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Original Paper Digestion 2010;81:252–264 DOI: 10.1159/000264649 Received: September 4, 2009 Accepted: November 26, 2009

Original Paper

Original Paper Digestion 2010;81:252–264 DOI: 10.1159/000264649 Received: September 4, 2009 Accepted: November 26, 2009

Digestion 2010;81:252–264 DOI: 10.1159/000264649

Received: September 4, 2009 Accepted: November 26, 2009 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 Crohn’s disease Immunoglobulin G antibodies Food antigen 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-

clusion: A nutritional intervention based on circulating IgG 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 Division of Gastroenterology and Hepatology

© 2010 S. Karger AG, Basel

Gerhard Rogler, MD, PhD Division of Gastroenterology and Hepatology Department of Internal Medicine University Hospital of Zurich Rämistrasse 100, CH–8091 Zurich (Switzerland) Tel. +41 44 255 9579, Fax +41 44 255 9497, E-Mail gerhard.rogler@usz.ch

0012–2823/10/0814–0252$26.00/0

Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com

Accessible online at:

www.karger.com/dig

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 (T regs ) [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 T regs 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 T regs 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) Enrollment
Assessed for eligibility from the
German IBD Competence Network,
Ratisbona, Germany (n = 100)
Enrollment
Randomized (n = 40)
Germany (n = 100) Enrollment Randomized (n = 40) Excluded (n = 60) Not meeting inclusion

Excluded (n = 60)

Not meeting inclusion criteria

(n = 32)

Refused to participate

(n = 28)

Allocated to intervention 1 (specific diet, 6 weeks)

(n = 20)

Received allocated intervention

(n = 20)

Did not receive allocated intervention

(n = 0)

(n = 20) Did not receive allocated intervention (n = 0) Allocated to intervention 2 (sham

Allocated to intervention 2 (sham diet, 6 weeks)

(n = 20)

Received allocated intervention

(n = 12)

Did not receive allocated intervention

(n = 8)

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

diet too restrictive: 4 low compliance: 2 other reasons: 2 Analyzed (n = 12) Excluded from

Analyzed (n = 12) Excluded from analysis (n = 0)

Allocation

Analysis

Allocated to intervention 2 (sham diet, 6 weeks)

(n = 20)

Received allocated intervention

(n = 20)

Did not receive allocated intervention

(n = 0)

(n = 20) Did not receive allocated intervention (n = 0) Allocated to intervention 1 (specific

Allocated to intervention 1 (specific diet, 6 weeks)

(n = 20)

Received allocated intervention

(n = 11)

Did not receive allocated intervention

(n = 9)

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

9) Reasons diet too restrictive: 6 low compliance: 1 other reasons: 2 Analyzed (n = 11)

Analyzed (n = 11) 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-

254 Digestion 2010;81:252–264

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.

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 azathioprine, glibenclamide budesonide budesonide, azathioprine, mesalazine, hydro- cortisonacetate, loperamide azathioprine, prednisolone azathioprine, prednisolone mesalazine budesonide azathioprine azathioprine cholestyramine azathioprine infliximab, azathioprine, prednisolone none cholestyramine mesalazine, azathioprine, hydrocortisone azathioprine, azulfidine infliximab azathioprine

2

w

55

cecum to sigma

3

w

24

colon, terminal ileum

4

w

37

terminal ileum, cecum

5

m

37

terminal ileum, proximal colon, proctitis

6

m

44

terminal ileum, cecum

7

m

41

ileum resection, multiple perianal fistulae

8

w

37

anastomosis

9

m

37

distal ileum

10

m

27

terminal ileum, cecum

11

m

54

neoterminal ileum, colon, sigma, anal stenosis

12

m

55

terminal ileum

13

w

31

esophagus lesions

14

m

54

stenosis terminal ileum, ileocecal resection

15

w

57

sigma segment resection, stenosis colon descendens

16

w

38

colon, sigma, rectum, stenosis terminal ileum

17

w

43

terminal ileum

18

w

54

colon

19

w

54

colon

20

w

31

neoterminal ileum, colon

6-mercaptopurine

21

w

50

fistulae

azathioprine infliximab mesalazine budesonide, azathioprine azathioprine infliximab, azathioprine, azulfidine, mesalazine, cholestyramine prednisolone, azathioprine methotrexate budesonide azathioprine, loperamide prednisolone, mesalazine, hydrocortisone azathioprine mesalazine budesonide prednisolone, ciprofloxacine, metronidazole cholestyramine infliximab methotrexate, azathioprine infliximab azathioprine

22

w

33

neoterminal ileum

23

w

25

colon, distal ileum

24

w

46

terminal ileum, caecum, sigma, rectum

25

w

49

ileotransversostomy

26

w

57

fistulae

27

m

41

colon

28

w

48

colon, ileocecal resection

29

m

51

colon

30

m

35

rectum

31

m

29

colon

32

w

59

colon, neoterminal ileum

33

w

43

terminal ileum, colon

34

w

48

neoterminal ileum

35

m

42

rectum, sigma, fistulae

36

w

22

terminal ileum, colon

37

w

28

colon

38

w

44

terminal ileum, sigma

39

m

41

terminal ileum, colon

40

m

35

colon

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-

Food Antigens in Crohn’s Disease

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

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 7.5–12.49 g/ml 12.519.99 g/ml 20.0–49.99 g/ml 50 g/ml

0 (0.0–0.9)

negative

1 (1.0–1.9)

weak

2 (2.0–2.9)

increased

3 (3.0–3.9)

high

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 T regs 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 ! 10 6 T regs were iso- lated from 50 ml with the AutoMACS programs Depl05 and Pos- seld2. Normally, T regs 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% CO 2 (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 T regs For suppression assays, 0.5–1 ! 10

5

CD4+CD25– T cells were

cultured in the absence or presence of 0.5–1 ! 10 5 autologous T regs /well in 96-well plates and in the presence of 2 ! 10 5 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 Dynabeads CD3/

CD28 T Cell Expander (Dynal , 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% CO 2 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 20 15 Specific diet Sham diet 0 2 4 6 8 10 12 Week
25
20
15
Specific diet
Sham diet
0
2
4
6
8
10
12
Week
Mean stool frequency

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 60 50 Specific diet Sham diet 40 0 2 4 6 8 10
80
70
60
50
Specific diet
Sham diet
40
0
2
4
6
8
10
12
Mean total score

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

 

10

3

CD4–CD25+ CD4+CD25+ 2.4% 62.6% CD4– CD4+CD25– 1.7% 33.3%
CD4–CD25+
CD4+CD25+
2.4%
62.6%
CD4–
CD4+CD25–
1.7%
33.3%
 

10

3

CD4–CD25+ CD4+CD25+ 0.1% 5.8% CD4– CD4+CD25– 3.3% 90.8%
CD4–CD25+
CD4+CD25+
0.1%
5.8%
CD4–
CD4+CD25–
3.3%
90.8%

10

2

10

2

CD25-PE

10

1

CD25-PE

10

1

10

0

10

0

 

10 0

10 1

10 2

10 3

 

10 0

10 1

10 2

10 3

a

CD4-FITC

b

CD4-FITC

Fig. 4. FACS analysis of CD4+CD25– T cells and T regs . a Staining of T regs . b Staining of CD4+CD25– T cells (effector T cells). The purity of CD4+CD25– effector T cells was 90.8% and of T regs 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

T regs

Isolation of CD4+CD25– effector T cells and T regs was controlled by FACS analysis. The purity of CD4+CD25– effector T cells was 90.8 and 62.6% for T regs ( fig. 4 a, b). CD4+CD25– effector T cells and APC were incubated in the absence or presence of T regs 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

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.

Fig. 5. Stimulation of CD4+CD25– T cells 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- 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- cates significance (obtained difference in mean ranks was greater than the 2 val- ue); u indicates outliers.

Fig. 6. Stimulation of CD4+CD25– T cells and APC in the presence of T regs with food antigens or negative control solution. Comparison of IFN secretion (pg/ml) be- tween base value (beginning of interven- 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 T regs secreted more IFN during 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- ic and sham diets, respectively. Kruskal- Wallis test; # indicates significance (ob- tained difference in mean ranks was great- er than the 2 value); u indicates outliers.

Food Antigens in Crohn’s Disease

Stimulation with Stimulation with food antigens negative control # 1,500 # 1,000 500 0 Base
Stimulation with
Stimulation with
food antigens
negative control
#
1,500
#
1,000
500
0
Base
Specific
Sham
Base
Specific
Sham
value
diet
diet
value
diet
diet
Stimulation with
Stimulation with
food antigens
negative control
2,000
#
1,500
1,000
500
0
Base
Specific
Sham
Base
Specific
Sham
value
diet
diet
value
diet
diet
Digestion 2010;81:252–264
259
IFN (pg/ml)
IFN (pg/ml)

Fig. 7. Stimulation of CD4+CD25– T cells, APC and T regs with antiCD3/antiCD28 so- lution. Comparison of IFN secretion (ng/ ml) between base value (beginning of in- tervention), specific diet and sham diet (after 6 weeks of intervention, respective- ly). There was 1,000-fold higher IFN se- cretion when cells were stimulated with antiCD3/antiCD28 solution than with food antigen/negative control solution. There was higher IFN secretion of CD4+CD25– T cells (left panel) between base value and specific diet. The mixture of CD4+CD25– T cells, APC and T regs 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); u indicates outliers.

CD4+CD25– and APC

Mixture CD4+CD25–, APC, T regs

800 600 # # 400 200 0 Base Specific Sham Base Specific Sham value diet
800
600
#
#
400
200
0
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Specific
Sham
Base
Specific
Sham
value
diet
diet
value
diet
diet
IFN (ng/ml)

Stimulation of the Co-Culture of CD4+CD25–, APC and T regs with Food Antigens and Negative Control Solu- tion

The co-culture of CD4+CD25– T cells and APC with T regs 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 T regs 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 T regs .

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 T regs ( fig. 7 ) showed high-

260 Digestion 2010;81:252–264

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 T regs (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 T regs .

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
Cell count

10 0

10 1

10 2 CFSE intensity r

10 3

10 4

Fig. 8. CFSE proliferation in the mixture of CD4+CD25– T cells, APC and T regs measured by FACS analysis. CD4+CD25– T cells and APC in the presence of T regs 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 T regs 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
Cell count

10 0

10 1

10 2 CFSE intensity r

10 3

10 4

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 T regs 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

EDN (ng/ml)

2,500

2,000

1,500

1,000

500

0

EDN (ng/ml) 2,500 2,000 1,500 1,000 500 0 Base Specific Sham value diet diet

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 had IgG antibodies against yeast. The IgG-antibody 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 T regs . 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 T regs after stimulation

with antiCD3/antiCD28 solution was also analyzed by Earle et al. [34]. In this issue, T regs 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 T regs resulted in reduced IFN production. In contrast to the findings of Earle, Nakamura et al. [35] postulated that T regs 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

References

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.

1

Hugot JP, et al: Prevalence of CARD15/ NOD2 mutations in Caucasian healthy peo- ple. Am J Gastroenterol 2007;102:1259–

7

Hafner S, et al: The role of domestic hygiene in inflammatory bowel diseases: hepatitis A and worm infestations. Eur J Gastroenterol

12

Awazuhara H, Kawai H, Maruchi N: Major allergens in soybean and clinical signifi- cance of IgG4 antibodies investigated by

   

1267.

   

Hepatol 2008;20:561–566.

   

IgE- and IgG4-immunoblotting with sera

2

Ogura Y, et al: A frameshift mutation in NOD2 associated with susceptibility to

8

Papp M, Altorjay I, Lakatos PL: Relevance of serologic studies in inflammatory bowel dis-

   

from soybean-sensitive patients. Clin Exp Allergy 1997;27:325–332.

   

Crohn’s disease. Nature 2001;411:603–606.

   

eases (in Hungarian). Orv Hetil 2007;148:

13

el Rafei A, et al: Diagnostic value of IgG4

3

Rogler G, Andus T: Cytokines in inflamma-

   

887–896.

   

measurements in patients with food allergy.

   

tory bowel disease. World J Surg 1998;22:

9

Ferguson LR, et al: Genes, diet and inflam-

   

Ann Allergy 1989;62:94–99.

   

382–389.

   

matory bowel disease. Mutat Res 2007;622:

14

Host A, et al: Prospective estimation of IgG,

4

Regueiro M, et al: Cigarette smoking and age

   

70–83.

   

IgG subclass and IgE antibodies to dietary

 

at diagnosis of inflammatory bowel disease. Inflamm Bowel Dis 2005;11:42–47.

10

Mekkel G, et al: Increased IgE-type antibody response to food allergens in irritable bowel

   

proteins in infants with cow milk allergy. Levels of antibodies to whole milk protein,

 

5

Kane SV, Flicker M, Katz-Nelson F: Tobacco use is associated with accelerated clinical re-

   

syndrome and inflammatory bowel diseases (in Hungarian). Orv Hetil 2005;146:797–

   

BLG and ovalbumin in relation to repeated milk challenge and clinical course of cow

   

currence of Crohn’s disease after surgically

   

802.

   

milk allergy. Allergy 1992;47:218–229.

   

induced remission. J Clin Gastroenterol

11 Crowe SE, Perdue MH: Gastrointestinal

15

Barnes RM, et al: Human serum antibodies

 

2005;39:32–35.

food hypersensitivity: basic mechanisms of

   

reactive with dietary proteins. IgG subclass

6

Picco MF, Bayless TM: Tobacco consump-

pathophysiology. Gastroenterology 1992;

   

distribution. Int Arch Allergy Appl Immu-

   

tion and disease duration are associated with

   

103:1075–1095.

 

nol 1988;87:184–188.

   

fistulizing and stricturing behaviors in the first 8 years of Crohn’s disease. Am J Gastro- enterol 2003;98:363–368.

       

16

Lessof MH, Kemeny DM, Price JF: IgG anti- bodies to food in health and disease. Allergy Proc 1991;12:305–307.

Food Antigens in Crohn’s Disease

Digestion 2010;81:252–264

263

17

Husby S, et al: Oral tolerance in humans. T cell but not B cell tolerance after antigen feeding. J Immunol 1994;152:4663–4670.

255.

25

Van Den Bogaerde J, et al: Gut mucosal re- sponse to food antigens in Crohn’s disease. Aliment Pharmacol Ther 2002;16:1903–

34

Earle KE, et al: In vitro expanded human CD4+CD25+ regulatory T cells suppress ef- fector T cell proliferation. Clin Immunol

18

Haddad ZH, et al: Detection and kinetics of

   

1915.

   

2005;115:3–9.

   

antigen-specific IgE and IgG immune com-

26

Zeger SL, Liang KY: Longitudinal data anal-

35

Nakamura K, Kitani A, Strober W: Cell

644.

   

plexes in food allergy. Ann Allergy 1983;51:

   

ysis for discrete and continuous outcomes. Biometrics 1986;42:121–130.

   

contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is medi-

19

Husby S, et al: Humoral immunity to dietary

27

Jones VA, et al: Food intolerance: a major fac-

   

ated by cell surface-bound transforming

   

antigens in healthy adults. Occurrence, iso- type and IgG subclass distribution of serum

   

tor in the pathogenesis of irritable bowel syn- drome. Lancet 1982;2:1115–1117.

   

growth factor beta. J Exp Med 2001;194:629–

   

antibodies to protein antigens. Int Arch Al- lergy Appl Immunol 1985;77:416–422.

28

Nanda R, et al: Food intolerance and the ir- ritable bowel syndrome. Gut 1989;30:1099–

36

Saitoh O, et al: Fecal eosinophil granule-de- rived proteins reflect disease activity in in-

20

Jonuleit H, Adema G, Schmitt E: Immune

   

1104.

   

flammatory bowel disease. Am J Gastroen-

   

regulation by regulatory T cells: implications

29

Niec AM, Frankum B, Talley NJ: Are adverse

   

terol 1999;94:3513–3520.

   

for transplantation. Transpl Immunol 2003;

   

food reactions linked to irritable bowel syn-

37

Atkinson W, et al: Food elimination based

   

11:267–276.

   

drome? Am J Gastroenterol 1998;93:2184–

   

on IgG antibodies in irritable bowel syn-

21

Spoettl T, et al: Serum soluble TNF receptor

   

2190.

   

drome: a randomised controlled trial. Gut

   

I and II levels correlate with disease activity

30

Burden S: Dietary treatment of irritable bow-

   

2004;53:1459–1464.

   

in

IBD patients. Inflamm Bowel Dis 2007;13:

   

el syndrome: current evidence and guide-

38

Baklien K, Brandtzaeg P: Comparative map-

457.

   

727–732.

   

lines for future practice. J Hum Nutr Diet

   

ping of the local distribution of immuno-

22

Podolsky DK: Inflammatory bowel disease.

   

2001;14:231–241.

   

globulin-containing cells in ulcerative coli-

   

N

Engl J Med 2002;347:417–429.

31

Darroch CJ, Barnes RM, Dawson J: Circulat-

   

tis and Crohn’s disease of the colon. Clin Exp

23

Beissert S, Schwarz A, Schwarz T: Regulatory

   

ing antibodies to Saccharomyces cerevisiae

   

Immunol 1975;22:197–209.

   

T

cells. J Invest Dermatol 2006;126:15–24.

   

(bakers’/brewers’ yeast) in gastrointestinal

39

Baklien K, Brandtzaeg P: Immunohisto-

24

Wing K, et al: CD4 T cell activation by my-

   

disease. J Clin Pathol 1999;52:47–53.

   

chemical characterization of local immuno-

   

elin oligodendrocyte glycoprotein is sup-

32

Elson CO, et al: Immuno-bacterial homeo-

   

globulin formation in Crohn’s disease of the

   

pressed by adult but not cord blood CD25+ T cells. Eur J Immunol 2003;33:579–587.

 

stasis in the gut: new insights into an old enigma. Semin Immunol 2001;13:187–194.

   

ileum. Scand J Gastroenterol 1976;11:447–

         

33

Breese E, et al: Interleukin-2- and interferon- gamma-secreting T cells in normal and dis- eased human intestinal mucosa. Immunol- ogy 1993;78:127–131.

     

264 Digestion 2010;81:252–264

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IRRITABLE BOWEL SYNDROME

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Food elimination based on IgG antibodies in irritable bowel syndrome: a randomised controlled trial

W Atkinson, T A Sheldon, N Shaath, P J Whorwell

See end of article for authors’ affiliations

Correspondence to:

Dr P J Whorwell, Department of Medicine, University Hospital of South Manchester, Manchester M20 2LR, UK; peter.whorwell@ smuht.nwest.nhs.uk

Revised version received

13 April 2004

Accepted for publication

13 April 2004

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. Patients: A total of 150 outpatients with IBS were randomised to receive, for three months, either a diet 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. Methods: Primary outcome measures were change in IBS symptom severity and global rating scores. Non- colonic symptomatology, quality of life, and anxiety/depression were secondary outcomes. Intention to treat analysis was undertaken using a generalised linear model. Results: After 12 weeks, the true diet resulted in a 10% greater reduction in symptom score than the sham diet (mean difference 39 (95% confidence intervals (CI) 5–72); p = 0.024) with this value increasing to 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

(p = 0.006, NNT = 2.5). All other outcomes showed trends favouring the true diet. Relaxing the diet led to a

24% greater deterioration in symptoms in those on the true diet (difference 52 (95% CI 18–88); p = 0.003).

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. 610 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 antigen 11 and have been implicated in some cases of food hypersensitivity. 1214 However, this mechanism is controversial and is considered by some to be

physiological 1517 especially as IgG food antibodies can be present in apparently healthy individuals. 1820 It has pre- viously been suggested that IgG food antibodies may have a

role in IBS 21 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

blood was taken and sent, with only a numerical identifier, to YorkTest Laboratories Ltd (York, UK) where an enzyme linked immunosorbant assay (ELISA) test was performed to

detect the presence of IgG antibodies specific to a panel of 29 different food antigens. This test has been described in detail elsewhere 23 and involves specimens being diluted 1/50, 1/150, and 1/450 with each dilution applied to an allergen panel. Each test was calibrated using 0 arbitrary unit (AU) and

25 AU standards prepared from a serum with a high IgG titre

to a cow’s milk allergen extract. A positive control serum at

45 AU was applied to each test. The test results were obtained

from the 1/150 dilution of the specimen. Where a high specimen background was observed, the test results were obtained from the 1/450 dilution. The threshold for a positive (reactive) result was selected as three times the background

signal obtained by the same sample against a no food allergen coated control well equivalent to 3 AU. Test results were scored as positive or negative only, relative to this cut off.

Staff based at the YorkTest Laboratories produced a true and sham diet sheet for each patient. The sham diet eliminated the same number of foods to which a patient exhibited IgG antibodies but not those particular foods. The goal was to try and include in the sham diet an equally difficult to eliminate staple food for every staple food in the true diet. Thus cow’s milk was (generally) replaced with potato, wheat with rice, and yeast with whole egg, where this was possible. Nut reactivities were replaced with other nuts in the sham diet, and legumes with other legumes, but this was not systematised. The true and sham diet sheets for each patient were sent to the University of York, again with only a number for identification. Patients were allocated to one of the two diet sheets based on a randomisation schedule developed using a random computer number generator. Thus patients would receive either an elimination diet based on their true sensitivity results (true diet) or a sham diet. All patients and clinical staff in the Gastroenterology Research Department and YorkTest Laboratory were blinded to the 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- inate the indicated foods from their diet for a period of 12 weeks. They also received a booklet with advice on eliminating the different foods and the telephone contact details of a free nutritional advisor whom they were able to contact for further advice if necessary. Symptoms were assessed using a questionnaire scoring 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 75–175, 175–300, and .300, respectively. A reduction in score of 50 or over is regarded as a clinically significant improvement. 24 Non-colonic symptomatology, 25 such as lethargy, backache, nausea, and urinary symptoms, was assessed and scored using visual analogue scales (range 0– 500). Quality of life (QOL) was measured using an instru-

ment proven to be sensitive to change in IBS (range 0–500). 2628 Anxiety and depression were evaluated using the hospital anxiety and depression scale (HAD). 29 This instrument scores anxiety and depression up to a maximum score of 21 for each

parameter, with a score above 9 indicating significant psychopathology. Data on these measures were recorded at baseline and after 4, 8, and 12 weeks of the dietary intervention period. In addition, at 4, 8, and 12 weeks, 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,

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Atkinson, Sheldon, Shaath, et al

slightly worse, no change, slightly better, better, or excel- lent?’’ The atopic status of all patients entering the study was also assessed.

During the treatment phase, patients were allowed to take concomitant medication provided it had been constant for six months prior to the start of the study. They were encouraged not to alter medication use during the course of the trial but any changes were recorded. Any patient withdrawing from the study was encouraged to complete a final symptom questionnaire at week 12 and their reasons for withdrawal were recorded. At the end of 12 weeks, patients were asked to resume consumption of the foods they had been advised to eliminate in order to assess the effect of their reintroduction. Patients were then reassessed after four weeks using the same measures and the result compared with their scores at the end of the elimination phase.

Data analysis Questionnaires were scored by an assessor blinded to the randomisation. The primary outcome measures were changes in IBS symptom severity score and global impact score at 12 weeks. Changes in non-colonic symptoms, QOL, and HAD scores were regarded as secondary outcome measures. Two sample t tests were used to establish whether there was an overall difference in the change in continuous outcome measures between the two groups of patients. Patients were analysed according to the group to which they were randomised, independent of their adherence to the diet. The global impact score, an ordered categorical variable, was analysed using a Wilcoxon Mann-Whitney test to compare the numbers in the active and sham groups showing significant improvement (‘‘better’’ or ‘‘excellent’’), no sig- nificant change (‘‘slightly worse’’, ‘‘no change’’, or ‘‘slightly better’’), and significant deterioration (‘‘worse’’ or ‘‘terri- ble’’). The number needed to treat (NNT) was calculated from the global impact score by calculating the reciprocal of the difference in probability of a significant improvement between the treatment and control groups. General linear modelling in SPSS was used to explore whether there was a

modelling in SPSS was used to explore whether there was a      

 

   

 

 
         
 

   

 

 
          Figure 1 Study flow diagram.

Figure 1

Study flow diagram.

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) No of males (%) No of foods to which sensitive Symptom duration (y) IBS symptom severity score Non-colonic features score Quality of life score HAD anxiety score HAD depression score No of diarrhoea predominant patients (%) No of constipation predominant patients (%) No of alternating predominant patients (%)

44 (17–72; 12.9) 7 (9.3%) 6.65 (3.66) 11.5 (9.9) 331.9 (70.8) 459.1 (160.7) 640.1 (252.6) 9.5 (4.6) 5.3 (3.4) 37 (52.1%) 19 (26.8%) 15 (21.1%)

44 (19–74; 15.2) 13 (17.3%) 6.63 (4.1) 10.1 (7.5) 309.0 (78.5) 452.6 (170.1) 639.3 (222.3) 9.5 (4.5) 6.0 (3.6) 41 (56.9%) 16 (22.2%) 15 (20.8%)

Results are expressed as mean (SD). HAD, hospital anxiety and depression scale.

relationship between the change in symptoms from baseline and treatment group, patient characteristics (for example, IBS subtype, history of atopy, number of foods to which sensitive, and concomitant medication) and adherence to the diet. 30

Sample size calculation It was estimated that approximately 40% of the placebo arm would report a significant improvement in symptoms. It was calculated that a sample size of 55 patients would be required in each group to detect, with 90% power, a difference of 30% points in the proportion reporting such an improvement (that is, 70% in the treatment arm) as statistically significant at the 5% level. Assuming a 20% dropout rate, a minimum of 138 patients would need to be entered into the trial. Thus we aimed to recruit a total of 150 patients into the study.

RESULTS

Recruitment of patients and their flow through each stage of the study is illustrated in fig 1, as recommended by the

Table 2

Frequency of foods excluded from the diet (% of

patients)

Food

Treatment group

Sham group

Barley

26.7

9.3

Corn

22.7

14.7

Rice

8

54.7

Rye

8

25.3

Wheat

49.3

8

Milk

84.3

1.3

Beef

24

9.3

Chicken

21.3

13.3

Pork

5.3

36

Cabbage

12

24

Celery

5.3

21.3

Haricot bean

17.3

14.7

Pea

38.6

1.3

Potato

9.3

61.3

Soy bean

22.7

10.7

Tomato

4

44

Apple

1.3

33

Orange

6.7

29.3

Strawberry

0

20

Almond

28

12

Brazil nut

22.7

17.3

Cashew nut

49.3

8

Peanut

10.7

20

Walnut

2.7

29.3

Cocoa bean

1.3

21.3

Shellfish

21.3

10.7

Fish mix

17.3

28

Whole egg

57.3

26.7

Yeast

86.7

0

CONSORT statement. 31 In summary, between January 2001 and July 2002, 176 patients were eligible for the study, of which 26 (15%) were excluded from participation, leaving 150 patients who were all found to be sensitive to at least one food. Seventy five of these were randomised to receive an elimination diet based on their true food sensitivity results and 75 patients to a sham diet. Data from 131 (87%) patients who gave 12 week data were available for the intention to treat analysis: 65 and 66 patients from the true and sham groups, respectively.

Patient characteristics The patients were typical of those with IBS in secondary care practice, the majority being women. Patients, on average, had experienced symptoms of IBS for over a decade and were found to be sensitive to approximately 6–7 foods (range 1– 19). Baseline demographic and clinical characteristics of the two groups, including the use of concomitant medication, were found to be similar with the exception of the IBS symptom severity score which was slightly higher in the treatment group (table 1). Thirty per cent of patients were found to be atopic. The frequency of foods excluded from the diet is shown in table 2. Adherence was lower in those on the true diet although no specific adverse events were recorded in either group. Twenty four patients withdrew from the study in the true diet group (mainly because of difficulty in following the diet) and 13 from the sham diet group (for a variety of reasons). However, 12 week data were obtained from 14 of those who withdrew in the true diet group and four in the sham diet group. There were no significant differences

the sham diet group. There were no significant differences Figure 2 according to degree of adherence.

Figure 2

according to degree of adherence. Difference between the groups with

high adherence: 101 (95% confidence interval 54, 147); ***p,0.001.

Mean change in symptom severity scores at 12 weeks

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1462

1462   Figure 3 (A) Average symptom severity scores over time for the group as a

1462   Figure 3 (A) Average symptom severity scores over time for the group as a

 

1462   Figure 3 (A) Average symptom severity scores over time for the group as a

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

Atkinson, Sheldon, Shaath, et al

between atopic and non-atopic patients. There was however 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 number of foods who fully adhered to their allocated diet, a 26% difference in reduction in symptom severity score was observed in favour of the true diet (a difference in score of 98 (95% CI 52, 144), p,0.001: a standardised effect size of 1.3). This benefit increased by a further 39 points (12%) (95% CI 7, 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 which the final score is the dependent variable and the baseline score is included as a covariate) instead of modelling change in scores. 30 The interaction between treatment group and adherence is demonstrated in fig 2 which shows a 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 12 weeks for the group as a whole and for those who fully adhered, respectively. This reveals that most improvements in symptoms are fully achieved within two months.

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 No significant change Significantly improved Total

3 (4.7)

8 (12.1)

44 (67.2)

47 (71.2)

18 (28.1)

11 (16.7)

65

66

NNT = 9

Patients fully adhering to the diet Significantly worse No significant change Significantly improved Total

1 (4.2)

5 (12.5)

10 (41.7)

29 (72.5)

13 (54.1)

6 (15)

24

40

NNT = 2.5

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Food elimination based on IgG antibodies in IBS

Food elimination based on IgG antibodies in IBS Figure 4 (A) Mean change in the secondary
Food elimination based on IgG antibodies in IBS Figure 4 (A) Mean change in the secondary

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

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Table 4

relative to the end of the elimination phase

Global rating following reintroduction of foods

 

Treatment group

True diet group (n (%))

Sham diet group (n (%))

Significantly worse No significant change Significantly improved Total

17 (41.5)

13 (25)

23 (56.1)

35 (67.3)

1 (2.4)

4 (7.7)

41 (100)

52 (100)

worsening of health compared with the sham diet group (p = 0.047).

DISCUSSION

A clinically significant improvement in IBS symptomatology was observed in patients eliminating foods to which they were found to exhibit sensitivity, as identified by an ELISA test for the presence of IgG antibodies to these foods. The 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 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 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 method of classifying global improvement as any value exceeding adequate relief of symptoms, we used a much stricter definition requiring patients to report symptoms as being either ‘‘better’’ or ‘‘excellent’’ compared with pretreat- ment levels. Despite this, the diet still achieved a significant improvement. However, as might be expected, the placebo response using this end point was somewhat lower than that usually reported in IBS treatment trials which have used less demanding criteria. The observation that patients on the sham diet also improved, although to a lesser extent, emphasises the importance of conducting double blind randomised controlled trials of such non-drug interventions in order to avoid overestimating their potential.

Most patients with IBS have attempted at least some form of dietary modification, which in some cases can be very extreme. Conflicting results have been reported using exclusion diets 4 5 3336 and this approach also suffers from the limitation that it has to be empirical. Thus potentially offending foods can only be identified after their elimination and subsequent reintroduction. This time consuming process would be much reduced if the offending foods could be identified beforehand. Attempts to do this using IgE antibodies have been disappointing 810 but the results of this 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 which was found to be no greater than that occurring in the

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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50, which is regarded as being of clinical significance both in

validation studies 24 and clinical practice. 2628 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. 3842 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

REFERENCES

1 Drossman DA, Camilleri M, Mayer EA, et al. American Gastroenterological Association Technical Review on Irritable Bowel Syndrome. Gastroenterology

2002;123:2108–31.

2 Lea R, Whorwell PJ. Quality of life in irritable bowel syndrome. Pharmacoeconomics 2001;19:643–53.

3 Talley NJ, Spiller R. Irritable bowel syndrome: a little understood organic bowel disease? Lancet 2002;360:555–64.

4 Jones VA, McLaughlan P, Shorthouse M, et al. Food intolerance: a major factor in pathogenesis of irritable bowel syndrome. Lancet 1982;2:1115–17.

5 Nanda R, James R, Smith H, et al. Food intolerance and the irritable bowel syndrome. Gut 1989;30:1099–104.

6 Zwetchkenbaum J, Burakoff, R. The irritable bowel syndrome and food hypersensitivity. Ann Allerg 1988;61:47–9.

7 Zar S, Kumar D, Benson M. J. Review article: food hypersensitivity and irritable bowel syndrome, Aliment Pharm Ther 2001;15:439–43.

8 Petitpierre M, Gumowski P, Girard JP. Irritable bowel syndrome and hypersensitivity to food. Ann Allergy 1985;54:538–40.

9 Barau E, Dupont C. Modifications of intestinal permeability during food provocation procedures in pediatric irritable bowel syndrome. J Pediatr Gastroenterol Nutr 1990;11:72–7.

10 Roussos A, Koursarakos P, Patsopoulos D, et al. Increased prevalence of irritable bowel syndrome in patients with bronchial asthma. Respir Med

2003;97:75–9.

www.gutjnl.com

Atkinson, Sheldon, Shaath, et al

11 Crowe SE, Perdue MH. Gastrointestinal food hypersensitivity: basic mechanisms of pathophysiology. Gastroenterol 1992;103:1075–95.

12 el Rafei A, Peters SM, Harris N, et al. Diagnostic value of IgG4 measurements in patients with food allergy. Ann Allergy 1989;62:94–9.

13 Host A, Husby S, Gjesing B, et al. Prospective estimation of IgG, IgG subclass and IgE antibodies to dietary proteins in infants with cow’s milk allergy. Levels of antibodies to whole milk protein, BLG and ovalbumin in relation to repeated milk challenge and clinical course of cow’s milk allergy. Allergy

1992;47:218–29.

14 Awazuhara H, Kawai H, Maruchi N. Major allergens in soybean and clinical significance of IgG4 antibodies investigated by IgE and IgG4 immunoblotting with sera from soybean-sensitive patients. Clin Exp Allergy 1997;27:325–32.

15 Barnes RMR, Johnson PM, Harvey MM, et al. Human serum antibodies reactive with dietary proteins: IgG subclass distribution. Int Arch Allergy Appl Immunol 1988;87:184–8.

16 Lessof MH, Kemeny DM, Price JF. IgG antibodies to food in health and disease. Allergy Proc 1991;12:305–7.

17 Husby S, Mestecky J, Moldoveanu Z, et al. Oral tolerance in humans. T cell but not B cell tolerance after antigen feeding. J Immunol 1994;152:4663–70.

18 Haddad ZH, Vetter M, Friedmann J, et al. Detection and kinetics of antigen- specific IgE and IgG immune complexes in food allergy. Ann Allergy

1983;51:255.

19 Husby S, Oxelius VA, Teisner B, et al. Humoral immunity to dietary antigens in healthy adults. Occurrence, isotype and IgG subclass distribution of serum antibodies to protein antigens. Int Arch Allergy Appl Immunol

1985;77:416–22.

20 Kruszewski J, Raczka A, Klos M, et al. High serum levels of allergen specific IgG-4 (asIgG-4) for common food allergens in healthy blood donors. Arch Immunol Ther Exp 1994;42:259–61.

21 Finn R, Smith MA, Youngs GR, et al. Immunological hypersensitivity to environmental antigens in the irritable bowel syndrome. Br J Clin Pract

1987;41:1041–3.

22 Drossman DA, Corazziari E, Talley NJ, et al. Rome II: a multinational consensus document on functional gastrointestinal disorders. Gut

1999;45:1–81.

23 Foster AP, Knowles TG, Hotston Moore A, 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.

24 Francis CY, Morris J, Whorwell PJ. The irritable bowel scoring system: A simple method of monitoring IBS and its progress. Aliment Pharmacol Therap

1997;11:395–402.

25 Whorwell PJ, McCallum H, Creed FH, et al. Non-colonic features of irritable bowel syndrome. Gut 1986;27:452–6.

26 Houghton LA, Heyman DJ, Whorwell PJ. Symptomatology, quality of life and economic features of irritable bowel syndrome—the effect of hypnotherapy. Aliment Pharmacol Ther 1996;10:91–5.

27 Gonsalkorale WM, Toner BB, Whorwell PJ. Cognitive change in patients undergoing hypnotherapy for irritable bowel syndrome. J Psychosom Res

2004;56:271–8.

28 Gonsalkorale WM, Houghton LA, Whorwell PJ. Hypnotherapy in irritable bowel syndrome: a large scale audit of a clinical service with examination of factors influencing responsiveness. Am J Gastroenterol 2002;97:954–61.

29 Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand 1983;67:361–70.

30 Everitt BS, Pickles A. Statistical aspects of the design and analysis of clinical trials. London: Imperial College Press Publishers, 2003:108–42.

31 Altman DG, Schulz KF, Moher D, et al. The revised CONSORT statement for reporting randomized trials: explanation and elaboration. Ann Intern Med

2001;134:663–94.

32 Novick J, Miner P, Krause R, et al. A randomised, double blind, placebo controlled trial of tegaserod in female patients suffering from irritable bowel syndrome with constipation. Aliment Pharmacol Ther 2002;16:1877–88.

33 Niec AM, Frankum B, Talley NJ. Are adverse reactions to food linked to irritable bowel syndrome? Am J Gastroenterol 1998;93:2184–90.

34 Burden S. Dietary treatment of irritable bowel syndrome: current evidence and guidelines for future practice. J Hum Nutr Diet 2001;14:231–41.

35 Bentley SJ, Pearson DJ, Rix KJB. Food hypersensitivity in irritable bowel syndrome. Lancet 1983;2:295–7.

36 McKee AM, Prior A, Whorwell PJ. Exclusion diets in irritable bowel syndrome:

Are they worthwhile? J Clin Gastroenterol 1987;9:526–8.

37 Durham SR, Church MK. Principles of allergy diagnosis. In: Holgate ST, Church MK, Lichtenstein LM, eds. Allergy, 2nd edn. London: Mosby,

2001:3–16.

38 Spiller RC, Jenkins D, Thornley JP, et al. Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome. Gut 2000;47:804–11.

39 Gonsalkorale WM, Perrey C, Pravica V, et al. Interleukin 10 genotypes in irritable bowel syndrome: evidence for an inflammatory component? Gut

2003;52:91–3.

40 Collins SM, Piche T, Rampal P. The putative role of inflammation in the irritable bowel syndrome. Gut 2001;49:743–5.

41 Collins SM. A case for an immunological basis for irritable bowel syndrome. Gastroenterology 2002;122:2078–80.

42 Chadwick VS, Chen W, Shu D, et al. Activation of the mucosal immune system in irritable bowel syndrome. Gastroenterology 2002;122:1778–83.

43 Mertz H. Review article: visceral hypersensitivity. Aliment Pharmacol Ther

2003;17:623–33.

44 Francis CY, Houghton LA, Whorwell PJ. Enhanced sensitivity of the whole gut in patients with irritable bowel syndrome. Gastroenterology

1995;108:601(abstract).

Future Directions

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Food Exclusion Based on IgG Antibodies Alleviates Symptoms in Ulcerative Colitis: A Prospective Study

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/m 2 , 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

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) 13 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

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

between gut antigens and host immunity. 6, 7 When certain 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. 1012 However, although some prospective studies have focused on the effect of dietary components on the disease 1315 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

1918

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

Food Exclusion Alleviates Ulcerative Colitis

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in patients with UC. The methods of these studies were empir-

100 U/mL (a grade of 1-plus) could be eaten on a 5-day rota-

ical and time-consuming; however, the results indicated that

tion

cycle. Foods with IgG antibody titers of less than 50 U/

IgG-based exclusion diets may benefit UC patients and provide

mL

(a grade of no-plus) and foods whose antigens were not

personalized and precise therapies. In this study, we aimed to evaluate whether an IgG-guided exclusion diet would improve

tested were categorized as normal, as long as they were nutri- tious (Table 1). Patients in the sham diet group were asked to

symptoms and QoL in patients with UC.

maintain their healthy diet as normal. The diet intervention did

not

influence the individual medical treatment, which mainly

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

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