacterial chal-
lenge and proinflammatory cytokines.33 34 41These molecules
are produced by the inflammatory infiltrate and by the
epithelium itself30directly affecting expression levels of
inducible hBD in epithelial cells. IL-1 has been shown to
induce upregulation of hBD2 in gastric and colonic epithelial
cell lines.34 41
In this study, a direct relation between mRNA synthesis for
inducible hBD and severity of UC (SS) has been shown for
the first time, together with the relationship between
inducible hBD and histology scores in UC patients. We were
unable to demonstrate a significant difference between the
inducible hBD in the post-synbiotic group versus the post-
placebo group but the proinflammatory cytokines TNF-a and
IL-1a were significantly reduced in the synbiotic group
compared with the placebo group. This can be explained by
the relationship between SS and levels of inducible hBD. As
our placebo group had lower clinical scores than the synbiotic
group, resulting from the small sample size and random
assignment to each group, we have inadvertently biased
against finding a significant difference in inducible hBD
between the two groups due to the direct linear relationship
between SS and hBD. This is not true for the inflammatory
cytokines that have a more all or nothing expression pattern
and can be produced by any infiltrating cell in the biopsy.
Furthermore, they are compared against the usual house-
keeping genes (GAPD/b-actin) for biopsy size standardisa-
tion, unlike hBD which are only produced by colonic
epithelial cells and epithelial cell numbers can be accurately
standardised by hBD1 levels.
The functions of these induced hBDs are unknown in UC
disease progression. However, hBD are antimicrobial peptides
that are particularly good at destroying Gram negative
bacteria33so their production may be an attempt by the
epithelium to modify the composition of the mucosal biofilm.
The bifidobacterial probiotic strain used in this study was
found to be insensitive to hBD mediated killing by
recombinant hBD1, 2, and 3 (results not shown), and
therefore, as part of a therapy for UC this organism would
exhibit better survival characteristics on the inflamed mucosa
and interact directly with the epithelial surface and modify
the immune response. We detected higher numbers of total
bifidobacteria on the mucosal surface in patients fed the
synbiotic compared with those taking placebo. We cannot
separately identify the probiotic organism from the indigen-
ous bifidobacteria that may have been stimulated by
inclusion ofa prebiotic
Bifidobacterium longum is a very complicated organism with
at least five different subgroups contained within the species
when 16S rDNA sequences are compared (data not shown).
With the present technology it is impossible to distinguish
the probiotic strain from other Bifidobacterium longum in a
small pinch biopsy. It is unknown what direct effects
probiotic bacteria have on the inflamed epithelium. They
could simply be out competing microorganisms involved in
disease aetiology for microniches on the gut wall, thereby
removing inflammatory stimuli. However, they may also be
directly influencing the expression of inflammatory cytokines
by the epithelium through ligation of particular pattern
recognition receptors on the luminal cell surface, as sup-
ported by studies using HT29 cells in preliminary screening
experiments with the probiotic. Alternatively, the probiotic
could be directly influencing dendritic cells that are present
in high numbers in the lamina propria42with access to the
inthe feeding regimen.
cytokine pathways and stimulating a more immunomodula-
tory and tolerant immune response.
In conclusion, this short term pilot study has provided the
first evidence that synbiotics have the potential to be
developed into acceptable therapies for patients suffering
from acute UC. A large scale clinical trial is now needed to
investigate the long term effects of synbiotic use in inducing
and maintaining remission in patients with active disease.
by downregulating proinflammatory
We wish to acknowledge the help of Dr Douglas Steinke as a
statistical consultant. The work was funded by the Medical Research
E Furrie, S Macfarlane, A Kennedy, G T Macfarlane, MRC Microbiology
and Gut Biology Group, University of Dundee, Ninewells Hospital
Medical School, Dundee, UK
J H Cummings, Division of Pathology and Neuroscience, University of
Dundee, Ninewells Hospital Medical School, Dundee, UK
S V Walsh, Department of Pathology, Ninewells Hospital and Medical
School, Dundee, UK
D A O’Neil, Immunology Unit, Rowett Research Institute, Aberdeen, UK
Conflict of interest: None declared.
1 Macdonald TT, Monteleone G, Pender SLF. Recent developments in the
immunology of inflammatory bowel disease. Scand J Immunol 2000;51:2–9.
2 Campieri M, Gionchetti P. Bacteria as the cause of ulcerative colitis. Gut
3 Schultsz C, Van Den Berg FM, Ten Kate FW, et al. The intestinal mucus layer
from patients with inflammatory bowel disease harbors high numbers of
bacteria compared with controls. Gastroenterology 1999;117:1089–97.
4 Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to
specificity and diversity. Cell 1991;67:1033–6.
5 Duchmann R, Kaiser I, Hermann E, et al. Tolerance exists towards resident
intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin
Exp Immunol 1995;102:448–55.
6 Sadlack B, Merz H, Schorle H. Ulcerative colitis-like disease in mice with a
disrupted interleukin-2 gene. Cell 1993;75:253–61.
7 Taurog JD, Richardson JA, Croft JT, et al. The germ free state prevents
development of gut and joint inflammatory disease in HLA-B27 transgenic
rats. J Exp Med 1994;180:2359–64.
8 Matsuda H, Fujiyama Y, Andoh A, et al. Characterisation of antibody
responses against rectal mucosa-associated bacterial flora in patients with
ulcerative colitis. J Gastroenterol Hepatol 2000;15:61–8.
9 Hartley MG, Hudson MJ, Swarbrick ET, et al. The rectal mucosa associated
microflora in patients with ulcerative colitis. J Med Microbiol
10 Poxton IR, Brown R, Sawyer A, et al. Mucosa-associated bacterial flora in the
human colon. J Med Microbiol 1997;46:85–91.
11 Macfarlane S, Furrie E, Cummings JH, et al. Chemotaxonomic analysis of
bacterial populations colonizing the rectal mucosa in patients with ulcerative
colitis. Clin Infect Dis 2004;38:1690–9.
12 Dickinson RJ, O’Connor HJ, Pinder I, et al. Double blind controlled trial of oral
vancomycin as adjunctive treatment in acute exacerbations of idiopathic
colitis. Gut 1985;26:1380–4.
13 Mantzaris GJ, Hatzis A, Kontogiannis P, et al. Intravenous tobramycin and
metronidazole as an adjunct to corticosteroids in acute, severe ulcerative
colitis. Am J Gastroenterol 1994;89:43–6.
14 Turunen UM, Farkkila MA, Hakala K, et al. Long-term treatment of ulcerative
colitis with ciprofloxacin: a prospective, double-blind, placebo-controlled
study. Gastroenterology 1998;115:1072–8.
15 Present DH. Ciprofloxacin as a treatment for ulcerative colitis—not yet.
16 Cummings JH, Macfarlane GT. Is there a role for microorganisms? In:
Jewell DP, Warren BF, Mortensen NJ, eds. Challenges in inflammatory bowel
disease. Oxford: Blackwell Science, 2001:47–8.
17 Macfarlane GT, Cummings JH. Probiotics, infection and immunity. Curr Opin
Infect Dis 2002;15:501–6.
18 Kruis W, Schutz E, Fric P, et al. Double blind comparison of an oral
Escherichia coli preparation and mesalazine in maintaining remission of
ulcerative colitis. Aliment Pharmacol Ther 1997;11:853–8.
19 Rembacken BJ, Snelling AM, Hawkey PM, et al. Non-pathogenic Escherichia
coli versus mesalazine for the treatment of ulcerative colitis: a randomised
trial. Lancet 1999;354:635–9.
20 Guslandi M, Mezzi G, Sorghi M, et al. Saccharomyces boulardii in
maintenance treatment of Crohn’s disease. Dig Dis Sci 2000;45:1462–4.
21 Roberfroid M. Prebiotics and synbiotics: concepts and nutritional properties.
B J Nutr 1998;80:S197–202.
248Furrie, Macfarlane, Kennedy, et al
22 Venturi A, Gionchetti P, Rizzello F, et al. Impact on the composition of the
faecal flora by a new probiotic preparation: preliminary data on maintenance
treatment of patients with ulcerative colitis. Aliment Pharmacol Ther
23 Prantera C, Scribano ML, Falasco G, et al. Ineffectiveness of probiotics in
preventing recurrence after curative resection for Crohn’s disease: a
randomised controlled trial with Lactobacillus GG. Gut 2002;51:405–9.
24 Laake KO, Line PD, Aabakkan L, et al. Assessment of mucosal inflammation
and circulation in response to probiotics in patients operated with ileal pouch
anal anastomosis for ulcerative colitis. Scand J Gastroenterol
25 Hart AL, Stagg AJ, Kamm MA. Use of probiotics in the treatment of
inflammatory bowel disease. J Clin Gastroenterol 2003;36:111–19.
26 Gionchetti P, Rizzello F, Helwig U, et al. Prophylaxis of pouchitis onset with
probiotic therapy: a double blind, placebo controlled trial. Gastroenterology
27 Mimura T, Rizzello F, Helwig U, et al. Once daily high dose probiotic therapy
(VSL#3) for maintaining remission in recurrent or refracrory pouchitis. Gut
28 Borruel N, Carol M, Casallas F, et al. Increased mucosal tumour necrosis
factor a production in Crohn’s disease can be down regulated ex vivo by
probiotic bacteria. Gut 2002;51:659–64.
29 Woywodt A, Ludwig D, Neustock P, et al. Mucosal cytokine expression,
cellular markers and adhesion molecules in inflammatory bowel disease.
Eur J Gastroenterol Hepatol 1999;11:267–76.
30 Daig R, Rogler G, Aschenbrenner E, et al. Human intestinal epithelial cells
secrete interleukin-1 receptor antagonist and interleukin-8 but not interleukin-
1 or interleukin-6. Gut 2000;46:350–8.
31 Zhao C, Wang I, Lehrer RI. Widespread expression of beta defensin hBD-1 in
human secretory glands and epithelial cells. FEBS Lett 1996;396:319–25.
32 Yamaguchi Y, Nagase T, Makita R, et al. Identification of multiple novel
epididymis-specific beta-defensin isoforms in humans and mice. J Immunol
33 O’Neil DA. Regulation of expression of beta-defensins: endogenous enteric
peptide antibiotics. Mol Immunol 2003;40:445–50.
34 O’Neil DA, Porter EM, Elewaut D, et al. Expression and regulation of the
human beta-defensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol
35 Wehkamp J, Harder J, Weichenthal M, et al. Inducible and constitutive beta
defensins are differentially expressed in Crohn’s disease and ulcerative colitis.
Inflamm Bowel Dis 2003;9:215–23.
36 Fellerman K, Wehkamp J, Herrlinger KR, et al. Crohn’s disease: a defensin
deficiency syndrome? Eur J Gastroenterol Hepatol 2003;15:627–34.
37 Daniel WW. Biostatistics: A foundation for analysis in the health sciences, 4th
edn. New York: John Wiley and Sons Inc, 1987.
38 Walmsley RS, Ayres RCS, Pounder RE, et al. A simple clinical colitis activity
index. Gut 1998;43:29–32.
39 Baron JH, Connell AM, Lennard-Jones JE. Variation between observers in
describing mucosal appearances in proctocolitis. BMJ 1964;1:89–92.
40 Cummings JH, Christie S, Cole TJ. A study of fructoloigosaccharides in the
prevention of travellers’ diarrhoea. Aliment Pharmacol Ther
41 O’Neil DA, Cole SP, Martin-Porter E, et al. Regulation of human beta-
defensins by gastric epithelial cells in response to infection with Helicobacter
pylori or stimulation with IL-1. Infect Immun 2000;68:5412–15.
42 Stagg AJ, Hart AL, Knight SC, et al. The dendritic cell: its role in intestinal
inflammation and relationship with gut bacteria. Gut 2003;52:1522–9.
43 Rescigno M, Urbano M, Valzasina B, et al. Dendritic cells express tight
junction proteins and penetrate gut epithelial monolayers to sample bacteria.
Nat Immunol 2001;2:361–7.
EDITOR’S QUIZ: GI SNAPSHOT ..................................................................
From question on page 206
Computed tomography scan of abdomen revealed a large collection of intrahepatic gas
peripherally (fig 1) and dilated loops of intestine with pneumatosis (fig 2). Hypotension
persisted despite aggressive volume resuscitation. Exploratory laparotomy revealed necrotic
intestine from the proximal jejunum to the hepatic flexure. The necrotic intestine was
resected, and jejunotransversostomy was performed.
Intrahepatic gas may represent either an incidental finding or a life threatening condition.
Intrahepatic gas may be present in either the biliary or portal venous systems. Computed
tomography scan may help differentiate gas in the biliary system from that of the portal
venous vasculature. Biliary gas tends to be located centrally in the hilar region in the larger
bile ducts. In contrast, gas in the portal system extends to the smaller portal venous
branches, resulting in a less localised peripheral distribution. Mucosal and submucosal
bowel wall damage may allow some intraluminal air to enter the damaged or dissected
bowel wall, leading to pneumatosis, and also to enter some intramural veins, leading to
portal venous gas. Therefore, portal venous gas may be observed in any patient with severe
inflammation of the intestinal walls—for example, necrotising enterocolitis, ulcerative
colitis, Crohn’s disease, large ulcerating gastric carcinoma, appendicitis, and diverticulitis.
the intestinal wall (black arrows), and a thickened oedematous intestinal
wall (arrowhead). White arrow represents luminal air.
Computed tomography scan demonstrating gas collections in
Synbiotic therapy improves active ulcerative colitis 249