Original Paper

Digestion 2010;81:252–264 Received: September 4, 2009

Accepted: November 26, 2009
DOI: 10.1159/000264649
Published online: January 30, 2010

Clinical Relevance of IgG Antibodies against

Food Antigens in Crohn’s Disease: A Double-Blind
Cross-Over Diet Intervention Study
S. Bentz a M. Hausmann a H. Piberger d S. Kellermeier a S. Paul c L. Held b
W. Falk d F. Obermeier d M. Fried a J. Schölmerich d G. Rogler a
a
Division of Gastroenterology and Hepatology, University Hospital Zurich, and b University of Zurich,
Institute of Social and Preventive Medicine, Biostatistics Unit, Zurich, Switzerland; c Evomed MedizinService
GmbH, Darmstadt, and d Department of Internal Medicine I, University of Regensburg, Regensburg, Germany

Key Words clusion: A nutritional intervention based on circulating IgG

Crohn’s disease ⴢ Immunoglobulin G antibodies ⴢ Food
antigen ⴢ Interferon
Abstract
Background: Environmental factors are thought to play an
important role in the development of Crohn’s disease (CD).
Immune responses against auto-antigens or food antigens
may be a reason for the perpetuation of inflammation. Meth-
ods: In a pilot study, 79 CD patients and 20 healthy controls
were examined for food immunoglobulin G (IgG). There-
after, the clinical relevance of these food IgG antibodies
was assessed in a double-blind cross-over study with 40 pa-
tients. Based on the IgG antibodies, a nutritional interven-
tion was planned. The interferon (IFN)␥ secretion of T cells
was measured. Eosinophil-derived neurotoxin was quanti-
fied in stool. Results: The pilot study resulted in a significant
difference of IgG antibodies in serum between CD patients
and healthy controls. In 84 and 83% of the patients, respec-
tively, IgG antibodies against processed cheese and yeast
were detected. The daily stool frequency significantly de-
creased by 11% during a specific diet compared with a sham
diet. Abdominal pain reduced and general well-being im-
proved. IFN␥ secretion of T cells increased. No difference for
eosinophil-derived neurotoxin in stool was detected. Con-
antibodies against food antigens showed effects with re-
spect to stool frequency. The mechanisms by which IgG an-
tibodies might contribute to disease activity remain to be
elucidated. Copyright © 2010 S. Karger AG, Basel
Introduction
Genetic influences [1, 2], cytokine activation [3] and
various specific and nonspecific environmental factors
like hygiene, social standard, climatic factors, environ-
mental pollution, smoking, stress and nutrition [4–7]
have been considered to be associated with the induction
and/or exacerbation of inflammatory bowel disease
(IBD), such as Crohn’s disease (CD).
Serologic markers for IBD, like anti-Saccharomyces
cerevisiae antibodies and atypical perinuclear antineu-
trophil cytoplasmic antibodies, remain to play a role in
the pathophysiology of IBD. There is a wide range of
other antibodies including outer-membrane porin C,
anti-Pseudomonas fluorescens and antiglycan antibodies
(anti-laminaribioside carbohydrate antibody, anti-chito-
bioside carbohydrate antibody, anti-mannobioside car-
bohydrate antibody), and anti-CBir1. The latter is the

© 2010 S. Karger AG, Basel Gerhard Rogler, MD, PhD

0012–2823/10/0814–0252$26.00/0 Division of Gastroenterology and Hepatology
Fax +41 61 306 12 34 Department of Internal Medicine University Hospital of Zurich
E-Mail karger@karger.ch Accessible online at: Rämistrasse 100, CH–8091 Zurich (Switzerland)
www.karger.com www.karger.com/dig Tel. +41 44 255 9579, Fax +41 44 255 9497, E-Mail gerhard.rogler @ usz.ch
first bacterial antigen found to induce colitis in animal
models of IBD and also leads to a pathological immune
response in IBD patients [8].
Frequently, IBD patients report that dietary intoler-
ance significantly contributes to their symptomatology.
The benefit from eliminating certain foods [9] from dai-
ly diet was refocused in the present study. Attempts to test
for food intolerance in IBD have largely focused on classic
food allergies based on the presence of immunoglobulin
E (IgE)-mediated antibody responses, although these re-
actions appear probably quite rare in IBD [10]. It is there-
fore possible that adverse reactions in IBD might be due
to some reactions mediated by IgG antibodies, which
characteristically give a more delayed response following
exposure to a particular antigen [11] and have been im-
plicated in some cases of food hypersensitivity [12–14].
However, this mechanism is controversial and is consid-
ered to be physiological [15–17], as IgG food antibodies
can be present in apparently healthy individuals [18, 19].
It has been assumed that chronic inflammation in IBD is
due to an imbalance between inflammatory and anti-in-
flammatory mechanisms like regulatory CD4+CD25+ T
cells (Tregs) [20–22].
High IgG levels against certain food components in
blood and the inflammatory response of T cells to food
antigens and the regulatory effect of Tregs in vitro was as-
sessed. As IgG food antibodies may play a role during the
initiation or perpetuation of IBD, we first investigated the
presence of IgG antibodies in CD patients and healthy
controls. In a second approach, the therapeutic potential
of an elimination diet based on the presence of IgG anti-
bodies to food in patients with CD in a randomized con-
trolled trial was investigated. Primary outcome parame-
ters were stool frequency, abdominal pain and general
well-being. The possible activation of T-effector cells
through IgG antibodies was measured by interferon
(IFN)␥ secretion. For the evaluation of disease activity,
eosinophil-derived neurotoxin (EDN) was quantified in
stool. A secondary outcome parameter was the total score
built from stool frequency, abdominal pain and general
well-being.
Methods
Study Design 1: Pilot Study
Initially, 20 healthy volunteers without history of food intoler-
ance and 79 CD patients with different disease status were in-
cluded in a pilot study. Forty-seven of them had clinical and en-
doscopic signs of acute inflammation (i.e. diarrhea and mucosal
ulcerations). Twenty-four CD patients presented with chronically
Food Antigens in Crohn’s Disease
active disease and 8 were in remission. Patients were recruited
from the German IBD Competence Network serum bank and ex-
amined for food specific IgG by the ImuPro300 test (Evomed,
Darmstadt, Germany). Disease activity was assessed by the pa-
tient’s medical record.
Study Design 2: Following Intervention Study
Consecutively in a randomized, double-blind, cross-over in-
tervention study, the clinical relevance of IgG antibodies against
food antigens in 40 CD patients was tested. Not all patients from
the previous pilot study were willing to participate in the follow-
ing intervention study; therefore, new patients were also tested for
food IgG antibodies in serum. Patients were not selected for IgG
levels in serum. A sample size calculation was not performed. Fi-
nally, the specific antibody pattern of 40 patients was determined
in serum samples by the ImuPro300 test system. The reactivity of
Tregs and CD4+CD25– T cells to the patient-specific food antigens
was determined in vitro (mixed lymphocyte stimulation assay)
and correlated with in vivo changes on the basis of a nutritional
record and a patient diary. The diary contained questions about
stool frequency, abdominal pain and general well-being. Patients
validated their pain perception with scores of 0, 1, 2 and 3 which
represented no pain, slight pain, moderate pain and severe pain,
respectively. The values were accumulated after each week. Ad-
ditionally, the patients rated their general well-being. The patients
assessed general well-being by a score of 0, 1, 2, 3 and 4, which
represented good, worse, bad, very bad and terrible, respectively.
To get an overall impression of the symptoms, stool frequency,
abdominal pain and general well-being, a total score was calcu-
lated. Each subject recorded his eating habits and disease symp-
toms over a period of 12 weeks and followed a specific or sham
diet. Each diet was followed for 6 weeks (fig. 1). The definition of
specific and sham diet was based on similarity of excluded food
components. If, for example, IgG against hazelnut was detected,
then almond was excluded in the sham diet; if cauliflower IgG was
found, broccoli was excluded. Patients were concealed and allo-
cated to one of the two diet sheets based on a randomization
schedule using a random computer number generator. Thus, pa-
tients received either an elimination diet based on their true sen-
sitivity results (specific diet) or a sham diet. Baseline demograph-
ic and clinical characteristics of the two groups, including the use
of concomitant medication, were found to be similar. All patients
and clinical staff in the gastroenterology research department
were blinded to the group assignment of all patients for the dura-
tion of the study. Patients were given their allocated diet sheet by
staff at the gastroenterology department and asked to eliminate
the indicated foods from their diet for a period of 12 weeks. They
also received a booklet with advice on how to eliminate the dif-
ferent foods (recipes and menus). This was explained by an expe-
rienced nutritionist. Furthermore, the telephone contact details
of a free nutritional advisor who they could contact for further
advice if necessary was given to each patient.
Subjects
There were 16 male and 24 female subjects. Ultimately, data
analysis of 23 patients was performed. Patients between the ages
of 18 and 60 years were considered eligible. In this study, the pa-
tients were between 21 and 59 (mean 41 8 11) years of age. Both
active and inactive patients were included, and diagnosis was
manifested at least 6 months before onset of the trial. Duration of
Digestion 2010;81:252–264 253
 Consort Flowchart

Assessed for eligibility from the

German IBD Competence Network,
Ratisbona, Germany (n = 100)

Excluded (n = 60)
Not meeting inclusion criteria
Enrollment (n = 32)
Refused to participate
(n = 28)
Randomized (n = 40)

Allocated to intervention 1 (specific diet, 6 weeks) Allocated to intervention 2 (sham diet, 6 weeks)
(n = 20) (n = 20)
Received allocated intervention Received allocated intervention
Allocation
(n = 20) (n = 20)
Did not receive allocated intervention Did not receive allocated intervention
(n = 0) (n = 0)

Allocated to intervention 2 (sham diet, 6 weeks) Allocated to intervention 1 (specific diet, 6 weeks)
(n = 20) (n = 20)
Received allocated intervention Received allocated intervention
(n = 12) (n = 11)
Did not receive allocated intervention Did not receive allocated intervention
(n = 8) (n = 9)

Reasons Reasons
diet too restrictive: 4 diet too restrictive: 6
low compliance: 2 low compliance: 1
other reasons: 2 other reasons: 2

Analyzed (n = 12) Analyzed (n = 11)

Analysis
Excluded from analysis (n = 0) Excluded from analysis (n = 0)

Fig. 1. Study flow of the intervention trial. Patients were allocated to one of the two diets: either an elimination
diet based on their true sensitivity results (specific diet) or a sham diet and followed for 6 weeks. 17 patients did
not finish the trial.

disease was between 2 and 39 years (mean 14.9 8 10.6). Patients
were excluded from participating if they had any significant co-
existing disease. During screening some patients had to be ex-
cluded due to a lack of cooperation (n = 3), severe concomitant
disease (n = 10), abscesses (n = 15) or for C-reactive protein 1150
(n = 4). Twenty-eight of the patients refused to participate. During
the intervention phases the patients were allowed to take con-
comitant medication provided it had been constant for the 12
weeks of intervention (table 1). The constant medication over
time was requested to determine a specific effect of nutritional
intervention and not of higher doses of medication. This study
was approved by the ethics committee of the University of Re-
gensburg and performed according to the declaration of Helsinki.
Informed consent was obtained from all patients.

254 Digestion 2010;81:252–264 Bentz et al.

Table 1. Baseline characteristics of the patients: 16 males (m) and 24 females (w) participated in the study

No. Gender Age Disease localization Treatment

1 m 21 terminal ileum, cecum mesalazine, dimeticone, diltiazem

2 w 55 cecum to sigma azathioprine, glibenclamide
3 w 24 colon, terminal ileum budesonide
4 w 37 terminal ileum, cecum budesonide, azathioprine, mesalazine, hydro-
cortisonacetate, loperamide
5 m 37 terminal ileum, proximal colon, proctitis azathioprine, prednisolone
6 m 44 terminal ileum, cecum azathioprine, prednisolone
7 m 41 ileum resection, multiple perianal fistulae mesalazine
8 w 37 anastomosis budesonide
9 m 37 distal ileum azathioprine
10 m 27 terminal ileum, cecum azathioprine
11 m 54 neoterminal ileum, colon, sigma, anal stenosis cholestyramine
12 m 55 terminal ileum azathioprine
13 w 31 esophagus lesions infliximab, azathioprine, prednisolone
14 m 54 stenosis terminal ileum, ileocecal resection none
15 w 57 sigma segment resection, stenosis colon descendens cholestyramine
16 w 38 colon, sigma, rectum, stenosis terminal ileum mesalazine, azathioprine, hydrocortisone
17 w 43 terminal ileum azathioprine, azulfidine
18 w 54 colon infliximab
19 w 54 colon azathioprine
20 w 31 neoterminal ileum, colon 6-mercaptopurine
21 w 50 fistulae azathioprine
22 w 33 neoterminal ileum infliximab
23 w 25 colon, distal ileum mesalazine
24 w 46 terminal ileum, caecum, sigma, rectum budesonide, azathioprine
25 w 49 ileotransversostomy azathioprine
26 w 57 fistulae infliximab, azathioprine, azulfidine, mesalazine,
cholestyramine
27 m 41 colon prednisolone, azathioprine
28 w 48 colon, ileocecal resection methotrexate
29 m 51 colon budesonide
30 m 35 rectum azathioprine, loperamide
31 m 29 colon prednisolone, mesalazine, hydrocortisone
32 w 59 colon, neoterminal ileum azathioprine
33 w 43 terminal ileum, colon mesalazine
34 w 48 neoterminal ileum budesonide
35 m 42 rectum, sigma, fistulae prednisolone, ciprofloxacine, metronidazole
36 w 22 terminal ileum, colon cholestyramine
37 w 28 colon infliximab
38 w 44 terminal ileum, sigma methotrexate, azathioprine
39 m 41 terminal ileum, colon infliximab
40 m 35 colon azathioprine

Patients were between 21 and 59 years of age (42 8 12), treated with diverse drugs (treatment) and had different disease localization.

ImuPro300 Test
Sera from 40 patients was examined for food specific IgG by
an enzyme-linked immunosorbent assay (ELISA) ImuPro300 test
according to the manufacturer’s recommendations (R-Biopharm,
Darmstadt, Germany). Specific IgG antibodies against 271 food
allergens (online suppl. table 1, for all online suppl. material see
www.karger.com/doi/10.1159/000264649) are possible to deter-
mine in human serum. The content of IgG antibodies is demon-
strated in table 2. Only high values (score 3) and very high values
(score 4) of IgG were excluded from the diet of the patients. Di-
luted human serum was incubated in three different 96-well
plates, each well coated with a different food extract. After wash-
ing the plates 3 times with diluted washing buffer, a polyclonal
anti-human IgG antibody (sheep; R-Biopharm) conjugated to al-
kaline phosphatase was added. After washing with phosphate-
buffered saline (PBS), substrate solution (pnpp, R-Biopharm) was

Food Antigens in Crohn’s Disease Digestion 2010;81:252–264 255

Table 2. IgG antibodies in patients’ serum by ImuPro300
IgG-class Allergen-specific
IgG content
<7.49 ␮g/ml 0 (0.0–0.9) negative
7.5–12.49 ␮g/ml 1 (1.0–1.9) weak
12.5–19.99 ␮g/ml 2 (2.0–2.9) increased
20.0–49.99 ␮g/ml 3 (3.0–3.9) high
≥50 ␮g/ml 4 (4.0–4.9) very high
In patient-specific diets, only foods with score 3 (IgG content
20–49.49 ␮g/ml) and score 4 (IgG content ≥50 ␮g/ml) were ex-
cluded.
added to reveal the presence of IgG in the serum. Color develop-
ment is proportional to the quantity of bound antibodies. After
addition of a stop solution (NaOH; R-Biopharm), optical densities
were measured photometrically (405/620 nm, Tecan Sunrise; Te-
can GmbH, Crailsheim, Germany). IgG concentrations were cal-
culated using a standard curve.
Collection of Peripheral Blood and Isolation of Tregs and
CD4+CD25– T Cells
50 ml of peripheral blood were obtained from each patient at
the beginning of the trial and after 6 and 12 weeks. Blood was di-
luted with RPMI 1640 (Sigma-Aldrich Chemie, Steinheim, Ger-
many) in a ratio of 1: 2. Peripheral blood mononuclear cells
(PBMCs) were isolated from the diluted blood by lymphocyte sep-
aration medium (PAA Laboratories GmbH, Pasching, Austria).
20 ml of lymphocyte separation medium were carefully covered
with a layer of diluted blood and centrifuged at 400 g for 20 min
at room temperature.
CD4+ T cells were isolated from PBMC using AutoMACS
(Miltenyi Biotec, Bergisch Gladbach, Germany) with a CD4+
CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) follow-
ing the manufacturer’s instructions. 0.4–1 ! 106 Tregs were iso-
lated from 50 ml with the AutoMACS programs Depl05 and Pos-
seld2. Normally, Tregs make up to 0.7–5.5% of PBMCs and 5–10%
of T-helper cells [20, 23, 24]. CD4– cells in the remaining negative
fraction were used to isolate antigen presenting cells (APC). CD4–
cells were allowed to adhere to 96-well tissue culture plates (Fal-
con, Becton Dickinson, Heidelberg, Germany) for 2 h in an incu-
bator at 37 ° C, 5% CO2 (Heraeus 6000, Sepatech, Osterode, Ger-
many). Nonadherent cells were removed by washing the wells
repeatedly with prewarmed RPMI 1640. Adherent cells were used
as APC.
Assay of Suppressor Function by Tregs
For suppression assays, 0.5–1 ! 105 CD4+CD25– T cells were
cultured in the absence or presence of 0.5–1 ! 105 autologous
Tregs/well in 96-well plates and in the presence of 2 ! 105 adher-
ent APC in RPMI 1640 medium with 1% nonessential amino ac-
ids (100!) and 1 mM sodium pyruvate (PAA Laboratories GmbH,
Pasching, Austria), 1% MEM vitamins (100!; Biochrom, Berlin,
Germany), 25,000 U penicillin and 25 mg streptomycin (Gibco
BRL Life Technologies, Eggenstein, Germany), ␤-mercaptoetha-
256 Digestion 2010;81:252–264
nol (final concentration 3 ! 10 –5 M; Gibco, Invitrogen, Karlsruhe,
Germany), and 10% AB-serum (Cambrex Corporation, Europe).
Cells were stimulated with food antigens (20 ␮g/ml; HAL Allergie
GmbH, Düsseldorf, Germany), negative control solution (diluent
without antigen, HAL Allergie GmbH) or Dynabeads쏐 CD3/
CD28 T Cell Expander (Dynal쏐, Hamburg, Germany). These an-
tigen solutions (as well as negative control solution) are common-
ly used for prick test analysis as previously described by Van Den
Bogaerde [25]. The cells were cultured at 37 ° C, 5% CO2 for 24 and
72 h, respectively.
Fluorescence-Activated Cell Sorting (FACS)
To determine the purity of the isolated T cell fractions, cells
were stained with CD25-PE and CD4-FITC. The following anti-
bodies were used: 10 ␮l of anti-human CD4-FITC, clone M-T466,
isotype mouse IgG1 and 10 ␮l anti-human CD25-PE, clone 7D4,
isotype mouse IgG2b (Miltenyi Biotec) according to the manufac-
turer’s instructions. The antibodies were incubated for 15 min on
ice and washed twice with PBS (PAA Laboratories GmbH). Sub-
sequently, the cells were fixed with 3.7% paraformaldehyde, cen-
trifuged at 300 g for 5 min and resuspended in 100 ␮l of PBS.
Stimulation of T cells with food antigens results in cell divi-
sion with distinct fluorescence peaks, allowing determination of
the number of cell divisions calculated by carboxyfluorescein di-
acetate succinimidyl ester (CFSE) fluorescence (Sigma-Aldrich
Chemie, Taufkirchen, Germany). CFSE is a fluorescein derivative
which passively diffuses through the cell membrane and binds
irreversibly to cytoplasmatic proteins. 24 and 72 h after in vitro
stimulation, cells were analyzed by FACS. Proliferation is shown
as a decrement of fluorescence because CFSE is distributed among
the daughter cells. The cells were examined with an EPICS XL-
MCL (Coulter Immunotech, Hamburg, Germany).
Determination of IFN␥
IFN␥ secretion in cell supernatants was quantitatively mea-
sured by ELISA (IFN␥-ELISA Set; Biosciences, San Diego, Calif.,
USA) according to the manufacturer’s protocol.
Determination of EDN
For the evaluation of a potential food allergy and disease activ-
ity, EDN was detected in 80 g of stool by ELISA (Immundiagnos-
tik, Bensheim, Germany) according to the manufacturer’s proto-
col.
Data Analysis
Statistical analysis was carried out using SPSS, R and Sigma
Stat. Weekly counts of stool frequency were analyzed with Pois-
son regression using generalized estimating equations (GEEs,
[26]) to account for correlations between observations made from
the same individual. This method provides a robust standard er-
ror for the treatment effect which was used to calculate confi-
dence intervals and p values. Tests for cross-over effects were also
performed by GEEs. Moreover, GEEs with normal outcomes have
been used to analyze the total score. Data were not analyzed ac-
cording to the intention-to-treat principle.
The application of all tests was verified by normality tests
(Kolmogorov-Smirnov test, Shapiro-Wilk test). For statistical
analysis of the pilot study, the quantity of patients and healthy
controls with IgG antibody levels (in percent) was assessed by a t
test. Statistical analysis of IFN␥ secretion was performed by the
Bentz et al.
 25
Mean stool frequency
20
15
Specific diet
Sham diet
0 2 4 6 8 10 12
Week
Fig. 2. Progression of stool frequency. Stool frequency was moni-
tored per week. Only those patients who first followed the spe-
cific diet had a significant reduction in stool frequency. Subjects
who first followed the sham diet had no significant change in their
stool frequency (GEE analysis).
Kruskal-Wallis test based on non-normally distributed data. Val-
ues were significantly different when the obtained difference in
mean ranks was greater than the ␹2 value (in all figures indicated
with #). Values are expressed as the mean [minimum, maximum].
Statistical analysis of EDN in stool was performed by analysis of
variance (ANOVA) for normally distributed data. Statistical sig-
nificance was based on a p value smaller than 0.05.
Results
Pilot Study-IgG Antibodies in CD Patients and
Healthy Controls
The pilot study resulted in a significant difference of
IgG antibodies in serum between CD patients and healthy
controls (p ! 0.0001, t test). All detected IgG antibody
reactions are presented online (online suppl. table 2). The
ten most frequently measured IgG antibodies in CD pa-
tients were against processed cheese (84%), yeast (83%),
agave syrup (78%), camembert cheese (76%), poppy seeds
(74%), aloe vera (74%), bamboo sprouts (73%), kamut (du-
rum wheat, 70%), unripe spelt grain (69%) and wheat
(60%). More CD patients showed reactions against the
evaluated food components than healthy controls, i.e.
35% of healthy controls had IgG antibodies against wheat
in contrast to 60% of CD patients. Moreover, 39% of CD
Food Antigens in Crohn’s Disease
80
70
Mean total score
60
50
Specific diet
Sham diet
40
0 2 4 6 8 10 12
Week
Fig. 3. Progression of total score. An average reduction of the total
weekly score of 6.5 points was estimated for the specific diet group
compared with the sham diet group (GEE analysis).
patients had IgG antibodies against hazelnut in contrast
to only 15% of healthy controls. This was even more pro-
nounced in IgG antibodies against linseed, where 70% of
CD patients and only 10% of healthy controls showed IgG
antibodies. The same was seen with processed cheese
(60% of healthy controls vs. 84% of CD patients).
The most frequently detected IgG antibodies in healthy
controls were against yeast (66%), Aspergillus niger (60%),
whey (60%), processed cheese (60%), bamboo sprouts
(55%), paprika spice (55%), crawfish meat (50%), cottage
cheese (45%), yoghurt (45%) and zander (45%).
Effects of the Nutritional Intervention on Stool
Frequency, Abdominal Pain and General Well-Being
There was no evidence for a cross-over effect in the
analysis of the weekly stool frequency counts (p = 0.08).
In the specific diet group, a significant reduction in the
daily stool frequency by 11% was achieved compared to
the sham diet group (p = 0.004, 95% CI: 4%, 18%). How-
ever, the effect was confounded by a significant increase
in stool frequency of 9% in the second intervention phase
of the study, regardless of type of diet (p = 0.025, 95% CI:
1%, 18%; fig. 2). The comparison of loose stools during
the specific and sham diets of each patient demonstrated
that, surprisingly, only those patients who first followed
Digestion 2010;81:252–264 257
 103
CD4–CD25+ CD4+CD25+
2.4% 62.6%
102
CD25-PE
101
100 CD4– CD4+CD25–
1.7% 33.3%
100 101 102
a CD4-FITC
Fig. 4. FACS analysis of CD4+CD25– T cells and Tregs. a Staining of Tregs. b Staining of CD4+CD25– T cells
(effector T cells). The purity of CD4+CD25– effector T cells was 90.8% and of Tregs 62.6%.
the specific diet had a significant reduction in stool fre-
quency. The group of subjects who first followed the sham
diet and then specific diet had no significant change in
their stool frequency.
Patients were asked to rate their pain perception and
general well-being. The given points were accumulated
after each week. To obtain an overall impression of stool
frequency, abdominal pain and general well-being, a total
score was calculated. There was neither evidence for a
cross-over effect nor an intervention phase in the analysis
of the total score. An average reduction of the total week-
ly score (fig. 3) of 6.5 points was estimated for the spe-
cific diet group compared with the sham diet group (95%
CI: –0.6, 13.6 points). The estimated effect seems to have
a clinically relevant effect, but is not significant (p =
0.07).
IFN␥ Secretion of CD4+CD25– Effector T Cells and
Tregs
Isolation of CD4+CD25– effector T cells and Tregs was
controlled by FACS analysis. The purity of CD4+CD25–
effector T cells was 90.8 and 62.6% for Tregs (fig. 4a, b).
CD4+CD25– effector T cells and APC were incubated in
the absence or presence of Tregs and stimulated with food
antigens, negative control solution or antiCD3/antiCD28
solution. The effect of food antigens on CD4+CD25– T
258 Digestion 2010;81:252–264
103
CD4–CD25+ CD4+CD25+
0.1% 5.8%
102
CD25-PE
101
100 CD4– CD4+CD25–
3.3% 90.8%
103 100 101 102 103
b CD4-FITC
cell activation was evaluated by quantification of IFN␥ in
cell supernatants by ELISA (fig. 5–7). All obtained IFN␥
values were not normally distributed; therefore, a Krus-
kal-Wallis test was performed.
Stimulation of CD4+CD25– and APC with Food
Antigens and Negative Control Solution
The incubation of CD4+CD25– T cells and APC with
food antigens caused an increase in IFN␥ secretion (fig. 5,
left panel). The amount of IFN␥ at base value (time point
zero) was 170.5 pg/ml [16.5; 495.3] and increased during
specific diet (411.1 pg/ml [16.9; 1,117.0]) and sham diet
(481.5 pg/ml [1.5; 1,234.0]). Unfortunately, there was no
significant difference between the three time points.
Stimulation of CD4+CD25– T cells and APC with
negative control solution (fig. 5, right panel) showed
IFN␥ secretion comparable to food antigen solution. The
amount of IFN␥ at base value was 153.1 pg/ml [5.8; 587.6]
and increased during specific diet (308.3 pg/ml [61.0;
1,220.0]) and sham diet (681.6 pg/ml [126.0; 1,347.0]).
There was a significantly higher IFN␥ secretion during
sham diet than during specific diet or base value, when
cells were stimulated by negative control solution.
In summary, CD4+CD25– T cells were not clearly
more stimulated by food antigen solution than by nega-
tive control solution.
Bentz et al.
 Stimulation with Stimulation with
food antigens negative control

1,500 #
#

1,000
Fig. 5. Stimulation of CD4+CD25– T cells

IFN␥ (pg/ml)
and APC with food antigens or negative
control solution. Comparison of IFN␥ se-
cretion (pg/ml) between base value (be-
ginning of intervention), specific diet and
sham diet (after 6 weeks of intervention,
respectively). Stimulation with food anti- 500
gens (left panel) increased IFN␥ secretion
of CD4+CD25– T cells cultivated with
APC during intervention. Stimulation
with negative control solution (right pan-
el) also increased IFN␥ secretion during
intervention compared with food antigen
stimulation. Kruskal-Wallis test; # indi- 0
cates significance (obtained difference in Base Specific Sham Base Specific Sham
mean ranks was greater than the ␹2 val- value diet diet value diet diet
ue); u indicates outliers.

Stimulation with Stimulation with

2,000 food antigens negative control

Fig. 6. Stimulation of CD4+CD25– T cells 1,500

and APC in the presence of Tregs with food
IFN␥ (pg/ml)

antigens or negative control solution.

Comparison of IFN␥ secretion (pg/ml) be-
tween base value (beginning of interven- 1,000
tion), specific diet and sham diet (after 6
weeks of intervention, respectively). Stim-
ulation with food antigens (left panel):
CD4+CD25– T cells co-cultivated with
APC and Tregs secreted more IFN␥ during 500
specific diet and sham diet in contrast to
base value. Stimulation with negative con-
trol solution (right panel): less IFN␥ secre-
tion between base value and the specif-
0
ic and sham diets, respectively. Kruskal-
Base Specific Sham Base Specific Sham
Wallis test; # indicates significance (ob- value diet diet value diet diet
tained difference in mean ranks was great-
er than the ␹2 value); uindicates outliers.

Food Antigens in Crohn’s Disease Digestion 2010;81:252–264 259


800
Fig. 7. Stimulation of CD4+CD25– T cells,
APC and Tregs with antiCD3/antiCD28 so- 600
lution. Comparison of IFN␥ secretion (ng/
ml) between base value (beginning of in-
IFN␥ (ng/ml)
tervention), specific diet and sham diet
(after 6 weeks of intervention, respective-
ly). There was 1,000-fold higher IFN␥ se- 400
cretion when cells were stimulated with
antiCD3/antiCD28 solution than with
food antigen/negative control solution.
There was higher IFN␥ secretion of 200
CD4+CD25– T cells (left panel) between
base value and specific diet. The mixture
of CD4+CD25– T cells, APC and Tregs
showed a significant increase only between
base value and the sham diet. Kruskal-
Wallis test; # indicates significance (ob-
tained difference in mean ranks was great-
er than the ␹2 value); uindicates outliers.
Stimulation of the Co-Culture of CD4+CD25–, APC
and Tregs with Food Antigens and Negative Control Solu-
tion
The co-culture of CD4+CD25– T cells and APC with
Tregs and stimulation with food antigens (fig. 6, left panel)
also resulted in an increase in IFN␥ secretion from base
value (169.5 pg/ml [42.7; 543.1]) to specific diet (683.8
pg/ml [37.4; 1,311.0]) or sham diet (609.8 pg/ml [34.0;
1,209.0]). Unfortunately, there was no significant differ-
ence between the three time points.
There was lower IFN␥ secretion when co-cultivated
CD4+CD25– T cells, APC and Tregs were stimulated with
negative control solution (fig. 6, right panel). Significant-
ly lower IFN␥ secretion was only received between base
value (294.4 pg/ml [10.1; 1,587.0]) and sham diet (21.2 pg/
ml [1.9; 53.8]). Specific diet (71.6 pg/ml [2.9; 231.4]) re-
sulted in no significant difference.
In summary, there was no clear difference between
the culture of CD4+CD25– T cells and the culture of
CD4+CD25– T cells with Tregs.
Stimulation with AntiCD3/AntiCD28 Solution
The stimulation with antiCD3/antiCD28 beads of
CD4+CD25– T cells and APC, or the co-cultivation of
CD4+CD25– T cells, APC and Tregs (fig. 7) showed high-
260 Digestion 2010;81:252–264
CD4+CD25– and APC Mixture CD4+CD25–, APC, Tregs
#
#
0
Base Specific Sham Base Specific Sham
value diet diet value diet diet
er IFN␥ secretion in contrast to the stimulation with food
antigens or negative control solution. Therefore, cells
were more effectively stimulated with antiCD3/antiCD28
solution than with food antigen/negative control solu-
tion, as seen in 1,000-fold higher IFN␥ secretion.
In case of CD4+CD25– T cells (fig. 7, left panel), a sig-
nificant increase in IFN␥ secretion was detected between
base value (48.2 ng/ml [0.8; 151.0]) and specific diet (169.3
ng/ml [19.6; 307.0]). There was no significant difference
between base value and sham diet (204.6 ng/ml [12.10;
791.0]).
The mixture of CD4+CD25– T cells, APC and Tregs
(fig. 7, right panel) showed only a significant increase be-
tween base value (51.9 ng/ml [0.1; 271.0]) and sham diet
(135.9 ng/ml [49.20; 309.0]). The increase during specific
diet (157.9 ng/ml [26.20; 621.0]) was not significant.
In summary, none of the three different stimulation
methods led to a significant difference in IFN␥ secretion
of CD4+CD25– T cells or the mixture of CD4+CD25– T
cells and Tregs.
Time Course of Cell Division after Cell Stimulation
The proliferative response of the isolated T lympho-
cytes to stimulation with food antigens, negative control
solution and antiCD3/antiCD28 solution was investigated
Bentz et al.
 Cell count
100 101 102 103 104
CFSE intensity r
Fig. 8. CFSE proliferation in the mixture of CD4+CD25– T cells,
APC and Tregs measured by FACS analysis. CD4+CD25– T cells
and APC in the presence of Tregs did not demonstrate any in vitro
proliferative responses to food antigens. After 24 h: stimulation
with food antigens (red), negative control solution (green) and
antiCD3/antiCD28 solution (blue); after 72 h: stimulation with
food antigens (orange), negative control solution (purple) and an-
tiCD3/antiCD28 solution (black); graphs were recorded separate-
ly and are shown in overlay mode.
by CFSE (fig. 8, 9). Each of the curves reflects a measure-
ment of the fluorescence intensity caused by cell division.
CD4+CD25– T cells and APC in the absence or presence
of Tregs did not demonstrate any in vitro proliferative re-
sponses to food antigens. Hence, the isolated T cells do not
proliferate in vitro after stimulation with food antigens.
Quantification of EDN in Stool
The disease activity was evaluated by the quantifica-
tion of EDN in stool samples (fig. 10). The samples were
normally distributed and, therefore, ANOVA was per-
formed. The concentration of EDN at the beginning of
intervention (base value) was 1,536 8 405 ng/ml. The
concentration declined during specific diet (1,228 8 530
ng/ml) but also during sham diet (1,355 8 373 ng/ml).
There was no significant difference between the three
time points. EDN concentration dropped in the same de-
gree under specific diet and sham diet. No difference for
EDN in stool indicated an absence of eosinophil-medi-
ated reactions.
Food Antigens in Crohn’s Disease
Cell count
100 101 102 103 104
CFSE intensity r
Fig. 9. CFSE proliferation of CD4+CD25– T cells and APC mea-
sured by FACS analysis. CD4+CD25– T cells and APC in the
absence of Tregs did not demonstrate any in vitro proliferative
responses to food antigens. After 24 h: stimulation with food
antigens (red), negative control solution (green) and antiCD3/
antiCD28 solution (blue); after 72 h: stimulation with food anti-
gens (orange), negative control solution (purple) and antiCD3/
antiCD28 solution (black). Graphs were recorded separately and
are shown in overlay mode.
Discussion
In the present study, we have shown that IgG antibod-
ies against food antigens are elevated in patients with CD
in contrast to healthy controls. A clinically significant
improvement in IBD symptoms was observed in patients
eliminating foods to which they were found to exhibit
sensitivity. IFN␥ secretion by T cells was increased after
specific diet, but also after sham diet. There was a reduc-
tion of EDN concentration in stool during specific diet
and sham diet, but no significant difference between the
two diets.
Forty-eight percent of patients in the present interven-
tion study had an improvement in stool frequency and
general well-being (total score). Only 9% of patients de-
scribed opposite effects.
The study results are encouraging; however, they have
to be interpreted with care as results may have been in-
fluenced by several confounders: The daily reporting of
consumed foods and the attachment to the diet recom-
Digestion 2010;81:252–264 261

2,500
2,000
EDN (ng/ml)
1,500
1,000
500
0 had IgG antibodies against yeast. The IgG-antibody
Base Specific Sham
value diet diet
Fig. 10. Detection of EDN in stool samples. Comparison between
base value (beginning of intervention), specific diet and sham diet
(after 6 weeks of intervention, respectively). EDN concentration
(ng/ml) declined during intervention in comparison to base val-
ue. There was no significant difference between the two diets.
ANOVA test.
mendations required much time and discipline from the
study subjects. Therefore, the problem of under-report-
ing (consumed foods and beverages were not listed cor-
rectly) is evident. We tried to reduce this problem by ex-
plaining the list of foods to be avoided in great detail to
each patient and showing ‘hidden sources’. This was done
by one well-trained person.
Basically, the main limitation of this cross-over study
was the high dropout rate (n = 17), which was a result of
the length of the study, the changes in CD and voluntary
withdrawals.
In addition, the 40 patients initially included in this
study were on different medications. Due to low numbers
of individuals in this study, stratification according to the
different treatments would have caused inability to do a
statistical analysis. However, strong immunosuppres-
sants certainly could have influenced the study results,
especially with respect to T cell function. We tried to re-
duce this problem by keeping the medication constant for
the time of intervention. In addition, many of the patients
with CD already kept to some individual forms of diet,
avoiding bloating foods such as onions or garlic [27–30].
In addition, it may be argued that the sham diet was
too similar to the specific diet, as the definition of spe-
262 Digestion 2010;81:252–264
cific and sham diet was based on the similarity of exclud-
ed food components. If for example IgG against hazelnut
was detected, then almond was excluded in the sham diet;
if cauliflower IgG was found, broccoli was excluded.
There may be some cross-reactivity of the respective an-
tigens, which could explain some effects of the sham diet
on IBD symptoms, T cell cytokine secretion and stool
EDN levels.
In addition, we did not have a washout phase at the
cross-over point, which may have led to some transmis-
sion of effects into the sham arm of the study.
More than 80% of CD patients in the pilot study and
more than 30% of CD patients in the intervention study
reactions against food antigens were also investigated in
CD patients by Van Den Bogaerde [25]. Increased sensi-
tization against yeast was demonstrated in vivo and in
vitro as in the present study. Additionally, a study from
Darroch et al. [31] pointed out that antibodies against
Saccharomyces cerivisiae are significantly increased in
patients with CD in comparison to patients with ulcer-
ative colitis and that they play a role in the function of T
and B cells in patients with CD. In the present study, the
difference in T cell function is shown by higher IFN␥ se-
cretion. Elevated IFN␥ levels after nutritional interven-
tion were detected in supernatants of cultures of
CD4+CD25– T cells and APC in the presence or absence
of Tregs. The mucosa of CD patients is dominated by T
cells of the T-helper cell phenotype 1 [32].
These cells are characterized by the secretion of IFN␥.
A greater number of cells secreting IFN␥ in CD in con-
trast to ulcerative colitis and healthy controls was found
[33]. IFN␥ is involved in specific cell-mediated immunity
and causes the secretion of IgG2 antibodies during de-
layed-type hypersensitivity. Van Den Bogaerde et al. [25]
illustrated, both in vitro and in vivo, a more pronounced
reaction of T cells against food antigens. They tested the
proliferation of peripheral blood lymphocytes after stim-
ulation with different food antigens like cereal, cabbage,
citrus fruits, yeast and nuts in 10 CD patients and 10
healthy controls [25]. They used commercial prick solu-
tion (just like in the present study) for cell stimulation.
The authors concluded that in vivo sensitization against
food antigens exists. The mechanism has to be further
elucidated, perhaps due to a defective epithelial barrier
function, which might allow infiltration of food antigens
to the mucosa. IFN␥ secretion of Tregs after stimulation
with antiCD3/antiCD28 solution was also analyzed by
Earle et al. [34]. In this issue, Tregs secreted no IFN␥ in
contrast to PBMC which secreted a concentration of 500
Bentz et al.
pg/ml. In addition, a co-cultivation of PBMC and Tregs
resulted in reduced IFN␥ production. In contrast to the
findings of Earle, Nakamura et al. [35] postulated that
Tregs produce significantly less IFN␥ than CD4+CD25– T
cells when stimulated adequately with antiCD3/antiCD28
solution. However, a limitation of the current study was
that no FoxP3 staining was performed.
In the context of this study, a significant reduction of
EDN concentration in stool during intervention could be
detected. Values up to 1,300 ng/ml were examined. These
findings are in line with results from Saitoh et al. [36] who
found values up to 3,500 ng/ml in active CD and 910 ng/
ml in inactive CD.
Similar to our data, an elimination diet in patients
with irritable bowel syndrome was effective. One hun-
dred fifty irritable bowel syndrome patients were tested
and got a ‘true’ or a ‘sham’ diet. The true diet excluded
foods against which IgG antibodies were found, the sham
diet excluded foods against which no IgG antibodies were
found (as in the present study). After 12 weeks of inter-
vention, patients with the true diet had a 10% greater im-
provement of their symptoms than those patients with
sham diet. Most of the patients within this study had IgG
antibodies against yeast, followed by milk, chicken, egg,
wheat and cashew nuts [37]. This study lacks a cross-over
design in contrast to the present study. Moreover, immu-
nohistochemical research found IgG producing B cells in
the colon and ileum of CD patients [38, 39]. The authors
claimed that the raised IgG antibodies are due to a failure
of intestinal barrier function. This circumstance could be
one reason for the effectiveness of an IgG-based diet in-
tervention.
In conclusion, a nutritional intervention diet based on
circulating IgG antibodies against food antigens showed
effects with respect to stool frequency, abdominal pain
and general well-being in this double-blind cross-over
study with 40 CD patients. Stool frequency and total
score during the specific diet were significantly lower in
contrast to the sham diet. Stimulation of T cells with the
specific antigens was followed by an increase in IFN␥ se-
cretion. However, there was no difference in T cell prolif-
eration in response to different antigens. The concentra-
tion of EDN in stool declined during the specific diet, but
also during sham diet. The mechanisms by which IgG
antibodies might contribute to disease activity remain to
be elucidated.
Acknowledgements
The authors thank the study nurses and the blood bank of the
German Competence Network for Inflammatory Bowel Disease
(KN-CED) for providing us with serum samples and we are grate-
ful for the patients’ contribution of the blood samples. This study
was supported by the German Competence Network for Inflam-
matory Bowel Disease (KN-CED) and the Evomed MedizinSer-
vice GmbH.

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 1459

IRRITABLE BOWEL SYNDROME

Food elimination based on IgG antibodies in irritable bowel

syndrome: a randomised controlled trial
W Atkinson, T A Sheldon, N Shaath, P J Whorwell
...............................................................................................................................
Gut 2004;53:1459–1464. doi: 10.1136/gut.2003.037697

Background: Patients with irritable bowel syndrome (IBS) often feel they have some form of dietary
intolerance and frequently try exclusion diets. Tests attempting to predict food sensitivity in IBS have been
disappointing but none has utilised IgG antibodies.
Aims: To assess the therapeutic potential of dietary elimination based on the presence of IgG antibodies to
food.
See end of article for Patients: A total of 150 outpatients with IBS were randomised to receive, for three months, either a diet
authors’ affiliations
....................... excluding all foods to which they had raised IgG antibodies (enzyme linked immunosorbant assay test) or
a sham diet excluding the same number of foods but not those to which they had antibodies.
Correspondence to: Methods: Primary outcome measures were change in IBS symptom severity and global rating scores. Non-
Dr P J Whorwell,
Department of Medicine, colonic symptomatology, quality of life, and anxiety/depression were secondary outcomes. Intention to
University Hospital of treat analysis was undertaken using a generalised linear model.
South Manchester, Results: After 12 weeks, the true diet resulted in a 10% greater reduction in symptom score than the sham
Manchester M20 2LR, UK;
peter.whorwell@ diet (mean difference 39 (95% confidence intervals (CI) 5–72); p = 0.024) with this value increasing to
smuht.nwest.nhs.uk 26% in fully compliant patients (difference 98 (95% CI 52–144); p,0.001). Global rating also significantly
improved in the true diet group as a whole (p = 0.048, NNT = 9) and even more in compliant patients
Revised version received (p = 0.006, NNT = 2.5). All other outcomes showed trends favouring the true diet. Relaxing the diet led to a
13 April 2004
Accepted for publication 24% greater deterioration in symptoms in those on the true diet (difference 52 (95% CI 18–88); p = 0.003).
13 April 2004 Conclusion: Food elimination based on IgG antibodies may be effective in reducing IBS symptoms and is
....................... worthy of further biomedical research.

I
rritable bowel syndrome (IBS) is a common disorder which
causes abdominal pain, abdominal distension, and bowel
dysfunction, characterised by loose bowels, constipation, or
a fluctuation between these two extremes.1 This condition
significantly impairs quality of life and places a large burden
on health care resources.2 Treatment of IBS is largely based
on the use of antispasmodics, antidepressants, and medica-
tions that modify bowel habit, depending on whether
constipation or diarrhoea is the predominant problem.1 The
notorious inadequacies of current drug therapy lead to much
patient dissatisfaction and a tendency for patients to seek a
variety of alternative remedies, especially of a dietary nature.
IBS is likely to be a multifactorial condition involving a
number of different mechanisms although the prominence of
any particular factor may vary from patient to patient.1 3
However, patients often strongly believe that dietary intoler-
ance significantly contributes to their symptomatology and
some sufferers seem to benefit from eliminating certain foods
from their diet. Detection of food intolerance is often difficult
due to its uncertain aetiology, non-specific symptomatology,
and relative inaccessibility of the affected organ. Thus most
previous studies have relied on the use of exclusion diets,
which are extremely labour intensive and time consuming.4 5
Attempts to ‘‘test’’ for food intolerance in IBS have largely
focused on ‘‘classic’’ food allergy based on the presence of IgE
mediated antibody responses, although it appears that these
‘‘immediate type’’ reactions are probably quite rare in this
condition.6–10 It is therefore possible that adverse reactions to
food in patients with IBS might be due to some other form of
immunological mechanism, rather than dietary allergy. Such
reactions could be mediated by IgG antibodies, which
characteristically give a more delayed response following
exposure to a particular antigen11 and have been implicated
in some cases of food hypersensitivity.12–14 However, this
mechanism is controversial and is considered by some to be
physiological15–17 especially as IgG food antibodies can be
present in apparently healthy individuals.18–20 It has pre-
viously been suggested that IgG food antibodies may have a
role in IBS21 and it was therefore the purpose of this study to
formally evaluate, in a randomised controlled trial, the
therapeutic potential of an elimination diet based on the
presence of IgG antibodies to food in patients with IBS.
PATIENTS AND METHODS
Patients
All patients with uncomplicated IBS (all bowel habit
subtypes) attending the Gastroenterology Department at
the University Hospital of South Manchester were considered
eligible for the study, and those aged between 18 and
75 years, who satisfied the Rome II criteria,22 were invited to
participate. Tertiary care patients were excluded from the
study. All patients had normal haematology, biochemistry,
and endoscopic examination when indicated. Coeliac disease
was excluded using the tissue transglutaminase test and a
hydrogen breath test was used for excluding lactose intoler-
ance. Patients were also excluded from participating in the
study if they had any significant coexisting disease or a
history of gastrointestinal surgery, excluding appendicect-
omy, cholecystectomy, and hiatus hernia repair. The study
was approved by the local ethics committee and all patients
provided written informed consent.
Methods
The study used a double blind, randomised, controlled,
parallel design in which patients were randomised to either a
‘‘true’’ diet or a ‘‘sham’’ diet control group. At screening,
Abbreviations: IBS, irritable bowel syndrome; ELISA, enzyme linked
immunosorbant assay; AU, arbitrary unit; HAD, hospital anxiety and
depression scale; QOL, quality of life; NNT, number needed to treat

www.gutjnl.com
1460 Atkinson, Sheldon, Shaath, et al

blood was taken and sent, with only a numerical identifier, to slightly worse, no change, slightly better, better, or excel-
YorkTest Laboratories Ltd (York, UK) where an enzyme lent?’’ The atopic status of all patients entering the study was
linked immunosorbant assay (ELISA) test was performed to also assessed.
detect the presence of IgG antibodies specific to a panel of 29 During the treatment phase, patients were allowed to take
different food antigens. This test has been described in detail concomitant medication provided it had been constant for six
elsewhere23 and involves specimens being diluted 1/50, 1/150, months prior to the start of the study. They were encouraged
and 1/450 with each dilution applied to an allergen panel. not to alter medication use during the course of the trial but
Each test was calibrated using 0 arbitrary unit (AU) and any changes were recorded. Any patient withdrawing from
25 AU standards prepared from a serum with a high IgG titre the study was encouraged to complete a final symptom
to a cow’s milk allergen extract. A positive control serum at questionnaire at week 12 and their reasons for withdrawal
45 AU was applied to each test. The test results were obtained were recorded. At the end of 12 weeks, patients were asked to
from the 1/150 dilution of the specimen. Where a high resume consumption of the foods they had been advised to
specimen background was observed, the test results were eliminate in order to assess the effect of their reintroduction.
obtained from the 1/450 dilution. The threshold for a positive Patients were then reassessed after four weeks using the
(reactive) result was selected as three times the background same measures and the result compared with their scores at
signal obtained by the same sample against a no food the end of the elimination phase.
allergen coated control well equivalent to 3 AU. Test results
were scored as positive or negative only, relative to this cut Data analysis
off. Questionnaires were scored by an assessor blinded to the
Staff based at the YorkTest Laboratories produced a true randomisation. The primary outcome measures were changes
and sham diet sheet for each patient. The sham diet in IBS symptom severity score and global impact score at
eliminated the same number of foods to which a patient 12 weeks. Changes in non-colonic symptoms, QOL, and HAD
exhibited IgG antibodies but not those particular foods. The scores were regarded as secondary outcome measures. Two
goal was to try and include in the sham diet an equally sample t tests were used to establish whether there was an
difficult to eliminate staple food for every staple food in the overall difference in the change in continuous outcome
true diet. Thus cow’s milk was (generally) replaced with measures between the two groups of patients. Patients were
potato, wheat with rice, and yeast with whole egg, where this analysed according to the group to which they were
was possible. Nut reactivities were replaced with other nuts randomised, independent of their adherence to the diet.
in the sham diet, and legumes with other legumes, but this The global impact score, an ordered categorical variable, was
was not systematised. analysed using a Wilcoxon Mann-Whitney test to compare
The true and sham diet sheets for each patient were sent to the numbers in the active and sham groups showing
the University of York, again with only a number for significant improvement (‘‘better’’ or ‘‘excellent’’), no sig-
identification. Patients were allocated to one of the two diet nificant change (‘‘slightly worse’’, ‘‘no change’’, or ‘‘slightly
sheets based on a randomisation schedule developed using a better’’), and significant deterioration (‘‘worse’’ or ‘‘terri-
random computer number generator. Thus patients would ble’’). The number needed to treat (NNT) was calculated
receive either an elimination diet based on their true from the global impact score by calculating the reciprocal of
sensitivity results (true diet) or a sham diet. All patients the difference in probability of a significant improvement
and clinical staff in the Gastroenterology Research between the treatment and control groups. General linear
Department and YorkTest Laboratory were blinded to the modelling in SPSS was used to explore whether there was a
group assignment of all patients for the duration of the study.
Patients were given their allocated diet sheet by staff at the
Gastroenterology Research Department and asked to elim- Assessed for
inate the indicated foods from their diet for a period of eligibility
12 weeks. They also received a booklet with advice on (n=176) Excluded (n=26):
eliminating the different foods and the telephone contact Did not meet inclusion
details of a free nutritional advisor whom they were able to criteria (n=19)
Refused to participate (n=5)
contact for further advice if necessary. Randomised Other reasons (n=2)
Symptoms were assessed using a questionnaire scoring (n=150)
system validated for use in IBS, including the IBS symptom
severity score (range 0–500).24 This is a system for scoring
pain, distension, bowel dysfunction, and general well being,
with mild, moderate, and severe cases indicated by scores of Allocated to Allocated to
75–175, 175–300, and .300, respectively. A reduction in receive true receive sham
score of 50 or over is regarded as a clinically significant diet (n=75) diet (n=75)
improvement.24 Non-colonic symptomatology,25 such as
lethargy, backache, nausea, and urinary symptoms, was 24 Withdrew: 13 Withdrew:
assessed and scored using visual analogue scales (range 0– Diet too restrictive (n=11) Diet too restrictive (n=3)
Lack of efficacy (n=1) Lack of efficacy (n=3)
500). Quality of life (QOL) was measured using an instru- Not prepared to follow Not prepared to follow
ment proven to be sensitive to change in IBS (range 0–500).26–28 diet (n=6) diet (n=4)
Anxiety and depression were evaluated using the hospital Other reasons (n=6) Other reasons (n=3)
anxiety and depression scale (HAD).29 This instrument scores
anxiety and depression up to a maximum score of 21 for each 10 Lost to follow up 9 Lost to follow up
parameter, with a score above 9 indicating significant
psychopathology. Data on these measures were recorded at 65 Included 66 Included
baseline and after 4, 8, and 12 weeks of the dietary in the final in the final
intervention period. In addition, at 4, 8, and 12 weeks, intention to intention to
treat analysis treat analysis
patients were asked to give a global rating of their IBS using
the question, ‘‘Compared with your IBS before you started
the food elimination diet, are you now: terrible, worse, Figure 1 Study flow diagram.

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Food elimination based on IgG antibodies in IBS 1461

Table 1 Baseline characteristics of the patients

Group True diet (n = 75) Sham diet (n = 75)

Age (y) (range, SD) 44 (17–72; 12.9) 44 (19–74; 15.2)

No of males (%) 7 (9.3%) 13 (17.3%)
No of foods to which sensitive 6.65 (3.66) 6.63 (4.1)
Symptom duration (y) 11.5 (9.9) 10.1 (7.5)
IBS symptom severity score 331.9 (70.8) 309.0 (78.5)
Non-colonic features score 459.1 (160.7) 452.6 (170.1)
Quality of life score 640.1 (252.6) 639.3 (222.3)
HAD anxiety score 9.5 (4.6) 9.5 (4.5)
HAD depression score 5.3 (3.4) 6.0 (3.6)
No of diarrhoea predominant patients (%) 37 (52.1%) 41 (56.9%)
No of constipation predominant patients (%) 19 (26.8%) 16 (22.2%)
No of alternating predominant patients (%) 15 (21.1%) 15 (20.8%)

Results are expressed as mean (SD).

HAD, hospital anxiety and depression scale.

relationship between the change in symptoms from baseline CONSORT statement.31 In summary, between January 2001
and treatment group, patient characteristics (for example, and July 2002, 176 patients were eligible for the study, of
IBS subtype, history of atopy, number of foods to which which 26 (15%) were excluded from participation, leaving
sensitive, and concomitant medication) and adherence to the 150 patients who were all found to be sensitive to at least one
diet.30 food. Seventy five of these were randomised to receive an
elimination diet based on their true food sensitivity results
Sample size calculation and 75 patients to a sham diet. Data from 131 (87%) patients
It was estimated that approximately 40% of the placebo arm who gave 12 week data were available for the intention to
would report a significant improvement in symptoms. It was treat analysis: 65 and 66 patients from the true and sham
calculated that a sample size of 55 patients would be required groups, respectively.
in each group to detect, with 90% power, a difference of 30%
points in the proportion reporting such an improvement (that Patient characteristics
is, 70% in the treatment arm) as statistically significant at the The patients were typical of those with IBS in secondary care
5% level. Assuming a 20% dropout rate, a minimum of 138 practice, the majority being women. Patients, on average, had
patients would need to be entered into the trial. Thus we experienced symptoms of IBS for over a decade and were
aimed to recruit a total of 150 patients into the study. found to be sensitive to approximately 6–7 foods (range 1–
19). Baseline demographic and clinical characteristics of the
RESULTS two groups, including the use of concomitant medication,
Recruitment of patients and their flow through each stage of were found to be similar with the exception of the IBS
the study is illustrated in fig 1, as recommended by the symptom severity score which was slightly higher in the
treatment group (table 1). Thirty per cent of patients were
Table 2 Frequency of foods excluded from the diet (% of found to be atopic.
patients) The frequency of foods excluded from the diet is shown in
table 2. Adherence was lower in those on the true diet
Food Treatment group Sham group although no specific adverse events were recorded in either
Barley 26.7 9.3 group. Twenty four patients withdrew from the study in the
Corn 22.7 14.7 true diet group (mainly because of difficulty in following the
Rice 8 54.7 diet) and 13 from the sham diet group (for a variety of
Rye 8 25.3
Wheat 49.3 8
reasons). However, 12 week data were obtained from 14 of
Milk 84.3 1.3 those who withdrew in the true diet group and four in the
Beef 24 9.3 sham diet group. There were no significant differences
Chicken 21.3 13.3
Pork 5.3 36
Cabbage 12 24
Celery 5.3 21.3 0
Haricot bean 17.3 14.7
Pea 38.6 1.3
***
Potato 9.3 61.3 _
IBS symptom severity

50 Sham
Soy bean 22.7 10.7 diet
Tomato 4 44 (n = 66)
Apple 1.3 33
_
Orange 6.7 29.3 100
Strawberry 0 20
Almond 28 12
Brazil nut 22.7 17.3 _
150 True diet
Cashew nut 49.3 8
(n = 65)
Peanut 10.7 20
Walnut 2.7 29.3 _
Cocoa bean 1.3 21.3 200
Low Medium High
Shellfish 21.3 10.7
Fish mix 17.3 28 Level of adherence
Whole egg 57.3 26.7
Yeast 86.7 0 Figure 2 Mean change in symptom severity scores at 12 weeks
according to degree of adherence. Difference between the groups with
high adherence: 101 (95% confidence interval 54, 147); ***p,0.001.

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1462 Atkinson, Sheldon, Shaath, et al

between atopic and non-atopic patients. There was however a

400
A
statistically significant interaction between treatment group
and both adherence to the diet and number of foods to which
patients were sensitive. For patients sensitive to the average
Sham
IBS symptom severity
300 number of foods who fully adhered to their allocated diet, a
* diet
(n=66) 26% difference in reduction in symptom severity score was
observed in favour of the true diet (a difference in score of 98
200 True diet (95% CI 52, 144), p,0.001: a standardised effect size of 1.3).
(n=65)
This benefit increased by a further 39 points (12%) (95% CI 7,
100
70; p = 0.016) for each food to which they were sensitive
over and above the average number. These results were not
materially altered by carrying out an ANCOVA analysis (in
0 which the final score is the dependent variable and the
0 4 8 12
baseline score is included as a covariate) instead of modelling
Time (weeks) change in scores.30 The interaction between treatment group
and adherence is demonstrated in fig 2 which shows a
400
B greater reduction in symptoms with full adherence in the
true diet but not in the sham diet group. Figure 3A and 3B
show the average change in symptom severity score over
*** Sham
IBS symptom severity

300
200
100
0
0 4 8 12
Time (weeks)
Figure 3 (A) Average symptom severity scores over time for the group
as a whole. Difference in mean change from baseline at 12 weeks: true
versus sham 39 (95% confidence interval 5, 72); *p = 0.024. (B) Average
symptom severity scores over time for the full adherence group.
Difference in mean change from baseline at 12 weeks: true versus sham
98 (95% confidence interval 52, 144); ***p,0.001.
between baseline characteristics of the 19 who were lost to
follow up and those for whom 12 week data were obtained.
Primary outcomes
IBS symptom severity
Patients in the true diet group experienced a 10% greater
reduction in symptom severity than those allocated to the
sham diet, with change in scores of 100 and 61.5, respectively
(mean difference 39 (95% confidence interval (CI) 5.2, 72.3);
p = 0.024): a standardised effect size of 0.52 (see fig 3A).
There were no differences in the response to the diet in terms
of age, sex, IBS bowel habit subtype, or IBS duration. In
addition, there was no difference in response to the diet
12 weeks for the group as a whole and for those who fully
diet adhered, respectively. This reveals that most improvements in
(n=40) symptoms are fully achieved within two months.
True diet
(n=24) Global impact score
The reported global rating of change by treatment group is
shown in table 3. The difference in mean ranking (70.9 v
60.3) was statistically significant (p = 0.048). When this was
repeated including only patients who fully adhered to their
diets (table 3), a greater percentage difference favouring the
true diet was found (p = 0.001). The NNT was 9 in the group
as a whole and 2.5 in patients fully adherent to the diet.
Secondary outcome measures
As can be seen from fig 4A and 4B, all data show changes
favouring the true diet group and are consistent with the
results for the primary outcomes. These trends were further
strengthened after adjustment for adherence and number of
food sensitivities but only reached statistical significance for
non-colonic symptomatology (p = 0.05). There were no
significant changes in medication use during the course of
the trial.
Reintroduction of eliminated foods
Of the 131 patients who gave 12 week data, 93 (41 in the true
and 52 in the sham diet groups) agreed to attempt
reintroduction of foods they had been asked to eliminate
and provided further follow up data on the primary outcomes
measures. Of these, 62% reported full adherence and 37%
moderate adherence to the previous elimination diet. Mean
IBS symptom severity score increased (that is, worsening of
symptoms) by 83.3 in the true group and by 31 in the sham

Table 3 Global impact score at 12 weeks

Treatment group

True diet Sham diet

(n (%)) (n (%))

All patients
Significantly worse 3 (4.7) 8 (12.1)
No significant change 44 (67.2) 47 (71.2)
Significantly improved 18 (28.1) 11 (16.7)
Total 65 66 NNT = 9

Patients fully adhering to the diet

Significantly worse 1 (4.2) 5 (12.5)
No significant change 10 (41.7) 29 (72.5)
Significantly improved 13 (54.1) 6 (15)
Total 24 40 NNT = 2.5

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Food elimination based on IgG antibodies in IBS 1463

True diet Sham diet Table 4 Global rating following reintroduction of foods
relative to the end of the elimination phase
All patients Full adherence
A Treatment group
p=0.14 p=0.05
180 True diet group Sham diet group
n=24 (n (%)) (n (%))
Non-colonic symptoms

Significantly worse 17 (41.5) 13 (25)

No significant change 23 (56.1) 35 (67.3)
120 n=65 Significantly improved 1 (2.4) 4 (7.7)
n=40 Total 41 (100) 52 (100)
n=66
60

worsening of health compared with the sham diet group

(p = 0.047).
0

180 p=0.27 p=0.27 DISCUSSION

A clinically significant improvement in IBS symptomatology
n=24
was observed in patients eliminating foods to which they
n=65 were found to exhibit sensitivity, as identified by an ELISA
Quality of life

120
test for the presence of IgG antibodies to these foods. The
n=66 n=40 number needed to treat of 9 for the group as a whole and 2.5
for patients closely adhering to the diet are both considerably
60 better than the value of 17 achieved after three months of
treatment with tegaserod,32 a drug that has been recently
licensed in the USA for use in IBS. IBS symptom severity and
0 global rating scores were chosen as primary outcome
measures in this study as they represented the most direct
measure of clinical improvement in this condition based on
patient self assessment. Rather than using the traditional
True diet Sham diet method of classifying global improvement as any value
All patients Full adherence exceeding adequate relief of symptoms, we used a much
B stricter definition requiring patients to report symptoms as
p=0.18 p=0.18 being either ‘‘better’’ or ‘‘excellent’’ compared with pretreat-
2.5 n=24 ment levels. Despite this, the diet still achieved a significant
n=65 improvement. However, as might be expected, the placebo
2 response using this end point was somewhat lower than that
usually reported in IBS treatment trials which have used less
1.5 demanding criteria. The observation that patients on the
n=66 n=40
Anxiety

sham diet also improved, although to a lesser extent,

1 emphasises the importance of conducting double blind
randomised controlled trials of such non-drug interventions
0.5 in order to avoid overestimating their potential.
Most patients with IBS have attempted at least some form
0 of dietary modification, which in some cases can be very
extreme. Conflicting results have been reported using
2.5 exclusion diets4 5 33–36 and this approach also suffers from
p=0.78 p=0.26
the limitation that it has to be empirical. Thus potentially
2 n=24 offending foods can only be identified after their elimination
and subsequent reintroduction. This time consuming process
Depression

1.5 would be much reduced if the offending foods could be

1 n=65
n=66
0.5
0 which was found to be no greater than that occurring in the
Figure 4 (A) Mean change in the secondary outcome measures of non-
colonic symptoms and quality of life for the group as a whole and the full
adherence group. (B) Mean change in the secondary outcome measures
of anxiety and depression for the group as a whole and the full
adherence group.
group, a statistically significant difference of 52 (24%) (95%
CI 18, 86; p = 0.003). The change in global score following
reintroduction of foods is shown in table 4. This indicates a
reversal of the pattern observed during the active treatment
phase, with more patients in the true diet group showing
identified beforehand. Attempts to do this using IgE
antibodies have been disappointing8–10 but the results of this
n=40
study suggest that measuring IgG antibodies may be much
more rewarding. The response to the IgG based diet in our
trial did not correlate with atopic status, the prevalence of
general population.37
The observation that adherence to the diet is critical in
determining a good outcome in the ‘‘true’’ diet group but not
the ‘‘sham’’ group is indicative of the fact that the diet is an
‘‘active treatment’’ which if not adhered to, does not seem to
have an effect. This notion is further supported by the
observation that a significantly greater deterioration was
observed in subjects in the true diet group compared with
those in the sham group when they reintroduced eliminated
foods at the end of the diet phase of the trial. Furthermore,
the improvement of 98 in the symptom severity score in those
fully adherent in the true diet group is well above the value of

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1464
50, which is regarded as being of clinical significance both in
validation studies24 and clinical practice.26–28 It was interesting
to note that patients exhibiting a greater number of
sensitivities, as determined by the IgG test, experienced a
greater symptom reduction if they adhered to the true but not
the sham diet.
There is currently considerable interest in the concept that
at least in some patients, IBS may have an inflammatory
component.38–42 Most of the work in this area has centred on
post dysenteric IBS, with gut pathogens being viewed as the
initiators of this process which can be identified by subtle
changes on histology.38 However, if, as indicated in this study,
IgG antibodies to food are important in the pathogenesis of
IBS in some patients, they too may be of relevance. Not all
patients exhibiting histological features consistent with post
dysenteric IBS give a history of a previous dysenteric illness.
This is usually assumed to be due to the fact that this has
been forgotten by the patient but our results may suggest an
alternative mechanism for immune activation and inflam-
mation without the need for prior infection.
It is now well recognised that up to 70% of patients with
IBS have evidence of hypersensitivity of the rectum,43 which
probably extends to involve most of the gut in many
individuals.44 It is possible that this hypersensitivity renders
patients more reactive to a low grade inflammatory process
which would not necessarily cause symptoms in a normal
individual. This would explain why excluding foods to which
patients have IgG antibodies might be particularly beneficial
in IBS despite the fact that these antibodies may also be
present in the general population. Indeed, if this mechanism
is particularly important in IBS, it might be anticipated that
IgG food antibodies would be relatively common in this
condition, as was the case in our study.
Many patients with IBS would prefer a dietary solution to
their problem rather than having to take medication, and the
economic benefits of this approach to health services are
obvious. It is well known that patients expend large sums of
money on a variety of unsubstantiated tests in a vain attempt
to identify dietary intolerances. The results of this study
suggest that assay of IgG antibodies to food may have a role
in helping patients identify candidate foods for elimination
and is an approach that is worthy of further biomedical and
clinical research.
.....................
Authors’ affiliations
W Atkinson, N Shaath, P J Whorwell, Department of Medicine,
University Hospital of South Manchester, Manchester, UK
T A Sheldon, Department of Health Sciences, University of York, York,
UK
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Future Directions

Food Exclusion Based on IgG Antibodies Alleviates Symptoms in

Ulcerative Colitis: A Prospective Study

Downloaded from https://academic.oup.com/ibdjournal/article-abstract/24/9/1918/4996921 by Bora Laskin Law Library user on 01 October 2018
Liu Jian, PhD,* He Anqi, MM,* Liu Gang, PhD, Wang Litian, MM, Xu Yanyan, MM,
Wang Mengdi, MM and Liu Tong, PhD

Background:  Most patients with ulcerative colitis (UC) rely predominantly on medication for disease control. Diet interventions can reduce
pharmaceutical expenditures and prolong remission. We designed a prospective study to evaluate whether an immunoglobulin G (IgG)–guided
exclusion diet would improve symptoms and quality of life (QoL) in patients with UC.
Methods:  The 6-month diet intervention included 97 patients with UC, who were randomly divided into an intervention group (n = 49) and a
control (n = 48) group. Individual diet plans were created for the intervention group according to IgG titers; the control group ate a healthy diet
as normal. Observational indices included disease activity, extraintestinal manifestations, nutritional status, and QoL. Relationships between
food-specific IgG antibodies and these indices were also analyzed.
Results:  At baseline, there were no significant differences between the groups. Food-specific IgG antibodies were detected in 70.10% of partici-
pants. After intervention, the Mayo score was significantly lower in the intervention group than in the control group (2.41 ± 0.89 vs 3.52 ± 1.15,
P < 0.05). The number of patients with extraintestinal manifestations decreased from 7 to 2 in the intervention group and from 6 to 5 in the con-
trol group. As for nutritive indices, the intervention group had higher mean body mass index and albumin than the control group (23.88 ± 3.31 vs
21.50 ± 6.24 kg/m2, respectively, P < 0.05; 48.05 ± 6.39 vs 45.72 ± 5.48 g/L, respectively, P < 0.05), whereas prealbumin and transferrin were not
significantly different between the groups. QoL improved after food exclusion (P < 0.05).
Conclusions:  An IgG-guided exclusion diet ameliorated UC symptoms and improved QoL. Interactions between IgG-based food intolerance
and UC warrant further study.
Key Words: colitis, ulcerative, immunoglobulin G, food exclusion

INTRODUCTION between gut antigens and host immunity.6, 7 When certain

Ulcerative colitis (UC) is a nonspecific colorectal
inflammatory disorder that causes diarrhea, mucopurulent
bloody stool, abdominal pain, and malnutrition. This condi-
tion impairs quality of life (QoL)1–3 and may require lifelong
treatment.4 In addition to medication and surgery, diet mod-
ification is 1 component of a holistic approach to managing
UC.5 Therapeutic measures should be based on mechanisms
and etiologies, which are complex and uncertain in UC. Some
researchers have proposed that UC is related to interactions
Received for publications November 25, 2017; Editorial Decision February 23,
2018.
From the Department of General Surgery, Tianjin Medical University General
Hospital, Tianjin, People’s Republic of China
Conflicts of interest: None declared.
Supported by: This research was supported by the intestinal barrier research
fund of Academician JieShou Li (LJS_201008).
*Authors contributed equally to this work and should be regarded as co-first
authors.
Address correspondence to: Liu Gang, PhD, Department of General Surgery,
Tianjin Medical University General Hospital, Anshan Road No.154, Heping
District, Tianjin, People’s Republic of China (landmark1059@163.com).
© 2018 Crohn’s & Colitis Foundation. Published by Oxford University Press.
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
doi: 10.1093/ibd/izy110
Published online 16 May 2018
digestive enzymes are lacking, it is difficult to digest food into
small molecules such as glucose, amino acids, and glycerol.
Undigested food components are identified by the immune
system as foreign substances, resulting in the development
of diseases related to the mucosal immune system, epithelial
function, and the intestinal microbiome.8 These reactions may
lead to food intolerance.
Food intolerance is a delayed or even asymptomatic
immune response to certain food antigens. It is mediated by
immunoglobulin G (IgG), unlike food allergy, which is an IgE-
mediated immediate immune response. A  retrospective study
reported that patients with inflammatory bowel disease (IBD)
had higher levels of IgG antibodies to specific food allergens
than healthy people.9 That group speculated that the level of
serum IgG may be related to disease status. Further studies have
been performed to demonstrate that food-specific IgG-guided
exclusion diets improve symptoms in Crohn’s disease and that
this approach may be useful in clinical practice.10–12 However,
although some prospective studies have focused on the effect of
dietary components on the disease13–15 and other studies have
evaluated food exclusion in UC patients,16 diet therapy based
on IgG antibodies remains rarely reported in patients with UC.
In 1942, Andresen reported that more than half of UC
patients benefited from a milk-free diet. Subsequent studies
have intermittently reported the advantages of food exclusion

Inflamm Bowel Dis • Volume 24, Number 9, September 2018 1918

Inflamm Bowel Dis • Volume 24, Number 9, September 2018
in patients with UC. The methods of these studies were empir-
ical and time-consuming; however, the results indicated that
IgG-based exclusion diets may benefit UC patients and provide
personalized and precise therapies. In this study, we aimed to
evaluate whether an IgG-guided exclusion diet would improve
symptoms and QoL in patients with UC.
METHODS
Patient Recruitment
Consecutive patients with UC who were admitted to
Tianjin Medical University General Hospital from April 2012
to September 2015 were considered for inclusion. The diagno-
sis of UC had been confirmed previously according to clinical
manifestations, endoscopic appearance, and histopathology.
Patients with severe disease activity, those under 18  years of
age, and pregnant patients were excluded. Patients with a his-
tory of immune disease, lactose intolerance, other significant
gastrointestinal disorders, concurrent malignancy, previous
bowel resection, psychiatric problems, or clinically significant
hepatic or renal disease were also excluded.
This study was designed as an open-label, stratified,
prospective study. First, patients’ disease status was classified
according to Mayo score as “remission,” “mild activity,” or
“moderate activity.” Eligible patients were then allocated to a
food exclusion group or sham diet group; a random number
table was used to keep the numbers and characteristics in the 2
dietary groups approximately equal.
Food-Specific IgG Antibodies Test
Patients’ blood samples were taken in the normal state at
the beginning and at completion of the study. Samples were sent
with numerical identifiers to the laboratory of the Department
of Immunology at our hospital, where the presence of IgG
antibodies specific to 14 different food antigens was detected
with enzyme-linked immunosorbent assay (ELISA). These
foods included egg, wheat, milk, corn, tomato, crab, rice, soy-
bean, cod, shrimp, mushrooms, beef, chicken, and pork. An
ELISA kit (Biomerica, Inc., CA, USA) and a microplate reader
(ANTHOS 2010, AnthosLabtec Instrument, Austria) were
used. The details of this test have been described in other stud-
ies.13 The level of food reaction was measured according to the
presence of IgG antibodies.9
Diet Intervention
The diet intervention was designed to last for 6 months
and was managed by doctors, nurses, and nutritionists.
Individual diet plans were made for patients in the food exclu-
sion group. Patients were asked to completely exclude foods
with IgG antibody titers greater than 100 U/mL (a grade of
2- or 3-plus) from their diets during the 6 months and to avoid
accidental ingestion. Foods with IgG antibody titers from 50 to
Food Exclusion Alleviates Ulcerative Colitis
100 U/mL (a grade of 1-plus) could be eaten on a 5-day rota-
tion cycle. Foods with IgG antibody titers of less than 50 U/
mL (a grade of no-plus) and foods whose antigens were not
tested were categorized as normal, as long as they were nutri-
tious (Table 1). Patients in the sham diet group were asked to
maintain their healthy diet as normal. The diet intervention did
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not influence the individual medical treatment, which mainly
included mesalazine and probiotics.
The patients in both groups were followed up every 2
weeks by our outpatient service. In addition, patients could
contact the team by face-to-face communication, telephone, or
e-mail whenever they had queries. During the study, patients
were asked to record their diet, the frequency and character of
their stools, and their symptoms every day. At each contact,
patients were asked whether they had adhered to the diet, and
they showed their records of daily diet, stools, and symptoms to
the doctors. Medical treatment plan modifications were consid-
ered according to patients’ current symptoms. If patients expe-
rienced any problems, they could be discussed with the team.
Observational Indices
All indices were observed and recorded at the begin-
ning and end of the study. The overall observational indices
included:
Disease activity index
The Mayo score, also called the Southerland Activity
Index, is an internationally accepted, practical tool to assess
UC activity. The total scores corresponding to remission, mild
activity, moderate activity, and severe activity are 0–2, 3–5,
6–10, and 11–12, respectively. The scoring system includes the
following 4 aspects: stool frequency, rectal bleeding, mucosa
friability, and physician’s global assessment.14 Patients were
asked to record the frequency and character of their stools
daily and to report to the researchers every 2 weeks. The scores
for stool frequency and rectal bleeding were the average levels
of the initial 2 weeks and of the final 2 weeks. Mucosa friability
was measured with colonoscopy. Colonoscopy examinations
were performed within 2 weeks of onset and at the end of the
diet intervention. The endoscopists were blinded to the groups.
Physician’s global assessment was determined by 1 experi-
enced physician who had no knowledge of the patient’s group
assignment.
TABLE 1:  Classification of IGG Antibody Levels
Detection Value, U/mL Judgment of Results Grade
<50 Negative -
50–100 Mild +
100–200 Moderate ++
>200 Severe +++
1919
Jian
 et al
Extraintestinal manifestations
Extraintestinal manifestations (EIMs) in UC patients
include anemia, arthropathy (peripheral and axial), meta-
bolic bone disease (osteoporosis), cutaneous manifestations
(erythema nodosum, pyoderma gangrenosum, Sweet’s syn-
drome, and anti–tumor necrosis factor–induced skin inflam-
mation), ocular manifestations (episcleritis and uveitis), and
hepatobiliary disease (primary sclerosing cholangitis, peri-
cholangitis, steatosis, chronic hepatitis, and cirrhosis).15 If
patients suffered from the above diseases in the absence of
obvious predisposing causes, they were classified as having
EIMs.
Nutritional indices
Body mass index (BMI), albumin (ALB), transferrin
(TRF), and prealbumin (PA) are important reference indices
reflecting nutrition status. BMI was calculated using patients’
height and body weight (kg/m2). ALB, TRF, and PA reflect
nutrition status for the previous 2 months, 2 weeks, and 2 days,
respectively.
Health-related quality of life
The Inflammatory Bowel Disease Questionnaire
(IBDQ) is the most widely used instrument to measure
health-related QoL over the prior 2 weeks in IBD patients.
The IBDQ consists of 4 items and includes 32 questions in
total: bowel symptoms (10 questions), systemic symptoms
(5 questions), social functions (5 questions), and emotional
functions (12 questions).16 Each question is scored from 1 to
7, corresponding to the state from worst to best. Total scores
range from 32 to 224; the higher the score, the better the qual-
ity of life.
Statistical Analysis
Statistical Product and Service Solutions (SPSS), ver-
sion 17.0, was used for data processing. Continuous vari-
ables were presented as mean ± standard deviation ( x ± s).
Two-sample t tests were used to establish whether there was
an overall difference in the change in continuous outcomes
between the 2 patient groups. Levels of the 14 IgG antibodies
at baseline and at 6 months were evaluated with t tests in both
groups to establish whether there was an overall difference
in IgG antibody levels for the 14 foods. Categorical variables
were expressed as counts and percentages and were compared
with a χ2 test. A P value of less than 0.05 was considered sta-
tistically significant.
Ethical Considerations
Written informed consent was obtained from each partic-
ipant before enrollment in this study. This study was approved
by the Research Ethics Committee of the Tianjin Medical
University General Hospital.
1920
Inflamm Bowel Dis • Volume 24, Number 9, September 2018
RESULTS
Patient Recruitment and Characteristics
One hundred fifty-nine eligible patients were invited to
a meeting at which details of the study were provided. Fifty-
eight patients did not attend the meeting, and 4 patients chose
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not to enroll. The remaining 97 patients participated; none of
the participants dropped out of the study. A  balanced strat-
egy was applied to reduce the impact of differences in disease
activity on the trial. Thirty-one patients were in remission, 37
had mild activity, and 29 had moderate activity; patients were
divided into the 2 study groups randomly. The food exclusion
group included 49 patients, and the sham diet group included
48 (Fig. 1). The age and sex distributions in the food exclusion
group were matched to those in the sham diet group (Table 2).
Relationship Between Food-Specific IgG
Antibodies and UC at Baseline
At the beginning of the study, 68 patients (70.10%)
were food-specific IgG antibody positive; 54 (55.67% of total
patients) were extremely sensitive to a food (with a grade of
3-plus). The 5 foods that most often tested IgG antibody pos-
itive were egg (58/97, 59.79%), wheat (37/97, 38.14%), milk
(31/97, 31.96%), corn (29/97, 29.90%), and tomato (25/97,
25.77%). The 5 foods that were most often strongly positive
(with a grade of 3-plus) were egg (48/97, 49.48%), wheat (16/97,
16.49%), milk (15/97, 15.46%), soybean (11/97, 11.34%), and
tomato (10/97, 10.31%). None of the patients was sensitive to
chicken or pork (Table 3).
The average Mayo scores among the remission, mild
activity, and moderate activity patients were 0.63, 3.59, and
7.12, respectively. IgG antibodies were present in 51.61%
(16/31), 67.57% (25/37), and 93.10% (27/29) of these patients,
respectively (P < 0.05).
Thirteen patients had EIMs, accounting for 13.4% of the
total study population. Among these, 6 patients had peripheral
or axial arthropathy, 4 had primary sclerosing cholangitis, 2 had
anemia, and 1 had uveitis. The prevalence of food-specific IgG
antibodies among patients with EIMs was higher than among
those without EIMs (92.31% [12/13] vs 66.67% [56/84], P < 0.05).
Comparison Between Groups After Diet
Intervention
Food-specific IgG antibodies
There was no significant difference between the groups at
baseline or at 6 months, either in total positive or severe-pos-
itive rate (P > 0.05). However, there was an obvious tendency
in the food exclusion group that both the total and the strongly
positive rates for almost all 14 food-specific IgG antibodies had
decreased at 6 months, and the total positive rate achieved sta-
tistical significance (P < 0.05) (Table 4).
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FIGURE 1.  Study flow diagram.

decrease in Mayo score in the food exclusion group. At baseline,

TABLE 2:  Baseline Characteristics of Patients According most patients in both groups had mild to moderate inflamma-
to Group tory changes on endoscopy; the others had no obvious abnor-
Intervention Group Control Group
mality. There were no significant differences in endoscopic
Variable (n = 49) (n = 48) P
findings between the groups. After intervention, the overall
mucosal state had improved, with no significant difference in
Mean age (range), y 38 (21–65) 39 (20–68) 0.244 improvement between the groups. However, the endoscopic
Sex, No. (%) 0.922 appearance tended to be better in the food exclusion group
Male 26 (53) 24 (50) (Fig. 2).
Female 23 (47) 24 (50) The number of patients with EIMs in the sham diet group
Nutritional status changed from 6 to 5, whereas the number in the food exclusion
 BMI 21.38 ± 3.48 20.27 ± 4.56 0.090 group decreased from 7 to 2.
 ALB 44.78 ± 5.54 44.29 ± 5.09 0.326
 PA 258.07 ± 45.4 257.36 ± 38.59 0.467 Nutritional status
 TRF 243.82 ± 15.76 241.17 ± 14.21 0.193 After the diet intervention, BMI and ALB were higher
Mayo score 3.01 ± 0.78 3.25 ± 1.09 0.107 in the food exclusion group than in the sham diet group
EMI, No. (%) 7 (14.3) 6 (12.5) (23.88  ±  3.31 vs 21.50  ±  6.24  kg/m2, respectively, P  <  0.05;
48.05 ± 6.39 vs 45.72 ± 5.48 g/L, respectively, P < 0.05). There
were no significant differences in PA or TRF between the
Disease activity index and extraintestinal groups (264.56  ±  48.22 vs 256.93  ±  46.50  mg/L, respectively,
manifestations P > 0.05; 246.67 ± 14.52 vs 249.04 ± 22.54 mg/L, respectively,
At the beginning of the study, the Mayo scores were P > 0.05) (Table 5).
equivalent in the sham diet group and the food exclusion group
(3.01 ± 0.78 vs 3.25 ± 1.09, respectively; P > 0.05). The num- Health-related quality of life
ber of patients with EIMs was likewise equivalent between the There was no significant difference in baseline QoL
groups (6 vs 7, respectively) (Table  3). Six months later, the between the 2 groups. After intervention, the IBDQ scores in
Mayo score in the sham diet group was higher than that in the the food exclusion group were higher than those in the sham
food exclusion group (3.52  ±  1.15 vs 2.41  ±  0.89, P  <  0.05). diet group in all dimensions, especially in the emotional func-
Among the 4 subitems, stool frequency contributed most to the tion subitem (P  <  0.01). All subitems in the food exclusion

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 et al Inflamm Bowel Dis • Volume 24, Number 9, September 2018

TABLE 3:  Distribution of Positive IGG Antibody Tests Among All Patients at Study Entry
Grade
Food Items Total Patients + ++ +++ Positive Rate, %

Egg 58 4 6 48 59.79

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Wheat 37 14 7 16 38.14
Milk 31 7 9 15 31.96
Corn 29 15 6 8 29.9
Tomato 25 10 5 10 25.77
Crab 22 19 3 0 22.68
Rice 17 2 6 9 16.49
Soybean 16 4 1 11 15.46
Cod 9 2 2 5 9.28
Shrimp 3 1 0 2 3.09
Mushroom 2 1 1 0 2.06
Beef 2 0 2 0 2.06
Chicken 0 0 0 0 0
Pork 0 0 0 0 0

TABLE 4:  Distribution of Positive IGG Antibody Tests in Food Exclusion Group

Total Positive Severe Positive
Total Patients Total Patients Grade Rate,a % Rate,b %
Before After + ++~+++
Food Items Before After Before After Before After Before After

Egg 29 22 2 4 27 18 59.18 44.90 55.1 36.73

Wheat 19 13 7 3 12 10 38.78 26.53 24.49 20.41
Milk 17 15 4 3 13 12 34.69 30.61 26.53 24.49
Corn 14 10 7 6 7 4 28.57 20.41 14.29 8.16
Tomato 13 11 5 4 8 7 26.53 22.45 16.33 14.29
Crab 12 7 10 4 2 3 24.49 14.29 4.08 6.12
Rice 9 8 2 2 7 6 18.37 16.33 14.29 12.24
Soybean 9 10 2 3 7 7 18.37 20.41 14.29 14.29
Cod 5 6 2 3 3 3 10.20 12.24 6.12 6.12
Shrimp 2 1 1 0 1 1 4.08 2.04 2.04 2.04
Mushroom 1 2 1 1 0 1 2.04 4.08 0 2.04
Beef 1 2 0 1 1 1 2.04 4.08 2.04 2.04
Chicken 0 0 0 0 0 0 0 0 0 0
Pork 0 0 0 0 0 0 0 0 0 0

a
P = 0.03; bP = 0.136.

group had improved, whereas there was no obvious change in
the sham diet group (Fig. 3).
DISCUSSION
We drew on studies of IgG-guided exclusion diets in
patients with Crohn’s disease and irritable bowel syndrome to
design our experimental scheme. First, a balanced strategy was
applied to reduce the impact of differences in disease activ-
ity on the trial; patients with severe activity were excluded.
Patients with severe UC are often hospitalized and need total
parenteral nutrtion; therefore, these patients are not among
the target audience for IgG-guided exclusion diets. Second,

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FIGURE 2.  Typical endoscopic findings. A, Sham diet group before intervention. B, Food exclusion group before intervention. C, Sham diet group
after intervention. D, Food exclusion group after intervention.

TABLE 5:  Nutritional Status of Patients After Intervention, According to Group

Nutritional Items Food Exclusion Group Sham Diet Group t P

BMI 23.88 ± 3.31 21.50 ± 6.24 2.353 0.010

ALB 48.05 ± 6.39 45.72 ± 5.48 1.700 0.047
PA 264.56 ± 48.22 256.93 ± 46.50 0.793 0.215
TRF 246.67 ± 14.52 249.04 ± 22.54 –0.533 0.298

individual diets in the food exclusion group were determined
according to the results of IgG antibody levels, which is an
evidence-based approach. In addition, a multidisciplinary
team worked together to ensure the successful implementation
of the diet intervention. The doctors and nutritionists formu-
lated diets according to the results of IgG antibody testing;
the nurses guided the patients on how to strictly adhere to
the diet.
The prevalence of food-specific IgG antibodies among
patients with UC in this study was 70.10% (68/97), which is
twice as high as that among healthy Chinese people (prevalence
of 33.1% reported in a study by Cai et al.).9 This difference may
be explained by the hypothesis that UC patients have increased
permeability of the intestinal mucosa, so that food antigens are
more easily presented to the gut immune system, resulting in
increased IgG antibodies.17, 18 In addition, this study indicated
that disease activity and EIMs were closely related to the pres-
ence of food-specific IgG antibodies. The exact mechanisms of
the interaction between UC and IgG-based food intolerance
remain unclear.

1923
Jian
 et al
FIGURE 3.  Comparison of scores for 4 dimensions derived from the IBDQ, according to group. A, Before intervention. B, After intervention. *P < 0.05;
P < 0.01.
**
Other studies have provided information about the rela-
tionship between diet and IBD. Jorrit et al. reported that foods
containing high levels of protein and dietary calcium could
lead to the development of IBD.19 Malow et al. provided strong
evidence that intolerance to mustard, wasabi, and tomato in
patients with CD was significantly associated with the G allele
of FOXO3.20 In our test of 14 foods, the 5 foods that were most
often IgG antibody positive were egg, wheat, milk, corn, and
tomato, which is consistent with the findings of the above stud-
ies. However, studies in Germany showed that antifood IgG or
IgG subclass levels in serum or feces of patients with IBD did
not correlate with food intolerance.21, 22 As only 29 UC patients
were included and only 3 of the tested IgG antibodies corre-
sponded to food intolerance, their studies were insufficient to
show the relationship between antifood IgG antibodies and
food intolerance in UC, and their conclusions remained to be
discussed. We hypothesize that IgG-based food intolerance may
also derive from hereditary and immunological factors. Further
studies are needed to explore the exact relationship between UC
and food-specific IgG antibodies. However, we need to be aware
of the possibility of poor clinical manifestations resulting from
inappropriate diet in patients with UC.
The primary aim of this study was to provide evidence on
whether an IgG-based exclusion diet can improve symptoms in
patients with UC, as theory suggests. Our results are worth dis-
cussion. After 6 months of an exclusion diet, IgG antibody lev-
els did not fall significantly in the intervention group; however,
a decreasing trend was obvious. We infer that in a longer diet
intervention, a significant difference would be found between
the food exclusion group and the sham diet group. Dietary
treatment can be a lengthy process.
Although the changes in IgG antibody levels in this study
were conservative, clinical symptoms improved markedly in the
food exclusion group. Stool frequency, rectal bleeding, mucosal
characteristics, and physician’s global assessment, as quantified
by Mayo score, improved significantly in the food exclusion
group. Among these, stool frequency and rectal bleeding were
recorded by patients, and the former changed the most notably.
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Inflamm Bowel Dis • Volume 24, Number 9, September 2018
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The endoscopic appearance of the mucosa tended to be bet-
ter after dietary intervention in the food exclusion group, and
the endoscopic score in the food exclusion group was slightly
more decreased compared with the sham diet group; however,
the improvement did not reach statistical significance. One
experienced physician, who was blind to patient grouping, per-
formed all physician’s global assessments, which minimized the
influence of subjective factors. We believe that medical therapy
contributed to the improvement in disease activity; however,
significant differences present after 6 months also indicate the
importance of dietary intervention.
The EIMs also improved significantly after the dietary
intervention in the food exclusion group. This finding may
indicate that the presence of EIMs is associated with disease
activity.
In the evaluation of nutritional status, all indices in the
food exclusion group were higher than those in the sham diet
group after intervention. The differences in BMI and ALB
were statistically significant. This result may be explained by
the fact that patients had less gastrointestinal discomfort after
exclusion of the triggering food and were thus able to eat more,
even if food variety was limited. These patients also had more
improvement in disease activity, as indicated by Mayo score.
Unfortunately, we did not collect exact data on total intake.
However, although the improvements in BMI and ALB were
significant, the differences in PA and TRF were not. This result
may indicate that dietary intervention mainly changes long-
term nutritional status, whereas short-term nutritional status
is influenced by multiple confounding factors, corresponding
to the viewpoint that dietary intervention in UC patients is a
long-term battle.
IBDQ scores reflect the subjective assessment of
patients with IBD concerning their state of illness and QoL.
Improvements in disease activity and nutritional status ena-
bled patients in the food exclusion group to participate in more
activities of daily living, so that their scores of health-related
QoL were higher than those in the sham diet group. Among all
the dimensions of the IBDQ, the significant difference between
Inflamm Bowel Dis • Volume 24, Number 9, September 2018
the two groups in the emotional function subitem was impor-
tant. There is a consensus that IBD is a somatopsychic illness.
The improvement in emotional health is important for patients
and will make them more willing to adhere to the food exclu-
sion diet, further ameliorating their symptoms.
In addition to the differences between the 2 groups, the
changes in various indices in the sham diet group, though less
obvious than the changes in the food exclusion group, are also
worth attention. The slight improvements in the sham diet
group may have resulted from adherence to a healthy diet and
rational drug use under the supervision of doctors during the
6-month study period.
In conclusion, sticking to an IgG-based exclusion diet
effectively improved symptoms and health-related QoL in
patients with UC. An IgG-based exclusion diet may ulti-
mately reduce the economic burden of this disease. In the
future, dietary interventions will play an increasingly impor-
tant role in UC management, acting as a viable option for cer-
tain patients to reduce their symptoms, shorten the course of
disease, and speed up recovery. However, this study had some
limitations. The types of food tested and number of patients
enrolled were limited. The mechanisms by which IgG anti-
bodies influence the course of UC were not explored. Thus,
further biomedical studies among polyethnic and larger pop-
ulations are needed.
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symptoms in Crohn’s disease: a pilot study. Colorectal Dis. 2011;13:1009–13.
13. Foster AP, Knowles TG, Moore AH, et al. Serum IgE and IgG responses to food
antigens in normal and atopic dogs, and dogs with gastrointestinal disease. Vet
Immunol Immunopathol. 2003;92:113–24.
14. Sutherland LR, Martin F, Greer S, et al. 5-aminosalicylic acid enema in the treat-
ment of distal ulcerative colitis, proctosigmoiditis, and proctitis. Gastroenterology.
1987;92:1894–8.
15. Magro F, Gionchetti P, Eliakim R, et  al; European Crohn’s and Colitis
Organisation. Third European evidence-based consensus on diagnosis and man-
agement of ulcerative colitis. Part 1: definitions, diagnosis, extra-intestinal mani-
festations, pregnancy, cancer surveillance, surgery, and ileo-anal pouch disorders.
J Crohns Colitis. 2017;11:649–70.
16. Irvine EJ, Feagan B, Rochon J, et al. Quality of life: a valid and reliable meas-
ure of therapeutic efficacy in the treatment of inflammatory bowel disease.
Canadian Crohn’s Relapse Prevention Trial Study Group. Gastroenterology.
1994;106:287–96.
17. Chahine BG, Bahna SL. The role of the gut mucosal immunity in the develop-
ment of tolerance versus development of allergy to food. Curr Opin Allergy Clin
Immunol. 2010;10:394–9.
18. Atkinson W, Sheldon TA, Shaath N, et  al. Food elimination based on IgG
antibodies in irritable bowel syndrome: a randomised controlled trial. Gut.
2004;53:1459–64.
19. Opstelten JL, Leenders M, Dik VK, et al. Dairy products, dietary calcium, and
risk of inflammatory bowel disease: results from a European prospective cohort
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20. Marlow G, Han DY, Triggs CM, et  al. Food intolerance: associations with
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 ISSN 0017-8748
Headache doi: 10.1111/j.1526-4610.2012.02296.x
© 2012 American Headache Society Published by Wiley Periodicals, Inc.

Research Submission
IgG-Based Elimination Diet in Migraine Plus Irritable
Bowel Syndrome
Elif Ilgaz Aydinlar, MD; Pinar Yalinay Dikmen, MD; Arzu Tiftikci, MD; Murat Saruc, MD; Muge Aksu;
Hulya G. Gunsoy; Nurdan Tozun, MD

Objectives.—To evaluate therapeutic potential of the immunoglobulin G (IgG)-based elimination diet among migraine
patients with irritable bowel syndrome (IBS).
Background.—Food elimination has been suggested as an effective and inexpensive therapeutic strategy in patients with
migraine and concomitant IBS in the past studies.
Methods.—A total of 21 patients (mean [standard deviation] age: 38.0 [11.2] years; 85.7% females) diagnosed with
migraine and IBS were included in this double-blind, randomized, controlled, cross-over clinical trial composed of baseline
(usual diet), first diet (elimination or provocation diets), and second diet (interchange of elimination or provocations diets)
phases and 4 visits.
Results.—IgG antibody tests against 270 food allergens revealed mean (standard deviation) reaction count to be 23.1
(14.1). Compared with baseline levels, elimination diet per se was associated with significant reductions in attack count (4.8 [2.1]
vs 2.7 [2.0]; P < .001), maximum attack duration (2.6 [0.6] vs 1.4 [1.1] days; P < .001), mean attack duration (1.8 [0.5] vs 1.1 [0.8]
days; P < .01), maximum attack severity (visual analog scale 8.5 [1.4] vs visual analog scale 6.6 [3.3]; P < .001), and number of
attacks with acute medication (4.0 [1.5] vs 1.9 [1.8]; P < .001). There was a significant reduction in pain-bloating severity (1.8
[1.3] vs 3.2 [0.8]; P < .05), pain-bloating within the last 10 days (3.2 [2.8] vs 5.5 [3.1]; P < .05), and improvement obtained in
quality of life (3.6 [1.4] vs 2.9 [1.0]; P < .05) by the elimination diet as compared with provocation diet.
Conclusions.—Our findings indicate that food elimination based on IgG antibodies in migraine patients who suffer from
concomitant IBS may effectively reduce symptoms from both disorders with possible positive impact on the quality of life of the
patients as well as potential savings to the health-care system.

Key words: migraine, irritable bowel syndrome, elimination diet, immunoglobulin G antibody, food antigen

(Headache 2012;••:••-••)

From the Department of Neurology, Acibadem University School of Medicine, Istanbul, Turkey (E.I. Aydinlar and P.Y. Dikmen);
Department of Gastroenterology, Acibadem University School of Medicine, Istanbul, Turkey (A. Tiftikci, M. Saruc, and N. Tozun);
Department of Nutrition and Dietetics, Acibadem Health Group, Istanbul, Turkey (M. Aksu and H.G. Gunsoy).
Address all correspondence to E.I. Aydinlar, Department of Neurology, Acibadem University School of Medicine, Gulsuyu mah,
Fevzi Cakmak Cad. Divan Sok., Maltepe, 34848 Istanbul, Turkey, email: eilgaz@acibadem.com.tr

Accepted for publication October 1, 2012.

Conflicts of Interest: The authors declare that they have no conflict of interest.
Funding: The study is supported by Immuno Diagnostic Laboratories, Istanbul, Turkey.

1
2
Migraine is characterized by recurrent unilateral
headache usually accompanied by nausea/vomiting,
photophobia, and/or phonophobia,1 with 1-year-
period prevalence of 10-12% in adults, 6% among
men, and 15-18% in women.2 The mechanisms under-
lying primary migraine are still unknown.3 Sensitivity
to particular food is one of the most common triggers
of migraine.4,5 Immunoglobulin (IgG) antibodies
against various food antigens have been reported to
be associated with migraine.6 Accordingly, consump-
tion of IgG-reactive food elimination diets for a spe-
cific period provided decrease in headache attacks
and significant improvement in symptoms.1
Irritable bowel syndrome (IBS) is a multifactorial
condition involving a number of different mecha-
nisms.7 Patients with IBS often strongly believe that
dietary intolerance significantly contributes to their
symptomatology, and some benefit from eliminating
certain foods from their diet.8 Besides, several studies
reported significant improvement in IBS by food
elimination based on IgG antibodies against food
antigens.8-11
IBS patients are likely to suffer from migraine
more than control subjects.12 Past studies suggested
food elimination as an effective and inexpensive
therapeutic strategy in patients with migraine and
concomitant IBS.13 Therefore, the present study was
designed to evaluate therapeutic potential of the IgG-
based elimination diet among migraine patients with
IBS via a double-blind, randomized, controlled, clini-
cal trial with a cross-over design.
SUBJECTS AND METHODS
Subjects.—Of 28 patients enrolled, a total of 21
patients diagnosed with migraine according to the
criteria of the International Classification of Head-
ache Disorders14 and accompanying uncomplicated
IBS (all bowel habit subtypes) according to the
ROME III criteria,15 being followed by the Headache
Outpatient Clinic of Neurology Department and the
Gastroenterology Department of Acibadem Univer-
sity School of Medicine, Istanbul, Turkey, were
included in this double-blind, randomized, controlled,
cross-over, clinical trial. Seven patients were excluded
due to unplanned pregnancy, difficulty to maintain
the diet, or lack of keeping records.
To have migraine diagnosis for at least 6 months
and at least 2 migraine attacks, and 4 headache days
within the last month; to be aged 18-65 years; to have
abdominal discomfort for at least 12-week duration in
the previous year, which need not be continuous; and
to be treated with preventive medications unchanged
at least for 6 months or with acute attack medica-
tions only were the inclusion criteria. Patients who
had medication-overuse headache, pure menstrual
migraine, or any other associated headache disorder,
inflammatory bowel disease, celiac disease, known
lactose intolerance, previous major abdominal
surgery, or other significant gastrointestinal disorder
were excluded from the study. In addition,
“advanced” cardiac, respiratory, renal, or hepatic dis-
eases; and malignancy, major psychiatric disorders, or
a history of drug/alcohol abuse and pregnancy at the
time of study enrollment and during the study were
considered as exclusion criteria.
Ethics.—The authors had full access to all the data
in the study and take responsibility for the integrity of
the data and the accuracy of the data analysis. Written
informed consent was obtained from each subject fol-
lowing a detailed explanation of the objectives and
protocol of the study that was conducted in accor-
dance with the ethical principles stated in the
“Declaration of Helsinki” and approved by the insti-
tutional ethics committee.
Study Procedures and Assessments.—Our study
was designed as a double-blind, randomized, con-
trolled, cross-over, clinical trial composed of 3 phases,
including baseline (run-in) phase (usual diet), first
diet phase (elimination or provocation diets, custom-
ized based on sensitivity results), and second diet
(interchange of elimination or provocation diets)
phase with a total of 4 evaluation visits per patient
(Figure). The patients visited the same headache and
gastroenterology physician during the whole study,
and they were encouraged not to modify the medica-
tions used during the course of the trial.
At the first visit, patients who fulfilled the patient
selection criteria were asked to fill out a headache
diary and watch their symptoms of gastric discomfort
for 6 weeks. The diary included questions on attack
count, headache days, attack duration (days), and
headache severity (via a 0-10 pain numeric rating
Headache
Figure.—Study flow chart. IBS = irritable bowel syndrome.
scale), and acute and overall medication use. The
patients were kept on their usual daily diet during this
6-week baseline run-in period.
At the second visit, the diaries were returned by
the patients, and emotional well-being was evaluated
considering happiness at home, happiness at work,
quality of life (QoL), belief in IBS treatment (via a
0-5 numeric rating scale: 0, worst; 5, best); fear of
illness and objective clinical findings of IBS symp-
toms including pain-bloating frequency and severity
(within the last 10 days), number of defecation days
(per week), diarrhea-constipation severity, and symp-
toms of urgency, straining, and inability to fully empty
the bowel via an IBS symptoms severity scale modi-
fied from that first described by Francis et al16 were
recorded (0, best; 5, worst) (see Appendix A). Addi-
tionally, blood samples were collected from each
patient to detect IgG antibody titers to 270 food aller-
gens to identify mean reaction (abnormally high titer)
count by enzyme-linked immunosorbent assay
(ELISA).
During the first diet phase of 6 weeks of patients
were allocated to either an elimination (excluding;
3
n = 11) or a provocation (including; n = 10) diet via
simple randomization procedures using a computer-
generated list of random numbers based on their sen-
sitivity results for 6-week run-in period. During this
first diet phase of 6 weeks, they were also asked to fill
a headache diary.
At visit 3, performed at the end of the first diet
phase, patients returned their headache diaries for
evaluation, and data on emotional well-being, belief
in IBS treatment, and IBS symptoms during the first
diet phase were recorded. Then, the patients were
asked to return to their usual diets for 3 weeks of
washout period without keeping a diary.
After washout, the patients who were on elimi-
nation diet in the first diet phase were given provo-
cation diet and vice versa for 6 weeks during which
they were asked to fill a headache diary. Patients were
reassessed after the second diet phase at the 3rd and
the 4th visit.
All patients and clinical staff in the headache
clinic and gastroenterology department were blinded
to the group assignment of all patients during the
study. The allocation sequence was concealed from
4
the researcher enrolling and assessing participants in
sequentially numbered, opaque, sealed, and stapled
envelopes. Patients were given their allocated diet by
2 qualified dieticians. Because of the double-blind
nature of the study design, the patients have been
informed that some of the foods might be provoking
and encouraged to consume certain foods during
both diet periods. The diet codes were broken, and
patients were informed about their food allergy
results, the order of the types of their diets, and if they
prefer how to continue their diet according to the
results.
IgG Antibody Detection Against Food Anti-
gens.—IgG antibodies against 270 food antigens
were detected using a commercially available ELISA
test (ImuPro 300 test; Evomed/R-Biopharm AG,
Darmstadt, Germany), previously used by Wilders-
Truschnig et al.17 IgG calibration was performed
against the international reference material 1st World
Health Organization International Reference Prepa-
ration 67/86 for human IgG. Quantitative measure-
ments are reported in mg/L. Detection limit was
2.5 g/L, normalized cut-off value was 7.5 mg/L,
according to the validation protocol provided by the
manufacturer. All values above 7.5 mg/L were consid-
ered as positive reaction to the corresponding food.
Diet Preparation.—Being arranged according to
the IgG antibody results, the elimination diet con-
sisted of a defined panel of IgG-negative food, while
the provocation diet consisted of a panel of IgG-
positive food. Both elimination diet and provocation
diet did not differ in calorie content. In both diet
phases, patients were never forced to eat or avoid
from certain foods to protect the blindness of patients
and their physician for the type of diet phase (elimi-
nation or provocation). Instead, they were asked to
follow their specially arranged diet list exactly and
not to consume any other food in any diet phase. Both
patients and physicians were blind to the type of diet
and IgG tests.
Statistical Analysis.—Statistical analysis was made
using the computer software SPSS version 13.0
(SPSS, Inc., Chicago, IL, USA). For comparisons
between baseline phase and provocation phase, base-
line phase and elimination phase, and provocation
phase and elimination phase, paired t-test or Wil-
coxon signed-rank test were used; for comparisons
between patients whose first diet phase was elimina-
tion diet and patients whose second diet phase was
elimination diet (to test the “period effect”), unpaired
t-test or Mann–Whitney test were used according to
the distribution pattern of the data. Multiple compari-
sons were made by using repeated measures variance
analysis (RM-ANOVA). Diet sequence was selected
as a covariate in repeated measures, which may elimi-
nate the cross-over effect. If a main effect is observed,
subgroup analyses were also performed. Overall sig-
nificance level was set as 0.05, and Bonferroni meth-
odology was used for subgroups analyses.
Data are expressed as “mean (standard deviation
[SD]),” minimum-maximum, median (25th-75th per-
centile) and percent (%) where appropriate. P < .05
was considered statistically significant.
RESULTS
Study Population and Basic Demographic and
Clinical Features.—The mean (SD) age of the study
patients (n = 21) was 38.0 (11.2) years, and 85.7%
were females. Most of patients were university gradu-
ates (85.7%), while 4.8% and 9.5% were primary and
secondary school graduates, respectively.
The mean (SD) duration of migraine was 10.8
(9.8) (range 3-36) years and the duration of IBS was
10.8 (11.9) (range 2-53) years. All patients were using
acute attack medication, and for migraine attacks, 4
(19.0%) patients were also identified to be under pre-
ventive medication; but, none of the patients were on
a preventive medication for IBS symptoms. IgG anti-
body tests against 270 food allergens revealed mean
(SD) reaction count (abnormally high titer) to be 23.1
(14.1) (range 6-75). According to food categories
listed in Table 1, from the most to least frequent IgG
positivity, seeds and nuts (86.0), and grain with gluten
(76.0%) were the foods with most frequent IgG
positivity.
The patients were questioned regarding adverse
events. There was no adverse event leading to discon-
tinuation of the given diet.
Headache Parameters With Respect to Phases of
the Study.—Significant reductions were observed
with the elimination diet compared with the run-in
period in attack count, maximum attack duration,
Headache 5

Table 1.—The Food Categories From Most to Least Frequent Additionally, elimination of the cross-over effect
Immunoglobulin G Positivity in Patients (n = 21)
via RM-ANOVA with selection of diet sequence as a
covariate also revealed significant reductions in mean
Food Type n (%) (SD) pain-bloating within the last 10 days by means
of elimination diet when compared with baseline
Seeds and nuts 18 (86.0) (P < .001) and provocation diet (P = .019) (Table 3).
Grain with gluten 16 (76.0) Emotional Well-Being and Belief in IBS Treatment
Spices 15 (71.0)
Fruits 15 (71.0)
in Study Phases.—Elimination diet was associated
Vegetables 15 (71.0) with significantly higher scores for happiness at home
Seafood 12 (57.0) (4.1 [1.1] vs 3.6 [1.1]; P < .01), happiness at work (3.2
Eggs 12 (57.0)
Grain without gluten 10 (48.0) [1.6] vs 2.7 [1.3]; P < .02), and belief in IBS treatment
Milk products 10 (48.0) (4.3 [1.3] vs 3.9 [1.3]; P < .05), while lower scores for
Food additives 9 (43.0)
Leguminous seeds 9 (43.0)
fear of illness (1.3 [1.2] vs 1.8 [1.3]; P < .05) (Table 3).
Coffee infusions 7 (33.0) Headache and IBS Parameters Before and After
Yeast 6 (29.0) the Interchange of Diets.—There was no significant
Meat 5 (24.0)
Sugar products 5 (24.0) difference between patients allocated to provocation
Moss 4 (19.0) or elimination diet in the first diet phase in terms of
Mushrooms 2 (10.0)
Salads 1 (0.1)
headache parameters. Following the second diet
phase, mean (SD) attack count (4.1 [1.9] vs 2.2 [1.9];
P < .05) and number of attacks with acute medication
(3.4 [1.8] vs 1.5 [1.7]; P < .01) were significantly higher
mean attack duration, maximum attack severity, for patients in the “elimination-to-provocation”
number of attack that needed acute medication, and group compared with patients in the “provocation-to-
total medication intake (Table 2). elimination” group (Table 4). With the elimination
Additionally, elimination of the cross-over effect diet, percentage of patients with at least 30% reduc-
via RM-ANOVA with selection of diet sequence as a tion in migraine days was 66.7%, and that with at least
covariate also revealed significant reductions in mean 50% reduction was 47.6%.
(SD) attack count, number of headache days, and Patients allocated to elimination diet in the first
number of attacks with acute medication by means of diet phase were determined to have lower scores for
elimination diet compared with baseline values pain-bloating severity (1.1 [1.1] vs 3.2 [1.1]; P < .01),
(P < .001 for each) and also compared with provoca- pain-bloating for the last 10 days (3.2 [2.0] vs 6.8 [2.8];
tion diet values for attack count (P = .025) and P < .01), and better scores for the QoL (4.2 [0.6] vs 2.5
number of attacks with acute medication (P = .014) [1.1]; P < .001) than patients allocated to provocation
(Table 2). diet in the first diet phase (Table 4).
IBS Symptom Scores With Respect to Phases of Nevertheless, there was no significant difference
the Study.—Significant reductions were observed between patients in the “elimination-to-provocation”
with the elimination diet compared with the run-in vs “provocation-to-elimination” groups in terms of
period in all symptoms except number of defecation median (interquartile range [IQR]) % change in
days per week (Table 3). However, during provoca- headache and IBS parameters obtained from first to
tion diet use, no improvement was seen in pain- second diet phases (Table 4).
bloating severity, number of defecation days per
week, QoL, and pain-bloating (Table 3). Symptom DISCUSSION
scores were significantly lower with the elimination Although the mechanisms of IgG-mediated food
diet compared with provocation for pain-bloating allergy have not been fully elucidated, increase in the
severity, diarrhea-constipation severity, QoL, and production of IgG antibodies and cytokines18 via
pain-bloating (Table 3). food allergy antigens has been proposed to result in
 6

Table 2.—Headache Parameters with Respect to Phases of the Study (n = 21)

RM-ANOVAa Post hoc – Bonferroni

Baseline Provocation Diet Elimination Diet P Values

Attack count Mean (SD) 4.8 (2.1) 4.1 (1.6) 2.7 (2.0)***++ .001 Baseline vs Pro: .483
% change; median (min-max) -20.0 (-66.7; 300.0) -40.0 (-100.0; 100.0)++ Baseline vs Elim: <.001
Pro vs Elim: .017
# of headache days Mean (SD) 12.7 (8.3) 8.6 (5.8)** 7.0 (6.7)*** .020 Baseline vs Pro: .025
% change; median (min-max) -33.3 (-80.0; 87.5) -42.9 (-100.0; 13.3) Baseline vs Elim: <.001
Pro vs Elim: .393
Maximum attack duration (days) Mean (SD) 2.6 (0.6) 2.2 (0.7) 1.4 (1.1)***+ .255 —
% change; median (min-max) 0.0 (-66.7; 200.0) -66.7 (-100.0; 50.0)+
Mean attack duration (days) Mean (SD) 1.8 (0.5) 1.5 (0.4) 1.1 (0.8)** .908 —
% change; median (min-max) 0.0 (-63.6; 120.0) -37.5 (-100.0; 66.7)+
Maximum attack severity (0-10) Mean (SD) 8.5 (1.4) 8.1 (1.6) 6.6 (3.3)***+ .167 —
% change; median (min-max) -10.0 (-40.0; 42.9) -14.3 (-100.0; 12.5)+
# of attacks with acute medication Mean (SD) 4.0 (1.5) 3.4 (1.5) 1.9 (1.8)***++ .009 Baseline vs Pro: .472
% change; median (min-max) -25.0 (-60.0; 100.0) -66.6 (-100.0; 25.0)++ Baseline vs Elim: <.001
Pro vs Elim: .014
Total medication intake (tablets) Mean (SD) 11.5 (7.6) 8.7 (6.7)* 6.7 (10.1)** .354 —
% change; median (min-max) -33.3 (-83.3; 340.0) -53.3 (-100.0; 100.0)+

Univariate analysis:
*P < .05, **P < .01, and ***P < .001 compared with baseline (Wilcoxon test).
+
P < .05, ++P < .01 compared with provocation diet (Wilcoxon test).
a
RM-ANOVA = repeated measures analysis of variance with diet sequence as a covariate; SD = standard deviation; — = not available.
Headache 7

Table 3.—IBS Symptom Scores, Emotional Well-Being and Belief in IBS Treatment with Respect to Phases of the Study
(n = 21)

Baseline Phase Provocation Diet Elimination Diet

IBS Symptom Scores Mean (SD) Mean (SD) Mean (SD) P Valuesa

Pain-bloating severity 3.5 (0.8) 3.2 (1.2) 1.8 (1.3)***+ .361

# of defecation days (per week) 5.7 (5.4) 6.4 (4.1) 6.0 (2.7) .299
Diarrhea-constipation severity 3.5 (1.1) 2.8 (1.4)* 1.9 (1.5)***+ .347
Quality of life 2.9 (0.8) 2.9 (1.0) 3.6 (1.4)*+ .525
Pain-bloating (within the last 10 days) 6.5 (2.7) 5.5 (3.1) 3.2 (2.8)***+ .019
Baseline vs Pro: .211
Baseline vs Elim: <.001
Pro vs Elim: .019
Urgency 1.2 (1.6) 1.0 (1.7)* 0.7 (1.3)** .206
Straining 2.4 (1.8) 1.7 (1.8)* 0.9 (1.1)*** .292
Inability to fully empty the bowel 3.5 (1.2) 2.7 (1.7)* 1.8 (1.5)** .241
Emotional well-being
Happiness at home 3.6 (1.1) 3.8 (1.1) 4.1 (1.1)** .456
Happiness at work 2.7 (1.3) 3.0 (1.5) 3.2 (1.6)** .178
Fear of illness 1.8 (1.3) 1.4 (1.5) 1.3 (1.2)* .361
Belief in IBS treatment 3.9 (1.3) 4.1 (1.3) 4.3 (1.3)* .098

Univariate analysis:
*P < .05, **P < .01, and ***P < .001 compared with baseline phase (Wilcoxon test).
+
P < .05 compared with provocation diet (Wilcoxon test).
a
RM-ANOVA = repeated measures analysis of variance with diet sequence as a covariate.
IBS = irritable bowel syndrome; SD = standard deviation.

an inflammation response that seems to play an
important role in the pathophysiology of migraine
attacks.19
Likewise, offering a potential explanation for
how dietary antigens could trigger migraine attacks,
IBS patients were shown to have a greater area of
intestinal mucosa occupied by mast cells than do
healthy control individuals.20 Additionally, the gut
permeability defect identified in IBS patient was
shown to lead to increased intake of dietary antigens
to lamina propria that may ultimately result in raised
IgG antibody production.21
Hence, tailored food elimination diet was
reported to decrease lymphocyte proliferation
responses, improve clinical outcomes, and decrease
release of inflammatory mediators.10
According to our findings, IgG-based elimination
diet per se was associated with significant improve-
ment both in migraine (attack count, mean and
maximum attack duration, maximum attack severity,
and number of attacks with acute medication) and
IBS parameters (frequency and severity of pain-
bloating and QoL) when compared with baseline as
well as provocation diet phases.
Indeed, concomitant reduction evident in both
migraine and IBS symptoms via IgG-based elimina-
tion diet in our study population is compatible with
the statement that many of the clinical characteristics
of IBS are conceptually similar to those of migraine.
Besides, subjects with IBS have been shown to be at
higher risk than controls to suffer from migraine.12
Similarly, improvement in both migraine and colitis
after a month of specific diet was reported in the
literature for 6 patients with both diseases.1 Further-
more, the improvement in celiac sufferers’ migraine
after changing to a gluten-free diet was also
reported.3
Besides, efficacy of IgG-based elimination diet in
migraine was also reported based on significant
reduction obtained both in the number of headache
days and in the number of migraine attacks in the
elimination diet period compared with baseline.22
 8

Table 4.—Headache and IBS Parameters Among Patients Before and After the Interchange of Diets

Provocation-to-Elimination Group (n = 10) Elimination-to-Provocation Group (n = 11)

Provocation Elimination Elimination Provocation

Following First Following Second Following First Following Second

Diet Phase† Diet Phase‡ % Change§ Diet Phase† Diet Phase‡ % Change§

Headache Parameters Mean (SD) Mean (SD) Median (IQR) Mean (SD) Mean (SD) Median (IQR)

Attack count 4.2 (2.1) 2.2 (1.9) -55.0 (66.0) 3.1 (2.1) 4.1 (1.9)* -33.3 (91.7)
# of headache days 8.3 (5.7) 6.0 (8.1) -63.3 (118.6) 7.9 (5.4) 8.9 (6.1) -5.6 (70.8)
Maximum attack duration (days) 2.3 (0.8) 1.4 (1.2) -66.7 (87.5) 1.5 (1.1) 2.1 (0.7) -50.0 (66.7)
Mean attack duration (days) 1.6 (0.5) 1.2 (1.0) -36.5 (82.5) 1.1 (0.8) 1.5 (0.5) -16.7 (50.0)
Maximum attack severity (0-10) 7.8 (1.5) 6.2 (3.8) 0.0 (87.5) 6.9 (2.8) 8.5 (1.6) -11.1 (37.5)
# of attacks with acute medication 3.5 (1.1) 1.5 (1.7) -63.3 (31.3) 2.3 (1.9) 3.4 (1.8)** -33.3 (125.0)
Total medication intake (tablets) 8.8 (5.7) 6.7 (11.9) -13.8 (128.5) 6.7 (8.7) 8.5 (7.9) -30.0 (66.7)

IBS Parameters Mean (SD) Mean (SD) Median (IQR) Mean (SD) Mean (SD) Median (IQR)

Pain-bloating severity 3.2 (1.1) 1.9 (1.6) -33.3 (95.8) 1.7 (1.1)** 3.1 (1.3) -50.0 (66.7)
# of defecation days (per week) 5.5 (3.8) 5.3 (3.0) 0.0 (91.5) 6.5 (2.5) 7.2 (4.5) 0.0 (106.7)
Diarrhea-constipation severity 3.0 (1.8) 2.1 (1.7) -26.7 (81.3) 1.7 (1.3) 2.6 (0.9) -50.0 (66.7)
Quality of life 2.5 (1.1) 3.0 (1.8) 0.0 (87.5) 4.2 (0.6)*** 3.3 (0.9) 25.0 (100.0)
Pain-bloating (within the last 10 days) 6.8 (2.8) 3.2 (3.6) -43.8 (100.0) 3.2 (2.0)** 4.3 (2.9) 0.0 (95.0)
Urgency 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)
Straining 1.9 (1.9) 1.0 (1.2) 0.0 (43.8) 0.8 (1.0) 1.5 (1.8) 0.0 (66.7)
Inability to fully empty the bowel 3.3 (1.6) 2.0 (1.8) -46.7 (85.0) 1.6 (1.2)* 2.1 (1.6) 0.0 (50.0)

*P < .05, **P < .01, and ***P < .001 compared with values of patients in “provocation-to-elimination” group.
†Following the first study diet application.
‡Following the second study diet application preceded by a washout period.
§From first to second diet phase (P > .05 for “provocation-to-elimination” vs “elimination-to-provocation” diets).
IBS = irritable bowel syndrome; IQR = interquartile range; SD = standard deviation.
Headache
Additionally, in a past Mexican study, IgG-based
elimination diet was reported to be effective in symp-
tomatic improvement leading to complete remission
of migraine in 43 out of 65 patients without the need
of medication.1 Likewise, in 1 large-scale, double-
blind trial of an elimination diet involving 88 patients
treated with an oligoantigenic diet, 93% of patients
with severe frequent migraine were reported to
respond to the diet that eliminates all but a few sen-
sitizing food antigens and documented to be free of
headaches.23
As the cause of IBS remains unknown, there is no
curative therapy directed to a specific target. As a
result, the management of IBS has tended to focus on
the amelioration of symptoms rather than disease
prevention, modification, or cure.24 In correlation to
significant reduction in IBS symptoms obtained via
IgG-based elimination diet in our study population, a
clinically significant improvement in IBS symptoma-
tology was observed in patients eliminating foods to
which they were found to exhibit sensitivity, as iden-
tified by an ELISA test for the presence of IgG anti-
bodies to these foods.8 Accordingly, food-specific
IgG4 antibodies have been demonstrated to be asso-
ciated with IBS, while the 12-week exclusion diet
based on IgG4 titers was reported to improve symp-
toms.25 Besides, identifying and appropriately
addressing food hypersensitivity and abnormal bowel
microenvironment in IBS patients not previously
responding to standard therapy were reported to be
associated with significant clinical response. This
response included objective improvement including
reduction in pain and diarrhea, as well as subjective
improvement considering increased QoL as in our
patients.10
Improving QoL for the functional bowel patient
has been indicated as the most important benefit that
encompasses multiple domains or areas of well-being
including, at a minimum, physical, psychological, and
social functioning, as well as symptom improve-
ment.10 In this regard, significant amelioration in QoL
among patients enrolled in this trial via elimination
diet is worth noting.
Notably, albeit to a lesser extent than elimination
diet, beneficial effect of provocation diet on certain
migraine and IBS symptoms may be assumed to be
9
associated with the cross-over design of our study,
enabling consecutive use of both diets for all patients
yielding same individuals to be their own controls
instead of using other healthy volunteers.
In fact, reintroduction of eliminated foods was
reported to result in a greater deterioration in symp-
toms26 and relapse in patients who improved previ-
ously on the elimination diet27 that suggests a real
causal relation between the eliminated foods and the
symptoms.26
Accordingly, comparison of “elimination-to-
provocation” vs “provocation-to-elimination” groups
in terms of headache parameters at the end of the
interchange period revealed worsening in attack
count as well as number of attacks with acute medi-
cation in our study population. Lack of similar trig-
gering effect of provocation diet in case of IBS
symptoms following the interchange of diets may be
explained by the pronounced superiority of elimina-
tion diet to the provocation diet in the improvement
of IBS symptoms of our patients at the end of the first
diet phase.
Besides, elimination of the cross-over effect via
RM-ANOVA, with selection of diet sequence as a
covariate, also revealed significant reductions in mean
(SD) attack count, number of headache days, number
of attacks with acute medication, and pain-bloating
within the last 10 days by means of elimination diet
compared with baseline values as well as provocation
diet.
Albeit no significant difference was
evident between “elimination-to-provocation” vs
“provocation-to-elimination” groups in terms of
median (IQR) % change in headache and IBS param-
eters obtained from the first to the second diet phases,
the overall symptom reduction in both headache and
IBS parameters was the final outcome provided that
the provocation diet was followed by the elimination
diet. This also emphasizes the triggering role of
provocation diet in induction of symptom relapse in
migraine patients with IBS.
Food elimination diets and food challenges are
considered to be extremely time-consuming for the
patient and practitioner, and to require a high degree
of patient motivation and compliance.10 Accordingly,
identification of higher percentage of patients believ-
10
ing in IBS treatment during elimination diet is worth
noting considering the fact that the diet is an “active
treatment” that if not adhered to does not seem to
have an effect.
Use of exclusion diets tailored to the individual
patient based on the serum IgG antibody titers was
reported to have many advantages, including objec-
tivity to the process, higher patient compliance and
physician confidence, and individualization of the diet
to a given patient, thereby obviating the need for
excluding a large number of foods from the diet.9
Hence, the use of a specific diet rather than a
universal migraine diet with simultaneous elimination
of all known dietary triggers seems to offer a well-
balanced diet in terms of safety and nutritional
reasons.27
CONCLUSIONS
In conclusion, our findings indicate that food
elimination based on IgG antibodies in migraine
patients who suffer from concomitant IBS may effec-
tively reduce symptoms from both disorders with
potential savings to the health care system. In this
regard, albeit small sample size of the present study
necessitating caution on translation of our result to
daily clinical practice, assay of IgG antibodies to food
seems to have a role in helping patients with concomi-
tant presence of migraine and IBS to identify candi-
date foods for elimination.
Ideally, it would have been possible to check
whether the IgG response correlated with diet by a
second IgG measurement after elimination or provo-
cation. Furthermore, it would be of interest to evalu-
ate patients presenting with IBS who happen to get
migraine because patients who receive treatment for
migraine can improve for many reasons besides pre-
ventive medication – patient education (which should
include a focus on regular exercise, healthy meals, and
avoiding fasting), relaxation/biofeedback, elimination
of medication overuse, or just that fact that they tend
to come in at their worst. A similar study specifically
in chronic migraine patients would also be of interest.
Therefore, further studies are warranted in this area.
Acknowledgment: The study is supported by
Immuno Diagnostic Laboratories, Istanbul, Turkey.
Authors would like to thank to Prof. Mustafa Ertas, MD,
for his significant contribution to this paper by providing
consultancy and performing statistical analysis. Authors
would also like to thank to Prof. Şule Oktay, MD, PhD,
and Cagla Isman, MD, from KAPPA Consultancy Train-
ing Research Ltd (Istanbul, Turkey), who provided edito-
rial support.
STATEMENT OF AUTHORSHIP
Category 1
(a) Conception and Design
Elif Ilgaz Aydinlar; Murat Saruc
(b) Acquisition of Data
Elif Ilgaz Aydinlar; Pinar Yalinay Dikmen; Arzu
Tiftikci; Murat Saruc; Muge Aksu
(c) Analysis and Interpretation of Data
Elif Ilgaz Aydinlar
Category 2
(a) Drafting the Article
Elif Ilgaz Aydinlar
(b) Revising It for Intellectual Content
Elif Ilgaz Aydinlar; Nurdan Tozun
Category 3
(a) Final Approval of the Completed Article
Elif Ilgaz Aydinlar; Pinar Yalinay Dikmen; Arzu
Tiftikci; Murat Saruc; Muge Aksu; Nurdan Tozun
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12

APPENDIX A
IBS Scoring Scale

Name:. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Date . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Age, Gender:. . . . . . . . . . . . . . . . . . . . . . . . . Occupation:. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Treatment/Medication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abdominal pain, Bloating Pretreatment 0 1 2 3 4 5
Diet-1 0 1 2 3 4 5
Diet-2 0 1 2 3 4 5
How many days of last 10 days did you experience abdominal pain or bloating?
Pretreatment . . . .days/10 days
Diet-1 . . . .days/10 days
Diet-2 . . . .days/10 days
Diarrhea, Constipation Pretreatment 0 1 2 3 4 5
Diet-1 0 1 2 3 4 5
Diet-2 0 1 2 3 4 5
Quality of life (at work, family, social) (best 5, worst 0)
Pretreatment 0 1 2 3 4 5
Diet-1 0 1 2 3 4 5
Diet-2 0 1 2 3 4 5
No of Defecation in a week: Pretreat:. . . ./a week Diet-1:. . . ./a week Diet-2:. . . ./a week
Please give points between 0 and 5 (0 for no complaint, 5 for worst complaint)
Pretreat Urgency for bowel movement ( ) Fecal incontinence ( ) Straining ( ) Incomplete evacuation ( )
Diet-1 Urgency for bowel movement ( ) Fecal incontinence ( ) Straining ( ) Incomplete evacuation ( )
Diet-2 Urgency for bowel movement ( ) Fecal incontinence ( ) Straining ( ) Incomplete evacuation ( )
Stress at work: Yes No Little
Personality: Meticulous Emotional Stressful Ambitious Relaxed Normal
Happy at work Pretreatment 0 1 2 3 4 5
Diet-1 0 1 2 3 4 5
Diet-2 0 1 2 3 4 5
Absence from work; how many days in last 1 month
Pretreatment (. . .days) Diet-1 (. . .days) Diet-1 (. . .days)
Happy at home Pretreatment 0 1 2 3 4 5
Diet-1 0 1 2 3 4 5
Diet-2 0 1 2 3 4 5
Fear of having a serious illness Pretreatment 0 1 2 3 4 5
Diet-1 0 1 2 3 4 5
Diet-2 0 1 2 3 4 5
Do you believe that your disease can be cured/relieved?
Pretreatment 0 1 2 3 4 5
Diet-1 0 1 2 3 4 5
Diet-2 0 1 2 3 4 5
Are you satisfied with the treatment? Diet-1 0 1 2 3 4 5
Diet-2 0 1 2 3 4 5
Original Research

Treating Irritable Bowel Syndrome with a Food

Elimination Diet Followed by Food Challenge and
Probiotics

Jeanne Drisko, MD, CNS, Bette Bischoff, MD, RD, Matthew Hall, PhD, Richard McCallum, MD
Program in Integrative Medicine (J.D.), School of Medicine (B.B.), Preventive Medicine and Public Health (M.H.),
Gastrointerology and Hepatology (R.M.), University of Kansas Medical Center, Kansas City, KS
Objective: In Irritable Bowel Syndrome, the gut-associated immune system may be up-regulated resulting
in immune complex production, low-grade inflammation, loss of Class I bacteria, and translocation of inflam-
matory mediators and macromolecules outside of the GI lumen. Since food intolerance may be one of the reasons
for this upregulation, our goal was to investigate the role of food intolerance in IBS patients.
Methods: In this open label pilot study, we enrolled 20 patients with IBS by Rome II criteria (15 women,
ages 24 – 81) who had failed standard medical therapies in a tertiary care GI clinic. Baseline serum IgE and IgG
food and mold panels, and comprehensive stool analysis (CSA) were performed. Breath-hydrogen testing and
IBS Quality-of-Life (QOL) questionnaires were obtained. Patients underwent food elimination diets based on the
results of food and mold panels followed by controlled food challenge. Probiotics were also introduced. Repeat
testing was performed at 6-months. We followed up with this cohort at 1 year after trial completion to assess the
reported intervention and for placebo effect.
Results: Baseline abnormalities were identified on serum IgG food and mold panels in 100% of the study
subjects with significant improvement after food elimination and rotation diet (p ⬍ 0.05). Significant improve-
ments were seen in stool frequency (p ⬍ 0.05), pain (p ⬍ 0.05), and IBS-QOL scores (p ⬍ 0.0001). Imbalances
of beneficial flora and dysbiotic flora were identified in 100% of subjects by CSA. There was a trend to
improvement of beneficial flora after treatment but no change in dysbiotic flora. The 1-year follow up
demonstrated significant continued adherence to the food rotation diet (4.00 ⫾ 1.45), minimal symptomatic
problems with IBS (4.00 ⫾ 1.17), and perception of control over IBS (4.15 ⫾ 1.23). The continued use of
probiotics was considered less helpful (3.40 ⫾ 1.60).
Conclusion: These data demonstrate that identifying and appropriately addressing food sensitivity in IBS
patients not previously responding to standard therapy results in a sustained clinical response and impacts on
overall well being and quality of life in this challenging entity.

INTRODUCTION and indirect associated medical costs [6,7]. Successful thera-

Irritable Bowel Syndrome is the most common functional
gastrointestinal disorder with a reported prevalence in the gen-
eral population between 12%–22% [1– 4]. In fact, IBS is the
most common diagnosis made by gastroenterologists in the
United States, accounting for 12% of visits to primary care
providers [2]. IBS is a diagnosis of exclusion developed by
a consensus definition and criteria known as the Rome II
Criteria [5].
IBS is a disorder that is poorly understood with high direct
peutic options have been difficult to develop because of the
lack of pharmacological targets and wide range of symptom-
atology [3,8,9]. As a result, an attempt is made to suppress
symptoms with anti-cholinergic, anti-spasmodic, anti-diarrheal,
and serotonergic agents with variable success as symptoms are
not completely eliminated.
The gut is the largest lymphoid organ in the body [10]. In
IBS, the gut-associated immune system is up regulated as
evidenced by increased inflammatory cytokines such as inter-
leukin 1, 6, and 10 [3,11–13]. The etiology of this altered

Address reprint requests to: Jeanne Drisko, MD, University of Kansas Medical Center, 3901 Rainbow Blvd, Mail Stop, 2028, Kansas City, KS 66160. E-mail:
jdrisko@kumc.edu
Grant support was received from BioCommunications Research Institute, Wichita, Kansas.

Journal of the American College of Nutrition, Vol. 25, No. 6, 514–522 (2006)
Published by the American College of Nutrition

514
 Treating Irritable Bowel Syndrome

immunity is unclear but may be related to food hypersensitivity Table 1. Summary of Demographic Data
and/or altered GI microbial environment combined with altered
Male Female
enteric nervous system sensation. It is known that there is
abnormal fermentation in IBS [14] and this leads to immune Number Enrolled 5 15
Age range (mean) 43 yrs–77 yrs (57) 24 yrs–80 yrs (49)
up-regulation [15]. It is also known that there is change in
Duration of IBS Symptoms 3 yrs–50 yrs (22) 3 yrs–60 yrs (23)
symbiotic, commensal, and dysbiotic microbial gut colonies in Results of Breath Hydrogen
IBS [16 –20]. It has been reported that types of microflora at Baseline 2 positive 2 positive
colonizing the gut play a role in regulating immunity [21]. In
addition, upregulated GI associated immune tissue is known to
definable organic pathology in the gastrointestinal tract (por-
stimulate discharge of enterochromaffin cells and other cells,
phyria), and 1 did not fulfill the strict Rome II criteria. One of
which release serotonin and/or histamine resulting in GI symp-
the study subjects withdrew from the study after 2 months
toms [22–29]. Inflammation can result in opening of tight
stating refusal to adhere to dietary requirements. Data was
junctions between enterocytes with translocation of large pro-
analyzed on intent to treat basis.
teins across the GI lumen. These proteins act as antigens
Baseline requirements included a visit with the gastroenter-
systemically and antibody production results [21,22,30].
ologist, comprehensive IBS symptom and quality of life ques-
It is our hypothesis that correcting the luminal micro-envi-
tionnaires (University of North Carolina School of Medicine—
ronment will lead to improvements in IBS symptoms. This may
Chapel Hill GI Psychosocial Research Group), and hydrogen
be accomplished by a two-pronged approach. First, food and
breath testing to assess for small bowel bacterial overgrowth.
mold hypersensitivity [14,18,22,30 –33] contributes to the al-
Subjects had 7 visits, including the baseline visit and 6 monthly
tered inflammatory environment in the GI track and serum IgE
intervention visits; after completion of the study, a follow-up
and IgG food and mold panels can guide a food withdrawal
visit with a gastroenterologist was required. At baseline, the
diet, resulting in improved symptom complex [15,32–36]. If the
serum IgE and IgG food and mold antigen panels (Allos Ref-
results of the IgE and IgG food and mold panels are significant,
erence Laboratory—Hitachi Chemical Diagnostics Inc., Moun-
subsequent systematic food challenges should result in IBS
tain View, CA) and stool collection for comprehensive diges-
symptom recurrence. Secondly, altered microbial environments
tive stool analysis were obtained (Great Plains Laboratory,
that are related in IBS may be corrected by probiotic adminis-
Overland Park, KS).
tration [17,20,37– 43].
Study subjects each received a tailored food withdrawal diet
based on the results of the serum IgE and IgG food and mold
antigen panels. The food and mold withdrawal diet was fol-
METHODS lowed for 21–28 days with subsequent individual food chal-
lenges performed over several months. Food and symptom
Study Design diaries were kept during the challenge phase and reviewed by
the investigators. If a food was tolerated during the challenge
The reported prospective outcome study with multifactorial
phase, the food was reintroduced back into the diet with in-
intervention enrolled a cohort of diarrhea dominant irritable
structions to adhere to a rotation diet. If IBS symptoms returned
bowel syndrome patients from a tertiary care gastroenterology
with food challenge, the food was eliminated from the diet for
clinic. Prerequisite for entering into the study included a diag-
6 months with instructions to rechallenge at a later date. Study
nosis of IBS by Rome II criteria and evaluation by gastroen-
subjects were given probiotics (Vital 10 powder, 1⁄4 teaspoon
terologists at the University of Kansas Medical Center. The
2X/day, Klaire Labs, Solana Beach, CA) beginning at month 2
following laboratory tests were required to be within normal
after the food and mold elimination diet period. The probiotics
range: total blood count, erythrocyte sedimentation rate, bio-
were taken daily from months 2 through 6 followed by a 1
chemistry screen, routine stool evaluation including culture,
month washout period.
examination for occult blood, ova, and parasites, and a recent
At one year after trial completion, a follow up questionnaire
normal sigmoidoscopy or colonoscopy within 2 years of en-
to assess gastrointestinal status was obtained; this was to eval-
rollment. Persons were excluded if organic intestinal disease
uate for the role of placebo effect in this intervention, which is
was present. Subjects were also excluded if there had been
known to be quite high in IBS [29].
recent antibiotic use or recent or concurrent enrollment into an
The protocol was approved by the Investigational Review
IBS study.
Board of the University of Kansas Medical Center. All partic-
Twenty-five subjects were screened between December
ipants provided written informed consent.
2001 and October 2002 and 20 were enrolled; of those enrolled,
there were 5 men and 15 women consistent with national
statistics [2,3]. See Table 1. Three patients declined to partic-
Breath Hydrogen and Methane Testing
ipate (refusal to obtain colonoscopy/sigmoidoscopy, or refusal The importance of breath testing is acknowledged [44]. All
to adhere to dietary requirements); one was excluded based on subjects presented to the GI Motility and Functional Bowel

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 515

Treating Irritable Bowel Syndrome
Center at the University of Kansas Medical Center for the
hydrogen and methane breath test after an 8-hour fast. Subjects
ingested 30 cc of glucose syrup after a sample of their breath
was collected at baseline. Further breath samples were obtained
every 15 minutes for 3 hours. All breath samples were end-
expiratory and analyzed immediately by model DP Microlyzer-
chromatograph (Quintron Instrument Company, Milwaukee,
WI). The concentration of breath hydrogen and methane was
measured in parts per million (ppm). The measurements were
plotted graphically and analyzed. The diagnosis of small intes-
tine bacterial overgrowth was based on the following criteria: A
peak within 90 minutes of hydrogen or methane concentration
of ⬎ 20 ppm, or a baseline level of hydrogen or methane of ⬎
10 ppm, which subsequently continued to increase after glucose
challenge.
Comprehensive Digestive Stool Analysis
Each study subject collected stool samples for a compre-
hensive stool analysis (test kits provided by Great Plains Lab-
oratory, Overland Park, Kansas and conducted by Doctor’s
Data, West Chicago, Illinois) on 2 consecutive days at baseline
and again at the end of the intervention for comparison. Bac-
teriology, aerobic evaluated by BAP, Mac, CNA, and HEK
plates to identify Salmonella, Shigella, yersinia, vibrio, and
Aeromonas plus any other pathogenic bacteria. GN broth is
used to isolate pathogens in smaller quantities and API for an
identification system; results are reported in organism type and
level from 0, 1⫹, 2⫹, 3⫹, 4⫹ with 0 representing no colonies
and 4 the highest count. Bifidobacter is cultured on anaerobic
culture media (modified CNA plates from Oxyrace create an-
aerobic environment); results reported in organism and level 0,
1⫹, 2⫹, 3⫹, 4⫹. Enterohemorrhagic E. Coli, Giardia, E.
Histolytica, and Cryptosporidium are evaluated by EIA kit-
ProSpect from Alexon Trend by Remel with results reported as
positive or negative. Campylobacter is identified by EIA Kit-
ProSpect from Alexon Trend by Remel and cultured with
microphilic environment pouch on campy plates with results
positive or negative. Parasitology identification Trichrome
Stain, and concentrate are evaluated by microscopy and iden-
tified parasites reported. Yeast culture is expressed as definitive
identification. Disc susceptibilities for yeast and bacteria are
done by Kirby-Bauer and reported as sensitive or resistant.
Cholesterol in the stool is identified by colormetric chemistry
analyzer (Olympus AU600 —Kit reagents used are from DCL);
results are given in mg/dL. Chymotrypsin amount in the stool
is determined by colormetric chemistry analyzer (see above);
results are given in IU/g. Fecal Lactoferrin and Lysozyme are
evaluated by Latex Agglutination with results positive or
negative. Fecal secretory IgA is measured by EIA Kit from
ALPCO; results expressed in ng/mL. Meat fibers, red and white
blood cells are counted by direct microscopy and reported as
none, few, moderate, or many. Occult blood is evaluated by
guiac—Hemoccult and results reported as positive or negative.
516
The pH of the stool is measured by a pH meter and reported as
a number from 0 –14. Short Chain Fatty Acids in the stool to
include acetate, propionate, butyrate, and valerate are measured
by Gas Chromatography—Varian GC/MS and Acetate: ex-
pressed as percent of total of the total N-Butyrate expressed as
␮g/g. Steatocrit is measured by capillary microcentrifugation
and reported in ng/mL. Triglycerides in the stool are measured
by colormetric chemistry analyzer and reported in mg/dl.
NEFA (Non-Estrified Fatty Acids) are identified by colormet-
ric chemistry analyzer (off-label use of Wako kit for serum).
Test results were reported within 4 weeks and collected at
baseline and again at completion of the intervention.
Serum IgE and IgG Panels
Venous blood was collected in a 10 mL serum separator
tube. Blood was allowed to stand in the serum separator tube
for 20 minutes. The samples were centrifuged at 3,000. Sam-
ples were immediately placed cold packs and sent to the Allos
Reference Laboratory (Hitachi Chemical Diagnostics, Inc.,
Mountain View, California). The test was performed at base-
line and again at the end of the 6-month intervention, but only
the IgG was repeated at the completion of the trial since it was
assumed that the results of the IgE would not change in the time
period.
For analysis, the serum was drawn into a sealed test cham-
ber containing 36 threads coated with antigens specific for food
and mold. (The test chamber contains a negative control and a
positive control with IgG, for the IgG system, or IgE, for the
IgE system, covalently bound). The test chamber with the
serum was incubated at room temperature for 18 –24 hours; the
serum was drained from the holding chamber.
The test chamber was washed with wash buffer, drained,
and filled with antibody reagent, and incubated at room tem-
perature for 4 hours. After draining and washing, the photore-
agent mix was drawn into the test chamber and incubated for 10
minutes. After 10-minute incubation, photoluminescence was
measured and reported in luminescence units. Results are re-
ported using a classification system based on relative light unit
system. The luminescence units were reported as class values
and assigned a numerical rating from 0 – 4 based on the amount
of light admitted by the individual threads in the test chamber.
Class values of one or greater represents progressively increas-
ing concentrations of allergen specific antibodies. Class zero
represents an absence or nondetectable levels of allergen spe-
cific antibodies.
The sensitivity detection limit of the assay is ten lumines-
cence units. There is less than 1% cross reactivity with human
serum immunoglobulins IgA, IgM, IgG, or IgD at normal
physiologic levels. On average, concordance (calculated as
efficiency) between CLA allergen and alternative in-vitro assay
is approximately 95%; the range of concordance is 86%–100%.
There are no standardized reference allergens available for
VOL. 25, NO. 6

comparisons between methods, or for the great majority of
clinical relevant allergens.
Probiotic Supplement
Replacement of beneficial microflora was by probiotic sup-
plementation (Vital-10, Klaire Labs, Solana Beach, CA 92075).
The product contained Lactobacillus acidophilus, Bifidobacte-
rium baifidum, L. rhamnosus, L plantarum, B. infantis, L.
salivarius, L bulgaricus, L casei, L brevis, and Streptococcus
thermophilus. Each dose gave a total of 10⫹ billion colony
forming units and was taken twice each day with meals to assist
in adherence to the gut wall.
Outcome Variable: Irritable Bowel Quality of Life
Outcome Instrument
Permission was obtained to use the IBS specific symptom
diary and QOL instrument (University of North Carolina
School of Medicine—Chapel Hill GI Psychosocial Research
Group). IBS-QOL is a validated survey instrument [45,46]. The
original version of the IBS-QOL contains 34 question items
that ask about the patient’s feelings and response is measured
on a 5-point Likert-scale where 1 ⫽ not at all, 2 ⫽ slightly, 3 ⫽
moderately, 4 ⫽ quite a lot, 5 ⫽ a great deal/extremely. All
items are summed-scored to calculate total scores (overall
score). Subscales are collected for dysphoria, interference with
activity, body image, health worry, food avoidance, social
reaction, sexual, and relationship. The IBS-QOL was obtained
in all study subjects as a baseline survey and at completion of
this study.
Predictor Variables
A record of objective clinical findings of change in stool
frequency and pain and subjective quality of life were obtained
at baseline and at completion of the study. In addition, the
changes in IgG food and mold scores from baseline to com-
pletion were measured after the treatment intervention. Finally,
stool microflora counts were assessed at baseline and after
probiotic use by the comprehensive stool analysis.
1-Year Follow Up Questionnaire
To evaluate for the role of the placebo effect in this inter-
vention, a follow-up questionnaire was administered at 1-year
post intervention. There had been no significant contact with
the study subjects by the study team in the interval. All twenty
patients were contacted and completed the questions. Four
questions were asked to evaluate current IBS symptoms, ad-
herence to a rotation diet, use of probiotics, and attitude about
control over IBS symptoms. The questions were based on a
5-point Likert-scale ranging from 1 ⫽ strongly disagree and
5 ⫽ strongly agree.
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
Treating Irritable Bowel Syndrome
Statistical Analysis
The goal of this study was to examine the contribution of
serum IgG food and mold antigen levels for tailoring food and
mold withdrawal/rotation diets and its impact on IBS symp-
toms and QOL. In addition, it was necessary to assess the
importance of stool microflora colonies and the impact of
probiotic products on IBS symptoms and QOL scores. We first
summarized all measurements with their means and standard
deviations. Wilcoxon’s Signed Rank Test was applied to de-
termine if there is a significant change in each measure from
baseline to completion.
RESULTS
Patient Characteristics
Of the 20 patients enrolled in the study, 5 were male and 15
were female. The age range for the men was 43–77 (57) years
and for the women 24 – 80 (49) years. The duration of IBS
symptoms for the men was 3–50 (22) years and for the women
was 3– 60 (23) years. There were 2 positive breath hydrogen
tests at baseline in each group of men and women, which
correlated with abnormalities on comprehensive digestive stool
analysis. See Table 1.
Pain and Stool Frequency
In this prospective study in a cohort of diarrhea dominant
IBS subjects, systematic food withdrawal guided by the results
of the IgG and IgE food and mold panels resulted in significant
improvement in symptoms including stool frequency and se-
verity of pain. At baseline reported stool frequency was 4.29
(2.49) stools per day and at completion were 3.43 (⫾ 1.22)
stools per day (P ⬍ .05). Pain diary scores based on a pain scale
from 1 (none) to 5 (most severe) resulted in a significant
improvement from baseline of 3.65 (⫾ 1.12) to completion of
2.71 (⫾ 1.38) (P ⬍ .05). See Table 2.
IBS QOL
Response was measured on a 5-point Likert-scale where
1 ⫽ not at all to 5 ⫽ a great deal/extremely with all items
summed-scored to calculate total overall score from baseline to
completion. In final data analysis, the five responses are trans-
formed in order to obtain a 100-point overall QOL score and
eight 100-point subscales. After conversion, higher scores de-
note a higher quality of life and lesser degree of IBS symptoms.
This cohort demonstrated a significant improvement in overall
QOL scores [46.51 (⫾ 21.08) to 67.22 (⫾ 20.92); P ⬍ .001].
Subscales collected at baseline and completion for dysphoria
[37.66 (⫾ 23.64) to 66.28 (⫾ 24.58); P ⬍ .001], interference with
activity [40.54 (⫾ 21.81) to 65.23 (⫾ 24.60); P ⬍ .001], body
image [59.69 (⫾ 23.52) to 76.32 (⫾ 18.47); P ⬍ .001], health
worry [58.33 (⫾ 24.63) to 77.63 (⫾ 20.42); P ⫽ .002], food
517
Treating Irritable Bowel Syndrome

Table 2. Results

Baseline Mean (Std) Completion Mean (Std) Signed Rank P-Value

# of Stools/day 4.29 (2.49) 3.43 (1.22) ⬍0.05
Pain Scale 1 (none)–5 (most severe) 3.65 (1.12) 2.71 (1.38) ⬍0.05
IgG Food Positive # 10.05 (10.08) 6.47 (8.85) ⬍0.01
IgG Food High Positive # 0.10 (0.31) 0.71 (2.26) 0.500
IgG Mold Positive # 3.30 (1.26) 2.63 (1.42) ⬍0.05
IgG Mold High Positive # 1.35 (1.69) 1.79 (1.87) 0.069
IBS-QOL
Dysphoria 37.66 (23.64) 66.28 (24.58) ⬍0.001*
Interference w/Activity 40.54 (21.81) 65.23 (24.60) ⬍0.001*
Body Image 59.69 (23.52) 76.32 (18.47) ⬍0.001*
Health Worry 58.33 (24.63) 77.63 (20.42) 0.002*
Food Avoidance 30.42 (26.80) 38.16 (25.36) 0.362
Social Reaction 48.13 (32.64) 69.08 (24.07) 0.002*
Sexual 73.13 (27.89) 79.61 (29.82) 0.100
Relationships 55.00 (32.83) 70.18 (25.36) ⬍0.001*
Overall 46.51 (21.08) 67.22 (20.92) ⬍0.001*
Beneficial Bacteria (Class I) 2.07 (1.54) 2.67 (1.30) 0.250
Dysbiotosis/Parasitology 1.58 (0.84) 1.47 (0.91) 0.620
* Significant at the 0.0025 level.

avoidance [30.42 (⫾ 26.80) to 38.16 (⫾ 25.36); P ⫽ .36], social Microflora Colony Counts
reaction [48.13 (⫾ 32.64) to 69.08 (⫾ 24.07); P ⬍ .002], sexual
In the comprehensive digestive stool analysis, colony counts
[73.13 (⫾ 27.89) to 79.61 (⫾ 29.82); P ⫽ .10], and relationships
of microflora are expressed as a range of 0 to 4⫹ colony counts
[55.00 (⫾ 32.83) to 70.18 (⫾ 25.36); P ⬍ .001]. See Table 2.
with 0 being no colonies identified and 4⫹ as the maximal
colony count. At baseline prior to probiotic intervention, the
Serum IgG Food and Mold Antigens study subjects were found to have a trend to improvement in
Class 1 beneficial microflora at 2.07 (⫾ 1.54) colony counts
Baseline serum IgG food reactions were measured in lumi- and after probiotic supplementation, beneficial colony counts
nescence units (LU) with the range from 0 –11 ⫽ negative, rose to 2.67 (⫾ 1.30). Counts in dysbiotic microflora at base-
12–26 ⫽ equivocal, 27– 65 ⫽ low positive, 66 –142 ⫽ positive, line were found to be (1.58 (⫾ 0.84) and found not to clear with
and 143 ⱖ 242 ⫽ high positive. For the purpose of the food and probiotic replacement from 1.58 (⫾ 0.84) to 1.47 (⫾ 0.91).
mold elimination diet, only reactions that were positive to high
positive were considered. At baseline, there were 10.05 (⫾ 1-Year Follow-up Questionnaire after Trial Completion
10.08) positive IgG food reactions identified per patient and at
completion after food elimination, 6.47 (⫾ 8.85) P ⬍ 0.01. At One-year follow-up questionnaire had 4 questions with re-
baseline, there were 0.10 (⫾ 0.31) high positive IgG food sponses based on a 5-point Likert-scale ranging from 1 ⫽
reactions and at completion 0.71 (⫾ 2.26), which did not show
a significant change. At baseline, there were 3.30 (⫾ 1.26) Table 3. Most Frequent Positive Serologic IgG Antigen-
positive IgG mold reactions identified and after completion a Antibody Food and Mold Tests
reduction to 2.63 (⫾ 1.42) P ⬍ 0.05. At baseline there were 4 or more molds 14 70%
1.35 (⫾ 1.69) high positive IgG mold reactions and at comple- Baker’s yeast 17 85%
tion 1.79, which was not significant. Onion mix 13 65%
The most frequent positive serologic IgG antigen-antibody Pork 12 60%
Peanut 12 60%
complexes found on the food and mold tests were: 4 or more
Corn 11 55%
molds, 14 out of 20 patients (70%); baker’s yeast, 17 out of 20 Wheat 10 50%
(85%); onion mix, 13 out of 20 (65%); pork, 12 out of 20 Soybean 10 50%
(60%); peanut 12 out of 20 (60%); corn, 11 out of 20 (55%); Carrot 9 45%
wheat, 10 out of 20 (50%); soybean, 10 (50%); carrot, 9 out of Cheddar Cheese 8 40%
Egg White 8 40%
20 (45%); cheddar cheese, 8 out of 20 (40%); egg white, 8 out
Milk (dairy)* 5* 25%*
of 20 (40%). See Table 3. Only 5 out of 20 reacted by IgG
* Dairy is often considered to be one of the foods that should be eliminated.
antibody production to dairy; however the majority of patients However, this cohort did not show a high prevalence of IgG Ag-Ab complexes.
reported eliminating dairy prior to trial enrollment presumably It may be that many of these subjects had already eliminated dairy and cleared
clearing antigen-antibody complexes prior to testing. dairy specific IgG Ag-Ab complexes.

518 VOL. 25, NO. 6


strongly disagree and 5 ⫽ strongly agree. All 20 study subjects
responded. The questions included adherence to the food rota-
tion diet (4.00 ⫾ 1.45), minimal symptomatic problems with
IBS (4.00 ⫾ 1.17), and perception of control over IBS (4.15 ⫾
1.23). The ongoing use of probiotics at 1-year was found to be
less helpful (3.40 ⫾ 1.60).
DISCUSSION
The reported multifactorial intervention resulted in signifi-
cant improvements in irritable bowel symptom complex and
QOL in this cohort of IBS diarrhea dominant patients. The
patients enrolled in this study were on whole a difficult group
of patients to manage having found their way to the tertiary
care gastroenterology clinic. They were not felt to be ade-
quately improved by extensive use of standard medical therapy
and care given by a single gastroenterologist with expertise in
this area (RM). Identifying and appropriately addressing food
hypersensitivity and abnormal bowel microenvironment in IBS
patients not previously responding to standard therapy resulted
in a significant clinical response. This improvement was found
to be sustained at 1 year post-intervention during which time
there was no significant contact with the investigators; the
1-year follow-up was done to evaluate the role of the placebo
effect as a major factor in the improvement. The improvement
was found to be both objective with reduction in pain and
diarrhea as well as subjective with increased quality of life. The
majority of study subjects continued to adhere to the rotation
food diet at 1 year post intervention and felt they had reduced
symptoms and increased control over their IBS.
The gastrointestinal tract is the largest immune organ and
responsible for vigilance and surveillance of ingested materials.
Up-regulation of gut immunity, resulting in increases in inflam-
matory cytokines and other inflammatory mediators, is associ-
ated with IBS [3,10 –13]. To date, it is unclear specifically what
causes the immune stimulation in IBS and since IBS is a
complex chronic disorder, there may be several contributing
factors that lead to the change in immunity. Abnormalities in
microbial biomass with decrease in Class 1 symbiotic micro-
flora and increase in dysbiotic flora will cause changes in the
patterns of immunity as do food and mold related hypersensi-
tivity that results in increased immune complex formation.
Immunoglobulin G (IgG) may be a helpful marker of im-
mune response for food hypersensitivity and delayed food
reactions [33]. In a blinded trial, Atkinson and colleagues
reported a benefit in IBS symptoms when evaluating IgG food
withdrawal diets when compared to sham diets. The circulating
elevated IgG may or may not be the cause of the symptoms but
its ability to form immune complexes with antigens and to
activate complement certainly fulfills the condition for immu-
nopathologically-mediated inflammation [47]. Activation of
immune reactions by non-IgE immune complexes that result
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
Treating Irritable Bowel Syndrome
from delayed hypersensitivity may explain many of the ob-
served reactions to food such as asthma, migraines, headaches,
arthritis, gastrointestinal dysfunction, etc [48]. By focusing
solely on IgE mediated reactions and excluding other elements
of the immune response, important etiologies of patients’
symptoms are overlooked.
The parallel assays of specific IgG and IgE antibodies to
food and mold provide an approach to determining offending
foods in the clinical situation. After antibodies to specific foods
are detected, the patient is placed on a food elimination diet for
two to four weeks, after which foods that do not mediate IgE
reactions are systematically returned to the diet one at a time
[33,48 –50]. Patients with true food hypersensitivities should
have clear reactions with food challenges, but these reactions
may not occur until hours or days post ingestion. Detailed food
diaries are necessary during the challenge phase to assess for
delayed hypersensitivity reactions. Open food challenges are
usually accurate and sensitive for testing non-IgE mediated
food reactions in clinical practice and placebo effect is gener-
ally not a problem [48].
It should be noted that IgG food testing has not been
considered a particularly useful test by the general medical
community [47,53–55]. It is believed that IgG is formed uni-
versally after the ingestion of food; IgG is generally considered
to be a protective antigen and as a result the test is thought to
be non-specific. This conclusion is now being challenged and
re-evaluated [47]. IgG by itself may or may not be the mediator
of the symptoms, but it’s presence in measurable quantities may
serve as an indicator that a protective antibody is necessary.
The rationale for adding IgG testing is based on the findings
that certain subclasses of IgG or non-IgE associated reactions
have been associated with in vitro degranulation of basophils
and mast cells, the activation of complement cascade, and the
observation that high circulating serum concentrations of some
IgG have been measured in certain atopic individuals
[47,48,56]. Based on the results of the IgE and IgG mold
panels, an appropriate food elimination diet may be imple-
mented. It has been shown that decreased lymphocyte prolif-
eration responses, improved clinical outcomes, and decreased
release of inflammatory mediators followed the tailored food
elimination diet [36,48,49].
Food elimination diets and food challenges are extremely
time consuming for the patient and practitioner and the elimi-
nation/challenge diet requires a high degree of patient motiva-
tion and compliance [33,35,48]. Although the serum IgE and
IgG testing may help guide the food elimination diet initially,
the oral food challenge remains the only modality to identify a
true clinical reaction [48,51,52]. After the food challenge phase
is complete and the offending foods are identified, these foods
may be added back into the diet on a rotation basis of not more
than 3 to 4 days between ingestion. That is, no food may be
eaten repeatedly on successive days because antigen—antibody
complexes may again accumulate, which result in recurrent
symptoms of IBS or food intolerance. In a rotation diet for
519
Treating Irritable Bowel Syndrome
example, wheat (gliadin or gluten antigen) sensitive patient
may eat wheat containing foods once every 3 to 4 days with no
wheat products consumed in between those days. Some patients
find that if their initial reaction after challenge is severe, certain
food groups may never be tolerated again in the diet without
provoking symptoms. In any event, teaching patients to eval-
uate symptoms, correlate symptoms with food diaries, and manage
specific food withdrawal and rotation diets gives them some
measure of control over their functional bowel complaints.
Caveats regarding IgG food testing include a lack of intra-
laboratory reproducibility, skepticism concerning the role of
IgG food related antibodies in the pathophysiology of adverse
reactions to food, the possibility that many adverse reactions to
foods are pharmacologically or contaminant mediated and not
detectable through immunological assays, and the possibility
that digestion alters the protein make up and therefore its
allergenicity. Another valid concern is it is not known what
percentage of the population free of bowel symptoms has
elevated IgG food-related antibodies.
Recently, O’Mahony and colleagues (2005) demonstrated
improvements in IBS symptoms in a blinded trial with the
addition of Bifidobacterium infantis 35624 in the diet with
normalization of the ratio of anti-inflammatory to proinflam-
matory cytokines. These investigators did not find a similar
effect when Lactobacillus salivarius UCC4331 was added. In
other clinical trials, Lactobacillus plantarum 299v and DSM
9843 strains were shown to reduce abdominal pain, bloating,
flatulence, and constipation [17,57]. It was also observed that
Saccharomyces boulardii decreased only functional diarrhea in
irritable bowel syndrome but was not effective in alleviating
other symptoms of the syndrome [58]. In this trial, the reported
improvements by probiotics on symptoms may be related to
specific physiological effects of altered cytokine production,
microflora cross-talk, or other direct effects and should be
considered in an expanded evaluation. It was beyond the scope
of this study to test for the direct effects of probiotics on the
gastrointestinal tract.
At baseline, the cohort of patients in this study demonstrated
decreased Class 1 beneficial microflora with decreased colo-
nies of Lactobacillus sp., bifidobacteria sp., and beneficial
E-Coli. In addition, there were increased dysbiotic microflora
colonies and fungal species in a subset of patients with positive
breath testing. Of note, there was a trend to improvement in the
Class 1 microflora with probiotic supplementation over the
course of the trial but this was not significant. This may be related
to underestimating the amount of probiotic supplement necessary,
type of flora necessary, or the duration of time needed to effect
such a change [20,37,40]. It would be useful in the future to
evaluate the dose response of various preparations with various
colony counts and correlate this with the changes in colony counts
on follow up stool testing and changes in gut immunity.
Although there was not a significant improvement in ben-
eficial colony counts in this study, patients reported symptom
relief when using probiotics. However at the 1-year follow up,
520
the study subjects were only sporadically continuing to use
probiotic supplements. As stated, more aggressive replacement
may be warranted or investigation into the direct effects of
probiotics on immunity would be helpful.
Interestingly, probiotics alone were not sufficient to eradi-
cate dysbiotic flora or produce normalization in follow-up
breath-hydrogen testing in the patients that had positive breath-
hydrogen tests at baseline. After the trial was complete and not
at baseline, antibiotics, with documented sensitivities to the
abnormal flora, were given to eradicate the dysbiosis in this
group of patients. It should be noted in this subset of patients,
the majority of IBS symptoms improved prior to dysbiotic flora
eradication. Since the sample size was small, this cannot be
commented on further but should guide further investigation
when enrolling subjects who have positive breath-hydrogen
tests and dysbiotic flora found on stool testing.
The patients enrolled in this trial demonstrated significant
improvements in quality of life (QOL) assessment. Ultimately,
improving QOL for the functional bowel patient is the most
important benefit. QOL is a term that has been used to denote
outcomes as experienced by the patient and there has been
growing interest in the use of health-related quality of life
measures in gastrointestinal disorders. QOL measures in clin-
ically ill individuals encompasses multiple domains or areas of
well-being (including, at a minimum, physical, psychological
and social functioning, as well as symptoms) and the perspec-
tive of the patient is critical in any measurement of QOL. Func-
tional bowel disorders have been studied pre- and post-treatment
with health status outcome measures [45,46,59 – 61]. The validity
of using the outcome instruments has been documented and has
been a useful tool for following the therapeutic benefits of treat-
ment in clinical practice and in controlled trials. While patient
perspectives are important in any health condition, they become
particularly so in diseases that are chronic such as IBS.
The reported trial is small and the food challenges were
open and not blinded, although some believe that reactions
related to IgG delayed hypersensitivity and tracked over 72
hours after the food challenge can safely be attributed to that
specific food [48], however a repeat larger trial with blinding is
warranted. In addition, the sensitivity and specificity of IgG
food testing needs to be evaluated and labs need quality con-
trols instituted to assure reproducibility. Further trials with
blinded food challenges may be necessary to overcome the bias
against IgG food hypersensitivity testing. In addition, compar-
ison to normal controls would be helpful to assess the signifi-
cance of the IgG food related immune complexes. Furthermore,
IgG is only a subsection of the immunity and represents only a
small percentage of food hypersensitivity and there may be
other causes of food hypersensitivity or increased inflammation
in the GI tract besides the antigen/antibody complex formation.
What may be even more helpful in further studies would be a
more direct assessment of the bowel milieu after food challenge
for changes in inflammatory cytokines and other immune mes-
sengers like histamine. This of course, by necessity, would
VOL. 25, NO. 6

need to include assessment of neurotransmitter production in
the gut such as serotonin excretion by the enterochromaffin
cells. Other important questions to be answered would be the
role of microflora cross-talk and the interaction of microflora
populations on bowel immune messages.
As a result of the high numbers of the population affected
by IBS, if simple solutions such as food elimination and rota-
tion diets and maximizing bowel flora could result in signifi-
cant improvements then potential savings to the health care
system could result. IgG food testing would need to be vali-
dated with intra-lab reproducibility and then the extended
healthcare team could manage the follow up diet manipulation
and recommendations. This could reduce the time and expense
of physician involvement and the use of expensive pharmaco-
logic interventions. It is recommended that repeat evaluations
include cost analysis studies.
The complex role of abnormal bowel flora coupled with the
presence of food hypersensitivities on symptom production has
been noted in a previous clinical trial of IBS [18]. Since both
microflora and ingested materials display a direct effect on
immunity, it is not surprising that both would play a compli-
cated intertwined role in the production of symptoms in IBS. It
is difficult to determine at the present time if the food intoler-
ance precedes the altered gut microbial environment or if the
reverse is true. However, it may be neither but rather both
abnormalities contributing to the outcome of increased inflam-
mation and up-regulation of the immune system, altered enteric
neurotransmitter output, and abnormal fermentation, which all
results in gas, pain, bloating, and diarrhea. Irritable bowel
syndrome is a complex chronic disorder necessitating complex
interventions.
ACKNOWLEDGMENTS
This research was funded by BioCommunications Research
Institute, Wichita, Kansas. Product was supplied by Klaire
Labs, Solana Beach, California. Laboratory testing was pro-
vided by Great Plains Lab, Overland Park, Kansas, and Allos
Reference Laboratory, Mountain View, California. This re-
search project was designed, conducted, and analyzed without
input from either the BioCommunications Research Institute or
the suppliers of product and tests.
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Received February 2, 2005; revision accepted July 13, 2006.
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