Paper 01 Dietary advice based on food specific IgG Results Geoffrey Hardman, Gillian Hart, University of York, Heslington, York, UK Nutrition and food science Vol 37 No 1 2007 pp 16-23

Paper 02 Food elimination based on IgG antibodies in irritable bowel syndrome: a randomised controlled trial. W Atkinson, T A Sheldon, N Shaath, PJ Whorwell Gut 2004:53 1459-1464 doi: 10.1136

Paper 05 Food allergy in irritable bowel syndrome: new facts and old fallacies. E Isolauri, S.Rautava, M.Kalliomaki Gut 2004; 53 1391-1393 10.1136

Paper 06 A prospective audit of Food Intolerance among Migraine patients in primary care clinical practise. Trevor Rees, David Watson, Susan Lipscombe, Helen Speight, Peter Cousins, Geoffrey Hardman and Andrew J. Dowson. Headache care Vol.2 No 2 2005 105-110

Paper 07 Celiac Disease. Peter H.R Green M.D. and Christopher collier, M.D. PhD The New England Journal of Medicine 2007; 357:1731-43

Paper 08 Alterations of food antigen-specific serum immunoglobulins G and E in patients with irritable bowel syndrome and functional dyspepsia. X.L.Zuo, Y.Q. Li, W.J.Li, Y.T. Guo, X.F. Lu, J.M. Li and P.V. Desmond Clinical and Experimental Allergy, 37, 823-830

Paper 10 IgG Antibodies against Food Antigens are Correlated with Inflammation and Intima Media Thickness in Obese Juveniles M. Wilders-Truschnig, H.Mangge, C.Lieners, H.J.Gruber, C Mayer, W Marz Exp Clin Endocrinol Diabetes 2008; 116:241-245

Paper 12 A Vegan diet free of gluten improves the signs and symptoms of Rheumatoid Arthritis.. I Hafstöm, B.Ringertz, A. Spångberg, L. Von Zweigbergk, S. Brannemark, I. Nylander, J.Rönnelid, L.Laasonen, L.Klareskog. British Society of Rheumatology, 2001 pp 1175-1179

Paper 13 The gut-joint axis: cross reactive food antibodies in rheumatoid arthritis. M Hvatum, L Kanerud, R Hällgren, P Brandtzaeg Gut 2006:55 1240-1247 originally published online 16 feb 2006

Paper 14 Toward an understanding of Allergy and In-Vitro Testing By Mary James N.D Great Smokies Diagnostic Laboratory

Paper 18 Reported food Intolerance and respiratory symptoms R.K.Woods, M.Abramson, J.M.Raven, M Bailey, JM Weiner, E.H.Walters Eur Respir J 1998 11:151-155

V1.2 October 2009

Paper 19 A critical review of IgG Immunoglobulins and Food Allergy- Implications in systemic health Raymond M. Suen, MT (ASCP), Shalima Gordon, ND 13500 Linden Ave. N. Seattle, WA 98133 Ph: (206) 365-1256, (877) 318-8728, Fax: (206) 363-8790

Paper 20 The clinical relevance of IgG food allergy testing through ELISA (Enzyme-Linked Immunosorbent Assay). From: Townsend Letter for Doctors and Patients | Date: 1/1/2004 | Author: Suen, Raymond M.;Gordon, Shalima

Paper 21 IgG mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet. Faculty of Ludwig Maximilian University of Munich Mario Krause from Rotenburg a. d. Fulda 2005

Paper 22 Gastrointestinal Candida colonisation promotes sensitisation against food antigens by affecting the mucosal barrier in mice. N Yamaguchi, R Sugita, A Miki, N Takemura, J Kawabata, J Watanabe, K Sonoyama Gut2006;55:954-960. Doi: 10.1136/gut.2005.084954

Paper 23 Ovalbumin-specific immunoglobulin G and subclass responses through the first 5 years of life in relation to duration of egg sensitization and the development of asthma. G.H.S Vance*, C.A. Thornton*, T.N.Brynat, J.A.Warner * and J.O.Warner*Child Health, Infection, Infammation & Repair Division and Information & Computing Division, University of Southhampton, UK

V1.2 October 2009

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IRRITABLE BOWEL SYNDROME Food elimination based on IgG antibodies in irritable bowel syndrome: a randomised controlled trial W Atkinson, T A Sheldon, N Shaath, P J Whorwell ......

Gut 2004;53:1459–1464. doi: 10.1136/gut.2003.037697

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

rritable bowel syndrome (IBS) is a common disorder which physiological15–17 especially as IgG food antibodies can be causes abdominal pain, abdominal distension, and bowel present in apparently healthy individuals.18–20 It has pre- dysfunction, characterised by loose bowels, constipation, or viously been suggested that IgG food antibodies may have a I 1 21 a fluctuation between these two extremes. This condition role in IBS and it was therefore the purpose of this study to significantly impairs quality of life and places a large burden formally evaluate, in a randomised controlled trial, the on health care resources.2 Treatment of IBS is largely based therapeutic potential of an elimination diet based on the on the use of antispasmodics, antidepressants, and medica- presence of IgG antibodies to food in patients with IBS. tions that modify bowel habit, depending on whether constipation or diarrhoea is the predominant problem.1 The PATIENTS AND METHODS notorious inadequacies of current drug therapy lead to much Patients patient dissatisfaction and a tendency for patients to seek a All patients with uncomplicated IBS (all bowel habit variety of alternative remedies, especially of a dietary nature. subtypes) attending the Gastroenterology Department at IBS is likely to be a multifactorial condition involving a the University Hospital of South Manchester were considered number of different mechanisms although the prominence of eligible for the study, and those aged between 18 and any particular factor may vary from patient to patient.13 75 years, who satisfied the Rome II criteria,22 were invited to However, patients often strongly believe that dietary intoler- participate. Tertiary care patients were excluded from the ance significantly contributes to their symptomatology and study. All patients had normal haematology, biochemistry, some sufferers seem to benefit from eliminating certain foods and endoscopic examination when indicated. Coeliac disease from their diet. Detection of food intolerance is often difficult was excluded using the tissue transglutaminase test and a due to its uncertain aetiology, non-specific symptomatology, hydrogen breath test was used for excluding lactose intoler- and relative inaccessibility of the affected organ. Thus most ance. Patients were also excluded from participating in the previous studies have relied on the use of exclusion diets, study if they had any significant coexisting disease or a which are extremely labour intensive and time consuming.45 history of gastrointestinal surgery, excluding appendicect- Attempts to ‘‘test’’ for food intolerance in IBS have largely omy, cholecystectomy, and hiatus hernia repair. The study focused on ‘‘classic’’ food allergy based on the presence of IgE was approved by the local ethics committee and all patients mediated antibody responses, although it appears that these provided written informed consent. ‘‘immediate type’’ reactions are probably quite rare in this 6–10 condition. It is therefore possible that adverse reactions to Methods food in patients with IBS might be due to some other form of The study used a double blind, randomised, controlled, immunological mechanism, rather than dietary allergy. Such parallel design in which patients were randomised to either a reactions could be mediated by IgG antibodies, which ‘‘true’’ diet or a ‘‘sham’’ diet control group. At screening, characteristically give a more delayed response following 11 exposure to a particular antigen and have been implicated Abbreviations: IBS, irritable bowel syndrome; ELISA, enzyme linked 12–14 in some cases of food hypersensitivity. However, this immunosorbant assay; AU, arbitrary unit; HAD, hospital anxiety and mechanism is controversial and is considered by some to be depression scale; QOL, quality of life; NNT, number needed to treat

www.gutjnl.com 1460 Atkinson, Sheldon, Shaath, et al blood was taken and sent, with only a numerical identifier, to slightly worse, no change, slightly better, better, or excel- YorkTest Laboratories Ltd (York, UK) where an enzyme lent?’’ The atopic status of all patients entering the study was linked immunosorbant assay (ELISA) test was performed to also assessed. detect the presence of IgG antibodies specific to a panel of 29 During the treatment phase, patients were allowed to take different food antigens. This test has been described in detail concomitant medication provided it had been constant for six elsewhere23 and involves specimens being diluted 1/50, 1/150, months prior to the start of the study. They were encouraged and 1/450 with each dilution applied to an allergen panel. not to alter medication use during the course of the trial but Each test was calibrated using 0 arbitrary unit (AU) and any changes were recorded. Any patient withdrawing from 25 AU standards prepared from a serum with a high IgG titre the study was encouraged to complete a final symptom to a cow’s milk allergen extract. A positive control serum at questionnaire at week 12 and their reasons for withdrawal 45 AU was applied to each test. The test results were obtained were recorded. At the end of 12 weeks, patients were asked to from the 1/150 dilution of the specimen. Where a high resume consumption of the foods they had been advised to specimen background was observed, the test results were eliminate in order to assess the effect of their reintroduction. obtained from the 1/450 dilution. The threshold for a positive Patients were then reassessed after four weeks using the (reactive) result was selected as three times the background same measures and the result compared with their scores at signal obtained by the same sample against a no food the end of the elimination phase. allergen coated control well equivalent to 3 AU. Test results were scored as positive or negative only, relative to this cut Data analysis off. Questionnaires were scored by an assessor blinded to the Staff based at the YorkTest Laboratories produced a true randomisation. The primary outcome measures were changes and sham diet sheet for each patient. The sham diet in IBS symptom severity score and global impact score at eliminated the same number of foods to which a patient 12 weeks. Changes in non-colonic symptoms, QOL, and HAD exhibited IgG antibodies but not those particular foods. The scores were regarded as secondary outcome measures. Two goal was to try and include in the sham diet an equally sample t tests were used to establish whether there was an difficult to eliminate staple food for every staple food in the overall difference in the change in continuous outcome true diet. Thus cow’s milk was (generally) replaced with measures between the two groups of patients. Patients were potato, wheat with rice, and yeast with whole egg, where this analysed according to the group to which they were was possible. Nut reactivities were replaced with other nuts randomised, independent of their adherence to the diet. in the sham diet, and legumes with other legumes, but this The global impact score, an ordered categorical variable, was was not systematised. analysed using a Wilcoxon Mann-Whitney test to compare The true and sham diet sheets for each patient were sent to the numbers in the active and sham groups showing the University of York, again with only a number for significant improvement (‘‘better’’ or ‘‘excellent’’), no sig- identification. Patients were allocated to one of the two diet nificant change (‘‘slightly worse’’, ‘‘no change’’, or ‘‘slightly sheets based on a randomisation schedule developed using a better’’), and significant deterioration (‘‘worse’’ or ‘‘terri- random computer number generator. Thus patients would ble’’). The number needed to treat (NNT) was calculated receive either an elimination diet based on their true from the global impact score by calculating the reciprocal of sensitivity results (true diet) or a sham diet. All patients the difference in probability of a significant improvement and clinical staff in the Gastroenterology Research between the treatment and control groups. General linear Department and YorkTest Laboratory were blinded to the modelling in SPSS was used to explore whether there was a group assignment of all patients for the duration of the study. Patients were given their allocated diet sheet by staff at the Gastroenterology Research Department and asked to elim- Assessed for inate the indicated foods from their diet for a period of eligibility (n=176) 12 weeks. They also received a booklet with advice on Excluded (n=26): eliminating the different foods and the telephone contact Did not meet inclusion details of a free nutritional advisor whom they were able to criteria (n=19) Refused to participate (n=5) contact for further advice if necessary. Randomised Other reasons (n=2) Symptoms were assessed using a questionnaire scoring (n=150) system validated for use in IBS, including the IBS symptom severity score (range 0–500).24 This is a system for scoring pain, distension, bowel dysfunction, and general well being, with mild, moderate, and severe cases indicated by scores of Allocated to Allocated to 75–175, 175–300, and .300, respectively. A reduction in receive true receive sham score of 50 or over is regarded as a clinically significant diet (n=75) diet (n=75) improvement.24 Non-colonic symptomatology,25 such as lethargy, backache, nausea, and urinary symptoms, was 24 Withdrew: 13 Withdrew: Diet too restrictive (n=11) Diet too restrictive (n=3) assessed and scored using visual analogue scales (range 0– Lack of efficacy (n=1) Lack of efficacy (n=3) 500). Quality of life (QOL) was measured using an instru- Not prepared to follow Notpreparedtofollow ment proven to be sensitive to change in IBS (range 0–500).26–28 diet (n=6) diet (n=4) Anxiety and depression were evaluated using the hospital Other reasons (n=6) Other reasons (n=3) anxiety and depression scale (HAD).29 This instrument scores 10 Lost to follow up 9Losttofollowup 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 65 Included 66 Included in the final in the final baseline and after 4, 8, and 12 weeks of the dietary intention to intention to intervention period. In addition, at 4, 8, and 12 weeks, treat analysis treat analysis patients were asked to give a global rating of their IBS using the question, ‘‘Compared with your IBS before you started the food elimination diet, are you now: terrible, worse, Figure 1 Study flow diagram.

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Table 1 Baseline characteristics of the patients

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

Age (y) (range, SD) 44 (17–72; 12.9) 44 (19–74; 15.2) No of males (%) 7 (9.3%) 13 (17.3%) No of foods to which sensitive 6.65 (3.66) 6.63 (4.1) Symptom duration (y) 11.5 (9.9) 10.1 (7.5) IBS symptom severity score 331.9 (70.8) 309.0 (78.5) Non-colonic features score 459.1 (160.7) 452.6 (170.1) Quality of life score 640.1 (252.6) 639.3 (222.3) HAD anxiety score 9.5 (4.6) 9.5 (4.5) HAD depression score 5.3 (3.4) 6.0 (3.6) No of diarrhoea predominant patients (%) 37 (52.1%) 41 (56.9%) No of constipation predominant patients (%) 19 (26.8%) 16 (22.2%) No of alternating predominant patients (%) 15 (21.1%) 15 (20.8%)

Results are expressed as mean (SD). HAD, hospital anxiety and depression scale. relationship between the change in symptoms from baseline CONSORT statement.31 In summary, between January 2001 and treatment group, patient characteristics (for example, and July 2002, 176 patients were eligible for the study, of IBS subtype, history of atopy, number of foods to which which 26 (15%) were excluded from participation, leaving sensitive, and concomitant medication) and adherence to the 150 patients who were all found to be sensitive to at least one diet.30 food. Seventy five of these were randomised to receive an elimination diet based on their true food sensitivity results Sample size calculation and 75 patients to a sham diet. Data from 131 (87%) patients It was estimated that approximately 40% of the placebo arm who gave 12 week data were available for the intention to would report a significant improvement in symptoms. It was treat analysis: 65 and 66 patients from the true and sham calculated that a sample size of 55 patients would be required groups, respectively. in each group to detect, with 90% power, a difference of 30% points in the proportion reporting such an improvement (that Patient characteristics is, 70% in the treatment arm) as statistically significant at the The patients were typical of those with IBS in secondary care 5% level. Assuming a 20% dropout rate, a minimum of 138 practice, the majority being women. Patients, on average, had patients would need to be entered into the trial. Thus we experienced symptoms of IBS for over a decade and were aimed to recruit a total of 150 patients into the study. found to be sensitive to approximately 6–7 foods (range 1– 19). Baseline demographic and clinical characteristics of the RESULTS two groups, including the use of concomitant medication, Recruitment of patients and their flow through each stage of were found to be similar with the exception of the IBS the study is illustrated in fig 1, as recommended by the symptom severity score which was slightly higher in the treatment group (table 1). Thirty per cent of patients were Table 2 Frequency of foods excluded from the diet (% of found to be atopic. patients) The frequency of foods excluded from the diet is shown in table 2. Adherence was lower in those on the true diet Food Treatment group Sham group although no specific adverse events were recorded in either Barley 26.7 9.3 group. Twenty four patients withdrew from the study in the Corn 22.7 14.7 true diet group (mainly because of difficulty in following the Rice 8 54.7 diet) and 13 from the sham diet group (for a variety of Rye 8 25.3 reasons). However, 12 week data were obtained from 14 of Wheat 49.3 8 Milk 84.3 1.3 those who withdrew in the true diet group and four in the Beef 24 9.3 sham diet group. There were no significant differences Chicken 21.3 13.3 Pork 5.3 36 Cabbage 12 24 Celery 5.3 21.3 0 Haricot bean 17.3 14.7 Pea 38.6 1.3 *** _ Potato 9.3 61.3 50 Sham Soy bean 22.7 10.7 diet Tomato 4 44 (n=66) Apple 1.3 33 _ Orange 6.7 29.3 100 Strawberry 0 20 Almond 28 12 _ Brazil nut 22.7 17.3 150 True diet

Cashew nut 49.3 8 IBS symptom severity (n=65) Peanut 10.7 20 Walnut 2.7 29.3 _ 200 Cocoa bean 1.3 21.3 Low Medium High Shellfish 21.3 10.7 Fish mix 17.3 28 Level of adherence Whole egg 57.3 26.7 Yeast 86.7 0 Figure 2 Mean change in symptom severity scores at 12 weeks according to degree of adherence. Difference between the groups with high adherence: 101 (95% confidence interval 54, 147); ***p,0.001.

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A between atopic and non-atopic patients. There was however a 400 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 300 Sham * diet number of foods who fully adhered to their allocated diet, a (n=66) 26% difference in reduction in symptom severity score was observed in favour of the true diet (a difference in score of 98 200 True diet (n=65) (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, 100 70; p = 0.016) for each food to which they were sensitive

IBS symptom severity over and above the average number. These results were not materially altered by carrying out an ANCOVA analysis (in 0 which the final score is the dependent variable and the 04812 baseline score is included as a covariate) instead of modelling Time (weeks) change in scores.30 The interaction between treatment group and adherence is demonstrated in fig 2 which shows a B 400 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 300 *** Sham 12 weeks for the group as a whole and for those who fully diet adhered, respectively. This reveals that most improvements in (n=40) symptoms are fully achieved within two months. 200 True diet (n=24) Global impact score The reported global rating of change by treatment group is 100

IBS symptom severity shown in table 3. The difference in mean ranking (70.9 v 60.3) was statistically significant (p = 0.048). When this was 0 repeated including only patients who fully adhered to their 04812 diets (table 3), a greater percentage difference favouring the Time (weeks) 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. 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 Secondary outcome measures versus sham 39 (95% confidence interval 5, 72); *p = 0.024. (B) Average As can be seen from fig 4A and 4B, all data show changes symptom severity scores over time for the full adherence group. favouring the true diet group and are consistent with the Difference in mean change from baseline at 12 weeks: true versus sham 98 (95% confidence interval 52, 144); ***p,0.001. 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 between baseline characteristics of the 19 who were lost to non-colonic symptomatology (p = 0.05). There were no follow up and those for whom 12 week data were obtained. significant changes in medication use during the course of the trial. Primary outcomes IBS symptom severity Reintroduction of eliminated foods Patients in the true diet group experienced a 10% greater Of the 131 patients who gave 12 week data, 93 (41 in the true reduction in symptom severity than those allocated to the and 52 in the sham diet groups) agreed to attempt sham diet, with change in scores of 100 and 61.5, respectively reintroduction of foods they had been asked to eliminate (mean difference 39 (95% confidence interval (CI) 5.2, 72.3); and provided further follow up data on the primary outcomes p = 0.024): a standardised effect size of 0.52 (see fig 3A). measures. Of these, 62% reported full adherence and 37% There were no differences in the response to the diet in terms moderate adherence to the previous elimination diet. Mean of age, sex, IBS bowel habit subtype, or IBS duration. In IBS symptom severity score increased (that is, worsening of addition, there was no difference in response to the diet symptoms) by 83.3 in the true group and by 31 in the sham

Table 3 Global impact score at 12 weeks

Treatment group

True diet Sham diet (n (%)) (n (%))

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

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

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

Significantly worse 17 (41.5) 13 (25) No significant change 23 (56.1) 35 (67.3) 120 n=65 Significantly improved 1 (2.4) 4 (7.7) n=40 Total 41 (100) 52 (100) n=66

60

Non-colonic symptoms worsening of health compared with the sham diet group (p = 0.047). 0

180 p=0.27 p=0.27 DISCUSSION n=24 A clinically significant improvement in IBS symptomatology was observed in patients eliminating foods to which they n=65 120 were found to exhibit sensitivity, as identified by an ELISA test for the presence of IgG antibodies to these foods. The n=66 n=40 number needed to treat of 9 for the group as a whole and 2.5 for patients closely adhering to the diet are both considerably 60 Qualityof life better than the value of 17 achieved after three months of treatment with tegaserod,32 a drug that has been recently licensed in the USA for use in IBS. IBS symptom severity and 0 global rating scores were chosen as primary outcome measures in this study as they represented the most direct measure of clinical improvement in this condition based on patient self assessment. Rather than using the traditional True diet Sham diet method of classifying global improvement as any value All patients Full adherence exceeding adequate relief of symptoms, we used a much B stricter definition requiring patients to report symptoms as p=0.18 p=0.18 being either ‘‘better’’ or ‘‘excellent’’ compared with pretreat- 2.5 n=24 ment levels. Despite this, the diet still achieved a significant n=65 improvement. However, as might be expected, the placebo 2 response using this end point was somewhat lower than that usually reported in IBS treatment trials which have used less 1.5 n=66 n=40 demanding criteria. The observation that patients on the sham diet also improved, although to a lesser extent, 1 emphasises the importance of conducting double blind randomised controlled trials of such non-drug interventions 0.5 in order to avoid overestimating their potential. Most patients with IBS have attempted at least some form 0 of dietary modification, which in some cases can be very extreme. Conflicting results have been reported using 2.5 p=0.78 p=0.26 exclusion diets4 5 33–36 and this approach also suffers from the limitation that it has to be empirical. Thus potentially 2 n=24 offending foods can only be identified after their elimination and subsequent reintroduction. This time consuming process 1.5 would be much reduced if the offending foods could be identified beforehand. Attempts to do this using IgE 1 n=65 antibodies have been disappointing8–10 but the results of this

Depression Anxiety n=40 n=66 study suggest that measuring IgG antibodies may be much 0.5 more rewarding. The response to the IgG based diet in our trial did not correlate with atopic status, the prevalence of 0 which was found to be no greater than that occurring in the general population.37 Figure 4 (A) Mean change in the secondary outcome measures of non- The observation that adherence to the diet is critical in colonic symptoms and quality of life for the group as a whole and the full determining a good outcome in the ‘‘true’’ diet group but not adherence group. (B) Mean change in the secondary outcome measures the ‘‘sham’’ group is indicative of the fact that the diet is an of anxiety and depression for the group as a whole and the full ‘‘active treatment’’ which if not adhered to, does not seem to adherence group. have an effect. This notion is further supported by the observation that a significantly greater deterioration was group, a statistically significant difference of 52 (24%) (95% observed in subjects in the true diet group compared with CI 18, 86; p = 0.003). The change in global score following those in the sham group when they reintroduced eliminated reintroduction of foods is shown in table 4. This indicates a foods at the end of the diet phase of the trial. Furthermore, reversal of the pattern observed during the active treatment the improvement of 98 in the symptom severity score in those phase, with more patients in the true diet group showing 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 11 Crowe SE, Perdue MH. Gastrointestinal food hypersensitivity: basic 24 26–28 mechanisms of pathophysiology. Gastroenterol 1992;103:1075–95. validation studies and clinical practice. It was interesting 12 el Rafei A, Peters SM, Harris N, et al. Diagnostic value of IgG4 measurements to note that patients exhibiting a greater number of in patients with food allergy. Ann Allergy 1989;62:94–9. sensitivities, as determined by the IgG test, experienced a 13 Host A, Husby S, Gjesing B, et al. Prospective estimation of IgG, IgG subclass greater symptom reduction if they adhered to the true but not 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 the sham diet. milk challenge and clinical course of cow’s milk allergy. Allergy There is currently considerable interest in the concept that 1992;47:218–29. at least in some patients, IBS may have an inflammatory 14 Awazuhara H, Kawai H, Maruchi N. Major allergens in soybean and clinical 38–42 significance of IgG4 antibodies investigated by IgE and IgG4 immunoblotting component. Most of the work in this area has centred on with sera from soybean-sensitive patients. Clin Exp Allergy 1997;27:325–32. post dysenteric IBS, with gut pathogens being viewed as the 15 Barnes RMR, Johnson PM, Harvey MM, et al. Human serum antibodies initiators of this process which can be identified by subtle reactive with dietary proteins: IgG subclass distribution. Int Arch Allergy Appl 38 Immunol 1988;87:184–8. changes on histology. However, if, as indicated in this study, 16 Lessof MH, Kemeny DM, Price JF. IgG antibodies to food in health and IgG antibodies to food are important in the pathogenesis of disease. Allergy Proc 1991;12:305–7. IBS in some patients, they too may be of relevance. Not all 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. patients exhibiting histological features consistent with post 18 Haddad ZH, Vetter M, Friedmann J, et al. Detection and kinetics of antigen- dysenteric IBS give a history of a previous dysenteric illness. specific IgE and IgG immune complexes in food allergy. Ann Allergy This is usually assumed to be due to the fact that this has 1983;51:255. 19 Husby S, Oxelius VA, Teisner B, et al. Humoral immunity to dietary antigens in been forgotten by the patient but our results may suggest an healthy adults. Occurrence, isotype and IgG subclass distribution of serum alternative mechanism for immune activation and inflam- antibodies to protein antigens. Int Arch Allergy Appl Immunol mation without the need for prior infection. 1985;77:416–22. It is now well recognised that up to 70% of patients with 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 43 IBS have evidence of hypersensitivity of the rectum, which Immunol Ther Exp 1994;42:259–61. probably extends to involve most of the gut in many 21 Finn R, Smith MA, Youngs GR, et al. Immunological hypersensitivity to individuals.44 It is possible that this hypersensitivity renders environmental antigens in the irritable bowel syndrome. Br J Clin Pract 1987;41:1041–3. patients more reactive to a low grade inflammatory process 22 Drossman DA, Corazziari E, Talley NJ, et al. Rome II: a multinational which would not necessarily cause symptoms in a normal consensus document on functional gastrointestinal disorders. Gut individual. This would explain why excluding foods to which 1999;45:1–81. 23 Foster AP, Knowles TG, Hotston Moore A, et al. Serum IgE and IgG responses patients have IgG antibodies might be particularly beneficial to food antigens in normal and atopic dogs, and dogs with gastrointestinal in IBS despite the fact that these antibodies may also be disease. Vet Immunol Immunopathol 2003;92:113–24. present in the general population. Indeed, if this mechanism 24 Francis CY, Morris J, Whorwell PJ. The irritable bowel scoring system: A simple method of monitoring IBS and its progress. Aliment Pharmacol Therap is particularly important in IBS, it might be anticipated that 1997;11:395–402. IgG food antibodies would be relatively common in this 25 Whorwell PJ, McCallum H, Creed FH, et al. Non-colonic features of irritable condition, as was the case in our study. bowel syndrome. Gut 1986;27:452–6. 26 Houghton LA, Heyman DJ, Whorwell PJ. Symptomatology, quality of life and Many patients with IBS would prefer a dietary solution to economic features of irritable bowel syndrome—the effect of hypnotherapy. their problem rather than having to take medication, and the Aliment Pharmacol Ther 1996;10:91–5. economic benefits of this approach to health services are 27 Gonsalkorale WM, Toner BB, Whorwell PJ. Cognitive change in patients obvious. It is well known that patients expend large sums of undergoing hypnotherapy for irritable bowel syndrome. J Psychosom Res 2004;56:271–8. money on a variety of unsubstantiated tests in a vain attempt 28 Gonsalkorale WM, Houghton LA, Whorwell PJ. Hypnotherapy in irritable to identify dietary intolerances. The results of this study bowel syndrome: a large scale audit of a clinical service with examination of suggest that assay of IgG antibodies to food may have a role factors influencing responsiveness. Am J Gastroenterol 2002;97:954–61. 29 Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta in helping patients identify candidate foods for elimination Psychiatr Scand 1983;67:361–70. and is an approach that is worthy of further biomedical and 30 Everitt BS, Pickles A. Statistical aspects of the design and analysis of clinical clinical research. 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. Authors’ affiliations 32 Novick J, Miner P, Krause R, et al. A randomised, double blind, placebo W Atkinson, N Shaath, P J Whorwell, Department of Medicine, controlled trial of tegaserod in female patients suffering from irritable bowel University Hospital of South Manchester, Manchester, UK syndrome with constipation. Aliment Pharmacol Ther 2002;16:1877–88. 33 Niec AM, Frankum B, Talley NJ. Are adverse reactions to food linked to T A Sheldon, Department of Health Sciences, University of York, York, irritable bowel syndrome? Am J Gastroenterol 1998;93:2184–90. UK 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 REFERENCES syndrome. Lancet 1983;2:295–7. 1 Drossman DA, Camilleri M, Mayer EA, et al. American Gastroenterological 36 McKee AM, Prior A, Whorwell PJ. Exclusion diets in irritable bowel syndrome: Association Technical Review on Irritable Bowel Syndrome. Gastroenterology Are they worthwhile? J Clin Gastroenterol 1987;9:526–8. 2002;123:2108–31. 37 Durham SR, Church MK. Principles of allergy diagnosis. In: Holgate ST, 2 Lea R, Whorwell PJ. Quality of life in irritable bowel syndrome. Church MK, Lichtenstein LM, eds. Allergy, 2nd edn. London: Mosby, Pharmacoeconomics 2001;19:643–53. 2001:3–16. 3 Talley NJ, Spiller R. Irritable bowel syndrome: a little understood organic 38 Spiller RC, Jenkins D, Thornley JP, et al. Increased rectal mucosal bowel disease? Lancet 2002;360:555–64. enteroendocrine cells, T lymphocytes, and increased gut permeability 4 Jones VA, McLaughlan P, Shorthouse M, et al. Food intolerance: a major following acute Campylobacter enteritis and in post-dysenteric irritable bowel factor in pathogenesis of irritable bowel syndrome. Lancet 1982;2:1115–17. syndrome. Gut 2000;47:804–11. 5 Nanda R, James R, Smith H, et al. Food intolerance and the irritable bowel 39 Gonsalkorale WM, Perrey C, Pravica V, et al. Interleukin 10 genotypes in syndrome. Gut 1989;30:1099–104. irritable bowel syndrome: evidence for an inflammatory component? Gut 6 Zwetchkenbaum J, Burakoff, R. The irritable bowel syndrome and food 2003;52:91–3. hypersensitivity. Ann Allerg 1988;61:47–9. 40 Collins SM, Piche T, Rampal P. The putative role of inflammation in the irritable 7 Zar S, Kumar D, Benson M. J. Review article: food hypersensitivity and bowel syndrome. Gut 2001;49:743–5. irritable bowel syndrome, Aliment Pharm Ther 2001;15:439–43. 41 Collins SM. A case for an immunological basis for irritable bowel syndrome. 8 Petitpierre M, Gumowski P, Girard JP. Irritable bowel syndrome and Gastroenterology 2002;122:2078–80. hypersensitivity to food. Ann Allergy 1985;54:538–40. 42 Chadwick VS, Chen W, Shu D, et al. Activation of the mucosal immune system 9 Barau E, Dupont C. Modifications of intestinal permeability during food in irritable bowel syndrome. Gastroenterology 2002;122:1778–83. provocation procedures in pediatric irritable bowel syndrome. J Pediatr 43 Mertz H. Review article: visceral hypersensitivity. Aliment Pharmacol Ther Gastroenterol Nutr 1990;11:72–7. 2003;17:623–33. 10 Roussos A, Koursarakos P, Patsopoulos D, et al. Increased prevalence of 44 Francis CY, Houghton LA, Whorwell PJ. Enhanced sensitivity of the whole gut irritable bowel syndrome in patients with bronchial asthma. Respir Med in patients with irritable bowel syndrome. Gastroenterology 2003;97:75–9. 1995;108:601(abstract).

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LEADING ARTICLE Food allergy in irritable bowel syndrome: new facts and old fallacies

E Isolauri, S Rautava, M Kallioma¨ki ......

Gut 2004;53:1391–1393. doi: 10.1136/gut.2004.044990 The notion of food allergy in irritable bowel syndrome (IBS) the presence or absence of disease, but the complex cascade of events determining their use. is not new. However, recent evidence suggests significant The notion of food allergy in irritable bowel reduction in IBS symptom severity in patients on elimination syndrome (IBS) resurfaces in scientific thinking diets, provided that dietary elimination is based on foods in this issue of Gut3 on the basis of a solid randomised placebo controlled trial conducted by against which the individual had raised IgG antibodies. Atkinson and colleagues (see page 1459). These findings should encourage studies dissecting the Determination of serum IgG antibodies against mechanisms responsible for IgG production against dietary foods was used to guide the construction of elimination diets. antigens and their putative role in IBS The presence of specific IgG class antibodies is ...... often accepted as uncommitted or protective ‘‘altered reactivity’’, unlike those of the IgE class. Detection of antigen specific IgE is invariably ringing empirical observations ad fontes taken as an attribute of causality, a condition advances science. In astrophysics, the term called ‘‘IgE mediated disease’’ and, more speci- ‘‘black hole’’ was introduced to describe an B fically, of ‘‘allergy’’.1 However, empirical data are extremely dense star which had collapsed into a accumulating to suggest that transient increases singularity under its own gravity. A black hole in antigen specific IgE antibodies prevail in most radiates nothing; it absorbs all matter and energy healthy asymptomatic children during the first falling within its sphere. The name was coined five years of life.4 Secondly, generation of these only after revisiting the initial theoretical antibodies (sensitisation) on antigen exposure achievements of Karl Schwarzschild, when may not necessarily induce clinical disease observations made outside the earth’s atmos- (atopic disease).5 Thirdly, reducing the risk of phere gave astrophysicists empirical ray data on x atopic disease does not necessitate reduction of a new type of cosmic object. In allergology, in sensitisation6–8 and, finally, resolution or aggra- contrast, adherence to a paradigm whereby vation of clinical disease is not invariably allergy is defined by the presence of specific IgE associated with a corresponding alteration in antibodies has hampered disentanglement. As a antibody concentration. Bearing these limita- result, allergy remains a dubiously defined term tions in mind, however, the clinician may with no unambiguous empirical content or successfully profit from determination of specific explanatory power. The time has come to seize IgE to complete the clinical history in an attempt upon the available empirical data and plunge to identify potential offending antigens in a into the original theory of Clemens von Pirquet. symptomatic patient’s diet for the explicit The term allergy was introduced by von diagnostic elimination-challenge procedure.9 Pirquet to denote a changed immunological This is precisely what Atkinson et al did, with reactivity which manifests itself on second specific IgG antibodies.3 They identified a sig- 1 exposure to an antigen (reviewed by Kay ). This nificant reduction in IBS symptom severity in altered reactivity is uncommitted, giving no patients on elimination diets, provided that indication of the direction of change; equally dietary elimination was based on foods against harmful and protective immune reactivity which the individual had raised IgG antibodies; reflects prior encounter (see fig 1). In modern fully compliant patients showed the best clinical terms, altered reactivity can be seen to evince improvement. The reverse pattern was observed See end of article for either the most common mode of immune after reintroduction of the respective foods. authors’ affiliations response elicited by the intestinal immune ...... system, tolerance, recently defined as any ‘‘IBS appears to result from an interplay Correspondence to: mechanism by which a potentially injurious Dr E Isolauri, Department immune response is prevented, suppressed, or between susceptibility genes and impaired of Paediatrics, Turku shifted to a non-injurious class of immune gut barrier functions, immunological dysre- University Central response,2 or abrogation of such an actively gulation, together with bacterial and viral Hospital, 20520 Turku, Finland; erika.isolauri@ maintained process, which is currently linked infections and other environmental factors’’ utu.fi to immunoinflammatory disease. Reassessment of the original theory of allergy is important as it In common with allergic disease, IBS appears Revised version received would appear that it is not the immunological to result from an interplay between susceptibility 10 June 2004 Accepted for publication resources gained during antigen exposure, mea- 19 June 2004 surable by specific antibodies or specifically Abbreviations: IBS, irritable bowel syndrome; PRR, ...... responding lymphocytes, which are decisive for pattern recognition receptor

www.gutjnl.com 1392 Isolauri, Rautava, Kallioma¨ki

Th2 Th1 Allergic IBD Inflammation disease IBS?

Normal range

Naïve Tolerance uncommitted Antigen Consolidation of Exposure immune reactivity

Figure 1 An attempt to reformulate the original conception of the immunological basis of allergy. On exposure to dietary and microbial antigens, the naı¨ve immune system matures either to establish appropriate immune reactivity and tolerance or, in the case of lack of suppressive and regulatory signals, develops inflammatory disease expressed as Th1 skewed autoimmune reactivity (for example, inflammatory bowel disease (IBD)) or Th2 skewed allergic disease. The exact nature of immune reactivity in irritable bowel syndrome (IBS) on this axis remains elusive. genes and impaired gut barrier functions, immunological addition to its effects on T cell function, transforming growth dysregulation, together with bacterial and viral infections factor b is a key factor in IgA production21 and thus and other environmental factors. It is no easy matter to contributes to maintenance of gut barrier function and to describe succinctly ‘‘gut barrier function’’. In the gastro- immune responses at other mucosal surfaces also. Taken intestinal tract, the external and internal environments are in together, ‘‘gut barrier function’’ strongly depends on antigen close proximity. The dilemma of the mucosal surface of the processing and presentation and the cytokine milieu in the intestine is to fend off the constant challenge from antigens, mucosal immune system, and determines the nature of the such as microorganisms, in mounting a brisk response to immune response (that is, tolerance or inflammation) pathogens, and to enable assimilation of innocuous antigens elicited to a particular antigen. derived from food. In order to perform these opposing functions, the intestine is in a state of continuous immune ‘‘Inflammation can cause profound alterations in the responsiveness, and a delicate balance is generated and function of smooth muscle and enteric nerves as well as maintained between concomitant facilitation and suppres- in deeper neuromuscular layers’’ sion of inflammatory responses. Gut barrier function consists of physiological and immu- In certain circumstances, such as metabolic stress, the nological factors which exclude and degrade antigens and peaceful coexistence across the barrier is disturbed and an restrict their adherence, penetration, and transfer. Antigen inflammatory response ensues.22 Abrogated barrier function presenting cells, and more precisely dendritic cells, are pivotal of the gut mucosa leads to greater antigen transfer when the 10–12 in directing mucosal immune responses. Three dendritic routes of transport are also altered, thereby evoking aberrant cell derived signals are required for an effective T cell immune responses and release of proinflammatory cytokines 11 response. The nature of signal 1 depends on the antigen with further impairment of barrier function. Inflammation in question and its processing; necessary costimulatory can cause profound alterations in the function of smooth molecules create the second signal and the pericellular muscle and enteric nerves as well as in deeper neuromuscular cytokine milieu is the basis of the third. On antigen layers.23 Indeed, a subtle inflammatory response and exag- recognition, maturation of dendritic cells and secretion of gerated sensitivity to that type of response has been cytokines and chemokines occur. These secretions direct the suggested to be causative in IBS. In view of recently reported polarisation of a naı¨ve T helper cell to type 1, type 2, or a alterations in the immunological defence in IBS,24 the regulatory T cell and thus regulate other adaptive immune trigger(s) of the vicious circle can be depicted among the responses, such as B cell derived immunoglobulin produc- intraluminal antigens. tion.11 Tolerance to lumenal dietary and microbial antigens is In this issue of Gut, Atkinson and colleagues3 describe IgG likely to be achieved through those dendritic cells which antibody responses to dietary antigens of clinical significance induce production of regulatory T cells secreting interleukin and an apparent causal relation to symptoms in IBS, in a 10 and transforming growth factor b.13 These cytokines fashion resembling the elimination-challenge procedure in promote gut barrier function by suppressing the production food allergy. To broaden this concept, it is intriguing to of both T helper 1 and 2 cytokines,14 overexpression of which speculate that IBS may perhaps also be associated with IgG is associated with increased gut permeability.15 16 Moreover, antibodies against other intraluminal antigens such as those the anergic T cells induced by interleukin 10 exposed from the indigenous microbiota, partially analogously to loss dendritic cells appear to be able to suppress other T cells in of tolerance to gut microbiota in inflammatory bowel an antigen specific manner.17 Transforming growth factor b disease.25 26 downregulates both T helper 1 and 2 responses directly18 and The human gastrointestinal tract harbours a complex indirectly by modulating the activity of antigen presenting collection of microorganisms which form the individual cells19 and favouring the development of regulatory T cells.20 microbiota typical for each person. Defence is facilitated by After intestinal priming, these cells migrate to the periphery, peristalsis, secretion of mucus and antimicrobial peptides thus mediating peripheral tolerance on reactivation. In such as defensins and cathelicidins, and commensal induced

www.gutjnl.com Food allergy in IBS 1393

IgA.27 28 Intestinal epithelial cells further contribute to the 5 Lau S, Illi S, Sommerfeld C, et al. Early exposure to house-dust mite and cat allergens and development of childhood asthma: a cohort study. Lancet homeostasis of gut barrier function by a scarcity of both 2000;356:1392–7. pattern recognition receptors (PRRs; for example, toll-like 6 Kallioma¨ki M, Salminen S, Kero P, et al. Probiotics in the primary prevention receptors and nucleotide binding oligomerisation domain of atopic disease: a randomised, placebo-controlled trial. Lancet proteins) and their coreceptors, expression of active negative 2001;357:1076–9. 7 Riedler J, Braun-Fahrla¨nder C, Eder W, et al. Exposure to farming in early life regulators of PRR signalling, and secretion of the suppressive and development of asthma and allergy: a cross-sectional survey. Lancet cytokines interleukin 10 and transforming growth factor b.13 2001;358:1129–33. All of these characteristics assist in preventing unnecessary 8 Jones CA, Holloway JA, Popplewell EJ, et al. Reduced soluble CD14 levels in amniotic fluid and breast milk are associated with the subsequent development and even hazardous systemic immunity to commensals while of atopy, eczema, or both. J Allergy Clin Immunol 2002;109:858–66. allowing local protective mucosal immune responses. In 9 Bock SA. Diagnostic evaluation. Pediatrics 2003;111:1638–44. addition, some specific strains of non-pathogenic bacteria 10 Pulendran B, Banchereau J, Maraskovsky E, et al. Modulating the immune response with dendritic cells and their growth factors. Trends Immunol have been shown to attenuate intestinal inflammation by 2001;22:41–7. selective inhibition of intracellular signalling pathways 11 Kapsenberg ML. Dendritic-cell control of pathogen-driven T-cell polarization. elicited by diverse potentially deleterious stimuli.29 30 A Nat Rev Immunol 2003;3:984–93. healthy gut microbiota is thus an indispensable component 12 Stagg AJ, Hart AL, Knight SC, et al. The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria. Gut 2003;52:1522–9. of ‘‘gut barrier function’’. 13 Nagler-Anderson C, Bhan AK, et al. Control freaks: immune regulatory cells. Nat Immunol 2004;5:119–22. 14 Rissoan MC, Soumelis V, Kadowaki N, et al. Reciprocal control of T helper cell ‘‘A healthy gut microbiota is thus an indispensable and dendritic cell differentiation. Science 1999;283:1183–6. component of gut barrier function’’ 15 Heyman M, Darmon N, Dupont C, et al. Mononuclear cells from infants allergic to cow’s milk secrete tumor necrosis factor a, altering intestinal function. Gastroenterology 1994;106:1514–23. The findings of Atkinson and colleagues3 should encourage 16 Berin MC, Yang PC, Ciok L, et al. Role for IL-4 in macromolecular transport studies dissecting the mechanisms responsible for IgG across human intestinal epithelium. Am J Physiol 1999;276:C1046–52. 17 Steinbrink K, Graulich E, Kubsch S, et al. CD4(+) and CD8(+) anergic T cells production against dietary antigens and their putative role induced by interleukin-10-treated human dendritic cells display antigen- in IBS. This may serve not only IBS research but also that into specific suppressor activity. Blood 2002;99:2468–76. allergy and allergic diseases. In the perspectives of both 18 Lu´dviksson BR, Seegers D, Resnick AS, et al. The effect of TGF-beta1 on immune responses of naive versus memory CD4+ Th1/Th2 T cells. normal gut barrier function and the vague findings in a few Eur J Immunol 2000;30:2101–11. 31–33 studies of probiotic supplementation in IBS, we suggest 19 Takeuchi M, Alard P, Streilein JW. TGF-beta promotes immune deviation by that the possible role of the gut microbiota in the pathogen- altering accessory signals of antigen-presenting cells. J Immunol esis of IBS may deserve closer attention. If the host-microbe 1998;160:1589–97. 20 Toms C, Powrie F. Control of intestinal inflammation by regulatory T cells. cross talk is misinterpreted in IBS, a working target for novel Microbes Infect 2001;3:929–35. therapeutic interventions beyond elimination diets could be 21 Stavnezer J. Regulation of antibody production and class switching by TGF- provided in modulating the composition and/or activity of the beta. J Immunol 1995;155:1647–51. 22 Nazli A, Yang PC, Jury J, et al. Epithelia under metabolic stress perceive gut microbiota and promoting gut immune defence. Research commensal bacteria as a threat. Am J Pathol 2004;164:947–57. interest in the science of nutrition is directed towards 23 Collins SM, Piche T, Rampal P. The putative role of inflammation in the irritable improvement of defined physiological functions beyond the bowel syndrome. Gut 2001;49:743–5. 24 Gonsalkorale WM, Perrey C, Pravica V, et al. Interleukin 10 genotypes in nutritional impact of food. The search for active non-nutritive irritable bowel syndrome: evidence for an inflammatory component? Gut compounds is also a focus for research in the treatment and 2003;52:91–3. prevention of allergic diseases. 25 Duchmann R, Kaiser I, Hermann E, et al. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin ...... Exp Immunol 1995;102:448–55. 26 Lodes MJ, Cong Y, Elson CO, et al. Bacterial flagellin is a dominant antigen in Authors’ affiliations Crohn disease. J Clin Ivest 2004;113:1296–306. E Isolauri, S Rautava, Department of Paediatrics, University of Turku and 27 Otte J-M, Kiehne K, Herzig K-H. Antimicrobial peptides in innate immunity of Turku University Central Hospital, Finland the human intestine. J Gastroenterol 2003;38:717–26. M Kallioma¨ki, Massachusetts General Hospital East, Combined Program 28 Macpherson AJ, Uhr T. Induction of protective IgA by intestinal dendritic cells in Pediatric Gastroenterology and Nutrition, Charlestown, carrying commensal bacteria. Science 2004;303:1662–5. 29 Haller D, Jobin C. Interaction between resident luminal bacteria and the host: Massachusetts, USA can a healthy relationship turn sour? J Pediatr Gastroenterol Nutr 2004;38:123–36. 30 Kelly D, Campbell JI, King TP, et al. Commensal anaerobic gut bacteria REFERENCES attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPAR-c 1 Kay AB. Concepts of allergy and hypersensitivity. In: Kay AB, ed. Allergy and and RelA. Nat Immunol 2004;5:104–12. allergic diseases. Oxford: Blackwell Science Ltd, 1997:23–35. 31 Nobaek S, Johansson M-L, Molin G, et al. Alteration of intestinal microflora is 2 Weiner HL. Oral tolerance: immune mechanisms and the generation of Th3- associated with reduction in abnormal bloating and pain in patients with type TGF-beta-secreting regulatory cells. Microbes Infect 2001;3:947–54. irritable bowel syndrome. Am J Gastroenterol 2000;95:1231–8. 3 Atkinson W, Sheldon TA, Shaath N, et al. Food elimination based on IgG 32 Sen S, Mullan MM, Parker TJ, et al. Effect of Lactobacillus plantarum 299v on antibodies in irritable bowel symdrome: a randomised controlled trial. Gut colonic fermentation and symptoms of irritable bowel syndrome. Dig Dis Sci 2004;53:1459–64. 2002;47:2615–20. 4 Kulig M, Bergmann R, Klettke U, et al. Natural course of sensitization to food 33 Kim HJ, Camilleri M, McKinzie S, et al. A randomized controlled trial of a and inhalant allergens during the first 6 years of life. J Allergy Clin Immunol probiotic, VSL#3, on gut transit and symptoms in diarrhoea-predominant 1999;103:1173–9. irritable bowel syndrome. Aliment Pharmacol Ther 2003;17:895–904.

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HEADACHE CARE 1742–3430 VOL. 2, NO. 2, 2005, 105–110 doi:10.1185/174234305X14962

© 2005 LIBRAPHARM LIMITED

ORIGINAL ARTICLE A Prospective Audit of Food Intolerance among Migraine Patients in Primary Care Clinical Practice Trevor Rees1, David Watson2, Susan Lipscombe3, Helen Speight4, Peter Cousins4, Geoffrey Hardman5 and Andrew J. Dowson6 1Hawthorns Surgery, Sutton Coldfield, West Midlands, UK 2Hamilton Medical Group, Aberdeen, Scotland 3Park Crescent New Surgery, Brighton, East Sussex, UK 4YORKTEST Laboratories Ltd (YTL), Osbaldwick, York, UK 5Centre for Health Economics, University of York, York, UK 6King’s Headache Service, King’s College Hospital, London, UK

Address for correspondence: Dr Andrew J. Dowson, The King’s Headache Service, King’s College Hospital, Denmark Hill, London SE5 9RS, UK. Tel./Fax: +44-1428-712546; email: [email protected] Key words: Diet – Food intolerance – Migraine – Primary care

SUMMARY

This prospective audit was set up to investigate took part in the audit and 39 completed 2 months whether migraine sufferers have evidence of of investigation. The mean number of foods IgG-based food intolerances and whether their identified in the IgG test was 5.3 for all condition can be improved by the withdrawal from participants and 4.7 for those successfully the diet of specific foods identified by intolerance altering their diet. About 90% of patients testing. Migraine patients were recruited from changed their diet to a greater or lesser primary care practices and a blood sample was extent following the identification of possible taken. Enzyme-linked immunosorbent assays food intolerances. A marked proportion of the (ELISA) were conducted on the blood samples to migraine patients benefited from the dietary detect food-specific IgG in the serum. Patients intervention, approximately 30% and 40% identified with food intolerances were encouraged reporting considerable benefit at 1 and 2 to alter their diets to eliminate appropriate foods months, respectively. Also, over 60% of patients and were followed up for a 2-month period. who reintroduced the suspect foods back into Endpoints included identification of the specific their diets reported the return of their migraine foods that the patients were intolerant to, symptoms. This investigation demonstrated assessing the proportion of patients who altered that food intolerances mediated via IgG may their diet and the benefit obtained by these be associated with migraine and that changing patients at 1 and 2 months. Patients reported the the diet to eradicate specific foods may be a level of benefit on a 6-point scale, where 0 = no potentially effective treatment for migraine. benefit and 5 = high benefit. Sixty one patients Further clinical studies are warranted in this area.

Paper 0047 105 Introduction The present audit was set up to investigate whether migraine sufferers have evidence of IgG-based food Dietary components are frequently proposed as precip- intolerances and whether their condition can be itating factors for migraine, particularly in children and improved by the withdrawal from the diet of specific adolescents1,2. Many different foods have been implic- foods identified by intolerance testing. ated as potential triggers for migraine attacks, including chocolate, cheese, red wine and many others3. However, evidence for this interaction is mostly anecdotal and Patients and Methods based on patient reports4. Open studies indicated that 5 6 low-fat and high carbohydrate diets could lead to Patients improvements in migraine frequency and/or severity. In contrast, no controlled study has confirmed the incidence Established adult migraine patients (age > 18 years) of food-evoked migraine attacks. A controlled study were recruited from primary care clinical practices by with chocolate failed to show that it provoked migraine their GPs. Patients were required to have high-impact attacks7. An alternative concept of the relationship of headaches. Patients were diagnosed with episodic food with migraine is that food cravings occur during migraine (≤ 15 days of headache per month) or chronic the prodrome phase; the food intake thus being a migraine (> 15 days of headache per month), according consequence of the attack rather than a cause of it8. to the GP’s usual practices. All patients provided their Migraine may be precipitated by food via chemical or written informed consent to take part in the audit. immunological mechanisms. Dietary components may affect phases of the migraine process by influencing Study Design release of serotonin and noradrenaline, causing vaso- constriction or vasodilatation, or by direct stimulation This prospective audit investigated whether migraine of trigeminal ganglia, brainstem and cortical neuronal patients identified in primary care clinical practice pathways1. Immunological reactions may be mediated exhibited food intolerances measured as elevated IgG by Immunoglobulin E (IgE [classical food allergies levels to specific foods. The audit also investigated the occurring immediately after eating]) or, more controv- effect of withdrawing foods associated with high IgG ersially, by Immunoglobulin G (IgG [food intolerance levels on patients’ migraine attacks over a 2-month involving a delayed allergic reaction 2–120 hours period. after eating]). Available evidence indicates that an Primary care physicians were briefed on the rationale IgE mechanism is relatively unimportant in food- and objectives of the audit at a meeting of the UK induced migraine9 and a review of the clinical literature charity Migraine in Primary Care Advisors (MIPCA) established no clear evidence of an immune dysfunction and agreed to participate. Each physician recruited in migraine sufferers10. However, the role of a putative up to 20 migraine patients and provided them with IgG mechanism is presently unknown. information about the audit. Before entering the study, The usual way to treat food intolerance is by food all patients completed a baseline questionnaire to elimination and re-challenge procedures, which record demography and allergy history and a Headache are imprecise, lengthy and inefficient. As a more Impact Test (HIT-613) questionnaire to record headache efficient alternative to this approach, an enzyme-linked severity. immunosorbent assay (ELISA) test to a panel of 113 Patients who completed the initial questionnaires food allergen extracts has been developed (YORKTEST were sent a validated blood testing kit by YTL. Patients Laboratories Ltd [YTL], York, UK). This detects raised took a blood sample by skin prick as detailed in the food-specific IgG in the serum of people with one or leaflet enclosed with the testing kit and returned the more, usually chronic, conditions. Patients with raised kit to YTL by mail. The blood samples were processed IgG levels to specific foods are advised to remove these by YTL on receipt of the questionnaires and blood kit. from their diets and their progress is monitored with ELISA tests on blood samples were used to detect food- a series of questionnaires. An independent audit of specific IgG in the serum of the blood samples. Results patients treated in this way between February 1998 and of the ELISA tests were sent directly to the patients by August 1999 showed that approximately 50% of all YTL, together with a guidebook on food intolerances patients reported a high or relatively high response and their treatment14. to dietary therapy, based on their levels of food- Patients were free to change their diets to eliminate specific IgGs11. A randomised, controlled clinical trial specific foods identified by the ELISA tests as possibly has demonstrated beneficial effects of this form of causing intolerance, either on their own initiative or dietary therapy on symptom relief for irritable bowel after consultation with their GP or other healthcare syndrome12. professional. Patients had access by telephone to a

106 A Prospective Audit of Food Intolerance among Migraine Patients in Primary Care Clinical Practice © 2005 LIBRAPHARM LTD – Headache Care 2005; 2(2) professional dietitian to help them with any dietary The majority of patients (78.0%) were in full-time alterations that they wished to implement. Follow-up education or employment. In examining the allergy questionnaires were sent to patients after 1 and 2 months history, 15 patients (24.6%) were aware of foods they to monitor their progress (investigation period). felt they were allergic to, 42 (68.9%) were in contact with pets, 12 (20.0%) were in contact with chemicals Study Endpoints and Statistical Analyses or occupational dust, 52 (86.7%) were currently taking medication and 21 (34.4%) knew about medications The main study endpoints were: they felt they were allergic to. Fifteen patients (24.6%) were current smokers and 18 (29.5%) had given up • Demographic data on the patient population, and smoking. Forty three patients (72.9%) drank alcohol but details of their allergy and headache histories, analysed only eight drank over seven units per week and only one as descriptive statistics. drank more than 14 units per week. • Identification of the specific foods to which the Most patients had suffered from headache for a patients could be intolerant, identified from the considerable time; 64% for ≥ 10 years, 20% for 5–10 ELISA tests of IgG levels and analysed as descriptive years and 16% for < 5 years. Patients were severely statistics. affected by their headaches (Table 2). Eighty two per • The proportion of patients who altered their diet due cent ‘very often’ or ‘always’ had severe pain, while to their ELISA test results, analysed as descriptive 67% were ‘very often’ or ‘always’ limited in their usual statistics. activities during their headaches. Between 87% and 90% • The benefit obtained at 1 and 2 months by the of patients were too tired to work, felt irritation and patients who altered their diet compared with the suffered from lack of concentration at least sometimes situation before diet alteration, analysed as descriptive during their attacks. Patients reported a mean of 10.1 statistics. Patients reported their level of benefit on a symptoms (range 1–24) associated with their headaches. 6-point scale, where 0 = no benefit and 5 = high Over 80% of patients reported that their headaches benefit. interfered with sleep, leisure and overall comfort. The mean weighted HIT score at baseline was 64.9 (range 48–78), corresponding to severe impact13. Results Table 1. Baseline demography (n = 61) Patient Disposition Gender Male 12 20.0% Female 48 80.0% Sixty-one patients from six UK GP practices (range Age group Under 30 10 16.7% 1–17 per centre) were recruited into the audit and 30 to 39 9 15.0% completed baseline assessments. In the investigation 40 to 49 14 23.3% period, 46 patients (75.4%) continued in the study to 1 50 to 59 21 35.0% 60 and over 6 10.0% month and 39 (63.9%) to 2 months. Employment status Retired 3 5.1% Sick/disabled 4 6.8% Housewife 3 5.1% Baseline Demography and Headache Part time 2 3.4% Severity Full time skilled 25 42.4% Full time semi-skilled 17 28.8% Table 1 shows the demography of the patients who Full time unskilled 2 3.4% took part in the study. The average age was 45.2 years Student 2 3.4% Unemployed 1 1.7% (range 21–68) and most patients (80%) were women.

Table 2. Severity of patients’ headaches: pain intensity, impact on daily activities and mood alterations

Headache severity Proportion of patients (%) Never Rarely Sometimes Very often Always Severe pain 2 0 16 61 21 Limit to usual activities 0 2 31 51 16 Desire to lie down 2 0 18 41 39 Too tired to work 5 8 51 31 5 Irritation 5 7 33 39 16 Lack of concentration 3 7 38 40 12

© 2005 LIBRAPHARM LTD – Headache Care 2005; 2(2) A Prospective Audit of Food Intolerance among Migraine Patients in Primary Care Clinical Practice Rees et al. 107 Identification of Food Intolerances had reactions to a total of 36 different foods, with an average of 4.7 (range 0–17) reactions per patient. Table 3 Food intolerances identified by IgG testing were analysed shows the distribution of food intolerances in these two for the 61 patients who took part in the study and for populations. The most frequently reported intolerances the 39 who completed the 2 months of investigation. In (in over 10% of patients in either population) were to the total study population, 60 of 61 patients (98.4%) had cow’s milk, yeast, egg white, egg yolk, wheat, gluten reactions to a total of 48 different foods, with an average (gliadin), corn, cashew nuts, mollusc mix, brazil nut, of 5.3 (range 0–17) reactions per patient. In the patients cranberry and garlic (Table 3), and were similar in who completed 2 months, 38 of 39 patients (97.4%) prevalence in the two populations.

Table 3. Food intolerances in the audit population: number and proportion of patients with a positive ELISA test to IgG from various foodstuffs

Food Positive ELISA test ( n [%]) Whole study population Patients completing 2 months ( n = 61) ( n = 39) Cow’s milk 52 (85.2%) 34 (87.2%) Yeast 37 (60.7%) 22 (56.4%) Egg white 34 (55.7%) 23 (59.0%) Egg yolk 20 (32.8%) 13 (33.3%) Wheat 19 (31.1%) 12 (30.8%) Gliadin 16 (26.2%) 10 (25.6%) Corn 15 (24.6%) 8 (20.5%) Cashew 12 (19.7%) 7 (17.9%) Mollusc mix 10 (16.4%) 3 (7.7%) Brazil nut 9 (14.8%) 6 (15.4%) Cranberry 7 (11.5%) 5 (12.8%) Garlic 5 (8.2%) 4 (10.3%) Beef 3 (4.9%) 2 (5.1%) Pork 3 (4.9%) 1 (2.6%) Ginger 3 (4.9%) 2 (5.1%) Buckwheat 4 (6.6%) 1 (2.6%) Crustacean mix 5 (8.2%) 1 (2.6%) Rye 2 (3.3%) 2 (5.1%) Millet 3 (4.9%) 2 (5.1%) Rice 1 (1.6%) 1 (2.6%) Soya bean 5 (8.2%) 3 (7.7%) Hazelnut 4 (6.6%) 3 (7.7%) Mustard seed 1 (1.6%) 1 (2.6%) Salmon/trout 2 (3.3%) 1 (2.6%) Plaice/sole 3 (4.9%) 1 (2.6%) Peanut 3 (4.9%) 2 (5.1%) Chicken 3 (4.9%) 1 (2.6%) Lentils 3 (4.9%) 1 (2.6%) Pea 2 (3.3%) 1 (2.6%) Almond 5 (8.2%) 3 (7.7%) Cola nut 3 (4.9%) 1 (2.6%) Duck 1 (1.6%) 0 Lamb 3 (4.9%) 1 (2.6%) Turkey 2 (3.3%) 0 White fish 3 (4.9%) 1 (2.6%) Kiwi 4 (6.6%) 2 (5.1%) Pineapple 2 (3.3%) 0 Sunflower seed 2 (3.3%) 0 Oat 2 (3.3%) 0 Haricot bean 3 (4.9%) 2 (5.1%) Coconut 1 (1.6%) 1 (2.6%) Tea 1 (1.6%) 0 Carrot 1 (1.6%) 0 Barley 1 (1.6%) 0 Tuna 1 (1.6%) 0 Sesame seed 1 (1.6%) 0 Coffee 1 (1.6%) 0 Avocado 1 (1.6%) 0

108 A Prospective Audit of Food Intolerance among Migraine Patients in Primary Care Clinical Practice © 2005 LIBRAPHARM LTD – Headache Care 2005; 2(2) Proportion of Patients who Altered their Diets migraine, reporting high levels of pain and impact on their everyday activities. This is a group of patients who are Of the 46 patients who returned the questionnaire after typically poorly managed in primary care15 and for whom 1 month of investigation, 41 (89.1%) patients changed new management initiatives would be welcome. their diets to eliminate foods identified by the IgG Almost all patients had multiple food intolerances testing and 5 (10.9%) did not. Of those who changed in this investigation, identified as positive food-specific their diet, 19 (46.3%) reported that they altered their IgG test results. Typically, individuals were positive to at diets a lot and 22 (53.7%) reported they had made a least one of cow’s milk, egg and yeast, together with a ‘reasonable attempt’ to avoid the specified foods. small number of more individual reactions. These results Of the 39 patients who returned the questionnaire are similar to those reported for other conditions11,12. Of after 2 months of investigation, 22 (56.4%) reported the patients who took part in the investigation, about that they altered their diets a lot and 13 (33.3%) 90% changed their diet to a greater or lesser extent at reported they had made a ‘reasonable attempt’ to avoid both 1 and 2 months. the specified foods. Two patients reported that they did A marked proportion of the migraine patients not change their diet at all. benefited from dietary intervention by cutting out foods for which they had an elevated IgG level. Approx- Benefit Obtained from Changing Diets imately 30% and 40% reported considerable benefit at 1 and 2 months, respectively. Reinforcing this is the fact Figures 1 and 2 show the level of benefit reported by that over 60% of patients who re-introduced the suspect patients after 1 and 2 months, respectively, using the 6- foods back into their diets reported the return of their point scale (0 = no benefit and 5 = high benefit). After migraine symptoms. These results are encouraging and 1 month, 27.5% of patients reported considerable benefit indicate that changing diet to counteract food intoler- (scoring 4 or 5), while 30.0% reported little or no benefit (scoring 0 or 1). Of 18 patients who had retried foods they had stopped taking, five (27.8%) reported a strong return of migraine symptoms and seven (38.9%) a slight return. After 25 22.5 2 months, 38.2% of patients reported considerable benefit 20.0 (scoring 4 or 5), while 32.4% reported little or no benefit 20 (scoring 0 or 1). Of 26 patients who had retried foods they 15.0 15 13.1 12.5 had stopped taking, seven (26.9%) reported a strong return 10.0 10 of migraine symptoms and 11 (42.3%) a slight return. % patients A limited post hoc analysis was conducted to investigate 5 the factors possibly associated with benefit. Of the 13 patients who reported considerable benefit from dieting 0 0 1 2 3 4 5 after 2 months, nine (69.2%) said they had dieted strictly No High after 1 month and 12 (92.3%) after 2 months. Of the 11 benefit benefit patients who reported little or no benefit after 2 months, Figure 1. Benefit of the diet after 1 month of the investigation: only two (18.2%) had dieted strictly after 1 month and proportion of patients reporting their level of benefit on a five (45.5%) after 2 months. Compared to those who did 6-point scale, where 0 = no benefit and 5 = high benefit not benefit, the patients who benefited were more likely to have suffered from bloating and sleep deprivation and to have never smoked (although all patients had given up at 25 least 10 years previously). Those who reported no benefit 20.6 20.6 20 17.6 17.6 from dieting were more likely to be trying other remedies 15 as well, including avoiding chocolate and taking sumatriptan 11.8 11.8 and homeopathic remedies. However, none of the above 10 differences was testable for statistical significance due to % patients the small number of patients involved. 5

0 0 1 2 3 4 5 Discussion No High benefit benefit

To our knowledge, this is the first investigation of possible Figure 2. Benefit of the diet after 2 months of the investigation: IgG-mediated food intolerances in migraine patients. The proportion of patients reporting their level of benefit on a 6- patients who took part were all severely affected by their point scale, where 0 = no benefit and 5 = high benefit

© 2005 LIBRAPHARM LTD – Headache Care 2005; 2(2) A Prospective Audit of Food Intolerance among Migraine Patients in Primary Care Clinical Practice Rees et al. 109 ances may be an effective treatment for at least some Acknowledgements migraine sufferers. However, it is not yet possible to recommend this The authors are pleased to acknowledge the help of Dr approach for general clinical use. This investigation was Frances Carter during the setting up and running of this a small audit to establish a possible relationship between audit. The audit was conducted by MIPCA, with help food intolerances and migraine. In this it was successful, and sponsorship from the Migraine Action Association although benefits experienced by patients may have and YORKTest Laboratories Ltd. been due (in part or in whole) to a placebo effect. There remains a series of questions that need to be answered before we have proof of this concept: References • Do migraine sufferers differ from unaffected people 1. Millichap JG, Yee MM. The diet factor in pediatric and adolescent migraine. Pediatr Neurol 2003;28:9-15 or people with other disorders in the pattern of IgG 2. Leira R, Rodriguez R. Diet and migraine. Rev Neurol 1996; that circulates? 24:534-8 • Do symptomatic reports of food intolerance correlate 3. Breslau N, Rasmussen BK. The impact of migraine: Epidemiology, risk factors, and co-morbidities. Neurology 2001;56(Suppl. 1): with the IgG data? 4-12 • Are migraine sufferers able to self-identify food 4. Peatfield RC. Relationships between food, wine, and beer- intolerances? precipitated migrainous headache. Headache 1995;35: 355-7 • Does allergen avoidance lead to an improvement 5. Bic Z, Blix GG, Hopp HP et al. The influence of a low-fat diet on in migraine and can this be confirmed by re- incidence and severity of migraine headaches. J Womens Health Gend Based Med 1999;8:623-30 challenge? 6. Hasselmark L, Malmgren R, Hannerz J. Effect of a carbohydrate- rich diet, low in protein-tryptophan, in classic and common We suggest two follow-up studies that may answer migraine. Cephalalgia 1987;7:87-92 7. Marcus DA, Scharff L, Turk D et al. A double-blind provocative these questions. Whether migraine patients differ from study of chocolate as a trigger of headache. Cephalalgia the general population and whether self-reported allergies 1997;17:855-62 8. Bedell AW, Cady RK, Diamond ML et al. Patient-centered correlate with food intolerances in migraine sufferers can strategies for effective management of migraine. Springfield, be examined in a blinded study investigating the pattern of Missouri: Primary Care Network, 2000 IgG-related food intolerances in migraine patients (with and 9. Pradalier A, Launay JM. Immunological aspects of migraine. Biomed Pharmacother 1996;50:64-70 without a history of allergy) and matched healthy controls 10. Kemper RH, Meijler WJ, Korf J et al. Migraine and function without migraine. A small placebo-controlled study can then of the immune system: a meta-analysis of clinical literature be used to study the effect of diet alteration on migraine published between 1966 and 1999. Cephalalgia 2001; 21:549-57 symptoms. The study requires a re-challenge phase, and 11. Sheldon TA. Independent audit of IgG food intolerance tested robust, validated endpoints, over a 3-month evaluation patient survey. British Allergy Foundation, 2000 time. 12. Atkinson W, Sheldon TA, Shaath N et al. Food elimination based on IgG antibodies in irritable bowel syndrome: a randomised In conclusion, this pilot audit demonstrated that controlled trial. Gut 2004;53:1459-64 migraine attacks may be related to food intolerances 13. Kosinski M, Bayliss MS, Bjorner JB et al. A six-item short-form survey for measuring headache impact: the HIT-6. Qual Life Res mediated via IgG and that changing the diet to eradicate 2003;12:963-74 specific foods may be a potentially effective treatment 14. YORKTEST guidebook on food intolerances and their treatment. for migraine. Further clinical studies are required York, UK: YORKTEST Laboratories Ltd, 2002 15. Lipton RB, Goadsby PJ, Sawyer JPC et al. Migraine: diagnosis to confirm these findings and examine the clinical and assessment of disability. Rev Contemp Pharmacother importance of this treatment approach. 2000;11:63-73

CrossRef links are available in the online published version of this paper: http://www.cmrojournal.com Paper HC-0047_2, Accepted for publication: 27 January 2005 Published Online: 18 February 2005 doi:10.1185/174234305X14962

110 A Prospective Audit of Food Intolerance among Migraine Patients in Primary Care Clinical Practice © 2005 LIBRAPHARM LTD – Headache Care 2005; 2(2) T h e new england journal o f medicine

review article

Medical Progress Celiac Disease

Peter H.R. Green, M.D., and Christophe Cellier, M.D., Ph.D.

eliac disease is a unique autoimmune disorder, unique because From the Department of Medicine, Colum- the environmental precipitant is known. The disorder was previously called bia University College of Physicians and Surgeons, New York (P.H.R.G.); and the celiac sprue, based on the Dutch word sprue, which was used to describe a dis- Department of Gastroenterology, Euro- C pean Georges Pompidou Hospital, René ease similar to tropical sprue that is characterized by diarrhea, emaciation, aphthous stomatitis, and malabsorption.1,2 Celiac disease is precipitated, in genetically pre- Descartes Paris V University, Assistance Publique–Hôpitaux de Paris, INSERM disposed persons, by the ingestion of gluten, the major storage protein of wheat U793, Paris (C.C.). Address reprint re- and similar grains.3 Originally considered a rare malabsorption syndrome of child- quests to Dr. Green at the Department of hood, celiac disease is now recognized as a common condition that may be diag- Medicine, Columbia University College of Physicians and Surgeons, 180 Fort nosed at any age and that affects many organ systems. The therapy for the disease is Washington Ave., Rm. 956, New York, NY a gluten-free diet; however, the response to therapy is poor in up to 30% of patients, 10032, or at [email protected]. and dietary nonadherence is the chief cause of persistent or recurrent symptoms. N Engl J Med 2007;357:1731-43. Small intestinal adenocarcinoma, refractory sprue, and enteropathy-associated T-cell Copyright © 2007 Massachusetts Medical Society. lymphoma are complications of celiac disease that must be ruled out when alarm- ing symptoms such as abdominal pain, diarrhea, and weight loss develop despite a strict gluten-free diet.

Pathogenesis

Celiac disease results from the interaction between gluten and immune, genetic, and environmental factors (Fig. 1).

The Role of Gluten Celiac disease is induced by the ingestion of gluten, which is derived from wheat, barley, and rye. The gluten protein is enriched in glutamine and proline and is poor- ly digested in the human upper gastrointestinal tract. The term “gluten” refers to the entire protein component of wheat; gliadin is the alcohol-soluble fraction of gluten that contains the bulk of the toxic components. Undigested molecules of gliadin, such as a peptide from an α-gliadin fraction made up of 33 amino acids, are resis- tant to degradation by gastric, pancreatic, and intestinal brush-border membrane proteases in the human intestine and thus remain in the intestinal lumen after gluten ingestion.4 These peptides pass through the epithelial barrier of the intes- tine, possibly during intestinal infections or when there is an increase in intestinal permeability, and interact with antigen-presenting cells in the lamina propria.

Mucosal Immune Responses In patients with celiac disease, immune responses to gliadin fractions promote an inflammatory reaction, primarily in the upper small intestine, characterized by infil- tration of the lamina propria and the epithelium with chronic inflammatory cells and villous atrophy (Fig. 1). This response is mediated by both the innate and the adaptive immune systems. The adaptive response is mediated by gliadin-reactive CD4+ T cells in the lamina propria that recognize gliadin peptides, which are bound

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Normal duodenal Duodenal mucosa Figure 1. Interaction of Gluten with Environmental, mucosa in celiac disease Immune,­ and Genetic Factors in Celiac Disease. Gluten is digested by luminal and brush-border enzymes into amino acids and peptides. The gliadin peptides in- duce changes in the epithelium through the innate im- mune system and, in the lamina propria, through the adaptive immune system. In the epithelium, gliadin damages epithelial cells, resulting in increased expres- sion of interleukin-15, which in turn activates intraepi- thelial lymphocytes. These lymphocytes become cyto- toxic and kill enterocytes that express MIC-A (a stress protein) on their surface. During infections or as the result of permeability changes, gliadin enters the lamina propria, where it is deamidated by tissue transglutamin- ase, allowing interaction with HLA-DQ2 (or HLA-DQ8) on the surface of antigen-presenting cells. Gliadin is presented to gliadin-reactive CD4+ T cells through a T-cell receptor, resulting in the production of cytokines that cause tissue damage. This leads to villous atrophy Gluten Other environmental and crypt hyperplasia, as well as the activation and ex- factors pansion of B cells that produce antibodies. Images of mucosa courtesy of Govind Bhagat, M.D. Gliadin Villous atrophy Increased intestinal Crypt hyperplasia Epithelial permeability Intraepithelial lymphocytosis absorptive proteinases and other tissue-damaging media- cells tors that induce crypt hyperplasia and villous in- jury.8 Gliadin peptides also activate an innate immune response in the intestinal epithelium that Overexpression of interleukin-15 is characterized by increased expression of inter- leukin-15 by enterocytes, resulting in the activa- tion of intraepithelial lymphocytes expressing the activating receptor NK-G2D, a natural-killer-cell Tissue marker.9 These activated cells become cytotoxic transglutaminase Intraepithelial and kill enterocytes with surface expression of lymphocytes major-histocompatibility-complex class I chain- related A (MIC-A), a cell-surface antigen induced Deamidated 10,11 gliadin by stress, such as an infection. The mecha- TCR CD4 Cytokine release T cell nism of the interaction between the processes in Lamina the epithelium and lamina propria has not been propria elucidated. HLA-DQ2 B cell Genetic Factors Antigen- Genetic The genetic influence in the pathogenesis of celiac factors presenting cell disease is indicated by its familial occurrence.12 Antibodies (antigliadin, antiendomysial, Celiac disease does not develop unless a person and tissue transglutaminase) has alleles that encode for HLA-DQ2 or HLA-DQ8 proteins, products of two of the HLA genes.13 However, many people, most of whom do not have to HLA class II molecules DQ2 or DQ8 on anti- celiac disease, carry these alleles; thus, their pres- gen-presenting cells; the T cells subsequently ence is necessary but not sufficient for the devel- 5 produce proinflammatory cytokines, particular­ opment of the disease.COLOR FIGURE Studies in siblings and 6 ly interferon-γ. Tissue transglutaminase is an en- identical twinsVersion 8suggest10/04/07 that the contribution of zyme in the intestine that deamidates gliadin HLA genesAuthor to theGreen genetic component of celiac dis- 7 Fig # 1 14 peptides, increasing their immunogenicity. The ease is lessTitle thanCeliac 50%. Disease Several non-HLA genes ensuing inflammatory cascade releases metallo- that mayME influenceIK susceptibility to the disease DE JRI Artist LAM AUTHOR PLEASE NOTE: Figure has been redrawn and type has been reset 1732 n engl j med 357;17 www.nejm.org october Please25, check2007 carefully Issue date 10/25/07

Downloaded from www.nejm.org by JUAN SABATER MD on November 14, 2007 . Copyright © 2007 Massachusetts Medical Society. All rights reserved. Medical Progress have been identified, but their influence has not more often diagnosed in women. The predomi- been confirmed. nance of the disease in women diminishes some- what after the age of 65 years.32 The classic pre- Environmental Factors sentation in adults is diarrhea, which may be Environmental factors that have an important role accompanied by abdominal pain or discomfort. in the development of celiac disease have been However, diarrhea has been the main presenting suggested by epidemiologic studies. These include symptom in less than 50% of cases in the past a protective effect of breast-feeding15 and the intro­ decade.33 Other, silent presentations in adults in- duction of gluten in relation to weaning.16-18 The clude iron-deficiency anemia, osteoporosis, and initial administration of gluten before 4 months incidental recognition at endoscopy performed of age is associated with an increased risk of dis- for other reasons, such as symptoms of gastro- ease development,18 and the introduction of glu- esophageal reflux.34 Less common presentations ten after 7 months is associated with a marginal include abdominal pain, constipation, weight risk.18 However, the overlap of gluten introduction loss, neurologic symptoms, dermatitis herpeti- with breast-feeding may be a more important pro- formis, hypoproteinemia, hypocalcemia, and ele- tective factor in minimizing the risk of celiac dis- vated liver enzyme levels.35 Substantial proportions ease.16 The occurrence of certain gastrointestinal of patients have received a previous diagnosis of infections, such as rotaviral infection, also increas- the irritable bowel syndrome32,36 and are over- es the risk of celiac disease in infancy.19 Further weight.37 Patients often have symptoms for a study of environmental factors might facilitate long time and undergo multiple hospitalizations the development of strategies for primary pre- and surgical procedures before celiac disease is vention of celiac disease.20 diagnosed.32,38,39 Some cases are diagnosed because of increased Epidemiology surveillance for celiac disease among people who have a family history of the disease24 and among Celiac disease occurs in adults and children at those with Down syndrome, Turner’s syndrome,40,41 rates approaching 1% of the population.21-25 The or type 1 diabetes, all of which are associated disease is recognized not only throughout Europe with celiac disease.42 Persons with celiac disease and in countries populated by persons of Euro- have an increased risk of autoimmune disorders pean ancestry but also in the Middle East,23,26 as compared with the general population.43-45 Asia,27 South America.28 and North Africa.29 In A case-finding study performed in multiple most affected people, celiac disease remains un- primary care practices in North America reported diagnosed,21 although the rate of diagnosis is a 43-fold increase in the rate of diagnosis of ce- increasing.30 liac disease over the 2 years of the study.46 The indications for screening in those who received Clinical Manifestations the diagnosis included bloating, the irritable bowel syndrome, thyroid disease, chronic unexplained Clinical manifestations of celiac disease vary diarrhea, chronic fatigue, and constipation. The greatly according to age group. Infants and young case-finding study shows that many patients with children generally present with diarrhea, abdom- celiac disease are seeking health care for a great inal distention, and failure to thrive. However, variety of common symptoms and that the more vomiting, irritability, anorexia, and even constipa- frequent use of screening is uncovering more tion are also common. Older children and adoles- cases of the disease. cents often present with extraintestinal manifesta­ tions, such as short stature, neurologic symptoms, Diagnosis or anemia.31 Among adults, two to three times as many The diagnosis of celiac disease requires both a duo­ women have the disease as men, for unknown denal biopsy that shows the characteristic find- reasons. In general, the prevalence of autoimmune ings of intraepithelial lymphocytosis, crypt hyper- diseases is higher in women than in men, and plasia, and villous atrophy and a positive response iron deficiency and osteoporosis, each of which to a gluten-free diet. The diagnostic criteria devel- prompts an assessment for celiac disease, are oped by the European Society for Pediatric Gastro-

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enterology and Nutrition require only clinical im- sive than the endomysial antibody test. Overall, provement with the diet,47 although histologic the sensitivity of the tests for both endomysial improvement on a gluten-free diet is frequently antibodies and anti–tissue transglutaminase anti­ sought and is recommended in adults because vil- bodies is greater than 90%,48 and a test for ei- lous atrophy may persist despite a clinical response ther marker is considered the best means of to the diet. In most patients, the diagnosis is eas- screening for celiac disease.48 The titers of endo- ily established. However, roughly 10% of cases are mysial antibodies and anti–tissue transglutamin- difficult to diagnose because of a lack of concor- ase antibodies correlate with the degree of mu­ dance among serologic, clinical, and histologic cosal damage51,52; as a result, the sensitivity of findings. these antibody tests declines when a greater number of patients with lesser degrees of vil- Serologic Testing lous atrophy are included in studies.53,54 The various commercially available assays for anti– Typical indications for serologic testing include tissue transglutaminase antibodies have differ- unexplained bloating or abdominal distress; chron­ ent characteristics and resultant sensitivities and ic diarrhea, with or without malabsorption or the specificities.55 irritable bowel syndrome; abnormalities on labo- Selective IgA deficiency is more common in ratory tests that might be caused by malabsorp- patients with celiac disease than in the general tion (e.g., folate deficiency and iron-deficiency population — 1 case in 40 as compared with 1 in anemia); first-degree relatives with celiac disease; 400. Consequently, patients with celiac disease and autoimmune diseases and other conditions and selective IgA deficiency lack IgA endomysial known to be associated with celiac disease (for antibodies and IgA antitissue antibodies against more information on indications for serologic test­ tissue transglutaminase. It is recommended that ing, see the table in the Supplementary Appendix, the test for anti–tissue transglutaminase anti- available with the full text of this article at www. bodies be used as a single screening test for ce- nejm.org). liac disease.48,56 If the levels of this marker are The most sensitive antibody tests for the diag- within the normal range (or if it is absent) and nosis of celiac disease are of the IgA class. The there is a high suspicion of celiac disease, selec- available tests include those for antigliadin anti- tive IgA deficiency needs to be ruled by measur- bodies, connective-tissue antibodies (antireticulin ing total IgA levels. In such cases, a test for IgG and antiendomysial antibodies), and antibodies antibodies against tissue transglutaminase should directed against tissue transglutaminase, the en- be performed.57 zyme responsible for the deamidation of gliadin These antibody tests fare less well in the clini- in the lamina propria. The antigliadin antibodies cal-practice setting than in the research setting.58,59 are no longer considered sensitive enough or spe- A recently developed rapid test for anti–tissue cific enough to be used for the detection of ce- transglutaminase antibodies that uses a sample liac disease, except in children younger than 18 of fingertip blood may be a convenient point-of- months of age,48 although new-generation anti- care test for the purpose of both case finding bodies to deamidated gliadin peptides appear to and dietary monitoring.60 be promising.49 Antireticulin antibodies are also rarely measured, having been surpassed in use by The Role of HL A-DQ2 endomysial and anti–tissue transglutaminase anti­ and HL A-DQ8 Assessment bodies. The diagnostic standard in celiac serologies re­ The HLA-DQ2 allele is identified in 90 to 95% of mains the endomysial IgA antibodies; they are patients with celiac disease, and HLA-DQ8 is iden- highly specific markers for celiac disease, ap- tified in most of the remaining patients.61 Be- proaching 100% accuracy. The recognition that cause these alleles occur in 30 to 40% of the gen- the enzyme tissue transglutaminase is the auto- eral population (with HLA-DQ2 more common than antigen for the development of endomysial anti- HLA-DQ8), the absence of these alleles is impor- bodies50 allowed development of automated tant for its high negative predictive value.62 Thus, enzyme-linked immunoassays that are less expen- the presence or absence of HLA-DQ2 and HLA-DQ8

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Downloaded from www.nejm.org by JUAN SABATER MD on November 14, 2007 . Copyright © 2007 Massachusetts Medical Society. All rights reserved. Medical Progress is important for determining which family mem- Table 1. Causes of Villous Atrophy Other Than Celiac Disease. bers should be screened with serologic testing and is useful for ruling out the disease in patients Giardiasis already on a gluten-free diet or for patients in Collagenous sprue whom the diagnosis is unclear. Common-variable immunodeficiency Autoimmune enteropathy Radiation enteritis Biopsy and Histologic Assessment Whipple’s disease Tuberculosis Biopsy of the small intestine remains the stan- Tropical sprue dard for diagnosing celiac disease, and it should Eosinophilic gastroenteritis always be performed when clinical suspicion is Human immunodeficiency virus enteropathy high, irrespective of the results of serologic test- Intestinal lymphoma ing. Biopsy confirmation is crucial, given the life- Zollinger–Ellison syndrome long nature of the disease and the attendant need Crohn’s disease for an expensive and socially inconvenient diet. Intolerance of foods other than gluten (e.g., milk, soy, chicken, tuna) Although no studies have examined the number of biopsies required for diagnosis, we believe that at least four to six endoscopic-biopsy speci- mens should be obtained from the duodenum, Table 2. Fundamentals of the Gluten-free Diet. given the patchy nature of the disease and the Grains that should be avoided difficulty of orienting the small pieces of tissue Wheat (includes spelt, kamut, semolina, triticale), rye, barley (including malt) taken during biopsy for assessment of villous Safe grains (gluten-free) 63,64 morphology. Rice, amaranth, buckwheat, corn, millet, quinoa, sorghum, teff (an Ethiopian Who should undergo endoscopic biopsy? In cereal grain), oats addition to patients whose serologic tests are Sources of gluten-free starches that can be used as flour alternatives positive, any patient who has chronic diarrhea, Cereal grains: amaranth, buckwheat, corn (polenta), millet, quinoa, sorghum, iron deficiency, or weight loss should undergo teff, rice (white, brown, wild, basmati, jasmine), montina (Indian rice grass) duodenal biopsy, irrespective of whether serolog­ Tubers: arrowroot, jicama, taro, potato, tapioca (cassava, manioc, yucca) ic testing for celiac disease has been performed. Legumes: chickpeas, lentils, kidney beans, navy beans, pea beans, peanuts, The recognition of endoscopic signs of villous soybeans atrophy, such as scalloping of mucosal folds, Nuts: almonds, walnuts, chestnuts, hazelnuts, cashews Seeds: sunflower, flax, pumpkin absent or reduced duodenal folds, or a mosaic pattern of the mucosa, should prompt biopsy.65 However, because these abnormalities are not sensitive markers of the presence of celiac dis- nosis is confirmed when there is a favorable re- ease,66 biopsy should be performed even if they sponse to the diet. are absent. The spectrum of pathologic changes in celiac Treatment disease ranges from near-normal villous architec- ture with a prominent intraepithelial lymphocy- Nutritional therapy, the only accepted treatment tosis to total villous atrophy.67 Pitfalls in the for celiac disease, involves the lifelong elimination pathological diagnosis include overinterpretation of wheat, rye, and barley from the diet. Clinical of villous atrophy in poorly oriented biopsy speci- studies suggest that oats are tolerated by most mens and inadequate biopsy sampling in patients patients with celiac disease and may improve the with patchy villous atrophy.63,64 The histologic nutritional content of the diet and overall quality findings in celiac disease are characteristic but of life.69 However, oats are not uniformly recom- not specific68; their presence permits a presump- mended, because most commercially available tive diagnosis of celiac disease and initiation of oats are contaminated with gluten-containing a gluten-free diet. Indeed, celiac disease is not the grains during the growing, transportation, and only cause of villous atrophy (Table 1). The diag- milling processes.70

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Although wheat, rye, and barley should be terfere with the immune response — by blocking avoided, there are other grains that can serve as the binding of deaminated gliadin to HLA-DQ2 substitutes as well as other sources of starch that or HLA-DQ8, for example, or by blocking the ac- can provide flours for cooking and baking (Ta- tion of tissue transglutaminase — are unlikely to ble 2). Because the substitute flours are not forti- be without side effects. fied with B vitamins, vitamin deficiencies may occur; they have been detected in patients who are Assessment of Cases with on the diet for a long time (more than 10 years).71 a Poor Response to Therapy Therefore, vitamin supplementation is advised. Meats, dairy products, and fruits and vegetables A gluten-free diet fails to induce clinical or histo- are naturally gluten-free and help to make for a logic improvement in 7 to 30% of patients,78 and more nutritious and varied diet. such lack of response should trigger a systematic After the diagnosis of celiac disease has been evaluation (Fig. 2). The first step is to reassess the established, the patient should be assessed for initial diagnosis, since villous atrophy with asso- deficiencies of vitamins and minerals, including ciated crypt hyperplasia is not exclusive to celiac folic acid, B12, fat-soluble vitamins, iron, and cal- disease (Table 1). Rare causes should at least be cium, and any such deficiencies should be treated. considered in people who do not have the expected All patients with celiac disease should undergo response to the diet. In patients with a question- screening for osteoporosis, which has a high able diagnosis, HLA-DQ2 or HLA-DQ8 typing may prevalence in this population.72,73 The health care be useful, since the negative predictive value of team should include a skilled dietitian who mon­ this test is almost 100%.62 itors the patient’s nutritional status and dietary The second step is to address the likelihood adherence on a regular basis. In children, ongo- of dietary nonadherence, the most common cause ing evaluation includes monitoring of growth and of unresponsive celiac disease. An experienced development. dietitian is required to assess the degree of ad- The elimination of gluten usually induces herence and possible reasons for nonadherence clinical improvement within days or weeks, though (Table 3). The highest rates of adherence are re- histologic recovery takes months or even years, ported among patients with a diagnosis in child- especially in adults, in whom mucosal recovery hood and those with severe symptoms at presen- may be incomplete.74 In rare cases, children toler- tation. In France and Belgium, less than half of ate the reintroduction of a normal diet after a adults with celiac disease who were studied ad- long-term clinical and histologic response.75 hered strictly to the diet for more than a year after Patient-support organizations are a valuable diagnosis.79 In a study in the United Kingdom, source of information about the disease and the the rate of adherence was low for both teenagers diet. Most countries have national support groups and adults,80 and in a study in Italy, adolescents in that are easily accessed on the Internet. The cost whom the diagnosis was established on the basis of the gluten-free products varies by country, but of mass serologic screening had poor adherence.81 the diet is usually expensive, making dietary treat- In another study, many people in whom the dis- ment problematic for patients with limited finan- ease was diagnosed in childhood became non- cial resources. Gluten-free products are particu- adherent to a strict gluten-free diet as adults.82 larly expensive and hard to find in developing The persistence of endomysial antibodies or anti– countries, whereas in other countries (including tissue transglutaminase antibodies in patients on the Netherlands, the United Kingdom, New Zea- a gluten-free diet for a year or more is suggestive land, Italy, Sweden, and Finland), the government of poor dietary adherence.79 Other causes of per- subsidizes these products. sistent symptoms in patients on a strict gluten- There is considerable interest in the develop- free diet are listed in Table 3. ment of nondietary therapies that might either replace or supplement the rigorous gluten-free Complications of Celiac Disease diet. Currently, the most attractive alternative in- volves the use of recombinant enzymes that di- Although the majority of patients who have re- gest the toxic gliadin fractions in the stomach or current or new symptoms when on a supposedly the upper small intestine.76,77 Therapies that in- gluten-free diet are in fact ingesting gluten, either

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Poorly responsive celiac disease

Exclusion of other causes of villous atrophy

Assessment of adherence to gluten-free diet

Good adherence Poor adherence

Evaluation Dietary counseling Exclusion of bacterial overgrowth Referral to support group and pancreatic insufficiency Regular follow-up by experienced Upper gastrointestinal endoscopy dietitian or colonoscopy and biopsy Intraepithelial-lymphocyte pheno- type on duodenal biopsy Enteroscopy (push or push–pull) Abdominal radiology (small bowel studies and CT scan) Video-capsule endoscopy

Enteropathy-associated Refractory celiac Adenocarcinoma T-cell lymphoma disease

Normal Abnormal clonal intraepithelial-lymphocyte intraepithelial-lymphocyte phenotype (type 1) phenotype (type 2)

Figure 2. An Assessment Plan for a Patient with Poorly Responsive Celiac Disease.

RETAKE 1st ICM AUTHOR: Green 2nd REG F FIGURE: 2 of 3 3rd intentionally or unintentionally,CASE a substantial pro- either intestinalRevised or extraintestinal; oropharyngeal portion may have a serious complicationEMail of celiac Lineand esophageal4-C SIZE adenocarcinoma; and cancers of ARTIST: ts H/T H/T 33p9 disease: intestinal adenocarcinoma,Enon enteropathy- Combothe small and large intestine, hepatobiliary sys- 83,84 86,87 associated T-cell lymphoma, or refractory sprue.AUTHOR, PLEASEtem, NOTE: and pancreas. The risk of breast cancer, Figure has been redrawn andhowever, type has beenappears reset. to be reduced.85,86 Adenocarcinoma of the Small IntestinePlease check carefully.In patients with celiac disease, the risk of ad- Patients with celiac diseaseJOB: have35717 an overall risk enocarcinomaISSUE: 10-25-07of the small intestine, generally a of cancer that is almost twice that in the general rare cancer, is increased manyfold as compared population. New population-based studies dem- with the risk in the general population88; still, onstrate that the risk is not as great as once con- the overall risk is very low given the rarity of this sidered.85 Reported cancers include both T-cell cancer. These carcinomas are most often located and B-cell non-Hodgkin’s lymphoma that may be in the jejunum and are more likely to develop as an

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toms of celiac disease after a period of good Table 3. Problems of Dietary Adherence and Poor Response in Celiac Disease. response to gluten withdrawal. Enteropathy-asso- Reasons for poor adherence to a gluten-free diet ciated T-cell lymphoma usually develops in the High cost jejunum but may also be found in the ileum or in Poor availability of gluten-free products (in developing countries) extraintestinal sites (e.g., liver, brain, chest, and Poor palatability bone) and is often multifocal. The prognosis is Absence of symptoms when dietary restrictions not observed poor; less than 20% of patients survive for 30 Inadequate information on gluten content of food or drugs months.91 The phenotype of enteropathy-asso­ Inadequate dietary counseling ciated T-cell lymphoma is consistent with a tu- Inadequate initial information supplied by diagnosing physician mor that derives from a clonal proliferation of in­ Inadequate medical or nutritional follow-up traepithelial lymphocytes. Immunohistochemical Lack of participation in a support group phenotyping indicates that this lesion is most Inaccurate information from physicians, dietitians, support groups, or Internet commonly CD3+, CD4–, CD8–, CD30+, and Dining out of the home CD103+.92 The treatment of this tumor is chemo- Social, cultural, or peer pressures therapy based, although there is a role for sur- Transition to adolescence gery in the treatment of localized or complicated Inadequate medical follow-up after childhood tumors.93 Successful autologous stem-cell trans- Causes of poorly responsive celiac disease plantation has been reported.94 Incorrect diagnosis Gluten ingestion (intentional or unintentional) Refractory Celiac Disease Microscopical colitis Approximately 5% of patients may have refractory Lactose intolerance celiac disease, defined as persistent symptoms Pancreatic insufficiency and villous atrophy despite scrupulous adherence Bacterial overgrowth to a gluten-free diet.95 The symptoms that usually Intolerance of foods other than gluten (e.g., fructose, milk, soy) develop in these patients include diarrhea, weight Inflammatory bowel disease loss, recurrence of malabsorption, abdominal pain, Irritable bowel syndrome bleeding, and anemia, and ulcerative jejunitis often Anal incontinence arises as well. This syndrome is also known as Collagenous sprue refractory sprue. The term was originally coined Autoimmune enteropathy because it was unclear whether patients with diar- Refractory celiac disease (with or without clonal T cells) rhea and villous atrophy whose condition did not Enteropathy-associated T-cell lymphoma improve with a gluten-free diet actually had celiac disease.2 adenoma–carcinoma sequence than as dysplasia Refractory celiac disease may be classified as in flat mucosa.89 Intuitively, video-capsule endos- type 1, in which there is a normal intraepithelial copy, which allows for visualization of the entire lymphocyte phenotype, or type 2, in which there small intestinal mucosal surface, would seem to is a clonal expansion of an aberrant intraepithe- be ideal in screening for cancers, but as yet no lial lymphocyte population. The identification of data support this approach. At present, video- an aberrant clonal population is primarily prog- capsule endoscopy plays a role in the evaluation nostic, since it is associated with a high risk of of celiac disease that is complicated by the devel- ulcerative jejunitis and frank enteropathy-associ- opment of abdominal pain or occult bleeding.90 ated T-cell lymphoma; the risk is so high that this condition has been described as a cryptic T-cell Enteropathy-Associated T-Cell Lymphoma lymphoma.83,96,97 The intraepithelial lymphocyte Enteropathy-associated T-cell lymphoma occurs expansion may be driven by overexpression of in adults, with the incidence peaking in the sixth interleukin-15 by the epithelium.9-11 Specific im- decade of life, and is usually at an advanced stage munophenotypic changes in the intraepithelial at diagnosis. Symptoms may include malaise, an- lymphocytes are seen in type 2 refractory celiac orexia, weight loss, diarrhea, abdominal pain, and disease. Intraepithelial lymphocytes in active ce- unexplained fever. The development of lympho- liac disease exhibit surface expression of CD3, ma is usually indicated by clinical relapse of symp- CD8, T-cell receptor (TCR) αβ, and TCRγδ, where­

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A

CD3+ +

Active Celiac Disease CD8+ +

B

CD3+ +

Refractory CD8– Celiac Disease -

Figure 3. Phenotyping Intraepithelial Lymphocytes in Active versus Refractory Celiac Disease.

In active celiac disease (Panel A, hematoxylinICM AUTHOR and eosin),green most intraepithelialRETAKE lymphocytes 1st express CD3 and CD8 (CD3+,CD8+) receptors, whereas inREG refractory F FIGURE celiac f3disease a_b (Panel B, hematoxylin 2ndand eosin), most of these lym- phocytes express CD3 but not CD8 CASE(CD3+,CD8−). In insets, brown color denotes positive3rd immunostaining. Images TITLE Revised provided by Dr. Diane Damotte, DepartmentEMail of Pathology, EuropeanLine 4-C Georges Pompidou Hospital. SIZE Enon ARTIST: mleahy H/T H/T FILL Combo 33p9 AUTHOR, PLEASE NOTE: Figure has been redrawn and type has been reset. Please check carefully.

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as intraepithelial lymphocytes in type 2 (clonal) intraepithelial lymphocytes and progression to an refractory sprue continue to express CD3 in the overt lymphoma have been reported in some treat­ cytoplasm but lack surface expression of CD8, ed patients, indicating that these drugs do not CD3, TCRαβ, and TCRγδ.92,96,98,99 Immunohis- cure the disease.106,107 Autologous hematopoietic tochemical studies demonstrating the presence stem-cell transplantation has been successful.108 of these cell-surface markers can be performed New therapeutic strategies, such as blocking on formalin-fixed biopsy specimens in most clin­ interleukin-15, should be aggressively investigat­ ical pathology laboratories. They allow for differ- ed, since the prognosis for patients with clonal entiation of type 2 refractory celiac disease, in refractory disease is poor, with a 5-year survival which an abnormal phenotype of the intraepi- rate of less than 50%.83 thelial lymphocytes (CD3+,CD8–), from type 1, in which there is a normal phenotype (CD3+,CD8+). Summary Patients who are not adherent to the diet will also express this normal phenotype (Fig. 3).100 Celiac disease occurs in nearly 1% of the popula- The development of new symptoms (e.g., weight tion in many countries. The diagnosis, which is loss, abdominal pain, or fever) or the recurrence straightforward in most cases, is usually estab- of diarrhea in patients who are on a strict gluten- lished on the basis of serologic testing, duodenal free diet often requires extensive investigation, biopsy, and observation of the response to a gluten- including the use of contrast radiology or com- free diet. A poor response to the diet is common puted tomographic (CT) enteroclysis (in which and requires extensive evaluation to rule out in- CT imaging is performed after infusion of a large testinal lymphoma and refractory sprue, compli- volume of contrast material — often 2 liters — cations that arise as the result of clonal expansion into the small intestine), video-capsule endoscopy, of intraepithelial lymphocytes. positron-emission tomographic scanning, extend­ Increasing awareness of the epidemiology and ed upper endoscopy (so-called push or push–pull diverse manifestations of the disease, as well as enteroscopy), and laparoscopy.90,101 the availability of sensitive and specific serologic Treatment of refractory celiac disease involves tests, especially among primary care physicians, nutritional support and repletion of vitamins and will lead to more widespread screening and diag- minerals, together with a strict gluten-free diet. nosis, which in turn will lead to greater availabil- In most cases, corticosteroids induce clinical im- ity of gluten-free foods and efforts to develop provement.83 Immunosuppressive drugs may be drug therapies that relieve patients of the burden beneficial102,103 but should be used with caution, of a gluten-free diet. In addition, earlier diagno- since they may promote the progression to lym- sis may lead to a reduction in the complications phoma.104 of the disease. The successful use of infliximab,105 an anti- CD52 monoclonal antibody, and cladribine (2-clo- Dr. Green reports receiving a consulting fee from Alvine Pharmaceuticals and lecture fees from Prometheus Laboratories. rodeoxyadenosine) has been reported, although No other potential conflict of interest relevant to this article was the persistence of clonally expanded, aberrant reported.

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Clinical and Experimental Allergy, 37, 823–830 c 2007 The Authors ORIGINAL PAPER Journal compilation c 2007 Blackwell Publishing Ltd

Alterations of food antigen-specific serum immunoglobulins G and E antibodies in patients with irritable bowel syndrome and functional dyspepsia w X. L. ZuoÃ,Y.Q.LiÃ,W.J.LiÃ,Y.T.GuoÃ,X.F.LuÃ,J.M.Lià and P. V. Desmond ÃDepartment of Gastroenterology, Qilu Hospital, Shandong University, Jinan, China and wDepartment of Gastroenterology, St Vincent’s Hospital, Fitzroy Vic., Australia

Clinical and Summary Background Post-prandial worsening of symptoms as well as adverse reactions to one or Experimental more foods are common in the patients with functional gastrointestinal diseases, such as irritable bowel syndrome (IBS) and functional dyspepsia (FD). However, the role played by Allergy true food allergy in the pathogenesis of these diseases is still controversial and there are no well-established tests to identify food allergy in this condition. Objective To investigate serum food antigen-specific IgG, IgE antibody and total IgE antibody titres in controls and patients with IBS and FD, and to correlate symptoms with the food antigen-specific IgG titres in IBS and FD patients. Methods Thirty-seven IBS patients, 28 FD patients and 20 healthy controls participated in this study. Serum IgG and IgE antibody titres to 14 common foods including beef, chicken, codfish, corn, crab, eggs, mushroom, milk, pork, rice, shrimp, soybean, tomatoes and wheat were analysed by ELISA. Serum total IgE titres were also measured. Last, symptomatology was assessed in the study. Results IBS patients had significantly higher titres of IgG antibody to crab (P = 0.000), egg (P = 0.000), shrimp (P = 0.000), soybean (P = 0.017) and wheat (P = 0.004) than controls. FD patients had significantly higher titres of IgG antibody to egg (P = 0.000) and soybean (P = 0.017) than controls. The percentage of individuals with detectable positive food antigen- specific IgE antibodies of the three groups did not show any significant differences (P = 0.971). There were no significant differences between IBS patients, FD patients and controls in the serum total IgE antibody titres (P = 0.978). Lastly, no significant correlation was seen between symptom severity and serum food antigen-specific IgG antibody titres both in IBS and FD patients. Correspondence: Conclusion Serum IgG antibody titres to some common foods increased in IBS and FD Prof. YanQing Li, Department of patients compared to controls. But there is no significant correlation between symptom Gastroenterology, Qilu Hospital of severity and elevated serum food antigen-specific IgG antibodies in these patients. Shandong University, Jinan 250012, China. Keywords food allergy, functional dyspepsia, IgE, IgG, irritable bowel syndrome E-mail: [email protected] Submitted 25 November 2006; revised 6 March 2007; accepted 16 March 2007

ing their symptoms to food allergy [2–4]. However, the Introduction role played by true food allergy in the pathogenesis of IBS Food allergy is a complex area of medicine. Up to 20% of and other functional diseases of the GI tract are still the population have adverse reactions to food and claim controversial and there are no well-established tests to to be of food allergy or food intolerance [1]. The percen- identify food allergy in this condition [5–7]. tage of food allergy is much higher in some functional Previously, food allergy was believed to be associated diseases of the gastrointestinal (GI) tract such as irritable with an IgE-mediated immune response to a particular bowel syndrome (IBS), with 20–65% of patients attribut- allergen in the diet. Therefore, the standardized skin prick 824 X. L. Zuo et al testing and RAST testing were frequently used to diagnose Controls food allergy [8, 9]. However, there is no evidence that The control group consisted of 20 healthy subjects (six men shows IgE does indeed play an important role in hyper- and 14 women, mean age 36.5 years) recruited from the sensitive reactions to food in IBS patients [10–14]. The community. All control subjects were free of GI symptoms gold standard in this condition is the double blind and had no evidence of acute or chronic illnesses. placebo-controlled food challenge test under careful None of the patients or healthy individuals was on any supervision in a hospital. But this test is cumbersome, medication at the time of the study. The patient groups time consuming and of poor patient compliance, which and the control group did not differ significantly with limits its use in clinical practise. Fortunately, accumulat- respect to mean age and sex ratio. ing data in recent years have indicated that IgG-mediated Informed, preferably written, consent was obtained immune response, which characteristically gives a more from each subject. The study has been performed accord- delayed response following exposure to a particular anti- ing to the Declaration of Helsinki and the procedures have gen, is of great importance in food allergy [15–17] and the been approved by Qilu hospital ethics committee. measurement of serum IgG titres opens up a new avenue for diagnosing food allergy in patients suffering adverse reactions to foods. Measurement of serum food antigen-specific immunoglo- Post-prandial worsening of symptoms as well as ad- bulins G and E antibodies. Serum samples were collected verse reactions to one or more foods are common [18, 19] from all subjects and stored at À20 1C for subsequent and dietary elimination can lead to symptomatic improve- analysis. Serum IgG and IgE antibody titres to 14 common ments in patients with IBS [3, 20–22]. Recently, Zar et al. foods including beef, chicken, codfish, corn, crab, eggs, [23] have shown elevated IgG titres in IBS patients while mushroom, milk, pork, rice, shrimp, soybean, tomatoes Atkinson et al. [24] have demonstrated that food elimina- and wheat were analysed by ELISA. tion based on IgG antibodies may be effective in reducing IBS symptoms. Other studies have shown food allergy is Serum food antigen-specific immunoglobulin G enzyme- also present in functional dyspepsia (FD), which has over- linked immunosorbent assay. Serum food antigen-specific lapping symptoms with IBS [25, 26]. However, fewer IgG ELISA was performed with Allerquant Food Allergy studies focus on the role of food allergy in the pathogen- Screening ELISA Kit (Biomerica, Inc. Newport Beach, CA, esis of FD and the level of serum food antigen-specific IgG USA). antibodies in FD has not been investigated. According to the instruction of the ELISA Kit, 50, 100, The aim of this study is to compare serum food antigen- 200 and 400 U/mL of Food IgG Calibrator were prepared specific IgG and IgE antibodies in controls and patients and added to the microplate together with blanks and with IBS and FD, and to correlate symptoms with the food positive controls. This is the calibration curve to be used in antigen-specific IgG and IgE titres in IBS and FD patients. the assay. Serum samples were diluted to 1/100 and 25 mL of each serum sample were taken and added to 2.5 mL of serum dilute regents. Then 100 mL of the diluted patient Materials and methods serum were placed into the microwells coated with 14 food antigens for 60 min at room temperature. And then, 100 mL Patients of food IgG–HRP conjugate were added to each well after Thirty-seven IBS patients (12 men and 25 women; mean washing and the plates were incubated for 30 min at room age 36 years) and 28 FD patients (nine men and 19 temperature. Another 100 mL of working substrate mix women; mean age 35 years) participated in this study. All (TMB and H2O2) were applied to each well and the plates patients were recruited from the Department of Gastro- were covered and incubated for 10 min at room tempera- enterology of Qilu Hospital, Shandong University and ture. The reaction was stopped using 50 mL per well of 1 N were diagnosed by using the Rome II criteria [27] for IBS sulphuric acid. At last, the plates were read using a Dynex (pain or abdominal discomfort accompanied by two or ELISA plate reader (Dynex Technologies, Inc., Chantilly, three symptoms, such as relief with defecation and/or with VA, USA) at 450 nm. The IgG concentration to each food alterations in the frequency of evacuations or in the shape antigen was expressed as units per millitre. of the feces for at least 12 weeks, which need not be Plates with microwells were washed three times with consecutive, in the preceding 12 months; in the absence of washing buffer between steps, and were incubated at room organic GI diseases) and for FD (persistent or recurrent temperature for each stage of the assay. symptoms, such as pain or discomfort in the upper abdo- men at least 12 weeks earlier, not necessarily serial, during Serum food antigen-specific immunoglobulin E and total the preceding 12 months). Organic GI disorders were immunoglobulin E enzyme-linked immunosorbent assay. excluded by routine laboratory tests and endoscopies with Serum food antigen-specific IgE and total IgE ELISA were biopsies. performed with the IVT Allergy Profile kit (In vitro

c 2007 The Authors Journal compilation c 2007 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 37 : 823–830 Food allergy in patients with IBS and FD 825

Technologies Inc. Arlington, TX, USA). It is a qualitative individuals with positive IgE antibodies between groups. test designed to identify IgE levels in the test samples. A Comparison between groups for interval data (age, positive result is identified visually by a gradual yellow to weight) was carried out with t-test. The correlation be- purple colour change around a reactive segment within tween the individual symptom scores and the IgG titres the capillary. This kit is also semiquantitative in that the was analysed by Pearson’s correlation test. Significance rate of conversion from yellow to purple and the intensity was accepted at 5% level (P o 0.05). The statistics package of the purple colour is proportional to the level of IgE SPSS v 13.0 running on Windows XP Professional was antibody in the patient sample. All components were used for the analyses. allowed to come to the room temperature before use. The test procedures are as follows. Firstly, each serum sample was added to a properly Results labelled multiple immunoassay device and allowed to react for 100 min at room temperature. Then was the Serum food antigen-specific immunoglobulin G sample expelled from the device and the device washed antibody titres with 1 mL wash solution. Secondly, the conjugate reagent was added to the device and allowed to react for 100 min. The serum IgG antibody titres of each of the IBS, FD and Then, was the conjugate reagent expelled from the device control groups to each food antigen are shown in this and the device washed with wash solution. Thirdly, the study together with the results of the ANOVA (Table 1). There substrate indicator was added to the device and the were significant differences between the three groups in characteristic yellow to purple colour change was within their IgG responses to crab, egg, shrimp, soybean and the observed next 30 min period. wheat. IBS patients had significantly higher titres of IgG Highly allergic reactions to a particular allergen caused antibody to crab, egg, shrimp, soybean and wheat than a rapid purple colouration around that specific segment controls and higher titres of IgG antibody to crab, egg, within 5 min. Weaker allergen took progressively longer shrimp and wheat than FD patients. FD patients had to effect the colour change. Negative reactions showed significantly higher IgG antibody titres to egg and soy- equivalent colour to the inert spacers. On the total IgE bean than controls and lower IgG antibody titres than IBS segment, low total IgE levels (430 IU/mL) were evident by patients to crab, egg, shrimp and wheat (Fig. 1). There a yellow to grey colouration, distinguishable from normal were no significant differences between the three groups IgE levels (60 Æ 30 IU/mL), which yield moderately purple in their IgG responses to beef, chicken, codfish, corn, colour throughout, and elevated IgE levels (>90 IU/mL), mushroom, milk, pork, rice and tomatoes. which yield intense purple colouration after 30 min in- cubation with the substrate indicator.

Symptom questionnaire. Symptomatology was assessed Table 1. Serum IgG antibody titres to food antigens showing the means in IBS, FD and control groups, the standard error of the mean (SEM) and in the study. All the patients completed a symptom P-value from the ANOVA assessment questionnaire based on Rome II criteria to evaluate the severity of symptoms for the 1 week period Mean Æ SEM (U/mL) before the interview. Questions targeting the presence and IBS (n = 37) FD (n = 28) Control (n = 20) P-value severity of abdominal pain/discomfort, bloating, bowel a a a urgency/diarrhoea, constipation, early satiety, nausea and Beef 32.86 Æ 0.46 32.43 Æ 0.44 32.10 Æ 0.60 0.556 Chicken 28.65 0.51a 27.68 0.58a 27.20 0.55a 0.171 belching were asked, for example ‘Have you experienced Æ Æ Æ Codfish 32.86 Æ 0.49a 32.89 Æ 0.52a 32.55 Æ 0.59a 0.902 abdominal pain/discomfort during this week?’ If the a a a Corn 31.87 Æ 0.43 31.82 Æ 0.46 31.29 Æ 0.60 0.617 subjects reported ‘yes’, they were then asked to grade the Crab 50.27 Æ 0.89a 37.53 Æ 0.95b 37.90 Æ 0.78b 0.000 severity of that symptom using the scale 1 = mild, Eggs 119.3 Æ 11.8a 69.71 Æ 4.63b 51.93 Æ 3.74c 0.000 2 = moderate, 3 = intense and 4 = severe. If the subjects Mushroom 27.78 Æ 0.51a 26.93 Æ 0.63a 27.25 Æ 0.49a 0.515 reported ‘no’, the score was recorded as 0. The total score Milk 33.14 Æ 0.51a 32.36 Æ 0.64a 32.41 Æ 0.98a 0.615 of each patient is the sum of each symptom score. Pork 31.54 Æ 0.45a 31.82 Æ 0.53a 30.80 Æ 0.59a 0.437 Rice 28.68 Æ 0.52a 29.54 Æ 0.75a 28.41 Æ 0.83a 0.493 Statistical analysis. Linear models were fitted to the log Shrimp 65.05 Æ 3.70a 45.39 Æ 1.62b 43.25 Æ 1.73b 0.000 a a b transformation of the variables, such as the IgG and total Soybean 55.83 Æ 3.35 53.29 Æ 1.89 43.60 Æ 1.95 0.017 Tomatoes 34.65 Æ 0.78a 33.79 Æ 0.98a 34.05 Æ 1.23a 0.782 IgE titres. A one-way ANOVA was carried out to test for Wheat 60.59 3.4a 49.39 2.05b 48.10 2.01b 0.004 differences in levels of antibody titres between the groups. Æ Æ Æ Tamhane’s T2 test was used to test for pairwise differences Means with different superscripts across rows were significantly different 2 between IBS, FD and control groups. The w tests were (P o 0.05). used to test for the differences of the percentage of IBS, irritable bowel syndrome; FD, functional dyspepsia.

c 2007 The Authors Journal compilation c 2007 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 37 : 823–830 826 X. L. Zuo et al

Fig. 1. Comparisons of serum IgG antibodies to crab, egg, shrimp, Fig. 2. Comparision of total IgE antibody titres to food antigens between soybean and wheat between irritable bowel syndrome (IBS), functional irritable bowel syndrome (IBS), functional dyspepsia (FD) and control dyspepsia (FD) and control groups. IBS patients have significantly higher groups. The data were expressed with the individual values in the three IgG antibodies to crab, egg, shrimp, soybean and wheat than controls. FD groups. Horizontal bars represent the means for every group. There were patients have significantly higher IgG antibodies to egg and soybean no significant differences between IBS patients, FD patients and controls than controls. IBS patients have significantly higher IgG antibodies to in the serum total IgE antibody titres (P = 0.978). crab, egg, shrimp and wheat than FD patients. The data were expressed à with the geometric means and the standard errors of the means. P o 0.05 compared with controls; #P o 0.05 compared with FD patients. against eggs, one against mushroom and four against wheat. Of 28 FD patients, one had positive IgE antibodies Serum food antigen-specific immunoglobulin E against beef, one against codfish, two against eggs, one antibody titres against soybean and three against wheat. In addition, one control had positive IgE antibodies against codfish, two In terms of serum food antigen-specific IgE antibodies, 10 against eggs, one against mushroom and two against of 37 IBS patients, eight of 28 FD patients and six of 20 wheat. The number of patients and controls with positive controls had detectable positive IgE ELISA results to beef, IgE antibodies against each individual food antigen was codfish, eggs, mushroom, soybean and wheat (Table 2). too small to apply a statistical test. The percentage of individuals with detectable positive IgE antibodies of the three groups did not show any signifi- cant differences (27.03% for IBS, 28.57% for FD and 30% Serum total immunoglobulin E antibody titres for controls, w2 = 0.059, P = 0.971). In IBS patients, only The serum total IgE antibody titres were also measured in two had positive IgE antibodies against codfish, three this study. There were no significant differences between IBS patients, FD patients and controls (means Æ SEM Table 2. Summary of the individuals with positive IgE ELISA results 66.87 Æ 7.65 for controls, 68.69 Æ 5.9 for FD patients and in IBS, FD and control groups 67.30 Æ 5.49 for IBS patients, P = 0.978, Fig. 2). IBS (n = 37) FD (n = 28) Control (n = 20) Beef 0 1 0 Correlation with symptoms Chicken 0 0 0 Codfish 2 1 1 The symptom severity of each FD and IBS patient was Corn 0 0 0 scored according to the symptom questionnaire. The Crab 0 0 0 relation between the symptoms and elevated IgG re- Eggs 3 2 2 sponses to some food antigens was observed and no Mushroom 1 0 1 significant correlation was seen between symptom sever- Milk 0 0 0 ity and serum food antigen-specific IgG antibody titres Pork 0 0 0 both in IBS and FD patients (Fig. 3). Rice 0 0 0 Shrimp 0 0 0 Soybean 0 1 0 Discussion Tomatoes 0 0 0 Wheat 4 3 2 This study demonstrated a significant increase in IgG antibody titres to several common foods in patients with IBS, irritable bowel syndrome; FD, functional dyspepsia. functional diseases of the GI tract compared with healthy

c 2007 The Authors Journal compilation c 2007 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 37 : 823–830 Food allergy in patients with IBS and FD 827

Fig. 3. The correlation between abdominal symptom scores and IgG titres to food antigen in irritable bowel syndrome (IBS) patients and functional dyspepsia (FD) patients. (a–e) The individual plots of values for food antigen-specific IgG titres and symptom scores in 37 IBS patients. No significant correlations are seen between symptom scores and IgG titres to crab (a, P = 0.426), eggs (b, P = 0.657), shrimp (c, P = 0.800), soybean (d, P = 0.854) and wheat (e, P = 0.794). (f) The individual plots of values for food antigen-specific IgG titres and symptom scores in 28 FD patients. No significant correlations were seen between symptom scores and IgG titres to eggs (P = 0.460) and soybean (P = 0.986).

subjects. In IBS patients, elevated food antigen-specific in China and they are used to consume less beef and more IgG antibodies to crab, egg, shrimp, soybean and wheat seafood. were observed while elevated food antigen-specific IgG Food allergy can involve different organs and systems antibodies to egg and soybean were observed in FD such as the digestive tract, the skin, the respiratory tract patients. It should be mentioned that, consistent with the and the cardiovascular system. While dermatologic, re- previous study [23], the antibodies against wheat were spiratory and systemic manifestations of food allergy are significantly elevated in IBS patients. However, we also well recognized, the reactions manifested primarily in the got some raised food antigen-specific antibodies different digestive tract can be difficult to recognize, diagnose and from the previous study [23], such as elevated antibody treat. This is due to the protean ways food can cause GI titres to crab and shrimp instead of beef. This might be due symptoms, the relatively poorly understood pathophysio- to the difference between a Chinese diet and a western logic mechanisms and the limited diagnostic methods diet. More specifically, most of the subjects in this study available to objectively identify afflicted individuals. are from a coastal area (where this study was performed) These deficiencies are, in part, a consequence of the

c 2007 The Authors Journal compilation c 2007 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 37 : 823–830 828 X. L. Zuo et al difficulty accessing the GI tract to establish mechanisms serum IgE measurements do not correlate well with the of disease and develop methods to diagnose and treat food mucosal allergic response in the intestine [28, 37]. This allergy [28, 29]. Often, patients of this nature are classified suggests that an IgE-mediated hypersensitivity response as being psychosomatic or being functional without to food is unlikely to play an important role in most of the defining the real problem. It has been recognized for some IBS and FD patients. time now that several ‘functional diseases’ might be Both IBS and FD patients reported that GI symptoms associated with food allergy [30–34]. often occur after certain food intake. There was also a The mechanisms that underlie these increases in IgG great overlap in the post-prandial dyspeptic symptoms in antibody responses to some common foods remain spec- the two groups of patients, such as gas problems, pain, ulative. The role of IgG and IgA antibodies in the coeliac nausea and upper abdominal discomfort. This came as no disease is well studied [35]. Coeliac disease occurs due to a surprise, as a high prevalence of overlap between FD and delayed immune reaction to gluten in wheat. This causes IBS has been universally reported and some shared intestinal membrane damage, IgG/IgA change and the common pathophysiological disturbances might exist in resultant diarrhoea, abdominal bloating and anaemia. these GI functional diseases, such as delayed gastric Raised IgG antibodies to food antigens have also been emptying, visceral hypersensitivity including food hyper- reported in patients with asthma caused by milk allergy sensitivity [38–40]. But, interestingly, we were unable to and patients with atopic dermatitis and/or bronchial correlate the level of food-specific IgG antibodies with the asthma caused by soybean allergy [17, 36]. Exclusions of severity of symptoms both in IBS and FD patients in this the offending foods from diet have shown to improve study. The underlying mechanisms of the pathogenesis symptoms in these diseases. Raised food-specific IgG have not yet been fully defined. Maybe some patients with antibodies may play a similar pathophysiological role in food sensitivities have non-allergic food reactions and the IBS and FD patients. The allergic food antigens trans- elevated IgG antibodies to food may be secondary to ported by way of M cells into the lamina propria activate ‘inflammation’ and therefore be more of an epi-phenom- T helper cells and B cells, increase the production of IgG enon. In these patients, there are no real food allergies or and cytokines. Then the increased IgG antibodies and immunity responses while experiencing symptoms. Psy- cytokines lead to the inflammation response of the gut, chological factors have also been suggested to be of great which is now believed to play an important role in the importance for the reported food intolerance in these pathogenesis of IBS through inducing alterations in GI patients [41, 42]. In addition, IBS and FD symptoms may peristalsis, abdominal discomfort and bowel dysfunction. also be related to abnormal intestinal bacteria, caffeine, In addition to the immune inflammation reaction ex- alcohol, low dietary fibre, overgrowth of intestinal yeasts planation, another possible mechanism that may account and excessive dietary sugars. for the elevated IgG antibodies is the alteration in the In conclusion, increased antigen-specific IgG antibody permeability of gut mucosa. Theoretically, any increases titres for some foods were found in IBS and FD patients in the gut mucosal permeability in IBS and FD patients compared with controls but there is no evidence that these might increase the uptake of undigested protein and findings contribute to the pathogenesis of these functional increase antigenic load presented to the mucosal immune GI diseases. Future studies along these lines are expected system. This may lead to the increased IgG antibody titres to lead to a better understanding about the role of elevated even with a normal physiological response of the gut food antigen-specific IgG antibodies in these functional immune system. If the above hypothesis is correct, then a GI diseases. generalized increase in the IgG antibodies to all 14 food antigens should have been observed. However, in this study we found that the food-specific IgG antibodies Acknowledgements increased to only some rather than to all food antigens. The possible explanation is that the patients might have The authors appreciate the considerable assistance from selective gut permeability to food antigens and the in- the Gastroenterology kinetic laboratory and the Central crease of food-specific IgG titres is a specific reaction Laboratory of Immunity in Qilu Hospital, Shandong Uni- rather than a non-specific response to increased gut versity. This study was founded by a research grant (NSFC, mucosal permeability. 30570831) from National Natural Science Foundation of In terms of serum food antigen-specific IgE antibody China and a grant (Y2005C02) from the Department of and total IgE antibody titres, no significant difference was Science and Technology of Shandong Province of China. found in IBS, FD patients and controls. Furthermore, there were fewer individuals who had positive food antigen- specific IgE responses compared with food antigen-specific References IgG responses in the studied population. This came as no 1 Young E, Stoneham MD, Petruckevitch A, Barton J, Rona R. A surprise, as several other studies also demonstrate that population study of food intolerance. Lancet 1994; 343:1127–30.

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Aliment Pharm Ther 2001; Gastroenterol Rep 2001; 3:351–7. 15:439–43. 30 Stefanini GF, Saggioro A, Alvisi V et al. Oral cromolyn sodium 12 Petitpierre M, Gumowski P, Girard JP. Irritable bowel syndrome in comparison with elimination diet in the irritable bowel syn- and hypersensitivity to food. Ann Allergy 1985; 54:538–40. drome, diarrheic type. Multicenter study of 428 patients. Scand 13 Barau E, Dupont C. Modifications of intestinal permeability J Gastroenterol 1995; 30:535–41. during food provocation procedures in pediatric irritable bowel 31 Niec AM, Frankum B, Talley NJ. Are adverse food reactions syndrome. J Pediatr Gastroenterol Nutr 1990; 11:72–77. linked to irritable bowel syndrome? Am J Gastroenterol 1998; 14 Roussos A, Koursarakos P, Patsopoulos D, Gerogianni I, Philip- 93:2184–90. pou N. Increased prevalence of irritable bowel syndrome in 32 Iacono G, Cavataio F, Montalto G et al. Intolerance of cow’s milk patients with bronchial asthma. Respir Med 2003; 97:75–79. and chronic constipation in children. N Engl J Med 1998; 15 Crowe SE, Perdue MH. Gastrointestinal food hypersensitivity: 339:1100–4. basic mechanisms of pathophysiology. Gastroenterology 1992; 33 Read NW. Food and hypersensitivity in functional dyspepsia. Gut 103:1075–95. 2002; 51 (Suppl. 1):i50–3. 16 Host A, Husby S, Gjesing B, Larsen JN, Lowenstein H. Prospective 34 Spanier JA, Howden CW, Jones MP. A systematic review of estimation of IgG, IgG subclass and IgE antibodies to dietary alternative therapies in the irritable bowel syndrome. Arch Intern proteins in infants with cow’s milk allergy. Levels of antibodies Med 2003; 163:265–74. to whole milk protein, BLG and ovalbumin in relation to repeated 35 O’Farrelly C, Kelly J, Hekkens W et al. Alpha gliadin antibody milk challenge and clinical course of cow’s milk allergy. Allergy levels: a serological test for coeliac disease. Br Med J (Clin Res 1992; 47:218–29. 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GUT IMMUNOLOGY The gut–joint axis: cross reactive food antibodies in rheumatoid arthritis M Hvatum, L Kanerud, R Ha¨llgren, P Brandtzaeg ......

Gut 2006;55:1240–1247. doi: 10.1136/gut.2005.076901

Background and aims: Patients with rheumatoid arthritis (RA) often feel there is an association between food intake and rheumatoid disease severity. To investigate a putative immunological link between gut immunity and RA, food antibodies were measured in serum and perfusion fluid from the jejunum of RA patients and healthy controls to determine the systemic and mucosal immune response. Methods: IgG, IgA, and IgM antibodies to dietary antigens were measured in serum and jejunal perfusion See end of article for fluid from 14 RA patients and 20 healthy subjects. The antigens originated from cow’s milk (a-lactalbumin, authors’ affiliations b-lactoglobulin, casein), cereals, hen’s egg (ovalbumin), cod fish, and pork meat...... Results: In intestinal fluid of many RA patients, all three immunoglobulin classes showed increased food Correspondence to: specific activities. Except for IgM activity against b-lactoglobulin, all other IgM activities were significantly Professor P Brandtzaeg, increased irrespective of the total IgM level. The RA associated serum IgM antibody responses were Institute of Pathology, Rikshospitalet, N-0027 relatively much less pronounced. Compared with IgM, the intestinal IgA activities were less consistently Oslo, Norway; per. raised, with no significant increase against gliadin and casein. Considerable cross reactivity of IgM and [email protected]. IgA antibodies was documented by absorption tests. Although intestinal IgG activity to food was quite low, no it was nevertheless significantly increased against many antigens in RA patients. Three of the five RA Revised version received patients treated with sulfasalazine for 16 weeks had initially raised levels of intestinal food antibodies; 24 November 2005 these became normalised after treatment, but clinical improvement was better reflected in a reduced Accepted for publication erythrocyte sedimentation rate. 1 February 2006 Conclusions: The production of cross reactive antibodies is strikingly increased in the gut of many RA Deceased Published online first patients. Their food related problems might reflect an adverse additive effect of multiple modest 16 February 2006 hypersensitivity reactions mediated, for instance, by immune complexes promoting autoimmune reactions ...... in the joints.

atients with rheumatoid arthritis (RA) often feel that reported in RA patients, but the levels were low compared there is an association between food intake and their with IgG and IgM directed against the same antigen in disease activity, but evidence to support such a connec- patients as well as controls.8 Raised IgG activity to gliadin P 1 tion has been contradictory. Reports are usually based on was found in 47% of 93 RA patients, and 41% concurrently diet experiments with quite different protocols, followed by had IgA rheumatoid factor (RF) and there was some some sort of food challenge. Food hypersensitivity in RA does association with duodenal villous atrophy.9 However, a not reflect IgE mediated allergy, and most studies have subsequent study was contradictory.10 concluded that food is unlikely to have a pathogenic effect in Overall, serum antibodies do not appear to provide an RA. Thus, Panush2 carried out blinded encapsulated chal- immunological link between diet and RA. The reason might lenges, and no more than 5% of the RA patients were deemed be that activation of the intestinal immune system is not to show immunological food sensitivity. Nevertheless, a reliably reflected in the serum, and the fact that circulating recent large European epidemiological study, determining IgA RF is predominantly polymeric supports a mucosal odds ratios (ORs) after adjusting for possible confounding origin.11 Moreover, RA patients may have occult small variables, suggested that there is a significant association intestinal inflammation and increased mucosal permeability between inflammatory polyarthritis and a high intake of red independent of the use of non-steroidal anti-inflammatory meat (OR = 1.9), meat and meat products combined drugs (NSAIDs).12 13 We therefore measured antibodies in (OR = 2.3), and total proteins (OR = 2.9).3 perfusion fluid from the jejunum of RA patients and healthy Few previous reports have considered that a pathogenic controls to investigate directly the mucosal immune response dietary effect on RA could depend on a persistent intake of to a variety of food proteins. food; a brief test challenge with a relatively small dose might not precipitate clinical symptoms. Studies considering the METHODS quantitative variable have in fact tended to suggest that food Study subjects does have pathogenic importance, at least in a significant 45 We studied 17 patients with seropositive RA diagnosed fraction (20–40%) of the patients. according to the criteria of the American College of Attempts to identify food sensitive RA patients by Rheumatology.14 Their mean age was 50 years (range 26 to measuring food specific antibodies or immune complexes in 70) and the mean duration of disease 10 years (range 6 serum have failed. Our previous investigation likewise 9 months to 30 years). Twenty healthy subjects served as concluded that serum antibody measurements seldom pre- dict or confirm food hypersensitivity in RA patients. Abbreviations: RA, rheumatoid arthritis; RF, rheumatoid factor; NSAID, Although increased IgM activities were found, there was no non-steroidal anti-inflammatory drug; ELISA, enzyme linked convincing association with clinical variables or dietetic immunosorbent assay; BSA, bovine serum albumin; OD, optical density; benefits.7 Raised serum IgA activity to gliadin has been SIgA, secretory IgA.

www.gutjnl.com Downloaded from gut.bmj.com on 17 October 2008 Intestinal food antibodies in rheumatoid arthritis 1241 controls; their sex distribution was similar to the RA patients solubilised in 0.02 M ammonium acetate, pH 7.0, while crude but because of ethical constraints we were unable to match antigens were extracted with ammonium acetate from the controls for age, although the ranges were highly wheat, oatS, soy, pork meat, or codfish. The antigen overlapping (mean age 29 years; range 23 to 39). RA patients preparations were frozen and thawed several times during and controls were all white and were taking a normal diet the extraction procedure to increase the protein output. with no restrictions; none complained of gastrointestinal symptoms. Antibody reagents for immunoassays The RA patients suffered from active disease as defined by Isotype specific rabbit antisera to human IgG and IgA were the presence of at least two of the following three criteria: prepared in our own laboratory (LIIPAT Nos 38 and 252). duration of morning stiffness >60 minutes, tenderness or Rabbit antibody to human IgM (Code No A0425) was swelling or both of six or more joints, and an erythrocyte obtained from Dako (Glostrup, Denmark). Alkaline phos- sedimentation rate (ESR) of .30 mm in the first hour. None phatase conjugated swine anti-rabbit IgG was obtained from of the patients had received treatment with gold, penicilla- Orion Diagnostica (Espoo, Finland). Total immunoglobulin mine, chloroquine, sulfasalazine, corticosteroids, or immuno- levels in serum and jejunal fluid were determined by enzyme suppressants for the three months before study inclusion. linked immunosorbent assay (ELISA), as previously Most of them were treated with NSAIDs, but in all except two 18 19 this treatment was withdrawn for at least three days before described. Albumin was measured by a commercial intestinal perfusion was undertaken. radioimmunoassay (Pharmacia, Uppsala, Sweden). Five of the patients were reinvestigated after treatment with sulfasalazine for 16 weeks; three of these had not Measurements of antibody activities received NSAIDs for at least one month. The dose of Relative levels of IgG and IgA antibody activities against sulfasalazine was increased by 0.5 g weekly from 1 g/day different food antigens were determined with an ELISA up to 2–3 g/day. slightly modified from our previous method.17 Briefly, The patient protocols as well as the sampling of serum and antigens in 0.02 M ammonium acetate were coated onto jejunal perfusion fluid were based on informed consent and Costar microtitre plates, No 3590 (Cambridge, were approved by the local ethics committee. Most of the Massachusetts, USA) and washed. Activities were deter- subjects were included in a previously reported study of mined in triplicates of perfusion samples, diluted 1/5 (IgG) or microbial antibodies.15 1/10 (IgA) in 0.02 M Tris buffer (pH 7.4) containing 0.05% Tween 20 and 0.5% (wt/vol) bovine serum albumin (BSA) for Jejunal perfusion fluid gliadin, wheat, oatE, oatS, soy, casein, b-lactoglobulin, and A segment of the jejunum was perfused, as detailed codfish, or 0.5% (wt/vol) gelatin for a-lactalbumin, ovalbu- elsewhere, by a small diameter tube 175 cm long and min, and pork meat, in addition to NaCl (29 g/l) and KCl containing six channels.16 The original report described the (0.2 g/l) for both procedures. The reactions with secondary insertion of the tube, gastric drainage, inflation of the (rabbit antibody to human IgG or IgA) and tertiary (alkaline balloons, and rinsing of the closed intestinal segment. C- phosphatase conjugated swine anti-rabbit IgG) immuno- labelled polyethylene glycol was used as a volume marker reagents took place in the same buffers as those used for the and phenolsulphonphthalein (phenol red) as a marker of the respective primary steps. The final step with alkaline patency of the proximal balloon. The recovery of the volume phosphatase substrate took place in diethanolamine buffer marker was (mean (SD)) 86 (10)% in RA patients and 89 (pH 9.6) before reading of optical densities (OD) at 405 nm. (6)% in controls. The fluid was collected on ice, centrifuged in After coating, the ELISA plates were treated with 0.5% BSA cold conditions at 2500 6g, and frozen in aliquots of 2 ml in ammonium acetate for three hours; also the successive until analysis. Successful sampling was achieved from 14 RA reaction steps took place in the presence of BSA, which made patients and all controls. it possible to store coated plates beneath a moist filter paper in the refrigerator for a couple of weeks. With gelatine, Antigens however, the plates were preferably used immediately after Antigen solutions were prepared as described.17 Briefly, coating. Deionised water was used for washing after the gliadin (Karl Roth, Karlsruhe, Germany) and oatE represent coating and the blocking steps. For washings between ethanol (70%) extractable prolamins from wheat and oat sequential antibody reaction steps, Tween 20 (500 ml/l) was flour, respectively, while wheat and oatS represent the added to the water to inhibit non-specific binding of proteins comparable water soluble antigens. Purified antigens such to the plates. It was ensured that the substances added to the as a-lactalbumin, b-lactoglobulin, casein, and ovalbumin perfusion fluid did not interfere with the measurements. (Sigma Chemical Company, St Louis, Missouri, USA) were IgM antibody activities to the same antigens were measured both in serum (1/400) and intestinal fluid (1/10) as described above for IgG and IgA, with the following Table 1 Total levels (mg/l) of immunglobulins, including exceptions: The first step (test sample) was reduced from secretory IgA (SIgA) in jejunal perfusion fluid overnight to two hours, while the concentration of BSA was increased to 2.5% (wt/vol) and gelatine to 1% (wt/vol) Subjects IgA SIgA IgM IgG because non-specific binding is a problem when IgM RA patients antibodies are determined in serum. Blocking with BSA Median 33 25 5.4 5.2 was omitted after coating with soy and both of the oats Range 11 to 70 13 to 50 0.3 to 9.0 1.9 to 14 Quartile 14; 42 13; 36 3.0; 7.8 4.1; 7.1 preparations. With codfish, the gelatine buffer was used instead of the BSA buffer. Healthy controls The results are expressed in units per ml (U/ml) related to a Median 24 20 1.6 5.1 serum pool from patients with untreated coeliac disease,17 Range 5.0 to 56 2.8 to 44 0.3 to 7.3 1.1 to 10.8 Quartile 14.6; 31 11.7; 28 0.8; 2.3 2.5; 7.5 arbitrarily taken to contain 1000 U/ml of IgG and IgA antibodies and 250 U/ml of IgM antibodies, thus providing Probability p,0.33 p,0.20 p,0.0003 p,0.51 incomparable isotype and specificity values. Standard curves were constructed from serial dilutions of the reference serum, and sample readings were carried out using an ELISACalc

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A Jejunal fluid (IgA) B Jejunal fluid (IgG) 30 150 Patients Patients

100 20

50 10

30 150 Controls Codfish Controls Codfish Pork meat Pork meat Ovalbumin Ovalbumin Casein Casein Σ lgA against food (units/ml) α-Lactalbumin Σ lgG against food (units/ml) α-Lactalbumin β β 100 -Lactoglobulin 20 -Lactoglobulin Soy Soy OatS OatS OatE OatE Gliadin Gliadin

50 10

Figure 1 Enzyme linked immunosorbent assay (ELISA) determinations of IgA (A) and IgG (B) antibody activities against various food antigens (see key) in jejunal perfusion fluid obtained from patients with rheumatoid arthritis (patients) (n = 14) and healthy controls (n = 20). Columns represent accumulative antibody levels, arranged in decreasing order for individual subjects. Note different scales on vertical axes. data program developed in our laboratory to provide even after full Bonferroni correction (table 2). Exceptions mathematical curve fitting.17 were IgM and IgG activities against b-1actoglobulin, and IgG activities against a-lactalbumin, ovalbumin, and soy. Statistical evaluation Likewise, intestinal IgA activity against gliadin and casein The Mann–Whitney two tailed non-parametric test was used showed no convincing increase (table 2). for statistical comparisons, with p,0.05 as the significance Interestingly, the two latter food proteins were the only level. Because the positive results obtained generally antigens against which the serum IgM activity was sig- appeared to be mutually correlated, as shown by Spearman nificantly raised in RA (fig 2B and table 3), perhaps reflecting correlation analyses (see later), full Bonferroni adjustment abundant antigen uptake owing to lack of a substantial for multiple tests would be too conservative (http://home.- mucosal IgA response. Also, serum and intestinal IgM clara.net/sisa/bonhlp.htm). The probability (p) values should activities to these two antigens were not correlated probably be adjusted somewhere between no correction and (r = 0.02); only IgM directed against b-1actoglobulin full; both these extremes are given in the tables, because it is (r = 0.75, p = 0.0005), oatS (r = 0.55, p = 0.023) and a- not possible to know exactly the extent to which the data lactalbumin (r = 0.54, p = 0.025) showed some relation in must be corrected. Notably, however, full Bonferroni correc- the two body fluids (uncorrected p values). Notably, the tion did not alter the main conclusions of the study. serum IgM activities to casein and ovalbumin tended to be correlated with the intestinal albumin level (r = 0.67, p,0025 and r = 0.59, p,0.05, respectively), possibly supporting the RESULTS idea that systemic immune activation depends on an Total immunoglobulin levels and food antibodies inadequate mucosal barrier function. Conversely, the intest- The total level of IgM in jejunal fluid of RA patients was inal IgM activities to all antigens were statistically unrelated significantly increased, whereas that of IgA and secretory IgA to any excessive leakage of albumin into the lumen (data not (SIgA) only showed a trend towards an increase (table 1). shown); and, as mentioned above, there was no indication of The median intestinal level of IgG was the same in RA a generally increased intestinal permeability for proteins in patients as in healthy control subjects, supporting the view the RA patients, because the average jejunal IgG level was that NSAID treatment had not caused any general increase in normal (table 1). mucosal permeability for intact proteins (see below). Jejunal IgA, IgG, and IgM activities to nearly all test antigens were highly or moderately increased in RA patients Antibody activities to food antigens are both related when compared with controls by ranking of accumulated and unrelated antibody levels (figs 1 and 2A). In particular, the IgM Because food intake and disease severity show an apparent activities were strikingly raised, and this elevation was connection in some RA patients only, we identified those unrelated to the total IgM levels (fig 2A). The antibody with increased intestinal antibody activities. In the control increases were generally significant or highly significant, group, IgA activities to most food antigens correlated with

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A Jejunal fluid (IgM) B Serum (IgM) 10 Patients Patients 300 2400 8

6 200 1600

4

100 800 2

Controls Wheat Controls Cod 300 Cod 2400 Pork Total IgM (mg/l) Total Pork 8 Ovalbumin Ovalbumin Casein

SlgM against food (units/ml) Casein SlgM against food (units/ml) α-Lactalbumin α β -Lactalbumin 6 -Lactoglobulin 200 β-Lactoglobulin 1600 Soy Soy OatS OatS Gliadin Gliadin 4

100 800 2

Figure 2 Enzyme linked immunosorbent assay (ELISA) determinations of IgM antibody activities against various food antigens (see key) in jejunal perfusion fluid (A) obtained from patients with rheumatoid arthritis (patients) (n = 13) and healthy controls (n = 20), and in serum (B) from patients (n = 17) and controls (n = 14). Columns represent accumulative antibody levels, arranged in decreasing order for individual subjects. Thin vertical bars in (A) represent total intestinal IgM concentrations for comparison with the individual IgM antibody levels. Note different scales on the three vertical axes. the total intestinal IgA levels. We therefore drew a linear (r = 0.61), soy (r = 0.67), cod fish (r = 0.74), and ovalbumin regression line for total IgA and IgA activity in controls and (r = 0.90); a-lactalbumin v ovalbumin (r = 0.55), casein considered RA activities to be increased when they were (r = 0.57), and oatE (r = 0.73); and oatE v gliadin above the limiting control lines, as suggested by Karol et al20 (r = 0.74). Conversely, IgA activities to most antigens were for serum IgE antibodies. However, IgG and IgM activities, as significantly correlated with each other in the controls. well as IgA antibodies to flour antigens (including soy), did Intestinal antibodies to one and the same antigen generally not show regression with the total isotype levels; for these did not correlate so well among the three Ig classes as did the activities the mean values for controls plus two standard activities of a specific isotype against different antigens, with deviations were considered the upper limit when recording the exception of antibodies to gliadin (r = 0.85). Otherwise an antibody increase in RA patients. the relations among different isotypes varied considerably The proportion of patients showing antibody increase (r = 0.06 to r = 0.61). depended both on the Ig class and on the type of food antigen. Thus patients deemed to have increased intestinal Effect of sulfasalazine IgA activity varied for flours from 7% (soy) to 21% (gliadin, Treatment of five RA patients with sulfasalazine for 16 weeks OatS), for cow’s milk proteins from none (casein) to 50% (a- reduced the increased food antibody levels seen initially in lactalbumin) or 57% (b-lactoglobulin), and for other types of three of them; this effect was striking in the two with the protein from 28% (ovalbumin) to 71% (cod fish) or 100% highest levels (fig 3), suggesting that sulfasalazine had an (pork meat). Increased IgG activity was less common and immunosuppressive effect on intestinal immune responses. It varied from 14–21% (a-lactalbumin, b-lactoglobulin, casein, has also been proposed that this drug can diminish mucosal ovalbumin, soy, oats) to 36–57% (pork meat, cod fish, permeability, but luminal albumin levels were not consis- gliadin). IgM activities were often increased, varying from tently decreased after the treatment, regardless of whether 36–50% (pork meat, cod fish, gliadin, b-1actoglobulin) to 57– the patients had received NSAIDs recently or not (fig 4); 86% (soy, OatS, a-lactalbumin, casein, ovalbumin). neither was the antibody decrease accompanied by any Individual intestinal IgM activities to different antigens apparent reduction of total Ig levels in jejunal fluid (data not were positively correlated in the RA patients (r = 0.68 to 0.99) shown)—in contrast to our observations after sulfasalazine and also in the controls, although at lower magnitude treatment in a contemporary study of patients with ankylos- (r = 0.33 to 0.93). IgG activities showed positive correlations ing spondylitis.19 In that disease, the suppressive effect on the for most antigens both in RA patients (r = 0.68 to 0.97) and intestinal IgM (p,0.01) and SIgA (p,0.002) levels was controls (r = 0.51 to 0.99), but usually of lower magnitude accompanied by clinical improvement and reduced ESR than for IgM activities in the patients. IgA activities did not (p,0.004).19 In the few RA patients subjected to sulfasalazine correlate in the RA patients except against some antigens: treatment, however, the clinical effect was associated with pork meat v a-lactalbumin (r = 0.53), b-lactoglobulin reduced ESR but not necessarily with decreased jejunal

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Table 2 Relative IgA, IgG, and IgM activity levels (ELISA units/ml, median and range) to various food antigens in jejunal perfusion fluid from patients with rheumatoid arthritis and healthy control subjects

Antigens Patients Controls Probability*

IgA Gliadin 5.3 (1.3 to 75) 2.8 (0.5 to 20.0) p = 0.068 (p = 0.68) OatE 7.0 (2.5 to 25.0) 3.8 (0.4 to 9.7) p = 0.029 (p = 0.29) OatS 2.1 (0.9 to 9.3) 0.9 (0.3 to 3.8) p = 0.0026 (p = 0.026) Soy 2.2 (0.7 to 6.2) 1.0 (0.1 to 5.8) p = 0.027 (p = 0.27) b-lactoglobulin 2.1 (0.9 to 6.3) 0.8 (0.1 to 2.3) p,,0.0004 (p,0.004) a-lactalbumin 4.6 (1.5 to 21.0) 1.8 (0.8 to 5.5) p = 0.0086 (p = 0.086) Casein 2.0 (0.3 to 5.7) 1.5 (0.1 to 7.5) p = 0.301 (p = 1.0) Ovalbumin 2.7 (0.9 to 11.0) 1.7 (0.1 to 4.8) p = 0.016 (p = 0.16) Pork meat 8.8 (3.3 to 25.0) 2.4 (0.3 to 7.5) p,,0.0004 (p = ,0.004) Codfish 5.3 (1.7 to 10.0) 1.9 (0.3 to 7.5) p = 0.0004 (p = 0.004)

IgG Gliadin 1.1 (0.3 to 4.5) 0.09 (0.03 to 1.1) p,0.0004 (p,0.004) OatE 1.0 (0.3 to 3.0) 0.20 (0.03 to 0.9) p,0.0004 (p,0.004) OatS 0.3 (0.03 to 1.1) 0.04 (0.03 to 0.8) p = 0.0086 (p = 0.086) Soy 0.4 (0.03 to 1.4) 0.08 (0.03 to 0.7) p = 0.0147 (p = 0.147) b-lactoglobulin 0.3 (0.03 to 1.9) 0.16 (0.03 to 0.6) p = 0.149 (p = 1.0) a-lactalbumin 0.7 (0.03 to 2.8) 0.15 (0.04 to 1.7) p = 0.086 (p = 0.86) Casein 0.5 (0.04 to 1.9) 0.04 (0.03 to 0.9) p = 0.0028 (p = 0.028) Ovalbumin 0.7 (0.04 to 4.3) 0.16 (0.03 to 1.8) p = 0.023 (p = 0.23) Pork meat 1.5 (0.1 to 5.8) 0.27 (0.04 to 0.4) p = 0.0006 (p = 0.006) Codfish 0.9 (0.07 to 3.5) 0.18 (0.03 to 0.6) p = 0.0004 (p = 0.004)

IgM Gliadin 10.9 (3.6 to 45.2) 3.0 (0.7 to 12.8) p,0.0004 (p,0.0036) OatE Not done Not done OatS 7.8 (3.5 to 33.7) 2.7 (1.2 to 6.8) p,,0.0004 (p,0.0036) Soy 5.3 (1.8 to 21.8) 1.7 (1.3 to 3.1) p,0.0004 (p,0.0036) b-lactoglobulin 3.8 (0.5 to 24.8) 1.2 (0.5 to 4.1) p = 0.031 (p = 0.279) a-lactalbumin 5.3 (2.3 to 37.1) 1.4 (0.5 to 2.3) p,,0.0004 (p,0.0036) Casein 4.3 (2.3 to 30.5) 0.6 (0 to 2.1) p,,0.0004 (p,0.0036) Ovalbumin 3.5 (1.6 to 26.8) 0.5 (0 to 1.3) p,,0.0004 (p,0.0036) Pork meat 9.8 (3.1 to 34.9) 2.9 (0.4 to 20.0) p = 0.0014 (p = 0.0126) Codfish 8.8 (3.0 to 40.0) 2.8 (0.1 to 22.0) p = 0.0016 (p = 0.0144)

*The probability that food specific IgA, IgG, or IgM activity is increased compared with matched controls (Mann–Whitney two tailed test). p Values in parentheses were subjected to full Bonferroni adjustment for multiple tests. ELISA, enzyme linked immunosorbent assay; OatE. oat flour extracted with 70% ethanol; OatS, oat flour extracted with ammonium acetate. antibody production (fig 3). Because a high protein intake had no effect on the readings, or could even increase them— gives an OR of only 2.9 for polyarthritis,3 a much larger study probably because of the formation of soluble immune group would clearly be required to demonstrate convincingly complexes which remained able to react with the coat. We an association between intestinal food antibodies and the therefore carried out antibody absorption with a large excess severity of RA. of gliadin, which is insoluble in aqueous solution. Thus intestinal fluid (fig 5A) and serum samples (fig 5B) were Cross reactivity of food antibodies revealed by tested in ELISA for remaining IgA and IgM antibody absorption test activities after being mixed with gliadin (0.1 g/ml) overnight Varying amounts of the coating antigen (1.56 to 10 mg/ml) on a shaker at room temperature. Residual IgM activity added to intestinal fluid the day before the ELISA generally against gliadin was only 0%–18% and against unrelated

Table 3 Relative IgM activity levels (ELISA units/ml, median and range) against various food antigens in serum samples from patients with rheumatoid arthritis and healthy control subjects

Antigens Patients Controls Probability*

Gliadin 597 (104 to 1087) 239 (651 to 1396) p,0.0004 (p,0.0036) OatS 270 (36 to 627) 136 (25 to 575) p = 0.057 (p = 0.513) Soy 159 (34 to 403) 131 (39 to 774) p = 0.575 (p = 1.0) b-lactoglobulin 61 (10 to 344) 32 (5 to 101) p = 0.074 (p = 0.666) a-lactalbumin 46 (2 to 103) 52 (18 to 158) p = 0.189 (p = 1.0) Casein 191 (43 to 523) 46 (1 to 204) p,,0.0004 (p,0.0036) Ovalbumin 71 (12 to 121) 49 (1 to 130) p = 0.18 (p = 1.0) Pork meat 54 (31 to 98) 109 (34 to 159) p = 1.0 Codfish 84 (35 to 200) 123 (39 to 381) p = 1.0

*The probability that food specific IgM activity is increased compared with matched controls (Mann–Whitney two tailed test). p Values in parentheses were subjected to full Bonferroni adjustment for multiple tests. ELISA, enzyme linked immunosorbent assay; OatS, oat flour extracted with ammonium acetate.

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Figure 3 Enzyme linked P1 P2 P3 P4 P5 immunosorbent assay determinations of IgM, IgA, and IgG antibody activities IgM against various food antigens as Gliadin 40 indicated (see key) in jejunal perfusion OatE fluid obtained from five patients with OatS rheumatoid arthritis (P1–P5) before (Be) Wheat and after (Af) treatment with Soy 30 β-Lactoglobin sulfasalazine for 16 weeks. P1, P3, and Casein P4 had not received NSAIDs for at least α-Lactalbumin one month before sampling (see fig 4). Ovalbumin The effect of sulfasalazine on the 20 Pork erythrocyte sedimentation rate (ESR) Codfish and clinical improvement (+ to ++)or worsening (2) following the treatment is indicated at the bottom. Note different scales on vertical axes. NSAID, non- 10 steroidal anti-inflammatory drug.

20 IgA

15

Specific antibody activity (units/ml) 10

5

6 IgG

4

2

Be Af Be Af Be Af Be Af Be Af

57ESR 119 70 34 35 7 48 16 38 18 Clinical _ +++++++ effect

80 Figure 4 Radioimmunoassay antigens 50–85%. Remaining IgA activity was measured only determinations of albumin in in one intestinal fluid (fig 5A); with most antigens the jejunal perfusion fluid obtained proportion of residual antibody activity was lower than that from the same patients with for IgM. 70 rheumatoid arthritis (P1–P5) reported in fig 3; those encircled had not received NSAIDs for at DISCUSSION 60 least one month. There was no P4 consistent difference in the This is the first extensive study of intestinal food antibodies albumin levels before (Be) or after in RA patients. Despite considerable variability—which can (Af) sulfasalazine treatment for be expected for antibody levels even in healthy adults 50 16 weeks (medians indicated by regardless of age and sex21—our results were remarkably horizontal bars). NSAID, non- positive in RA, particularly for the IgM class, and included P1 steroidal anti-inflammatory drug. most test antigens in a surprising manner. Absorption with 40 insoluble gliadin revealed a substantial level of cross mg/l P3 reactivity for both IgM and IgA antibodies. Notably, even in the normal state, SIgA in various human secretions has been 30 reported to show a relatively high level of cross reactivity, recognising both self and microbial antigens,22 but apparently P2 without involving peritoneally derived B1 cells, in contrast to 23 20 P5 the situation in mice. Rather, it reflects a substantial innate drive of the intestinal immune system.23 The IgM reactivity in jejunal fluid was only marginally 10 related to that in serum of the same patient, and it was neither related to total IgM levels nor to mucosal protein permeability as deemed by mucosal leakage of albumin or 0 IgG. Therefore, a truly RA related mucosal production of Be Af antibodies was strongly suggested, rather than excessive

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36 100 antibodies used in the assay. A panel of food antigens to A IgM Perf 455 IgM Perf 451 IgA Perf 451 document gut antibody cross reactivity in RA has apparently not been used before, although it should be pointed out that 80 we have not demonstrated polyreactivity in the formal sense. Intestinal IgM and IgA with RF activity have been observed 60 in patients with untreated coeliac disease, and the IgM RF level was quite high in coeliac patients with IgA deficiency.37 Mucosal RF synthesis is apparently linked to the gluten 40 response because RF in serum from patients with coeliac disease or dermatitis herpetiformis was carried only by IgA.38 20 Furthermore, SIgA RF has been detected in serum from RA patients, so some intestinal RF synthesis may take place also in RA.11 However, we found that intestinal fluid from RA 100 patients contained only low levels of IgA and IgM RF, some B IgM Ser 457 IgM Ser 451 IgM Ser 453 1000 times less than in serum.15 The IgA, but notably not the 80 IgM, RF activities were generally well correlated with the food antibody levels of all the three Ig classes (r = 0.65 to 0.94; p = 0.01 to 0.0001). 60 Multispecific antibodies may exist in antigen complexes.39 In the gut, such complex formation depends on antigen 40 stability and on pH dependent pepsin hydrolysis. Thus infants are prone to develop cow’s milk allergy while their gastric acidity is pH 3–4 (compared with pH 2 in adults); at

Remaining antibody activity (%) to food after gliadin inhibition 20 pH 4 the degradation of a-lactalbumin, BSA, and bovine IgG is markedly reduced in contrast to b lactoglobulin.40 Some 80% of untreated RA patients have been shown to have 41

Gil reduced maximum gastric acid output, which could Soy Cas Cod Pork Ova β -La α -La OatS OatE contribute to enhanced food immunoreactivity. Wheat Antigen A germ-free state prevents the development of gut and joint inflammation in HLA-B27 transgenic rats, thereby Figure 5 Enzyme linked immunosorbent assay determinations of IgA giving strong support to a connection between mucosal and IgM antibody activities (per cent of original levels) against various immunity and arthritis.42 Also, reactive arthritis in humans food antigens as indicated (see key), remaining in two different perfusion fluids (Perf 451 and Perf 455) obtained from the jejunum (A) and in three appears to be caused by a combination of a mucosa 43 serum samples (B) of patients with rheumatoid arthritis after absorption associated microbial impact and genetic predisposition. of the samples with an excess of insoluble gliadin. Interestingly, some 90% of patients with reactive arthritis or ankylosing spondylitis express HLA-B27, and these disorders can be associated with Crohn’s disease, ulcerative colitis, and immunostimulation after potentially NSAID induced absorp- jejuno-ileal bypass surgery43—again emphasising the putative tion of dietary antigens.12 Also, notably, two patients who had gut–joint axis which is also supported by shared homing not received NSAIDs for at least one month showed increased properties of activated intestinal immune cells.44 intestinal IgM and IgA food reactivity as well. Increased Moreover, animal experiments have demonstrated a wide- levels of circulating SIgA associated with IgA RF complexes spread tissue distribution of food antigens shortly after have been observed in RA patients,11 likewise suggesting that feeding,45 which could predispose to synovial immune intestinal immunity is overactivated in this disease. complex formation and thereby autoimmune joint reac- RA sera are known to contain increased amounts of so tions.27 We have previously reported that intestinal levels of called ‘‘natural antibodies’’, which are encoded by germ line IgM and IgA are increased in patients with ankylosing Ig variable genes with only few or no somatic mutations. spondylitis related to disease activity.19 Antigens from the gut Such antibodies display a broad array of mostly autoimmune microbiota rather than food are apparently involved in that activity, perhaps enabling them to clear waste products.24 In disease,43 44 because the IgM reactivity to dietary antigens was serum, this activity has been thought to represent low avidity not different from normal control levels (our unpublished IgM antibodies but is, instead, mainly of the IgG class.24 RF is observations), in striking contrast to the data presented here mostly of the IgM, IgG, or IgA class25 and appears to be for RA. Disparate antigenic or mitogenic stimulation in the subjected to antigen driven mutation in RA, but still showing gut might explain the different response to sulfasalazine substantial cross reactivity, like other autoantibodies.26 treatment noted in the two disorders with regard to reduction Therefore, it cannot be excluded that food antigens are of total intestinal immunoglobulin levels as mentioned in involved in the rheumatoid factor induction process, accord- Results. ing to the ‘‘multiple hit model’’ for RA.27 The underlying immunoregulatory defect may involve both poor activity of Conclusions regulatory T cells28 and impaired early B cell tolerance.29 Both systemic and intestinal humoral immunity was found to Polyreactive antibodies most probably have a flexible be aberrant in many RA patients, with a particularly striking antigen binding ‘‘pocket’’ that can accommodate different elevation of cross reactive food antibodies in proximal gut antigens.30 Polyreactivity of RF could hence explain the secretions. IgM reactivity against some food items was overall tendency to increased IgM activities in RA serum increased also in serum, but relatively much less so. irrespective of test antigen, for instance Proteus mirabilis, Measurements of serum antibodies (except for RF) appears staphylococcal enterotoxin B, b2 microglobulin, and cyto- to be of little informative value in RA. Conversely, measure- kines.31–34 It should also be noted that investigation of food ments of intestinal antibodies provide more striking results, antibodies in RA sera is difficult because RF of different Ig suggesting a connection between mucosal immune activation classes may cause assay interference.35 Another complication and the pathogenesis of RA, at least in some patients. Their is that IgM RF binds IgG from other species, including the food related problems may reflect the additive effect of

www.gutjnl.com Downloaded from gut.bmj.com on 17 October 2008 Intestinal food antibodies in rheumatoid arthritis 1247 multiple modest hypersensitivity reactions mediated—for 17 Hvatum M, Scott H, Brandtzaeg P. Serum IgG subclass antibodies to a variety of antigens in patients with coeliac disease. Gut 1992;33:632–8. instance, by immune complexes—which could predispose 18 Kanerud L, Engstro¨m GN, Tarkowski A. Evidence for differential effects of 27 the joints for autoimmune tissue destructive reactions. sulphasalazine on systemic and mucosal immunity in rheumatoid arthritis. Ann Rheum Dis 1995;54:256–62. 19 Feltelius N, Hvatum M, Brandtzaeg P, et al. Increased jejunal secretory lgA ACKNOWLEDGEMENTS and IgM in ankylosing spondylitis: normalization after treatment with Supported by the Research Council of Norway, the Norwegian sulfasalazine. J Rheumatol 1994;21:2076–81. Rheumatological Society, the Swedish Research Council, the Swedish 20 Karol MH, Kramarik JA, Ferguson J. Methods to assess RAST results in Association Against Rheumatism, the King Gustaf V’s 80 year Fund, patients exposed to chemical allergens. Allergy 1995;50:48–54. and Professor Nanna Svartz’ Foundation. Kathrine Hagelsteen is 21 Cummings JH, Antoine JM, Azpiroz F, et al. PASSCLAIM—gut health and thanked for excellent assistance with ELISA, and Hege Eliassen for immunity. Eur J Nutr 2004;43(suppl 2):II118–73. assistance with the manuscript. 22 Quan CP, Berneman A, Pires R, et al. Natural polyreactive secretory immunoglobulin A autoantibodies as a possible barrier to infection in humans. Infect Immun 1997;65:3997–4004...... 23 Brandtzaeg P, Johansen F-E. Mucosal B cells: phenotypic characteristics, Authors’ affiliations transcriptional regulation, and homing properties. Immunol Rev M Hvatum, P Brandtzaeg, Laboratory for Immunohistochemistry and 2005;206:32–63. Immunopathology (LIIPAT), Institute of Pathology, University of Oslo, 24 Avrameas S. Natural autoantibodies: from ‘‘horror autotoxicus’’ to ‘‘gnothi seauton’’. Immunol Today 1991;12:154–9. Rikshospitalet University Hospital, Oslo, Norway 25 Jefferis R. Rheumatoid factors, B cells and immunoglobulin genes. Br Med Bull L Kanerud, Department of Rheumatology, Karolinska Institute, Stockholm 1995;51:312–31. So¨der Hospital, Stockholm (current address: Rheumatology Unit, 26 Levinson SS. Humoral mechanisms in autoimmune disease. J Clin Karolinska Institute, Farsta La¨karhus, Farsta), Sweden Immunoassay 1994;17:72–84. RHa¨llgren, Department of Medical Sciences, University Hospital, 27 van Gaalen F, Ioan-Facsinay A, Huizinga TW, et al. The devil in the details: the emerging role of anticitrulline autoimmunity in rheumatoid arthritis. Uppsala, Sweden J Immunol 2005;175:5575–80. Conflict of interest: None declared. 28 Ehrenstein MR, Evans JG, Singh A, et al. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy. 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Enhanced jejunal production of connection between intestinal and articular inflammation. Aliment Pharmacol antibodies to Klebsiella and other Enterobacteria in patients with ankylosing Ther 1997;11(suppl 3):24–39. spondylitis and rheumatoid arthritis. Ann Rheum Dis 1997;56:421–5. 45 Gu¨tgemann I, Fahrer AM, Altman JD, et al. Induction of rapid T cell activation 16 Knutson L, Odlind B, Ha¨llgren R. A new technique for segmental jejunal and tolerance by systemic presentation of an orally administered antigen. perfusion in man. Am J Gastroenterol 1989;84:1278–84. Immunity 1998;8:667–73.

www.gutjnl.com Toward An Understanding of Allergy and In-Vitro Testing By Mary James, N.D.

Learning to recognize Food represents the largest anti- don’t will be discussed, or why a genic challenge facing the immune specific food elicits symptoms in and manage food system. Assuming complete diges- an individual at one point in time, allergies can go a long tion, an intact intestine, a sturdy yet appears to be well tolerated at constitution, and minimal anti- other times. We will examine why way in achieving better genic exposure such that the an individual sometimes feels bet- immune system is not over- ter from eating an allergenic food, clinical results whelmed, all goes well. Weaknesses but feels worse from eating it fol- with patients. in one or more of these areas, lowing a period of elimination. We however, can result in immune will look at why an allergy test attacks upon foods as if they were might be normal in an individual foreign invaders. A long list of who knows he experiences symp- conditions have been associated toms when ingesting certain foods, with food reactions, including or why an in-vitro test may show fatigue, migraines, irritable bowel elevated antibodies to foods in an syndrome, inflammatory bowel asymptomatic person. Finally, we disease, gallbladder disease, arthri- will discuss how to effectively man- tis, asthma, rhinitis, Attention age allergenic food elimination, Deficit Hyperactivity Disorder reintroduction and rotation. While (ADHD), enuresis, epilepsy, there are admittedly many useful eczema, psoriasis, aphthous ulcers, approaches to the diagnosis and and recurrent sinusitis, otitis media treatment of allergic disorders, the and other infections.(1) A patient following discussion will be limited with numerous and seemingly primarily to in-vitro assessment unrelated symptoms often moves and dietary management. from doctor to doctor in search of a diagnosis. When the inflammato- The Immune System ry response to an allergen takes Before embarking on a discussion hours or days to develop, the of allergy, let’s start with a few relationship between foods and basics of the immune response. symptoms is often hard to pin The following overview will provide down. Learning to recognize and a foundation for our discussion. manage food allergies can go a long way in achieving better clinical The principle cells of the immune results with these patients. The system are lymphocytes, plasma intent of this paper is to provide cells and macrophages, collectively such an understanding. organized into lymphoid tissue. Lymphocytes can be further divid- In this paper, we will focus on IgE- ed into B-cells and T-cells. and IgG-mediated immune reac- Interestingly, these cell names were tions. Reasons why some individu- based on parts of a chicken, as 2 als develop allergies while others studies of the thymus (T) and the bursa of Fabricius (B) (a lymphoid reactions to foods and other sub- organ near the cloaca) brought stances. Examples include lactose about the first understanding of intolerance, pharmacological their respective immunological responses to alkaloids in foods functions. The chief role for B-cells such as solanine (potato family), is the provision of humoral immu- salicylate sensitivity, and lectin nity. Upon exposure to an antigen, reactions, in which dietary lectins B-cells proliferate and evolve to interact with surface antigens on antibody-synthesizing plasma cells cells, causing them to agglutinate. that then produce antigen-specific Bacteria and bacterial toxins may immunoglobulins of different isotypes, called IgM, IgG, IgE, IgD and IgA. T-cells, on the Non-immune Mediated Reactions to Foods other hand, provide cell-mediat- ed immunity. • Lactase deficiency (dairy) ➔ bloating, flatulence, diarrhea, abdominal pain • Spoilage or contamination of food by bacteria (Proteus) or heat-stable toxins There is considerable interaction (tuna, bonita, mackerel) ➔ itching, rash, vomiting, diarrhea among components of the • Gallbladder disease ➔ abdominal pain (RUQ), nausea, flatulence, aggravation by fats immune system. Subsets of T- cells, function as: helper T-cells, • Vasoactive amines -phenylethylamine (chocolate, aged cheese, red wine) ➔ migraine which stimulate B-cell activity; -tyramine (cheddar cheese, French cheeses, brewer’s yeast, chianti, canned fish) ➔ or as suppressor T-cells which migraine, erythema, urticaria, hypertensive crises in patients on MAO inhibitors suppress both humoral and cell -histamine (fermented cheese, fermented foods (e.g. sauerkraut), pork sausage, mediated immune responses. canned tuna, anchovies, sardines) ➔ erythema, headache, hypotension Macrophages are released from -histamine-releasing agents (shellfish, chocolate, strawberries, tomatoes, peanuts, ➔ the bone marrow as monocytes, pork, wine, pineapple) urticaria, eczema, pruritis and develop into macrophages • Food additives (e.g. tartrazine, FD&C Yellow No. 5, sodium benzoate) ➔ hives, upon entering tissue. rash, asthma Macrophages serve to present • Sulfites (salad bar lettuce, shrimp, dried fruits and vegetables, wine, beer) ➔ asthma antigen to both T- and B-cells, or anaphylaxis, loss of consciousness as well as to clear antigen/anti- • Monosodium glutamate (Chinese and Japanese dishes) ➔ headache, facial tension, body complexes from the circu- sweating, chest pain, dizziness lation. The efficiency of this • “Nightshade” alkaloids (potatoes, tomatoes, eggplant, peppers, tobacco) ➔ joint function is critical in controlling pain food-induced hypersensitivity ➔ reactions, as we will discuss. • Hypoglycemia Fatigue, palpitations, shakiness, cognitive impairment, mood swings, poor memory, blurred vision, anxiety, dizziness, headache • Lectin reactions (wide variety of foods) ➔ wide variety of symptoms, depending on Getting Our Terms blood type compatibility. (Refer to: Eat Right 4 Your Type, by Peter D’Adamo, N.D.) Straight The terms "food intolerance," "allergy" and "hypersensitivity" are cause gastrointestinal and systemic often used interchangeably. For reactions, such as in scombroid clarification, it’s useful to poisoning from the ingestion differentiate among them… of contaminated tuna. Vasoactive amines (epinephrine, norepineph- Intolerance rine, tyramine, dopamine, hista- mine and 5-hydroxytryptamine) The term "intolerance" generally are found in bananas, tomatoes, applies to non-immune mediated avocados, cheeses, pineapples and 3 wines, and can contribute to •Type II immune reactions involve symptoms such as migraine antibody-mediated destruction of headache. Sulfite, used as a tissue following adherence of preservative in foods such as let- foreign material. This reaction is tuce, shrimp, dried fruit and wine, often referred to as a "cytotoxic can cause asthma and urticaria.(2) reaction." Examples of Type II Some compounds, such as alco- reactions include penicillin reac- Any food eliciting an hol, may even induce histamine tions and those resulting in red responses, although the reaction is cell or platelet destruction. adverse reaction should not immune mediated. •Type III reactions are mediated ideally be avoided, no Because of the conspicuous by mixed immunoglobulins, but matter what the relationship between ingestion primarily IgG. Complexes com- of the food and the onset of posed of antigen and antibody mechanisms, and symptoms, such reactions are fre- activate complement and cytokines quently mistaken for allergy, yet an in the body, resulting in an inflam- careful investigation allergy test may be negative for that matory response. Type III reactions aimed toward the food. Any food eliciting an adverse constitute the basis of "delayed- reaction should ideally be avoided, onset" food allergies. Symptoms causative factors. no matter what the mechanism, are delayed because of the time and careful investigation aimed required for the formation of toward the causative factors. complexes. These reactions will also be discussed in more detail.

Hypersensitivity •Type IV refers to cell-mediated Although the use of the term immune reactions, where T-cells "hypersensitivity" is sometimes act as the primary players. T-cells reserved only for Gell and Coombs’ become cytotoxic cells when acti- classification of Type III, IgG- vated by antigen, capable of killing mediated reactions, traditionally viruses, bacteria, tumor cells or the term is applied to all four types other target cells. Type IV reactions of tissue injury. Types I through IV play a significant role in tuberculo- all depend upon the interaction of sis, mycotic and viral infections, antigen with humoral antibody, contact dermatitis and allograft and result from an excessive rejection. These reactions may also immune reaction to antigen, be involved in some food allergies, leading to gross tissue changes such as protein-losing entero- and symptoms. pathies and celiac disease.

•Type I reactions are mediated by Allergy IgE antibodies, and are character- The definition of the term "allergy" ized by the release of histamine is much debated. Although many and other chemical mediators traditional allergists strictly reserve upon exposure to an allergen. the use of the term for Type I IgE- Type I reactions are responsible mediated reactions such as hay for "immediate-onset" allergies, fever, a more general definition of such as hay fever or anaphylaxis. "allergy" refers to any acquired They will be discussed in more hypersensitivity to an antigen that detail below. results in harmful immunologic 4 consequences. For our purposes, bronchial and cutaneous allergies we will apply this broader defini- show a familial tendency. This is par- tion to both Type I and Type III ticularly true for asthma, hay fever, immune reactions. recurrent rhinitis and bronchitis, and eczema; when present in a parent, Immediate-Onset IgE there is an increased prevalence of Reactions the same disorder in the child.(3) If neither parent is allergic, a patient’s When an antigen attaches to IgE IgE antibody levels may symptoms are less likely to reflect an antibodies already stationed on a IgE-mediated reaction. IgE allergies drop with avoidance mast cell or basophil, pre-formed are in place for life. Antibody levels bundles of histamine are released. may drop with avoidance of expo- of exposure, but Inhalation of antigens usually sure, but re-exposure will quickly leads to the symptoms we com- re-exposure will quickly result in the mobilization of IgE anti- monly associate with "allergy" bodies and the subsequent release of result in the such as sneezing, itching of the histamine. palate or ears, runny nose, itching mobilization of and tearing in the eyes, and fatigue. As mentioned above, IgE-mediated Ingestion of an antigen may lead to antibodies and the food allergies are usually easy to symptoms such as asthma, an itchy spot because of the immediate subsequent release rash, angioedema or gastrointesti- appearance of symptoms. Common nal symptoms such as abdominal culprits include peanuts and shell- of histamine. cramps and diarrhea. fish. Sensitivity to these substances is so extreme in some cases that Chronic IgE allergies may manifest anaphylaxis may result from the as sinusitis, recurrent ear or upper mere inhalation of vapors from respiratory infections, mouth food or contact with the skin. breathing and post-nasal drip. Needless to say, management of "Allergic shiners" under the eyes these allergies requires strict avoid- and a white line across the nose, ance of the offending substance. from repeated upward wipes of the IgE reactions triggered by inhalants nose with one’s hand, are common often show a seasonal pattern, with signs of IgE-mediated allergies in reactions to tree pollens children, although these signs have typically occurring in the spring, also been observed in individuals grass pollens in late spring and early with IgG allergies. Severe Type I summer, and weed pollens in late reactions may include anaphylaxis. summer and early fall. Reactions to The allergic reaction occurs imme- dust mites often appear in the win- diately upon exposure to the anti- ter, with the onset of home heating. gen (usually less than two hours); Allergy testing during a sympto- consequently, the person experi- matic period can help to identify encing the reaction usually easily the responsible antigens. Allergies recognizes the link between aller- to pet dander can occur at any gen and symptom. time, upon exposure. Specific IgE-mediated allergies are not inherited, although Type I aller- Mold and Fungi Reactions gic diseases in general tend to run Mold allergies tend to coincide in families, and end-organ sensitivi- with wet months that feature ties associated with nasal, above-freezing temperatures, e.g. 5 December through March in typically are Type III and IV and California, summer in the mid- involve IgG antibodies. Symptoms west, or year-round in Florida. Very may include flu-like illness with dry states, such as Arizona or fatigue, rash, muscle and joint Nevada, are unfortunately not pain, headache, fever and exempt from inhalant mold aller- nightsweats. Fungal colonization A food allergy test can gies, as the moisture in air condi- may be life-threatening in immuno- be most clinically tioning units invites the growth of compromised individuals. mold which then becomes dissemi- useful for measuring nated throughout the living space. Avoidance of allergen, in the case Delayed-Onset IgG IgG antibodies involved of molds and fungi, can be Reactions in delayed-onset extremely challenging since these Type III reactions involve the substances are so ubiquitous in the formation and deposition of Type III reactions. environment. Common food antigen/antibody (Ag/Ab) com- sources include fermented cheese, plexes, mostly involving IgG. In wine, beer and bread. Other com- contrast to the immediate IgE mon, but less suspected sources histamine-mediated reactions, include tea, processed foods, these reactions are delayed, since dough conditioner, commercial they involve the gradual formation fruit juices, citric acid, malt flavor- of immune complexes. Because ings, chocolate, soy sauce, tomato these reactions are delayed by products (crushed and left to sit hours or even days following the for better flavor), Lactaid and B exposure, the relationship between vitamins. Fungal colonization of food and symptoms is much more the skin or mucous membranes difficult to spot. It is these reac- represents a third source of expo- tions for which a food allergy test sure which may serve to amplify measuring IgG antibodies can be reactions to other fungi in the envi- most clinically useful. ronment. Since different fungi from different sources share common IgG-mediated reactions typically surface proteins, the immune result from exposure to an excess response to fungi may cross-react. of antigen over an extended period As a result, correcting conditions of time. In the case of food allergy, such as dandruff, athlete’s foot increased intestinal permeability and Candida overgrowth often coupled with repetitious ingestion helps to lessen reactions to the of particular foods causes excessive other sources. antigen to be presented to the immune system. Formation of Such reactions are all primarily insoluble antigen/antibody com- IgE-mediated and feature the plexes results in the activation of symptoms commonly associated complement and the subsequent with histamine, such as sneezing, respiratory burst in neutrophils, runny nose and asthma. More the release of proteolytic enzymes, severe illness may result when an mast cell mediators and vasoactive individual is exposed to a much peptides, and the aggregation of larger number of fungal particles, platelets. Although complement such as in occupational exposures. stimulates inflammation, it also Due to the massive amount of functions to prevent the progres- 6 antigen exposure, these reactions sion from small complexes to larger ones, a factor that helps may NOT elicit symptoms, despite minimize the severity of symptoms. the fact that an immune reaction is Macrophage activity triggers the occurring. An overload of antigen, release of inflammatory mediators however, will saturate the such as interleukin-1, tumor necro- macrophages’ capacity, resulting in sis factor, reactive oxygen species the circulation of complexes and and nitric oxide. their deposition in tissue. Immune compromise may lead to the same Symptoms are typically delayed end, resulting in symptoms. (This in onset, by hours or days, and process is quite different from the vary, not only according to the "loaded gun" IgE response which is specific nature of the immune elicited with every exposure.) This complex, but also according to "overload" phenomenon may help the tissue in which the complexes explain why reducing exposure to are deposited. Headache, vasculitis one allergen may result in an indi- or hypertension may result from vidual being better able to tolerate deposition in the vascular space; other allergens. Unlike Ag/Ab inter- asthma, alveolitis or recurrent actions, macrophage clearance is infection may result from deposi- NON-specific. This means that tion in respiratory tissue, dermato- ANY reduction in demand on logic changes from deposition in macrophages (including non-aller- the skin, and joint pain from gic conditions such as infection or deposition of complexes in the joint xenobiotic exposure) may serve to space. Symptoms such as rhinitis or reduce symptoms and allow other angioedema may also occur, since reactive foods to be eaten. Antigen “overload” two elements in the complement cascade (C3 and C5) are capable This concept of "total load" can- may help explain why of inducing histamine release. Any not be overemphasized. It was not system may be affected and any until the middle of the last century reducing exposure to one symptom is possible, depending on and the growth of the industrial allergen may result in an an individual’s susceptibilities. revolution that the diseases which Reactions may last for days. we now call atopic allergic diseases, individual being better able were recognized as entities. (4) to tolerate other allergens. Hypersensitivities involving Immune Competency and bronchial symptoms and asthma "Total Load" nearly doubled during the 1980s Although an immune interaction and early 1990s.(5) Air pollution, between antibody and antigen including the contribution by diesel occurs every time an individual is exhaust particle emissions, has exposed to an allergenic food, the been shown to enhance both nasal presence and the degree of symp- IgE production and the expression tomatology depends upon the sol- of Th2 cytokines (6), and may ubility of complexes and the reticu- serve as a carrier for pollen and loendothelial system’s ability to other compounds. clear them. Macrophages pick up Ag/Ab complexes immediately, but Food allergies can develop at any have a finite capacity. With an effi- point in one’s life, but an individ- cient immune response, the half- ual may also be born with them. life of a complex may only be a few Such allergies are usually to foods minutes, and exposure to allergens that the mother consumed fre- 7 quently during her pregnancy, symptoms, yet allergies exist, a test although the antibodies are the can help to identify those foods baby’s own. Since maternal dietary which to some degree are stressing proteins are capable of reaching the immune system. If a person is amniotic fluid, and since the fetus is allergic to numerous foods, the capable of mounting antibody and test can also help provide a start- other immune responses as early as ing place, in terms of elimination. the tenth week of gestation, it is pos- sible that fetal sensitization to these In-Vivo Skin Testing proteins begins as early as the first Type I allergies are often trimester of pregnancy.(7) Maternal diagnosed with skin prick testing, IgG antibodies traverse the placenta or with intradermal testing, often during pregnancy, but levels of these employed as a follow-up to a in the infant are typically down by 3- negative skin prick test. Advantages 6 months after birth.(8) Dietary of skin testing include rapid results, proteins from the mother’s diet good sensitivity, and the ability to are also transferred to breast milk. test any antigen. Disadvantages While a certain amount of antigen include the discomfort inherent in passage into the breast milk is prob- the procedure, ably important for the development possible danger of anaphylaxis,(9) of the infant’s tolerance the contraindicating effects of An allergy test to foods, excessive exposure can medications such as anti-hista- result in hypersensitivity. Breast-fed mines, decongestants, beta should always be infants whose mothers take dietary blockers, bronchodilators and theo- precautions during lactation are assessed in conjunction phylline, and occasional interfer- observed to have a markedly reduced ence from skin disease. Since skin with a patient’s incidence of atopic eczema.(7) testing only reflects IgE-mediated reactions, it also cannot inform clin- clinical picture. Unlike IgE allergies, those icians as to the potential for the mediated by IgG may be cured, delayed hypersensitivity reactions following a period of avoidance responsible for such a wide range of and attention to underlying con- symptoms seen clinically.(10) tributing factors. Although a food may be tolerated at some point on In-vitro Antibody a limited basis, the immune system Measurement "holds a grudge," in a sense. The measurement of antigen-spe- Because the hypersensitivity is cific antibodies is a useful tool for recorded in the body’s "memory assessment of allergies, particularly cells" (antigen-stimulated lympho- to foods. One study of young chil- cytes), the response may be reacti- dren found that 62.5% of children vated if exposure again becomes with symptoms had specific IgG excessive or too frequent. antibodies and 22.9% had specific IgE antibodies, while the children DIAGNOSIS without symptoms of food allergy An allergy test should always be had neither.(11) assessed in conjunction with a patient’s clinical picture. If an indi- Three widely used methods for vidual’s immune clearance mecha- measuring specific antibodies 8 nisms are effective in averting include ELISA, MAST and RAST/RASP. ELISA (enzyme-linked ing can provide a starting place for immunosorbent assay) can detect trial elimination programs. As a either IgG or IgE antibodies. MAST screen for an asymptomatic individ- and RAST (radioallergosorbent ual, in-vitro testing may reveal food procedure) measure IgE antibod- allergies which are currently being ies, although MAST testing for IgG effectively managed by the immune antibodies is currently being devel- system (hence, no symptoms), but oped. Some laboratories measure which could manifest symptomati- In-vitro allergy tests only IgG4 for foods; however, cally in the future, in the event of measurement of total IgG increased immune burden. offer the advantages of is recommended. Occasionally, a test will exhibit "across-the-board" low-level IgG convenience, safety (no Although all IgG subclasses are reactivities for an individual. Clinical danger of anaphylaxis), involved in the immune response, observation has suggested the pos- IgG1 is thought to be the main sibility of a chronically "leaky gut" lack of interference by instigator of inflammation. The in such situations, and the con- role of IgG4 in food allergy has comitant immune reaction to a medications or skin been debated. IgG4 is unable to large number of absorbed antigens. condition, and good activate complement, so does not contribute to a true inflammatory Disadvantages of in-vitro testing reproducibility. reaction. It is, however, able to include the requirement of serum precipitate the release of histamine collection, the sometimes lengthy from basophils (12), which might incubation and the possibility of partially explain the elevated levels some false negatives for IgE, which observed in atopic individuals and may result from a number of fac- amelioration of symptoms upon tors. Antibodies that are directed removal of the "positive" foods toward altered forms of antigen not from the diet.(13) At the same time, used in the test, e.g. cooked, it has been suggested that IgG4 spoiled or processed foods, might may function as a "blocking" anti- go undetected. False negatives may body to allergic reactions, particu- also follow immunotherapy, a larly since levels tend to increase result of IgG "blocking" antibody dramatically following successful production. (This is one of the immunotherapy for IgE-mediated mechanisms behind immunothera- pollen allergies.(14) Although IgG4 py’s effectiveness for IgE-mediated can induce histamine release, its allergies; antigen now preferentially release appears to be more delayed binds to IgG, rather than IgE, so and the reaction less acute than in that the immediate and acute hist- the Type I IgE reactions. amine reaction is prevented.) Finally, with the possible exception In-vitro tests offer the advantages of anaphylaxis-inducing allergens, of convenience, safety (no danger specific immunoglobulins tend to of anaphylaxis), lack of interfer- gradually diminish in response to ence by antihistimines or skin con- antigen elimination. Although the dition, and good reproducibility. half-life of IgE antibodies is only 3 In -vitro also allows the use of par- days and the half-life of IgG 23 allel controls with each run. A pos- days, absorbed antigens which have itive reaction on an in-vitro test sig- been sequestered by the liver may, nifies allergy in that individual. For in some cases, be slowly released the "global reactor," in-vitro test- over several months, resulting in 9 some persistent antibody ticular food may not, in fact, corre- production. The levels, however, late with improvement in, or aggra- will still decline over time, barring vation of, an individual’s level of any new exposure. Because foods reactivity. Even the broad cate- such as wheat, dairy and corn are gories of 0-3+ should be evaluated widely used as additives in against the patient’s clinical pic- processed foods or cosmetics, IgG ture. A 3+ IgG reaction will gener- levels are more likely to persist in ally imply a stronger immune reac- an individual who mistakenly tion (or a more severe allergy) than presumes that he has completely a 1+ reaction. However, that 3+ eliminated the foods. IgE antibod- reaction may never manifest as ies to seasonal allergens may be symptoms in a person whose sys- undetectable if measured during an tem is effectively neutralizing asymptomatic period. immune complexes, while a 1+ reaction may result in debilitating Interpreting the In-Vitro symptoms in a person whose retic- Test ulendothelial system is over- It should be noted that in-vitro whelmed. In other words, the best antibody tests are semi-quantita- use of the test is to identify reactive tive. All procedures involve the substances which can then be binding of specific anti-food anti- avoided or rotated in a clinical bodies to an antigen that is already trial. bound to a solid phase, e.g. a plate or test tube. Each procedure Elimination Diets includes a tag, or signal, which is Elimination and reintroduction of quantitated. The tag used in the foods is an invaluable means of RAST is a radioactive isotope, establishing a relationship between In-vitro antibody tests while MAST uses a luminescent sig- a symptom and a particular food. nal. In the ELISA test, the tag is an The typical protocol involves the are semi-quantitative. enzyme that induces a color elimination of all possibly aller- change which is then read photo- The best use of genic foods (commonly the routine metrically as "optical densities." foods in one’s diet) for 1-2 weeks the in-vitro allergy The more intense the color change, and, assuming any clinical the higher the concentration of improvement during this time, the test is to identify specific antibodies. Because of gradual reintroduction of one food inherent imprecision in the multi- reactive substances every 2-3 days. If a food in one step processes used in these proce- meal fails to produce symptoms, which can then be dures, there will always be some then a larger amount is eaten in natural "drift" in the quantifica- the next couple of meals. If symp- avoided or rotated in tion. It is for this reason that toms reappear within the 2-3 days reporting of broad categories for a clinical trial. of repeated ingestion of a food, reactivity, e.g. 0-3+, has become an that food is regarded as allergenic, industry standard. and eliminated from the diet. If no symptoms are induced, the food is Literal reliance upon the larger now included in the hypoallergenic optical density numbers (in the diet, and the next food is tested in case of ELISA) can be misleading, the same manner. as a follow-up test featuring higher or lower optical densities for a par- Needless to say, this process may 10 take several weeks to complete, Inhalant allergies are most often depending upon the number of treated with a combination of foods tested, and it requires avoidance, where possible, and more than a modicum of self- immunotherapy, which consists of discipline. The results, however, injections of serially increasing con- prove invaluable. The patient is centrations of antigen. As antigen not only able to ascertain which is introduced into a patient’s sys- foods produce which symptoms, tem, the immune system gradually but the clinical experience of develops a mixed IgG response to improvement on the elimination it, although never enough to cause diet and the aggravation upon tissue damage. The IgG antibodies ingestion of a food, often serves are thought to block the more to provide the needed incentive severe IgE reaction, and symptoms usually abate within a few weeks. for the subsequent long-term Combining a elimination or rotation of offend- Standard immunotherapy is not ing foods. The process assists in used for the desensitization to preliminary in-vitro revealing the NON-immune medi- foods and molds because the large ated reactions as well. As discussed amount of antigen exposure from test with an natural sources is usually already earlier, a relationship between food elimination diet and symptom does not automati- so high that severe reactions are cally mean "allergy." likely to be induced. It should be represents the most noted that, although IgE antibod- There are a few disadvantages to ies to a substance remain in the efficient approach of system, an IgE blood test may fail relying solely on the elimination all, and improves diet for diagnosis. The length of to pick them up following treat- time and discipline required are ment, due to the competitive patient compliance. mentioned above. Sub-clinical inhibition by IgG. This significant hypersensitivities will not be detect- a decline, however, appears to ed. Finally, the possibility exists occur only after about two years that some of the foods consumed of hyposensitization.(15) as components of the "hypoaller- genic" diet are antigenic for a given individual. In this case, ameliora- Enzyme-potentiated tion of symptoms on the diet does desensitization not occur, and the false conclusion Enzyme-potentiated desensitization is reached that foods play no part (EPD) also utilizes the periodic in the patient’s illness. Combining injection of antigen into the a preliminary in-vitro test with an patient’s skin, but the amount of elimination diet represents the antigen compared to standard most efficient approach of all, and immunotherapy is exceedingly improves patient compliance. small. Furthermore, the antigen is accompanied by the enzyme TREATMENT beta-glucuronidase which helps to activate T-suppressor (CD8) cells. Following is a brief discussion of a Unlike immunotherapy, IgG levels few of the most popular methods do NOT rise in response to the for managing allergies. treatment, although tolerance develops. Injections are adminis- Immunotherapy tered every two months and in most cases can eventually be (Hyposensitization) 111 spaced further apart, assuming greater likelihood that sensitivities improvement. EPD is used for food to oyster, scallop and squid (other and mold allergy and is preferred members of the "mollusk" family) by many physicians for treatment might be present. At the same time, of inhaled allergens, as well. such cross-reactions have been observed to be relatively infrequent, Medications and patient compliance is always a Other standard treatments for consideration. Care must also be inhalant allergies include the use taken that suspected foods are not of anti-histamines, decongestants, inadvertently consumed while hid- glucocorticosteroids, and sodium den in other foods, e.g. eggs con- cromoglycate. This last one serves tained in mayonnaise. to block mast cell degranulation, eosinophil and neutrophil chemo- Eliminating the antigenic food taxis and mediator release; howev- provides the immune system a rest. er, it is poorly absorbed orally, Levels of antibodies gradually so must be taken frequently in decline, immune complexes are order to maintain adequate cleared, and symptoms improve. mucosal concentrations. Quercitin Lesser reactive foods are often is a natural bioflavonoid with tolerated in the beginning if eaten similar activity.(16) All of these no more often than every four medications are merely palliative, days or so; however, this tolerance Eliminating the but of course can make a world varies between individuals, and in of difference in a patient who is the more symptomatic patient antigenic food experiencing paroxysmal sneezing even foods with 1+ reactivities or a frightening bout with asthma. may have to be temporarily provides the immune removed from the diet in order to system a rest. Levels of Allergen Elimination improve clinical outcome. The most important component antibodies of any allergy program is the reduc- Antigen/Antibody gradually decline, tion of exposure to the offending Equilibrium agent. As long as exposure is main- One of the most important deter- immune complexes are tained, antibodies will continue to minants of the pathogenicity of be produced and the immune sys- cleared, and the Ag/Ab complex and the clinical tem "primed" to react. IgE-mediat- course of the patient is the equilib- symptoms improve. ed allergies tend to be "fixed," thus rium between antigen and anti- avoidance is usually life-long. In body. At equivalent concentrations contrast, the IgG-mediated allergies (also called the "zone of equiva- may be reversed over time. Reactive lence"), complexes can grow to foods are ideally eliminated from enormous size. The larger and the diet for a minimum of three more numerous the complexes, the months, particularly those that more likely they are to overwhelm evoke the most severe symptoma- the reticuloendothelial system and tology or the highest scores on an deposit in bodily tissues. in-vitro test. Ideally exposure to Individuals with IgG allergies often foods within the same food family have the paradoxical experience of of a reactive food should be feeling immediately better, rather reduced as well. A sensitivity to than worse, after consuming an clam, for example, may indicate a allergenic food. What is happening 121 here is a shift into the zone of Plasma cells retain a memory of "antigen excess."(17) In this zone, that sensitivity, and new antibodies complexes become more soluble could be produced with any expo- and symptoms may diminish, that sure. Only a trial reintroduction is, until more antibody is produced will answer that question. If no and the "zone of equivalence" is symptoms are elicited when the

Antigen Excess Zone of Equivalence Antibody Excess

again reached, producing more food is reintroduced, the food can symptoms. Eating more of the usually be replaced into the diet, food once again brings relief, but ideally on a rotational basis. perpetuating a vicious cycle of Repetitive exposure to a food usu- addiction that is difficult to break. ally contributes to the development Following 7-10 days of strict of hypersensitivity in the first place, elimination of a reactive food, an thus a return to the same frequen- individual moves into a zone of cy of exposure often brings with it One of the "antibody excess." With less anti- an eventual return of symptoms. most important gen on board, complexes reduce in size and number, and symptoms determinants of the diminish. During this period, he Other Considerations pathogenicity of the or she is extremely sensitive (hence In the case of reactivity to molds, the conspicuous and diagnostic yeast or fungi, it is often useful to clinical course of aggravation from reintroductions treat any existing infections. of offending foods at this point), Examples include ringworm, ath- the patient is the and should stay with the elimina- lete’s foot or jock itch, as well as equilibrium between tion diet for three or more months, vaginal or intestinal yeast over- while antibody levels drop. growth. Doing so may help to antigen and antibody. eliminate cross-reactivity reactions After this point, a follow-up in- and lower the overall burden on vitro test might well show lower the immune system. reactivities for those foods that have been avoided. This fact does Because IgG allergies can be not guarantee that the food will be established as a result of the tolerated when reintroduced. translocation of food antigens 13 across a permeable gut wall (18), imbalances will greatly help to attention to gut repair is a prereq- reduce the likelihood of developing uisite for preventing further prob- additional allergies. lems. It is not uncommon for a person who is eating soy foods, Finally, it’s worth noting that e.g. as a substitute for foods such adjunctive nutritional support for as cow’s milk and wheat, to sud- the immune system, as well as Correcting imbalances denly find herself now unable to selective botanicals, homeopathic tolerate soy after a few months. remedies or acupuncture, may con- in the gastrointestinal Evaluation of intestinal permeabili- tribute significantly to the reduc- flora and providing ty as well as a comprehensive stool tion in immune sensitivity and the analysis can serve to establish the inflammatory response. nutritional support for presence of "leaky gut" and con- tributing factors such as maldiges- Acknowledgements: Special thanks the immune system can tion, malabsorption, imbalances in to Vincent Marinkovich, M.D., greatly help reduce flora, secretory IgA deficiency and (Diplomat, American Board of mucosal injury due to infection or Allergy and Clinical Immunology) immune sensitivity. agents such as non-steroidal anti- for his generous contribution of inflammatories. Correcting any information on the subject.

14 REFERENCES 1) Gaby AR. The role of hidden food allergy/intolerance in chronic disease. 10) El Rafei A, Peters SM, Harris N. Bellanti JA. Diagnostic value of IgG4 mea- Alt Med Review 1998;3(2):90-100. surement in patients with food allergy. Ann Allergy 1989;62:94-99.

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15 Copyright ©ERS Journals Ltd 1998 Eur Respir J 1998; 11: 151–155 European Respiratory Journal DOI: 10.1183/09031936.98.11010151 ISSN 0903 - 1936 Printed in UK - all rights reserved

Reported food intolerance and respiratory symptoms in young adults

R.K. Woods*+, M. Abramson‡, J.M. Raven*, M. Bailey‡, J.M. Weiner+, E.H. Walters*+

aa Reported food intolerance and respiratory symptoms in young adults. R.K. Woods, M. Depts of *Respiratory Medicine, ‡Epidemi- Abramson, J.M. Raven, M. Bailey, J.M. Weiner, E.H. Walters. ©ERS Journals Ltd 1998. ology and Preventive Medicine and +Medi- ABSTRACT: The aim of the study was to assess the ability of the European Commu- cine, Monash Medical School and Alfred nity Respiratory Health Survey (ECRHS) questionnaire to provide data on the preva- Hospital, Melbourne, Australia. lence, type and reported symptoms associated with food intolerance from a group of Correspondence: R.K. Woods, Dept of Res- young adults in Melbourne. piratory Medicine, Alfred Hospital, Com- Six hundred and sixty nine randomly selected subjects completed the questionnaire mercial Road, Prahran, Victoria 3181, with 553 attending the laboratory for skin-prick tests, anthropometry, and ventila- Australia. Fax: 00 613 9276 3434 tory function tests. A further 207 symptomatic participants completed the question- naire, with 204 of them attending the laboratory. Keywords: Atopy, dietary assessment, epi- Seventeen per cent of all respondents reported food intolerance or food allergy. A demiology, food intolerance, respiratory wide variety of food items was cited as being responsible for food-related illnesses. Those with current asthma did not report food-related illness more frequently than Received: December 30 1996 Accepted after revision July 8 1997 those without asthma. Respondents who reported respiratory symptoms following food ingestion were more likely to be atopic, to have used inhaled respiratory medica- This study was supported by Allen and tions in the previous 12 months, reported less exposure to regular passive smoking Hanbury's, Australia. Kabi-Pharmacia, Swe- over the past 12 months and weighed more. den provided the Phazets™. These associations between respiratory symptoms and food intolerance require further prospective investigation and verification. The importance of using appropri- The results presented here are from a local ate dietary methodology in future studies for determining diet-disease relationships analysis of data collected for the ECRHS. was highlighted by this study. Any final comparison may use a different Eur Respir J 1998; 11: 151–155. analysis.

Asthma is a common source of morbidity and is a ma- ness and asthma [9]. However, further well-conducted jor public health problem in Australia. It has previously observational studies and controlled clinical trials are re- been shown that the prevalence of wheeze that limits quired before any conclusions can be drawn. speech, frequent nocturnal wheezing and doctor-diagno- A large proportion of people with asthma perceive that sed asthma are higher in Australasian children than in diet plays an important part in their asthma control. In a Europe [1]. Until recently there has been little publish- recent survey of 135 Melbourne adults with asthma who ed information comparing the prevalence of adult asthma had attended an asthma and allergy clinic, 73% reported between countries using standardized epidemiological food-induced asthma [10]. Furthermore, 61% stated that methods. Analysis of the first phase of the European Com- they had tried to modify their diet in order to improve their munity Respiratory Health Survey (ECRHS), which was asthma control. By far the majority of dietary modifica- conducted in over 50 countries worldwide between 1992 tions were dietary restrictions. Other dietary perception and 1994, showed that respiratory symptoms are more studies, both in adults and children, have reported similar common in Australia, New Zealand, the west coast of the findings [11–13]. USA and the UK, than in continental Europe [2]. Indeed, Double-blind, placebo-controlled food challenges are more young adults from Melbourne, Australia reported an the "gold standard" for the diagnosis of adverse reactions attack of asthma during the preceding 12 months than to foods and food additives [14] and have shown that from any other centre [3]. The ECRHS was also concern- fewer than 5% of patients may have objective evidence of ed with risk factors that may explain the international var- food-induced asthma [15, 16]. iation in asthma prevalence. There are no published studies that have gathered data The relationship between diet and asthma is an area of contemporaneously from the community on people with controversy that has never been fully evaluated [4]. Epi- and without asthma in order to determine whether dietary demiological studies have, to date, provided conflicting manipulation is unique to those with asthma or whether it opinions with regard to diet as a risk factor for asthma. merely reflects general community concern and behaviour There have been reports that anti-oxidants, particularly in regard to diet and health. The aim of the present study vitamin C, magnesium, selenium and fish oils may be pro- was to assess the ability of the ECRHS questionnaire to tective factors for asthma [5–8]. Dietary sodium has been provide data on the prevalence, type and reported symp- implicated in the aetiology of bronchial hyperresponsive- toms associated with food ingestion among young adults 152 R.K. WOODS ET AL. in the Melbourne community. We also aimed to identify the measurement of spirometry and methacholine challen- characteristics of those who reported food-related respira- ge testing have been reported elsewhere [2, 17]. Bronchial tory symptoms. hyperreactivity (BHR) was defined as a provocative dose of methacholine causing a 20% fall in FEV1 (PD20) <2 Materials and methods mg. Current asthma was defined as the combination of wheezing in the last 12 months and BHR [19]. Study subjects The subjects were participants in the second phase of Skin-prick testing the ECRHS in Melbourne. The full details of the sampling The details of skin-prick testing with 11 common aer- protocol have been described elsewhere [2, 17]. Briefly, oallergens have been reported elsewhere [17]. Atopy was 4,500 adults aged 20–44 yrs were randomly selected from ≥ the electoral roll. Postal questionnaires were returned by defined as a positive reaction (a wheal 3 mm diameter) to 3,200 (72%) of subjects in the first phase of the ECRHS. any allergen, in the context of a positive histamine control Random and symptomatic samples were invited to the and negative reaction to the uncoated lancet. laboratory for the second phase of the study. From the ran- dom sample, 669 participants completed the questionnaire Statistical analysis with 553 attending the laboratory for further testing. An additional 207 symptomatic participants completed the Data from the questionnaires, ventilatory function tests questionnaire with 204 of these attending the laboratory and skin-prick tests were externally entered and verified. for further testing. Thus, a total of 757 participants com- Range and logic checks were performed to confirm the pleted the questionnaire in the laboratory with a further validity of the data. Association between categorical vari- 119 by telephone interview. ables was assessed by the Chi-squared test and compari- son of continuous variables by Student's t-test using the Statistical Package for the Social Sciences (SPSS) com- Questionnaire puter package (Norusis MJ, SPSS Inc 1995, Chicago, Participants completed the detailed second phase EC- IL, USA). All prevalence rates for reported illness from RHS questionnaire administered by one of three trained food were estimated using the random sample participants interviewers. The background and validity of this ques- only. tionnaire have been described elsewhere [2]. Briefly, the Univariate and multivariate analyses were conducted questionnaire covered: respiratory symptoms (including using the Statistical Analysis System (SAS) computer wheeze, shortness of breath, cough and phlegm produc- package (SAS Institute Inc, 1988; Cary, NC, USA). Multi- tion) during the previous 12 months; history of asthma; variate models of illness from food and symptoms were home and work environment; allergic symptoms; smok- fitted using stepwise multiple logistic regression. Univari- ing; demographic information; medications: and dietary ate and multivariate analyses used participants from both information. the random and symptomatic samples. There were only four questions relating to diet. The aim of the first three questions was to gather information on the amount of convenience food and "junk" food that the Results respondents were consuming, from which to gain an indi- Table 1 details the response rates and subject characte- cation of sodium and food additive intake. However, it ristics of the random sample and the combined random was not possible to calculate food additive and sodium and symptomatic samples. intakes from the data collected and, therefore, these res- ponses will not be considered further in this analysis. The Table 1. – Characteristics of subjects and response rates fourth question asked whether respondents had ever suf- for both random sample alone and combined random and fered any "illness/trouble" from food ingestion, and if so symptomatic samples to list the food(s) and the symptoms in order to distingu- Random Random + ish between symptoms of indigestion/food poisoning and sample symptomatic food allergy or intolerance. sample No. completing questionnaire n 669 876 Age* yrs 34.3 (6.81) 34.2 (6.83) Ventilatory function testing Females % 52 53 Valid skin-prick tests n 545 745 Spirometry was measured according to the American Underwent methacholine 509 (28) 675 (36) Thoracic Society (ATS) criteria using a computerized challenge n (% BHR positive) Fleisch number 3 pneumotachograph connected to a Hew- Wheeze in past 12 months % 35 45 lett Packard, Lung Function Analyser (Lexington, MA, Atopic % 51 56 "Allergy vaccination" 5 7 USA) [18]. The initial forced expiratory volume in one (immunotherapy) % second (FEV1) was recorded as the best of five expiratory Ever smoked % 51 51 manoeuvres. Methacholine (Provocholine™; Hoffman La Current smokers % 24 25 Roche, Basel, Switzerland) was delivered via a Mefar 3B Regularly exposed to passive 35 36 dosimeter (Mefar srl, Bovezzi, Italy) until the FEV1 fell smoking % by 20% from the initial value or until a cumulative dose Body mass index* kg·m-2 25.2 (3.8) 25.4 (4.1) of 2 mg (10 µmol) had been administered. The details of *: mean (SD). BHR: bronchial hyperresponsiveness. FOOD INTOLERANCE AND RESPIRATORY SYMPTOMS IN YOUNG ADULTS 153

Table 2. – Food items reported to have caused an illness Risk factors for reporting illness from food (random + after eating (n=128) symptomatic sample) Item Responses % No statistically significant associations were found be- Fruits, fresh/frozen/canned 14.5 tween reporting the same illness or trouble after eating Seafood/shellfish/fish 11.3 food and age, gender, BHR, atopy, asthma status, FEV1, Dairy products, milk/cheese/yoghurt/ice-cream 10.4 Herbs/spices/condiments/garlic/chilli 5.4 body mass index (BMI), smoking status or allergen im- Monosodium glutamate 5.0 munotherapy. Alcohol 5.0 Univariate analysis showed that respondents who repor- Eggs 4.1 ted respiratory symptoms nearly always after a particular Fats/oils, butter/margarine/cream/salad dressing 4.1 food(s) were more likely to be atopic, BHR positive, have Chocolate 3.6 current asthma, have wheezed or to have used inhaled Vegetables, fresh/frozen/canned 3.6 respiratory medications within the past 12 months, have a Red meat, fresh 3.6 lower per cent predicted FEV1, weigh more and were less High fat foods 3.2 likely to have reported exposure to passive smoking over Nuts, including peanut butter/coconut 3.2 Wheat products, bread/plain cereals 2.7 the past 12 months (table 4). No statistically significant as- Sugar, including golden syrup/jam 1.8 sociations were found between food-related respiratory Restaurant meals/take-away meals 1.8 symptoms and age, gender, BMI, active smoking status or Fruits, dried 1.4 allergen immunotherapy. Multivariate analysis confirmed Sauces, including tomato paste/seasonings 1.4 that atopy, use of inhaled respiratory medications in the Tea/coffee 1.4 past 12 months, increasing weight and less exposure to Poultry 1.4 regular passive smoking over the previous 12 months Spicy foods 1.4 were the only independent predictors for respiratory symp- Processed meats, including ham/bacon 1.4 Other (12 separate items cited) 8.8 toms following the ingestion of food (table 4). When cur- rent asthma was fitted in a multivariate model, and atopy or inhaled respiratory medications were excluded, current Reported illness from food (random sample) asthma was still not significantly associated with food- related respiratory symptoms. Twenty five per cent of respondents (n=167) indicated Those respondents who reported that respiratory symp- that they had experienced "illness or trouble" caused by toms alone nearly always occurred after a particular food(s), eating a particular food or foods. Seventy eight per cent were more likely to be atopic, to have used inhaled respi- of those reporting an illness (n=128, 19% of all respon- ratory medications in the past 12 months, had a lower per- dents) nearly always had the same illness or trouble after centage predicted FEV1, were taller and weighed more eating particular food(s). Excluding those who reported (table 4). Multivariate analysis found that impaired FEV1 nonspecific symptoms, such as lethargy/tiredness, tachy- was the only independent factor which predicted report- cardia or heartburn/indigestion, 17% (n=114) of all res- ing of respiratory symptoms alone, which nearly always pondents perceived that they had food intolerance or food occurred after eating food (table 4). allergy. The food items that were reported as causing illness or trouble when eaten are listed in table 2. Thirty four Discussion different items were cited as causing illness from food. Table 3 outlines the types of symptoms that were report- We found that 17% of young adults reported that a ed by eating particular foods. Only eight respondents re- particular food or foods nearly always caused "illness or ported that a particular food caused respiratory symptoms trouble" when eaten, presumably due to either food in- alone. tolerance or food allergy. These results are consistent Respondents with current asthma did not report more with other similar international studies which have been perceived food intolerance or food allergy than those with- conducted in the UK, USA, and the Netherlands which out asthma (χ2=0.005, p=0.94). found reported food intolerance prevalence rates of 20, 16

Table 3. – Reported symptoms that occur nearly every time following eating particular food(s) (n=128) Symptom type Subjects reporting Items Most commonly reported food items responsible for symptom symptoms n cited n (% of responses) Gastro-intestinal (nausea, 58 24 Dairy (16%), seafood (14%), fresh fruits (9%) vomiting, diarrhoea) Urticaria 31 18 Fresh fruit (25%), seafood (15%), nuts (10%), eggs (10%) Severe headache 22 16 Alcohol (14%), monosodium glutamate (14%) Rhinitis 18 17 Dairy (25%) Respiratory 17 14 Fresh fruit (9%), dried fruit (9%), dairy (9%), chocolate (9%), sauces (9%), alcohol (9%), high fat foods (9%), monosodium glutamate (9%) Nonspecific 15 13 Seafood (20%) Angio-oedema 11 7 Fresh fruit (31%), seafood (15%), nuts (15%), alcohol (15%) Lethargy/tiredness 5 8 Dairy (27%), sugar (18%) Heartburn/indigestion 4 5 Fresh fruit, dairy, nuts, pastry, fats/oils Tachycardia 3 5 Fresh vegetables, chocolate, eggs, herbs/spices, monosodium glutamate 154 R.K. WOODS ET AL.

Table 4. – Crude and adjusted associations between asthma-related and other variables and the reporting of food- related illness Outcome/predictors Crude Adjusted odds ratio odds ratio Respiratory symptoms nearly always reported after eating food (n=42) Current asthma 3.00 (1.59–5.65) Wheeze in past 12 months 6.46 (2.84–14.70) Exposure to passive smoking in past 12 months (less likely) 0.41 (0.19–0.89) 0.31 (0.10–0.94) Use of inhaled respiratory medications 5.39 (2.76–10.55) 3.63 (1.56–8.46) BHR (methacholine positive) 2.12 (1.13–3.96) Log dose-response slope (BR) 1.32 (1.09–1.59) FEV1 (lower) 0.97 (0.95–0.99) Atopy 4.77 (2.18–10.43) 3.36 (1.11–10.18) Weight (heavier) 1.02 (1.00–1.04) 1.03 (1.01–1.06) Respiratory symptoms alone nearly always reported after eating food (n=17) Atopy 3.49 (1.13–10.80) Use of inhaled respiratory medications 3.24 (1.22–9.60) FEV1 (lower) 0.95 (0.92–0.98) 0.95 (0.92–0.98) Height (taller) 1.06 (1.01–1.12) Weight (heavier) 1.03 (1.00–1.07) Values in parentheses are 95% confidence intervals. BHR: bronchial hyperresponsiveness; BR: bronchial responsiveness; FEV1: forced expiratory volume in one second. and 12%, respectively [20–22]. Whilst the true prevalence medications was also found to be an independent risk fac- of food intolerance remains unknown, double-blind, pla- tor, which provides some evidence that those with respira- cebo-controlled food challenge studies suggest that the tory symptoms requiring treatment are more likely to prevalence of food intolerance is closer to 2% [23–25]. report food intolerance. It is unclear why current asthma Obviously, there is a wide gap between the actual preva- (defined as positive BHR and reported wheeze in the past lence of food intolerance and public perceptions of it. The 12 months) alone was not associated with food intole- high level of perception of food intolerance does have sig- rance, although the numbers of people reporting food nificant potential consequences. It has been previously intolerance may have been insufficient for a small effect to documented in children that unnecessarily restrictive diets be detected. Another possible reason could be that the can result in nutritional deficiencies and may, in extreme wording of the relevant question did not specifically relate cases, be fatal [26–28]. Public education strategies are, food intake to respiratory symptoms alone. The general therefore, required to deal explicity with this disparity. wording of the question may have made it difficult to When interpreting the results of this study, account determine whether there was really any difference in per- must be taken of several factors that limit the extent to ceived food intolerance between people with asthma and which generalizations can be made about the community, those without asthma. in terms of symptom prevalence. The response rate was Less exposure to regular passive smoking and obesity poor, with only 42% of those invited to take part in the were also found to be independent risk factors for report- random sample completing the questionnaire. We have ing respiratory symptoms following food ingestion. These previously found that the randomly selected subjects who findings may reflect lifestyle or the fact that people with attended the laboratory were significantly more likely asthma tend to avoid smoky environments and exercise. than those who were interviewed by telephone to report Further research into this area is required before any con- wheeze or an attack of asthma over the preceding 12 clusions can be made. A larger study population would months, thus indicating some volunteer bias [29]. enable more comprehensive multivariate analyses to be This study used a cross-sectional design and asked performed, allowing a clearer insight into the possible re- whether respondents had "ever" experienced symptoms lationship between asthma and reported food intolerance. and was, thus, unable to investigate a temporal relation- It was interesting that fruits (fresh/frozen/canned) were ship between previous symptoms and current asthma sta- the most commonly reported food items causing illness. tus. The associations that were found with reporting of However, double-blind, placebo-controlled studies in adults food-related symptoms require further investigation and have not supported fruit being a common cause of food verification in prospective studies. Causal relationships intolerance [20, 23]. Although immunological cross reacti- should not be inferred from these results alone. vity of plant aeroallergens with fruits is well documented Participants with current asthma did not report a higher as the "oral allergy syndrome", the clinical relevance of this prevalence of food-related illness in general than those remains unclear [31]. Future studies should obtain further without asthma in this study. These results suggest that information by incorporating specific radioallergosorbent dietary manipulations are not unique to the asthma popu- tests (RASTs) for commonly reported food allergens. lation, but merely reflect general community concern We found significant limitations in the ECRHS ques- about diet and health. tionnaire for dietary analysis. Quantitative assessment of We did find that atopy was a risk factor for reporting dietary intake or exposure levels to food additives, can respiratory symptoms following food ingestion and it is only be obtained using accepted dietary assessment metho- well known that atopy is an important risk factor for dology such as dietary recall, quantification of intake or asthma in young adults [30]. Using inhaled respiratory semiquantitative food frequency questionnaires [32, 33]. FOOD INTOLERANCE AND RESPIRATORY SYMPTOMS IN YOUNG ADULTS 155

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Am Acknowledgements: The authors wish to acknowledge Rev Respir Dis 1992; 146: 633–637. the assistance of C. Hartley-Sharpe and A. Lanigan with 20. Young E, Stoneham MD, Petruckevitch A, Barton J, interviewing and testing in the Lung Function Laboratory, and of J. Kutin with data management and analysis. Rona R. A population study of food intolerance. Lancet 1994; 343: 1127–1130. 21. Altman DR, Chiaramonte LT. Public perception of food References allergy. J Allergy Clin Immunol 1996; 97: 1247–1251. 22. Jansen JJ, Kardinaal AF, Huijbers G, Vlieg-Boerstra BJ, 1. Pearce N, Weiland S, Keil U, et al. Self reported pre- Martens BP, Ockhuizen T. Prevalence of food allergy and valence of asthma symptoms in children in Australia, intolerance in the Dutch population. J Allergy Clin Immu- England, Germany and New Zealand: an international nol 1994; 93: 446–456. comparison using the ISAAC protocol. Eur Respir J 23. Sampson HA, Metcalfe DD. Immediate reactions to foods. 1993; 6: 1455–1461. 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Ad- Allergy 1995; 25 (Suppl. 1): 29–30. verse consequences arising from misdiagnosis of food 5. Hatch GE. Asthma, inhaled oxidants and dietary antioxi- allergy. Br Med J 1988; 297: 719–720. dants. Am J Clin Nutr 1995; 61 (Suppl. 1): 625S–630S. 27. Lloyd-Still JD. Chronic diarrhoea of childhood and the 6. Landon RA, Young EA. Role of magnesium in regulation misuse of elimination diets. J Pediatr 1979; 95: 10–13. of lung function. J Am Diet Assoc 1993; 93: 674–677. 28. Davidovitis M, Levy Y, Avramovitz T, Eisenstein B. Cal- 7. Flatt A, Pearce N, Thomson CD, Sears MR, et al. Re- cium-deficiency rickets in a four-year-old boy with milk duced selenium in asthmatic subjects in New Zealand. allergy. J Pediatr 1993; 122: 249–251. Thorax 1990; 45: 95–99. 29. Kutin J, Abramson M, Raven J, Lanigan A, Czarny D, 8. Schwartz J, Weiss ST. The relationship of dietary fish Walters EH. Non-responders and volunteers: can't live intake to level of pulmonary function in the first National with them: can't live without them. Am J Respir Crit Care Health and Nutrition Survey (NHANES I). Eur Respir J Med 1995; 151 (4,2): A567. 1994; 7: 1821–1824. 30. Abramson M, Kutin JJ, Raven J, Lanigan A, Czarny D, 9. Antonios TFK, MacGregor GA. Deleterious effects of Walters EH. Risk factors for asthma among young adults salt intake other than effects on blood pressure. Clin Exp in Melbourne, Australia. Respirology 1996; 1(4): 291–297. Pharmacol Physiol 1995; 22: 180–184. 31. Bock SA. Oral challenge procedures. In: Metcalfe DD, 10. Woods RK, Weiner J, Abramson M, Thien F, Walters EH. Sampson HA, Simon RA, eds. Food Allergy: Adverse Patients' perceptions of food-induced asthma. Aust NZ J Reactions to Foods and Food Additives. Boston, Black- Med 1996; 26: 504–512. well Scientific Publications, 1991; pp. 81–95. 11. Dawson KP, Ford RPK, Mogridge N. Childhood asthma: 32. Stamler J. Assessing diets to improve world health: nutri- what do parents add or avoid in their children's diets? NZ tional research on disease causation in populations. Am J Med J 1990; 103: 239–240. Clin Nutr 1994; 59 (Suppl.): 146S–156S. 12. Chiaramonte LT, Altman D. Food sensitivity in asthma: 33. Petersen BJ, Chaisson CF, Douglass JS. Use of food- perception and reality. J Asthma 1991; 28: 5–8. intake surveys to estimate exposures to nonnutrients. Am 13. Griffin T, Jones J, Stevens D, Henry RL. Dietary modifi- J Clin Nutr 1994; 59 (Suppl.): 240S–243S.

A Critical Review of IgG Immunoglobulins and Food Allergy –

Implications in Systemic Health

Raymond M. Suen, MT (ASCP), Shalima Gordon, ND

US BioTek Laboratories 2003

13500 Linden Ave. N. Seattle, WA 98133 Ph: (206) 365-1256, (877) 318-8728, Fax: (206) 363-8790

Food allergy is defined as an adverse clinical reaction due activation of FcgammaRI receptors on human mast cells, is to an immune-mediated hypersensitivity response resulting qualitatively indistinguishable from responses stimulated from the ingestion of a food. A wide variety of foods have through FcepsilonRI, the high affinity receptor for IgE.6 In been shown to produce allergic reactions including cow’s addition, FcgammaRI receptor expression on mast cells is milk; chicken eggs; legumes; fish and shellfish; and up regulated by IFN-gamma, allowing for recruitment of cereals.1 Depending on the speed of onset of symptoms, mast cells through IgG-dependent mechanisms into the less than 45 minutes to 2 hours to days, immediate and IFN-gamma-rich inflammatory locus.7,8,9 IgG-mediated delayed food allergies have been described throughout the immediate hypersensitivity, also known as IgG-mediated literature encompassing a variety of gastrointestinal, anaphylaxis, is not a new concept in allergy research. In respiratory, and cutaneous pathologies.2 The inflammatory conventional circles, anaphylaxis denotes an immediate response is the common theme to all allergic pictures and hypersensitivity reaction to an allergen, exclusively is characterized by the release of chemical mediators, mediated by IgE antibodies. Hence, the foundation of the vasodilation, increased vascular permeability, edema, and skin-scratch testing method, which detects IgE –induced tissue damage. histamine release through a wheal and flare response from antigen provocation, and IgE RAST quantification. The role of IgE in Type I immediate hypersensitivity However, as early as the 1970’s, Parish demonstrated the allergic reactions is well understood in the scientific presence of anaphylactic IgG antibodies in human sera.10 literature. In classic Type I IgE-mediated hypersensitivity, Halpern et al, later suggested that this IgG anaphylactic food-specific IgE antibodies bind to FcεRI and FcεRII antibody is indeed a subtype of IgG4. Further studies from receptors on the cell membranes of mast cells, basophils, Bryant et al, and Pepys have shown that IgG anaphylactic macrophages, monocytes, lymphoctes, eosinophils and antibody activity could not be removed through platelets. Inflammatory mediators including histamine, precipitation with anti-IgE but, only by precipitation with serotonin, and tumor necrosis factor alpha, are released anti-IgG, clearly indicating a novel mechanism for mast and induce symptoms upon exposure of these bound cell recruitment into inflammation.11,12 However, the antibodies with food antigens that have penetrated the potential for IgG4 to inhibit, or block IgE–mediated protective intestinal mucosal barrier. It is generally anaphylaxis is a clearly established theme in this line of understood that symptoms of an IgE-mediated allergy research, and some authors argue a correlation between manifest within 2 hours of consumption of the culpable increased IgE levels and IgG4 in atopic patients, where food. Classical atopic symptoms include; urticaria, IgG4 is thought to hamper antigen binding to cell-bound eczema, respiratory and nasal symptoms, and IgE which would otherwise promote a much stronger gastrointestinal distress. In the gut specifically, mast cell allergic reaction. Moreover, the basic principle behind degranulation and mediator release promotes muscle allergen immunotherapy (IT), is oral or intradermal contraction, stimulates pain fibers, increases mucus administration of the allergen to induce the development of production, recruits inflammatory cells, and increases a systemic immune response, including the production of permeability to macromolecules, the latter of which may systemic blocking antibodies. In an update on perpetuate a vicious cycle of food antigen exposure and immunotherapy, “Immunotherapy update: mechanisms of symptoms.3 Specific IgE has a half-life of only 1-2 days in action”, Greenberger concludes that the reduction of circulation however, exhibits residual activity on mast allergic symptoms, specifically of allergic rhinitis and cells of about 2 weeks with late phase reactions and asthma, reflect changes in the cytokine and inflammatory changes.4 immunoglobulin profile from intradermal allergen provocation. Most notably, intradermal grass injection It is strongly argued in the scientific literature that allergic resulted in a profound increase in antiallergen IgG (2-10 reactions may occur independent of antigen-specific IgE. fold), IgG4 (10-100 fold), a decline in antiallergen IgE High affinity receptors for IgG (FcgammaRI), on human antibodies, reduced numbers of nasal or bronchial mast mast cells and basophils, are activated in immediate cells and eosinophils, down-regulation of T-helper 2- hypersensitivity reactions, following receptor aggregation lymphocytes and IL-4, and a lack of increase in interferon through IgG binding. IgG-mediated immediate gamma.13 This study clearly establishes IgG4 as a blocking hypersensitivity results in degranulation, with the release antibody. Furthermore, in a study involving 42 children of histamine and arachidonic acid metabolites.5 Okayama with malabsorption disorders, those demonstrating high et al, have demonstrated that the mediator profile through levels of IgG antibodies to ovalbumin (Anti-OA) showed significantly lower serum concentrations of OA at both 2 macromolecules, are factors to consider influential in IgG4 and 8 hours after oral OA administration, compared to subclass expression and progression of disease. children with lower anti-OA levels. The researchers A distinguishing feature of IgG4 is its inability to activate conclude that the high levels of IgG antibodies to the classical complement pathway. This supports the role ovalbumin demonstrate blocking capacity in the of IgG4 as a blocking antibody. In a study of egg circulation. Such antibodies in the intestinal secretions and hypersensitivity, Nakagawa draws our attention to IgG1 in the gut wall would limit the quantity of antigen that can involvement in clinical egg hypersensitivity, suggesting penetrate the mucosa and enter circulation.14 However, that increased IgG4 reduces the effect of complement- other studies show elevated IgG4 in symptomatic atopic fixing antibodies like IgG1; a good prognostic sign as he patients without a concomitant rise in IgE levels.15,16,17 suggests.25 This is further supported by Van Der Zee, who shows that IgG4 antibodies inhibit complement activation Supporting evidence from Yoshida et al, demonstrate that of IgG1 antibodies, probably through competitive binding IgG4 is not only elevated in milk allergic children, for for related antigenic determinants.26 example, but diagnostic of milk allergy in atopic children independent of IgE.18 In another study, milk-specific IgG4 Like all immune mediated reactions, food allergies depend in particular, IgG4 to casein, has been shown to be on the intimate association between mature T-helper cells diagnostic of milk allergy causing eczema in adults.19 In and B-lymphocytes with the production and release of an elegant study by Eysink et al, atopy could be correctly inflammatory mediators and activation of food specific classified in 75.4% of young children studied with or antibodies. After B-cells are stimulated by antigen, they without eczema, through identification of high levels of are terminally differentiated into plasma cells of which IgG to certain foods. In particular, high levels of IgG secrete antigen-specific immunoglobulins. antibodies to egg, milk, orange, and a mixture of wheat and rice, were identified in atopic children compared to A review of T-helper 2 (TH2) and T-helper 1 (TH1)- nonatopic children. Further, this elevation served as a induced antibody production reminds us that interleukin-2 positive predictor of increased IgE antibodies to inhalant (IL2), interferon gamma (IFN gamma), and tumor necrosis allergens, namely cat, dog, and house dust mite. The factor beta (TNF beta), via TH1 T-helper cell activity, investigators of this study conclude that the association favors B-lymphocyte class switching to the production of drawn from these results may clearly identify children with IgG2a. Conversely, IL-4 and IL-5 secreted by TH2 T- an increased risk of developing future allergic disease.20 helper cells induce class switching to IgE and IgG1. The exact role of the different subclasses of IgG remains to be Other reports demonstrating the importance of IgG understood. Studies suggest sequential switching as a antibodies in food allergies include IgG- mediated allergy prerequisite for B-cell differentiation, most prominent in to casein and other milk proteins, which has been cells that switch to IgE. For instance, B-cells cultured with implicated in the development and progression of infantile interleukin-4 show IgG1/IgE double-positive staining, autism.21 Furthermore, one study involving rheumatoid which appear after 3 days of culture, after which arthritis, showed a decrease in gluten-specific IgG serum predominantly secrete IgE on reculture. This serves as an levels which correlated with an improvement in the interesting note suggesting an essential switch from IgG1 symptoms of this disease in 40% of subjects placed on a with possible prerequisite involvement in IgE production.27 gluten-free diet, compared to a 4% improvement in a The amount of antigen absorbed, the quantity of antibody control group, over a one-year period.22 required, the chronicity of antigen exposure, and the specific role of IgG subclasses in the pathogenesis of food The evidence from the above research suggests that IgG4 allergic illness, clearly influence progression of disease, antibodies may act as sensitizing as well as blocking and define the magnitude of the immune response to antibodies. This dual role of IgG4, anaphylactic or dietary antigen to warrant further investigation. blocking antibody, lends way to defining IgG4 subtypes 4a and 4b, as described by Halpern and Scott in their review, A key feature of food allergies that deserves close attention “Non-IgE mediated mechanisms in food allergy”, whereby is its implications in the development and maintenance of exposure to an allergen may lead to the production of the immune cell memory from chronic and repeated exposure anaphylactic or nonanaphylactic/blocking subtype of to food allergens, with resultant clinical consequences. which may depend on genetic predisposition and Food-induced immune reaction favors maturation and environmental factors.23 It is interesting to note that there proliferation of naïve T-cells into CD4 and CD8 effector is some structural homology between IgG4a, IgG1 and T- cell lines. Under chronic antigen exposure CD4 IgG3 immunoglobulins, and between IgG4b and IgG2.24 populations generate distinct populations, as mentioned IgG subclass antibodies and their role in the pathogenesis above, TH1 and TH2 of which TH2 promotes the of food allergic disease deserves considerable attention. activation of cytokines favoring immunoglobulin Chronicity of antigen exposure, a hyperactive mucosal production. From chronic antigen exposure there is a immune system and/or an increased permeability to pronounced alteration in the ratio between TH1 and TH2, suggesting immune imbalance, polyclonal B-cell

2 activation, and an exaggerated immunoglobulin response. cell activity, or suppressive intra-epithelial lymphocytes This mechanism of action has been argued for autoimmune (IEL), is the B-cell switching from IgE/IgG antibody disease due to chronic antigen exposure, and has production to IgA, under the influence of a novel cytokine interesting implications in food allergies, which also profile unlike that governing TH1/TH2 mechanisms. In represent a state of chronic antigen exposure. As such, the particular, transforming growth factor beta (TGF-B), the role of IgG in delayed-onset food allergies deserves close predominant oral tolerance cytokine, is released by attention for its implications in systemic disease. activated IEL’s and suppresses any potential for TH1 or TH2 response to dietary antigen, thereby favoring IgA There is no argument regarding the presence of serum IgG expression and active suppression. Bias towards immune reactive with different dietary proteins. Specific serum IgG response TH1/TH2, or immune tolerance TH3, is antibodies to different food proteins have been reported in dependent on the cytokine profile elaborated under the significant numbers of adults and children in cases of influence of the gastrointestinal mucosal immune milieu, celiac disease, dermatitis herpetiformis, and atopic defined by the individual’s defense factors, innate and eczema.28,29 Moreover, higher total and specific serum acquired. It is especially important to note that the immune IgG4 levels to common foods are raised in cases of atopic response favored in the adult will differ from that of the eczema compared to the healthy population.30,31 The neonate, where there is a predilection towards oral presence of elevated levels of IgG antibodies to food tolerance in the latter. In any event, local gastrointestinal antigens have been observed substantially in diseases with immunity, through the expression of a cytokine profile increased intestinal permeability, in particular, IgA indicative of either an immune response or active deficiency32 and inflammatory bowel disease.33 suppression, each being unique, will have systemic consequences, as these cytokines migrate to other mucosal The role of secretory IgA is clear; induction of mucosal sites and peripheral tissues, with implications in the onset immunity and establishment of oral tolerance through oral and progression of symptoms. 35 immunization with food antigens occurs in most normal individuals, and plays a major role in antigen handling and It is proposed that the breakdown of oral tolerance, and elimination. The elevation of IgG in IgA-deficient states hence sensitization to dietary antigens may occur early in suggests increased intestinal permeability to life during a viral or bacterial infection, under conditions macromolecules. In other words, a lack of sIgA may where the secretory immune system has not fully matured. permit the permeation of undigested food antigens into the By that means, a hyperresponsive state to dietary agents is blood stream, thereby allowing for immune complex set up, with inflammation and the development of formation and circulation for an uncertain period of time. inflammatory bowel disease; ulcerative colitis or Crohn’s Cunningham et al, conclude from their study involving disease.36 By definition, inflammatory bowel disease is the IgA-deficient subjects, that milk and other protein loss of tolerance to normal flora with consequential precipitins are a common feature in IgA deficiency, and hypersensitive food reactions and systemic sequelae.37 that a high proportion of these individuals may have These arguments are supported by many authors including circulating immune complexes of these food antigens.34 In Tahmeed and Fuchs, who comment in their review that addition, a review by Saavedra-Delgado and Metcalfe intestinal infections and reduced secretory IgA may alter explain from other studies, that the presence of circulating intestinal permeability resulting in an increased uptake of IgG-food antibodies is consistent with increased food antigens thereby initiating an abnormal mucosal absorption of food antigens and stimulation of antibody immune response and chronic enteropathy.38 The production, as a result of mucosal damage. These authors breakdown of oral tolerance and resultant disease is far note that circulating antigen-antibody complexes are more reaching and characterizes a number of conditions frequently detected in symptomatic cases of ulcerative including childhood onset Type I hypersensitivity, celiac colitis as evidenced by IgG - immune complex deposition disease, and a number of autoimmune conditions. 39 and complement in colonic epithelium along the basement membrane. Mucosal exclusion of dietary antigens other than via secretory IgA has also been demonstrated to occur through It is not difficult to conceive a rationale for the observed experimentally induced high titers of IgG antibodies. elevation in IgG antibody levels to food antigens in IgA- However, some in vitro experiments using everted gut sacs deficient states, when we consider the mechanisms of oral show the contrary, with enhanced intestinal absorption of tolerance. Oral tolerance lies at the heart of immunological antigen under the influence of high IgG titers.40,41 Tolo et theories and is the cornerstone of setting up a reaction or al, through in vitro studies of rabbit oral mucosal non-reaction against self and non-self (dietary challenge). membranes, found that serum-derived IgG antibody retards Factors influential in the induction of an immune response the penetration of corresponding antigen, however impairs with IgE and IgG expression, versus the induction of the mucosal barrier none the less, by allowing for active suppression, or immune tolerance, an IgA response, concurrent mucosal penetration of unrelated are cutting edge research and are under considerable macromolecules.42 It is not unreasonable to rationalize a investigation. Oral tolerance to dietary antigens via TH3- similar set of circumstances for intestinal epithelia given

3 sufficient experimental data. Immunohistochemical studies within the hour following consumption of cow’s milk with demonstrate an abundance of serum-derived IgG present in notable immune complexes appearing in the serum.46 the lamina propria of mucous membranes, traces of which diffuse into the intestinal epithelial interstices. Immune Since immunocomplexes, through Type III complex formation in these sites may on the one hand, hypersensitivity are free in circulation, the implications of perturb mucosal antigen uptake by signaling the multiple end organ pathology are not without possible emigration of neutrophils with phagocytosis and protein justifications. It may be possible to correlate such degradation. On the other hand, the local emigration of phenomena to the pathogenesis and etiology of certain other immune cells and the release of inflammatory autoimmune disease47, connective tissue disorders, and mediators may concomitantly enhance mucosal perhaps malignancy. permeability and penetration of other food macromolecules. As such, IgG may in effect compromise In addition to IgG involvement in Type I and III mucosal integrity through undue penetration of new food hypersensitivity reactions, there are concrete studies antigens thereby setting up a vicious cycle. To review, by demonstrating the role of specific IgG antibodies to food activating complement, IgG antibodies may promote allergens in Type II immune mechanisms as well. In Type increased mucosal permeability, tissue damage, and II, antibody-dependent cytotoxic hypersensitivity, specific persistent immunopathology. In addition, IgE antibodies IgG recognize and bind to food antigens that have adhered may enter the gut mucosa via mast cells causing their to the surface of cells. Antigen-antibody bound complex degranulation with histamine release and mucosal lesion activates the complement cascade and the release of formation as well. Such immunological mechanisms may cytotoxic substances from activated killer cells, with therefore perpetuate an inflammatory state of the bowels eventual cell death. Cow’s milk-induced thrombocytopenia with undue systemic exposure to dietary antigens.43 has been implicated by this type of reaction.48

It is important to note that intestinal hyperpermeability is Through Type IV, cell-mediated delayed hypersensitivity, not necessarily a prerequisite for penetration of food IgG antibody activity plays an integral role in this type of antigens into the lamina propria and systemic circulation. immune response as well. Type IV mechanisms represent a Kleinman and Walker mention that small, nutritionally major immunological pathway in conditions such as cow’s insignificant amounts of antigenically intact milk-induced enteropathy and celiac disease.49 Food macromolecules may be transferred across the gut lining induced gastroenteropathy, specifically celiac disease, has through simple mechanisms.44 With this in mind, when we been clearly defined through Type IV mechanisms with consider the absorption of pounds of foods on a daily the involvement of IgG specific antibodies to wheat basis, continuous exposure between food antigen, most gliadin. In this condition, abnormalities in intestinal often of the foods eaten regularly, and stimulation of permeability are associated with both inflammatory intestinal lymphoid follicles may very well provide the processes and loss of jejunal microvilli favoring the impetus for the development of a systemic immune absorption of large molecules that pose an allergenic response with circulating antibody complexes and atopic threat. Kemeny et al, have demonstrated increased IgG1 reactions. antibody levels to gliadin, ovalbumin, and casein in addition to elevated IgG4 to casein in untreated celiac In Type III, immune complex-mediated hypersensitivity, patients compared to healthy control subjects.50 In IgG antibodies combine with food antigen forming addition, in immediate-type egg allergic patients, these circulating immune complexes to which complement is investigators found raised IgG1 antibody levels to fixed. These immunocomplexes may circulate throughout ovalbumin, compared to healthy control subjects.51 In the the periphery and deposit in various tissues promoting an celiac subjects, in particular, both IgG1 and IgG4 antibody Arthus-like inflammatory reaction with vasculitis and levels to gluten fell from a gluten-free diet. tissue damage. Intestinal biopsy studies have shown evidence of this type of immune-mediated reaction in the As a clinical aside, anti-gliadin IgG antibodies are a pathogenesis of cow’s milk sensitive colitis.45 In another sensitive screening test for jejunal biopsy in patients with study, Lee et al, have demonstrated deposition of human suspected celiac disease. Also, the monitoring of the IgG and precipitins to cow’s milk in lung tissue specimens disappearance of gliadin antibodies during a gluten-free of infants with pulmonary hemosiderosis. diet can be used to indicate successful elimination of Immunofluorescence of snap-frozen biopsy tissue in these gluten from the diet. infants exceeded that in control lung biopsies. The researchers suggest a Type III mediated mechanism to Symptoms of delayed food allergies are diverse and may explain the presence of the milk-related pulmonary affect any system in the body. It can be argued that infiltrates. Absorption of the milk antigens through the delayed-onset food allergies are much more common than intestine with subsequent deposition of the circulating the more widely accepted IgE- mediated immediate immune complexes could not be discounted as there was hypersensitivity reactions. In addition, IgG-mediated food no evidence to suggest aspiration, and symptoms appeared allergies may account for variety of chronic health

4 conditions that have otherwise been misdiagnosed and thus immunoglobulin serum levels with activation of immune unresponsive to conventional medical care. Fatigue, mediators of inflammation. Research indicates that food irritability, aching joints, cognitive dysfunction, and and inhalant allergies are implicated in a number of health chronic migraines are a few known complications due to problems. Through ELISA testing we provide a useful tool food allergies, which suggest a strong IgG component due with which an individual’s sensitization to food and to their chronic nature. The diagnosis of food allergy is inhalant allergens can be assessed. made simple though serum ELISA analysis for IgG- specific food antigens. The condition is treated by Through this testing method, a multi-well ELISA plate is eliminating the allergenic food from the diet for an coated with purified food proteins and glycoproteins or, indefinite period of time. inhalants at a specific concentration. The patient’s serum sample is then added to the plate. If the patient’s serum Typically, IgG subclasses exhibit a half-life of about 21 contains antibodies to any of these specific and defined days with a residual time on mast cells of about 2 to 3 food or inhalant proteins, a binding reaction will occur. months.52 However, due to the long survival of IgG The degree of antibody-antigen binding is dependent on relative to other serum immunoglobulins, immune the concentration of antibodies present in the patient’s complex formation may persist in circulation for an serum. This reaction is detected through a color change indefinite period of time. As such, the time period required and assessed spectrophotometrically. to abstain from the allergenic foods is arguable, and may extend for up to 9-12 months as in the case of cow’s milk Our ELISA limit of detection for IgE is 391 picograms/ml, allergy.53 which is well below 200 nanograms that define normal total serum concentration, and 900 nanograms that define 54 The production of antibodies to dietary antigens, especially the majority of atopic individuals. Our limit of detection of the IgG class takes place as an immune response to food for specific IgG is 1.5 micrograms/ml which is also well antigens. This is true and well established through food below cited reference ranges in current literature. allergy provocation studies and quantification of IgG via ELISA methodology. Effector function of the different US BioTek ELISA tests for IgG4 and Total IgG in serum. subclasses varies. Chronicity of food antigen exposure, IgG pool testing ensures maximal recovery of both gastrointestinal mucosal integrity, and host immune symptom-provoking and potential “blocking” IgG competence, are a few of the physiological circumstances antibodies; key indicators of an immune response to a food that influence subclass IgG expression, activation of allergen. complement, and cellular immune mechanisms. Together, these functional components of the mucosal immune With our ELISA testing, we do each and every serum system orchestrate progression or neutralization of the specimen in complete duplicate when we perform the inflammatory process induced by food antigen exposure, analysis, assuring that there is no more than a 20% the former of which may promote varying symptomology. variance between each run. This allows us to use the patient as his or her own control.

An appreciation of the associated symptoms of food We also perform daily in-house blinded split sample allergy poses a unique challenge to the clinician both reproducibility checks using both positive and negative because of the variability in severity and onset. As such, controls, in accordance to Clinical Laboratory food allergy should not, by any means, be underestimated Improvement Amendments (CLIA), proficiency testing as a key etiological factor in disease and disease criteria, for acceptable analytical performance.55 Our goal progression. Gastrointestinal food allergy is a fascinating is to take every measure to ensure reproducible and line of research that merits close consideration in clinical reliable results. practice for the assessment and care of our patients.

We subscribe to the College of American Pathologists The US BioTek Advantage (CAP); certified and accredited under COLA (Commission

of Laboratory Accreditation). Under the oversight of the US BioTek employs Enzyme-Linked Immunosorbent Assay Washington State Department of Health, USBioTek methodology, or ELISA, a simple, safe and reliable test Laboratories is recognized and holds a Medical Test Site that demonstrates food-antigen-specific IgE and IgG in License, and is obligated to abide by the Federal serum. Government’s enacted rules and regulations for medical

ELISA is a semi-quantitative analysis designed to assess laboratories. We also participate with the American immediate (IgE) and delayed (IgG) immune reactivity to Association of Bioanalysts Proficiency Testing Service for food antigens. Our region-specific inhalant panels assess periodic blinded testing. for immediate IgE hypersensitivity. These measures and many other quality control Allergic reactions to foods and inhalants are procedures, assure us precision, consistency, and characterized by enhanced allergen-specific objectivity from day-to-day and week-to-week testing. 5

References

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Eur Cunningham-Rundles, op. cit. 35 J Immunol, 31(11): 3298-307. Plummer, Nigel, Oral Tolerance: Revolutionary Implications in 10 Parish, W.E, Short-Term Anaphylactic IgG Antibodies in Human Immunological Health and Disease. Pharmax LLC: Practical Sera. Lancet, 2:591, 1970. Solutions Seminar Series. Bellevue, WA. June 7-8, 2003. 36 11 Bryant, D.H., Burns, M.W., Lazarus, C., Identification of IgG Saavedra-Delgado, Ana Maria., Metcalfe, Dean D., Interactions Antibody as a Carrier of Reaginic Activity in Asthmatic Patients. J Between Food Antigens and the Immune System in the Pathogenesis Allergy Clin Immunology, 56:417, 1975. of Gastrointestinal Diseases. Annals of Allergy, 55:694-702, 1985. 37 12 Pepys, J., et al, Clinical Correlations Between Long-Term (IgE) Plummer, op. cit. 38 and Short-Term (IgG S-TS) Anaphylactic Antibodies in Atopic and Tahmeed, op. cit. 39 “Nonatopic” Subjects with Respiratory Allergic Disease. Clin Plummer, op. cit. 40 Allergy, 9:645, 1979. Tolo, K., Brandtzaeg, P., Jonsen, J., Mucosal Penetration of 13 Greenberger, PA., Immunotherapy Update: Mechanism of Action. Antigen in the Presence or Absence of Serum-Derived Allergy Asthma Proc, 23-(6): 373-6,2002. Antibody.Immunology, 33:733-743, 1977. 41 14 Dannaeus, A., Inganas, M., Johansson, S.G.O., Foucard, T., Quinti, I., et al, IgG Subclasses to Food Antigens. Allergie Intestinal Uptake of Ovalbumin in Malabsorption and Food Allergy Immunol, 20:41,1988. 42 in Relation to Serum IgG Antibody and Orally Administered Sodium Tolo, op. cit. 43 Cromoglycate. Clinical Allergy, 9:263-270, 1979. Brandtzaeg, P., et a, The Human Gastrointestinal Secretory 15 Furic, R., et al, The Development of Sensitive Radioimmunoassay Immune System in Health and Disease. Scand J Gastroenterol Suppl, to Detect IgG4 Immunogobulin. Clin Rev Allergy, 1:213-224, 1983. 114:17-38, 1985. 44 16 Halpern, Georges M., Scott, John R., Non-IgE Antibody Mediated Kleinman, Ronald E., Walker, Allan W., Antigen Processing and Mechanisms in Food Allergy. Annals of Allergy, 58:14-27,1987. Uptake from the Intestinal Tract. Clin Rev Allergy, 2:25-37,1984. 45 17 Paganelli, R., et al, Isotypic Analysis of Antibody Response to a Saavedra-Delgado, op. cit. 46 Food Antigen in Inflammatory Bowel Disease. Int Archs Allergy Lee, Sok Kyu., et al, Cow’s Milk-Induced Pulmonary Disease in apppl. Immun, 78:81-85, 1985. Children. Adv Pediatr, 25:399-57, 1978. 47 18 Yoshida, S., et al, Beta-lactoglobulin-Specific IgE and IgG in Sera Karjalainen, J., et al, A Bovine Albumin Peptide as a Possible of Patients with Milk Allergy. 5th International Food Allergy Trigger of Insulin-Dependent Diabetes Mellitus. N Engl J Med, Symposium. Atlanta, Georgia, 1984. 327(5): 302-7, 1992. 48 19 Shakib, F, Clinical Relevance of Food-Specific IgG4 Antibodies. N Caffrey, E., et al, Thrombocytopenia Caused by Cow’s Milk. Engl Reg Allergy Proc, 9(1): 63-6, 1988. Lancet, 2:316, 1981. 49 20 Eysink, P.E.D., et al, Relation Between IgG Antibodies to Foods Gershwin, Eric M., German, Bruce J., Keen, Carl L., eds. Nutrition and IgE Antibodies to Milk, Egg, Cat, Dog and/or Mite in a Cross- and Immunology: Principles and Practice. New Jersey: Humana Sectional Study. Clinical and Experimental Allergy, 29:604-610, Press, Inc., 2000. 50 1999. Kemeny, D.M., et al, Sub-Class of IgG in Allergic Disease I. IgG 21 Lucarelli, S., et al, Food Allergy and Infantile Autism. Panminerva Sub-class Antibodies in Immediate and Non-immediate Food Med, 37(3): 137-41,1995. Allergy. Clinical Allergy, 16: 571-581, 1986. 51 22 Hafstrom, I., et al, A Vegan Diet Free of Gluten Improves the Kemeny, op. cit. 52 Signs and Symptoms of Rheumatoid Arthritis: the Effects on Rafei, op. cit. 53 Arthritis Correlate with a Reduction in Antibodies to Food Antigens. Tahmeed, op. cit. 54 th Hematology (Oxford), 40(10): 1175-9,2001. Roitt, Ivan., Brostoff, Johnathan., Male, David. Immunology 5 Ed. 23 Halpern, op. cit. London: Mosby Int. Ltd., 1998. 55 24 Van Der Zee, J.S., Aalberse, R.C., Immunochemical CLIA Requirements for Analytical Quality. Westgard Quality Characteristics of IgG4 Antibodies. N Engl Reg Allergy Proc, 9(1): Corporation http://www.westgard.com/clia.htm 31-33, 1988. 25 Nakagawa, T, Egg White-Specific IgE and IgG Subclass Antibodies and their Associations with Clinical Egg Hypersensitivity. N Engl Reg Allergy Proc, 9(1): 67-73, 1988. 26 Van De Zee, op. cit.

6 The clinical relevance of IgG food allergy testing through ELISA (Enzyme-Linked Immunosorbent Assay).

From: Townsend Letter for Doctors and Patients | Date: 1/1/2004 | Author: Suen, Raymond M.;Gordon, Shalima

Allergic reactions to foods may be classified as either IgE-mediated or nonIgE-mediated--the role of the former in food allergy being well-established. However, interestingly enough, the majority of food allergies are associated with specific nonIgE-mediated immune sensitivities. As such, appropriate tests must be utilized to identify possible causes, including food- antigen specific IgG antibodies. There are many testing methods available for the detection of food allergies including the skin prick test and RAST, or radioallergosorbent test. Unfortunately, both of these methods only look for allergen-specific IgE antibodies from the patient's serum. This poses considerable limitations in the clinical assessment of the chronically unwell patient.

The Skin Prick Test--Pitfalls

With regards to IgE testing, the ELISA method offers an excellent confirmatory test for IgE- mediated food allergies when skin prick testing is equivocal or negative, as it is not unusual for a patient to be skin prick test negative and ELISA positive. Generally, the assumption in such a case is that the extracts used for IgE skin prick testing were defective, unstable or non-standardized. Conversely, a false positive skin test may be due to nonspecific enhancement of the hypersensitive reaction through an axon reflex of a neighboring strong wheal-and-flare reaction. In addition, skin prick testing does pose a health risk to the patient, as eluates of protein extracts are pierced through the skin. Anaphylaxis is a possibility and resuscitation equipment must be on hand. Furthermore, the results of skin prick testing do not exhibit a strong correlation to food allergy symptoms. ELISA is reported to be more sensitive than skin prick testing in the identification of IgE-mediated food allergies, and as most food allergies are nonIgE-mediated, skin prick testing is rather obsolete. (1-5)

The principle behind the skin prick testing method is simple. Sensitized tissue mast cells display IgE antibodies on their cell membranes, which through provocation by a recognized food antigen will promote the release of histamine and other inflammatory mediators from these immune cells. The result is a wheal-and-flare reaction marked by redness and swelling. However, identification of such mast cell dependent reactions for the detection of food allergies does have its pitfalls in addition to those mentioned. First, diseases such as eczema may attenuate the skin response. Second, there is decreased reactivity of the skin in infants and elderly patients making this testing method inappropriate for these populations. In addition, mast cells from different sites of the body (skin, lungs, and gastrointestinal tract) exhibit marked heterogeneity with respect to their functional properties. (6) This is of fundamental importance from a clinical perspective since one cannot simply extrapolate the results from a skin prick test and assume a direct correlation to that which is occurring in the gut. Furthermore, skin prick testing does not assess for delayed-onset food allergies mediated through IgG antibodies. IgG concentrations increase from repeated exposure to food antigens. (7) IgG-mediated food reactions occur hours to days after exposure to the incriminating foods, and unlike that of IgE, IgG related symptoms are cumulative in nature.

Since the discovery of IgE in 1967, conventional medical practice has focused chiefly on IgE-mediated allergies as identified primarily through skin prick testing. As our understanding of the disease model progresses with physiological mechanisms finding root in regulation of oral tolerance, the clinical importance of IgG antibodies is rapidly following suit as a key player in the allergic model of disease and chronic pathology. (8)

The IgG Immunoglobulin Class

The IgG immunoglobulin class has an exceptionally long half-life in circulation and makes up about 75% of the total serum immunoglobulin pool. This class is comprised of four known subtypes; IgG1, IgG2, IgG3, and IgG4. IgG1 constitutes about 68% of total IgG; IGg2, 20%; IgG3, 8%; and IgG4, 4%. IgG1 through IgG3 are capable of binding complement and initiating complement-mediated tissue injury, whereas IgG4 is not. (9) However, it is argued that altered IgG4 through immune complex formation may act as an autoantigen. Since IgG levels increase with exposure, these complexes may reach appreciable levels over time. Autoantibodies such as those of the IgM class, formed to these altered IgG4 autoantigens, may cross-link cell-bound IgG4 and activate complement. (10) Such a mechanism has been reported responsible for the exacerbation of symptoms in atopic eczema patients where high-molecular-weight (i.e. 21S or more) immune complexes have been identified. (11) An autoimmune process such as this clearly deserves considerable attention to its clinical implications in chronic allergic disease. Interesting among the IgG class of antibodies are the IgG receptors, Fc[gamma]R. Since IgG represents the dominant antibody class in plasma, receptors for IgG have been intensively studied over the years. (12) These receptors are found on a wide variety of immune cells and are said to serve as a bridge between the cellular and humoral parts of the immune system. Effector functions that can be triggered by Fc[gamma]R include; antibody- dependent cellular cytotoxicity (ADCC), antigen presentation, cytokine release, phagocytosis, degranulation, and regulation of antibody production. (13) With a constant stream of IgG antibodies in circulation due to chronic challenge, inappropriate regulation of Fc[gamma]R-mediated responses, or inefficient Fc[gamma]R function may lead to a hyperresponsive state with greatly magnified effector responses that may subsequently promote inflammatory disease and increase susceptibility to autoimmunity. (14-17)

The Gut Immune System

It is well known that a significant portion of ingested proteins from food reach the gut- associated lymphoid tissues (GALT), in an immunologically intact form capable of stimulating immune responses in the susceptible individual. This susceptibility rests in the competency of the immunoregulatory mechanisms of the GALT that normally prevent the induction of a hypersensitive response to otherwise innocuous food challenges. It is undesirable to be intolerant to the foods we eat. Mobilization of the GALT against food antigens defines loss of oral tolerance to foods, and may provoke injurious local and systemic immune responses. The gut mucosal response, particularly that involving an antibody response, is highly dependent on T cell help. (18) T helper pattern induced clonal expansion may proceed through a cell-mediated (Th1) response, humoral (Th2) response, or immune tolerance (Th3). (19) It is important to note that T cells of the mucosal lymphoid tissue are heavily biased toward a Th2 response. This accounts for the predominance of the protective IgA isotype in mucosal effector tissues. However, the phenotypic polarization of immunoglobulin producing B cells favoring IgA is heavily influenced by the cytokine profile present in the mucosal milieu. Interleukin-4 (IL-4) for example, promotes isotype switching to both IgG1 subclass and IgE (20) whereas, the cytokine transforming growth factor beta (TGF-[beta]) favors B cell class switching from IgG and IgE to IgA, thereby suppressing any potential for a Th1 or Th2 inflammatory response to dietary antigen.

Ideally the intestinal immune system can discriminate proteins in the food stream as innocuous and not of any pathogenic importance. It can be said in this case that a state of tolerance is achieved with suppression of IgE and IgG responses, and enhancement of a local secretory IgA antibody response. Certainly, the integrity of the mucosal barrier with its immune constituents in competent interplay is prerequisite for oral tolerance induction, and susceptibility to breakdown of oral tolerance varies individually. Loss of mucosal barrier integrity and genetic polymorphisms in markers of innate immunity, including that of the Fc[gamma]R class of IgG receptors, no doubt play a key role in abrogation of oral tolerance to dietary challenge. The biological mechanisms of food allergies are diverse and remain to be explored. Loss of tolerance, as exemplified by elevated food-specific IgG class antibodies is a breakdown of the GALT to distinguish antigens of non pathogenic importance, abrogating the metabolic usefulness of the foods we eat, and instigating the potential for inflammatory and autoimmune conditions.

Effective assessment of food allergies, especially through IgG testing should be as routine for the practitioner as ordering a CBC (Complete Blood Count). Identification of elevated food-specific IgG antibodies is a means to identify loss of tolerance to dietary proteins, and provides the practitioner with a tool to direct care in the appropriate manner. Once identified, treatment of food allergies includes dietary rotation of compatible foods and avoidance of allergenic ones, in addition to cogent measures to re-establish tolerance.

The ELISA Method

The ELISA colorimetric technique, or Enzyme-Linked Immunosorbent Assay, is a useful screen for immediate and delayed food allergies mediated through immunoglobulin E (IgE) and immunoglobulin G (IgG), respectively. Allergic reactions to foods are characterized by elevated allergen-specific immunoglobulin serum levels with activation of immune mediators of inflammation. Food allergies are implicated in intestinal pathology, as typified by celiac disease, and a number of systemic inflammatory conditions. (21), (22)

ELISA is a quantitative/semiquantitative in vitro analysis designed to detect and quantify IgG and IgE antibodies reactive to various food proteins. Through the ELISA testing method, lyophilized food proteins are immobilized by adsorption to plastic wells and reacted with the serum portion of the individual's blood sample. After washing, the plate is reacted with an HRP-labeled anti-human IgG or IgE antibody conjugate. The enzyme tag, HRP, or horseradish peroxidase, facilitates a color change upon addition of its substrate, a chromagen, to allow for easy detection of antigen-antibody interaction within the wells. The intensity of the color change is quantified through spectrophotometric analysis, and is proportional to the concentration of food antigen-specific IgG or IgE antibodies present in the serum sample. The ELISA Method--Reproducible, Reliable and Valid

There are several industry standards that should be considered for ELISA testing to allow its implementation as a routine method suitable for analysis of food allergies. Official criteria for any bioanalytical method includes clear demonstration of reproducibility and reliability for its intended use based on guidelines set by CLIA (Clinical Laboratory Improvement Amendments) Requirements for Analytical Quality. (23) Moreover, a laboratory implementing ELISA methodology for the detection of IgG and IgE food-specific antibodies must clearly identify its suitability for this purpose in yielding reproducible and consistent results for each patient tested on every occasion. Reproducibility as the name implies, is the ability of the test to reproduce the same test results for identical samples under identical test conditions. Identical testing conditions must be assured by the laboratory through day-to-day and run-to- run, for a dependable test, or a correct and precise testing procedure that has been exactly defined. Duplicate testing for example, provides an internal measure of control and assures reproducibility. If the testing method is precise there should be minimal variation between the duplicate runs. In addition to this, daily in-house blinded split sample reproducibility checks are on the onus of the lab and constitute good laboratory practice for quality assurance. Most often a laboratory also participates in periodic blinded testing through an approved accredited organization to further insure reproducibility of test results. These strict quality measures guarantee repeatability of the results; namely the presence of food-specific IgE/IgG antibodies will be consistently detected each and every time the patient's serum sample is tested.

In order for a laboratory to provide its testing services, it must hold a license and abide by federal CLIA rules, the governing body for analytical proficiency testing criteria for acceptable analytical performance. The purpose of CLIA is to promote good laboratory practices and to assure a reliable test with reproducible and consistent results. Under the government of CLIA, a diagnostic laboratory has demonstrated and documented participation in proficiency testing and quality assurance and control. CLIA certification and accreditation requires that the laboratory be inspected by a CLIA accredited non profit organization, and approved by the federal Centres for Medicare and Medicaid Services (CMS), formerly HCFA (Health Care Finance Administration). Inspections for this certification may be completed through COLA (Commission of Laboratory Accreditation) or CAP (College of American Pathologists), and are often more rigorous than CLIA regulations. CLIA governs all laboratory operations including; accreditation, proficiency testing, quality assurance, quality control, records and information systems, test methods, equipment, and instrumentation. Regulations set under these operations are designed to assure reliability and consistency of laboratory test results. In the strictest sense, a laboratory must establish and follow CLIA procedures for monitoring and evaluating the quality of the analytical testing process to assure reliability; a true and reputable test result. However, it is the responsibility of the laboratory in compliance with federal quality standards established by CLIA, to assure reliable laboratory results and documentation/records. More so, it is the responsibility of the health care practitioner to understand these criteria and to seek a reputable testing facility.

For ELISA food allergy testing to be valid it must accurately measure what it purports to measure, namely food-specific IgG and IgE antibodies. Only in this way can it be of any clinical worth to the practitioner and patient. With respect to accuracy, accuracy expresses the closeness, or degree of agreement between a measured and established reference value. What, however, defines the established reference value as that which to compare? The presumption is that there is a true "gold standard" or an accepted method to which a new method can and should be compared to define its accuracy and hence, credibility. With respect to IgG food allergy testing, there is no "gold standard" or accepted method available to define all others. Is IgG ELISA food allergy testing therefore accurate? It can be argued that this testing method is accurate if it yields similar results to that obtained from another lab using the same sample. However, each lab abides by their own in-house validation and quality assurance measures, which may vary from lab to lab. Strictly, this implies that the results from one laboratory cannot be compared justly to that of another lab, again because there is no "gold standard" for quantification. The onus of responsibility to provide the practitioner and patient with a valid IgG food allergy test therefore lies in the hands of the laboratory to uphold good laboratory practices in compliance with specific federal quality standards established by CLIA.

Validity is the predictive significance of a test for its intended purpose. That is, the correlation between the test results and some criterion to which this test is supposed to predict. In laboratory medicine this criterion refers to a disease state or condition. In conventional circles the validity of a test is justified by its positive predictive value (PPV). That is, a remarkable or positive test result will identify a particular diseased state in a large proportion of the population, with defined signs and symptoms. A true relationship between the PPV and the prevalence of the disease/condition in the population represents the diagnostic value of the test and hence its worth. That is, the test when applied to the general population can efficiently identify those individuals who are likely to have the disease in question while excluding those unaffected. The diagnostic efficiency of the test is improved by utilizing it only in patients with clear clinical features suggestive of the disease. Two major factors in improving the diagnostic worth of the test are sensitive and specific achievement. Test sensitivity is defined as the percentage of people in the population with the disease state in question that have a remarkable test result. The specificity on the other hand, is defined as the percentage of people in the population without the disease who have a normal test result. Ideally, the specificity of a test with regards to the general population should be equal to 97.5% with 2.5% representing "false positives." It is important to note however, that it is often difficult to reliably define sensitivity and specificity for a particular test, in part because of the challenge involved in defining the "reference population" on which to deduce a true generalization for the population at large. In addition, to what criteria do we define the presentation of the disease in question to justify its predictive value for substantiating test validity? This question needs to be addressed when considering the validity of IgG food allergy testing. First and foremost, as with all laboratory testing, it is a prudent assumption that a test supplement rather than substitute for clinical skills, and careful clinical assessment. No test should take the place of sound clinical decision-making. In addition, the clinician should understand the factors that influence the reliability of the test as such to guide valid decisions for patient care.

The purpose of IgG ELISA food allergy testing is to identify elevated IgG antibody levels to food antigens from a sample of the patient's serum. An antigen is any substance that is regarded as foreign by the immune system and therefore capable of stimulating an immune response. Elevated food-specific IgG antibody levels are understood to represent IgG immune-mediated allergies to these particular food antigens. An allergy is defined as a pathological immune reaction to an antigen. With this in mind, allergy should not be defined solely as an IgE-mediated hypersensitive atopic condition; allergic rhinitis, atopic dermatitis and asthma. Allergy is any abnormal immune reaction to an allergen that may result in a broad range of inflammatory responses, and elevated food-specific IgG antibodies may have far-reaching systemic consequences. It is well established in immunological circles that Fc[gamma]R polymorphisms play an important role in the pathogenesis of inflammatory disease. This, in association with the extended half-life of IgG antibodies, make insult through dietary challenge an important issue in the management of the chronically unwell patient. Assessment of elevated IgG food-specific antibodies provide a useful tool for patient-tailored diet therapy as a means to control in part, undue Fc[gamma]R-mediated effector functions in the patient with receptor polymorphisms that are implicated in disease susceptibility. Furthermore, one cannot discuss the clinical validity of IgG food allergy testing without discussing the mechanisms of oral tolerance. Oral tolerance lies at the heart of immunological theories and is the cornerstone of setting up a reaction or non-reaction against self and non-self (dietary challenge). It has been argued that oral tolerance to dietary antigens is the B cell switching from IgE/IgG antibody production to IgA, under the influence of a novel cytokine profile.

Abrogation of tolerance to otherwise innocuous food proteins may be involved in the pathogenesis of a variety of disease states. Loss of mucosal barrier integrity, excessive stimulation of antigen presenting cells, favor overstimulation of Th cells, and a cytokine profile that is incompatible with induction of tolerance. (24) This loss of tolerance is the model of a variety of pathologies from autoimmune-based disease to food allergies and enteropathies; the mechanisms of which are in the forefront of clinical research today.

Mucosal Tolerance

Mucosal tolerance represents the most important response to food antigens that affords systemic hyporesponsiveness or protection from inflammatory events and bodily disorder. A tolerogenic response to dietary challenge is critical to allow for competent digestion and absorption of nutrients for maintenance of normal structure and function of the body. Loss of tolerance on the other hand, is the unfavorable immune reaction with hyperresponsiveness to daily dietary challenge. As a result, mediators for enhanced inflammation and tissue damage, both local and systemic, predominate with sequelae both acute and chronic. Moreover, hypersensitivity to ingested foods, IgG and IgE-mediated food allergies, signifies loss of oral tolerance. Celiac disease for example, is loss of tolerance to wheat gliadin, a prolamine-derived peptide fraction of the cereal protein gluten. Multiple grain allergies result, with elevated IgG antibodies to other prolamines including that of rye (secalin), and barley (hordein). This is an abnormal immunemediated and cytotoxic reaction characterized by partial or total villous atrophy and lymphoid infiltration of the lamina propria. Crohn's disease and ulcerative colitis also represent inflammatory bowel diseases in which there is a loss of oral tolerance, namely to commensal bacteria. (25)

From each meal of the day the gut mucosa is bombarded with a myriad of potentially antigenic food proteins. Likewise, the diverse population of normal bacterial flora in the intestine poses an additional potential antigenic challenge. Yet, under normal and ideal circumstances, the body does not react unfavorably to these mucosal antigens. Resident microbial flora and food proteins result in immunologic silence, or tolerance. How the mucosal immune system is able to define these antigens as pathogenically important and mount an inappropriate response in any given circumstance in the susceptible individual is influenced by many factors. In the neonate for instance, improper establishment of oral immune tolerance may be influenced through genetic makeup, insufficient acquisition of microflora, (26) early introduction of solid foods, early cessation of breast-feeding, and maternal transfer of food antigens through the breast milk. (27) In the adult, breach of oral tolerance may be mediated through medication use; NSAIDs (non-steroidal anti- inflammatory drugs) and prednisone block oral tolerance induction. (28) Moreover, any trauma or insult to the protective mucosal barrier that increases permeability may abrogate a tolerogenic response.

As a clinician, a true understanding of the mucosal immune system of the gastrointestinal tract and the induction of oral tolerance, or lack thereof to dietary proteins, is key to developing a clear appreciation for the potential implications of food allergies in systemic health.

The body employs many mechanisms at the intestinal lumen-mucosa interface to prevent the induction of hypersensitivity to food proteins. The first level of protection against undue penetration of oral antigens involves non-immunological factors. These factors play a pivotal role in mucosal integrity and antigen exclusion and include: tight junctions and basement membranes that form the cohesive bonding among the mucosal epithelial cells, low luminal pH, digestive enzymes, peristalsis, mucus, enteric microflora, mucosal surface regeneration rate, and the glycocalyx. Breach of any of a number of these defense factors, and integrity loss of the mucosa allows for aberrant antigen handling, and consequent production of cytokines triggering a number of tissue damaging events.

The intestinal immune system offers a second line of defense against food antigens. Immunologic responses include local production of secretory IgA (sIgA) antibodies in the intestine; systemic priming with cell-mediated immunity and the generation of antibodies; or tolerance to subsequent antigen challenge. It is argued that IgA deficiency may predispose one to food hypersensitivity as sIgA is believed to serve as a barrier to absorption, preventing the uptake of food antigens. (29), (30) In addition, early studies rationalize a systemic decrease in specific IgE and IgG concomitant with a local increase in sIgA as an integral role in the induction of oral tolerance. (31), (32) The proposed mechanism was thought to be due to the influence of Th2 cytokines and TGF-[beta] which act to suppress IgG/IgE B cell differentiation, but at the same time enhance IgA B cell differentiation. In other words, oral tolerance was believed to be associated with concomitant local IgA immunity. (33) However, the prime importance of sIgA in oral tolerance is not without challenge. Experimental studies have proven it difficult to induce an IgA antibody response in animals immunized orally with protein antigens, and under normal circumstances there is negligible food specific IgA in the intestine. (34), (35) Shi et al, in particular, have demonstrated the suppression of OVA-specific IgA responses by fed antigen in experimentally bred mice deficient in Th1 and Th2 cells, but competent in TGF-[beta]-mediated oral tolerance. In other words, oral tolerance in these mice did not correlate with a concomitant elevation in OVA- specific IgA. On the contrary, the IgA response was suppressed compared to that observed in normal BALB/c control mice. (36) The reduction in IgA in other words, paralleled the reduction of systemic IgG and IgE in oral tolerance. The researchers conclude therefore a supporting role for Th1 and Th2 cytokines in regulating the induction of IgA immunity. Contrary to these findings, Kim et al, have shown TGF-[beta] to be co-stimulatory in IgA production, influencing B cell differentiation into IgA-producing cells. (37)

IgA is the predominant immunoglobulin secreted by the B cells of the gut. Constituting over 70% (38) of all immunoglobulin present in the intestinal mucosa, it obviously plays a key role in immune exclusion of food antigens as a "default" mucosal B cell response. However, its position in oral tolerance is less clear. The GALT is exquisitely sensitive to the residing cytokine milieu of which dysregulation alters mucosal responsiveness. TFG-[beta] and other immunosuppressive cytokines, including those of Th2, interact to maintain intestinal homeostasis and nonresponsiveness to innocuous food antigens. TFG-[beta] in particular, inhibits the proliferation of T and B cells, and decreases the secretion of IgG immunoglobulins, yet at the same time induces isotype IgA class switching. (39) Clearly, local IgA immunity alone is unlikely to account for the absence of food hypersensitivities, but does accompany and serves as a useful backup to other more pivotal immunoregulatory mechanisms.

The Gastrointestinal Mucosa

When we consider the cellular arrangements in the gastrointestinal system it is amazing how the epithelial lining of the mucosa, connected by tight junctions, represents the primary barrier to food antigen entry. The mucosal epithelium, comprised of absorptive cells, mucus producing goblet cells, intraepithelial lymphocytes (IEL's), and a basal membrane, is the interface between the external and internal environments of the body, and permits or excludes entry of various materials, appropriately under ideal conditions. The gastrointestinal mucosa is the largest surface of about 300[m.sup.2] that is in continuous contact with the external environment. (40) Rightly so, it houses over 60% of measurable immune parameters including; mesenteric lymph nodes, Peyer's patches (PP), isolated follicles, lamina propria lymphocytes, and IEL's. These immune components span the epithelial lining and lamina propria and constitute the gut associated lymphoid tissues (GALT). GALT is the largest lymphoid organ of our immune system comprising 80% of the immunoglobulin producing cells in the body and 75% of the entire T cell population, of which 60% is above the basal membrane. (41)

Antigen presentation in the intestinal mucosa includes; B cells, macrophages, and dendritic cells of which reside primarily in the lamina propria, PP, and mesenteric lymph nodes of the GALT. (42) Not limited to this repertoire, antigen presentation also occurs via; mucosal T cells, IEL's, and intraepithelial cells (IEC's). (43) It is clear from this list that antigen sampling does not solely occur via the M cells overlying PP. All cell types are implicated in the mechanisms of oral tolerance induction. The competency in antigen presentation, the dynamics in T cell trafficking, the dose and type of antigen, and changes in the cytokine milieu of the gut, together influence the antigen-specific T helper pattern activity; either towards down-regulation of the mucosal immune response to facilitate tolerance, or towards untoward inflammation.

Other factors influencing the predominant immune response to food antigen include; genetic background and indigenous gut flora. With regards to the former, celiac disease for example, is believed to be due in part to aberrant antigen presentation. Over 95% of patients with celiac disease carry a DQ2 (HLA-DQ2) gene that encodes MHC II markers that present gliadin to T cells in the lamina propria. (44), (45) Cytokine release increases the expression of HLA-II, thus amplifying the immune response with resulting cell damage. These inflammatory mediators also increase gut permeability and promote the differentiation of B cells into IgG-antigliadin antibody-producing plasma cells. (46)

Indigenous gut microflora has been strongly implicated in competent induction of oral tolerance. The gastrointestinal tract contains about 100,000 billion, or three and one- half pounds worth of viable microflora of which there is a variation in number and type in the different regions of the intestine. (47) Lactobaccilli predominate in the small intestine, particularly in the middle and distal ileum, whereas Bifidobacteria increase in prevalence from the cecum to large intestine. Gut microflora are compulsory to the development of mucosal immuno-responsiveness--humoral and cell-mediated immunity, during the neonatal period, and serves to prime the GALT throughout the life of the individual. (48) Following birth, in the absence or delay of colonizing microflora, oral tolerance may be abrogated. Specifically, there is incomplete maturation and development of Peyer's patches, intraepithelial and lamina propria lymphocytes, in addition to decreased levels of plasma cells and IgA antibody production. (49) Clearly, defective development of the mucosal immune system in this way will incite deregulated inflammation and negatively influence the immune response to dietary antigens.

The mucosal surface represents the interface between the internal and external environments of the body that is in continual contact with a myriad of food proteins, invasive pathogens and indigenous flora on a daily basis. Discernment between infectious and noninfectious agents is therefore key to survival of the individual in his environment. Under normal circumstances down-regulation of the immune response governs oral tolerance to dietary antigens and indigenous flora of the gastrointestinal tract. (50), (51) The precise mechanisms involved in inducing oral tolerance to dietary antigens are imperfectly known. It is important to keep in mind that oral tolerance is a complex immune response that involves a precarious balance among several immune-mediated parameters. A glimpse into the competency of tolerance through IgG food allergy testing via the ELISA method is a simple tool for the practitioner to visualize the immunological response to dietary challenge in the patient. In practice, this assessment may guide treatment to nullify undue mediators of inflammation in the body that may be perpetuating a disease process.

US BioTek Laboratories [c]2004

References

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(2.) Sampson, H.A., Role of immediate food hypersensitivity in the pathogenesis of atopic dermatitis. J. Allergy Clin. Immun. 71(5): 473-80, 1983.

(3.) Bock, S.A., et al. Appraisal of skin tests with food extracts for diagnosis of food hypersensitivity. Clin. Allergy, 8(6): 559, 1978.

(4.) Sampson, H.A., Albergo, R., Comparison of results of skin tests, RAST and double- blind, placebo-controlled food challenges in children with atopic dermatitis. J. Allergy Clin. Immunol. 74(1): 26-33, 1984. (5.) Campbell, D.E., et al. Indirect enzyme-linked immunosorbent assay for measurement of human immunoglobulins E and G to purified cow's milk proteins: application in diagnosis of cow's milk allergy. J Clin Microbiol. 25(11): 2114-2119, 1987.

(6.) Barrett, K.E., Metcalfe, D.D., The mucosal mast cell and its role in gastrointestinal allergic diseases. Clin. Rev. Allergy, 2:39-53, 1984.

(7.) Trotsky, Martin, Immunology for the otolaryngic allergist. Otolaryngic Allergy, 25(1): 151- 162, 1992.

(8.) Suen, Raymond, Gordon, Shalima, A critical review of IgG immunoglobulins and food allergy -implications in systemic health. Townsend Letter for Doctors & Patients, 241/242:134-38, 2003.

(9.) Trotsky, op. cit.

(10.) Shakib, F., The role of antiglobulins in IgG4-mediated allergic diseases. N. Engl. Reg. Allergy Proc. 9(1); 35-42, 1988.

(11.) Ferguson, A.C., Salinas, F.A., Elevated IgG immune complexes in children with atopic eczema. J. Allergy Clin. Immunol. 74:678-682, 1984.

(12.) Gessner, J.E., et al. The IgG Fc receptor family. Ann. Hematol. 76(6): 231-48, 1998.

(13.) Van der Pol, W., Van de Winkel, J.G., IgG receptor polymorphisms: risk factors for disease. Immunogenetics, 48(3): 222-32, 1998.

(14.) Van Sorge, N.M., Van Der Pol, W.L., Van De Winkel, J.G., FcgammaR polymorphisms: implications for function, disease susceptibility and immunotherapy. Tissue Antigens, 61(3): 189-202, 2003.

(15.) Loos, B.G., et al. Fcgamma receptor polymorphisms in relation to periodontitis. J. Clin. Periodontol. 30(7): 595-602, 2003.

(16.) Brun, J.G., Madland, T.M., Vedeler, C.A., Immunoglobulin G fc-receptor (FcgammaR) IIA, IIIA, and IIIB polymorphisms related to disease severity in rheumatoid arthritis. J. Rheumatol. 29(6): 1135-40, 2002. (17.) Dijstelbloem, H.M., et al. Fcgamma receptor polymorphisms in systemic lupus erythematosus: association with disease and in vivo clearance of immune complexes. Arthritis Rheum. 43(12): 2793-800, 2000.

(18.) Kawanishi, H., Saltzman, L.E., Strober, W., Mechanisms regulating IgA class-specific immunoglobulin production in murine gut-associated lymphoid tissues. I.T cells derived from Peyer's patches that switch sIgM B cells to sIgA B cells in vitro.'J. Exp. Med.'157(2): 433- 450, 1983.

(19.) Spiekermann, Gerburg, M., Walker, Allan, W., Oral tolerance and its role in clinical disease. Journal of Pediatric Gastroenterology and Nutrition, 32:237-255, 2001.

(20.) Rousset, F., Garcia, E., Banchereau, J., Cytokine-induced proliferation and immunoglobulin production of human B lymphocytes triggered through their CD40 antigen. J. Exp. Med. 173(3): 705-710, 1991.

(21.) Gershwin, Eric, M., German, Bruce, J., Keen, Carl, L., Eds. Nutrition and Immunology: Principles and Practice. New Jersey: Humana Press, Inc., 2000.

(22.) Suen, op. cit.

(23.) CLIA Requirements for Analytical Quality. Westgard Quality Corporation http://www.westgard.com/clia.htm

(24.) Brandtzaeg, P., Overview of the mucosal immune system. Current Topics in Microbiology and Immunology, 146:13-25, 1989.

(25.) Elson, C.O., et al. Experimental models of inflammatory bowel disease. Gastroenterology, 109(4): 1344-1367, 1995.

(26.) Plummer, Nigel, Oral Tolerance: Revolutionary Implications in Immunological Health and Disease. Pharmax LLC: Practical Solutions Seminar Series. Bellevue, WA. June 7-8, 2003.

(27.) Gershwin, op. cit.

(28.) Postlethwaite, Arnold, E., Can we induce tolerance in rheumatoid arthritis? Current Rheumatology Reports, 3:64-69, 2001. (29.) Cunningham-Rundles, C., et al. Milk precipitins, circulating immune complexes, and IgA deficiency. Proc. Natl. Acad. Sci. 75(7): 3387-3389, 1978.

(30.) Challacombe, S.J., Tomasi, Jr., T.B., Systemic tolerance and secretory immunity after oral immunization. J. Exp. Med. 152: 1459-1472, 1980.

(31.) Challacombe, op. cit.

(32.) Saklayen, Mohammad, G., et al. Kinetics of oral tolerance: study of variables affecting tolerance induced by oral administration of antigen. Int. Archs. Allergy appl. Immun. 73:5-9, 1984.

(33.) Challacombe, op. cit.

(34.) Mowat, A.M., Maloy, K.J., Donachie, A.M., Immune-stimulating complexes as adjuvants for inducing local and systemic immunity after oral immunization with protein antigens. Immunology, 80(4): 527-534, 1993.

(35.) O'Mahony, S., et al. Dissociation between systemic and mucosal humoral immune responses in coeliac disease. Gut, 32(1): 29-35, 1991.

(36.) Shi, Hai Ning, Grusby, Michael, J., Nagler-Anderson, Cathryn. Orally induced peripheral nonresponsiveness is maintained in the absence of functional Th1 or Th2 cells. The Journal of Immunology, 162:5143-5148, 1999.

(37.) Kim, P.H., Kagnoff, M.F., Transforming growth factor-beta I is a costimulator for IgA production. J. Immunol. 144(9): 3411-3416, 1990.

(38.) Abreu-Martin, Maria, T., Targan, Stephan, R., Regulation of immune responses of the intestinal mucosa. Critical Reviews in Immunology, 16:277-309, 1996.

(39.) Nagler-Anderson, Cathryn, Tolerance and immunity in the intestinal immune system. Critical Reviews in Immunolgy, 20:103-120, 2000.

(40.) Spiekermann, op. cit.

(41.) Plummer, op. cit. (42.) Wardrop, III, R.M., Whitacre, C.C., Oral tolerance in the treatment of inflammatory autoimmune diseases. Inflamm. Res. 48:106-119, 1999.

(43.) Abreu-Martin, op. cit.

(44.) Balas, A., et al. Absolute linkage of celiac disease and dermatitis herpetiformis to HLA- DQ. Tissue Antigens, 50(1): 52-56, 1997.

(45.) Godkin, A., Jewell, D., The pathogenesis of celiac disease. Gastroenterology, 115(1): 206-210, 1998.

(46.) Gershwin, op. cit.

(47.) Plummer, op. cit.

(48.) Plummer, op. cit.

(49.) Plummer, op. cit.

(50.) Plummer, op. cit.

(51.) Spiekermann, op. cit. by Raymond M. Suen, MT (ASCP) and Shalima Gordon, BSc, ND

Correspondence:

Raymond Suen, MT

U.S. Biotek Laboratories

13500 Linden Ave. North

Seattle, Washington 98133 USA

From the Inner City Medical Polyclinic of Ludwig Maximilian University in Munich, Chairman: Prof. Dr. med. D. Schlöndorff

IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Dissertation for acquisition of a Doctoral Degree in Medicine at the Medical Faculty of Ludwig Maximilian University in Munich

Submitted by Mario Krause from Rotenburg a. d. Fulda 2005

IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

With permission of the Medical Faculty of the University of Munich

Main supervisor: Prof. Dr. med. Herbert Kellner

Second supervisor: Priv. Doz. Dr. C. Otto

Support through the doctoral assistants:

Dean: Prof. Dr. med. D. Reinhard

Date of oral examination: October 6, 2005

2 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Contents

1. Introduction 1.1. Definitions 1.1.1. Food allergy 1.1.2. Anaphylaxis or anaphylactic shock 1.1.3. Food intolerance 1.1.4. Enzymatic food intolerance 1.1.5. Pharmacological food intolerance 1.1.6. Mucosal irritations 1.1.7. Toxic food intolerances 1.1.8. Food aversion 1.1.9. Psychological intolerance 1.1.10. Strain on the organism through food 1.2. Clinical symptoms of a food allergy 1.2.1. Sequence of allergic reactions 1.3. Food quantity and allergic reaction 1.4. Frequency 1.5. The importance of the elimination diet 1.6. Pathomechanism of a type III food allergy

Goal of the paper

2. Patient collective and research method 2.1. Sex 2.2. Age 2.3. Height, weight and BMI during admission visit 2.4. Classification of initial weight 2.5. Diagnosis 2.6. General risk factors

3. Results 3.1. Symptoms at start of treatment 3.1.1. Expressivity of symptoms at start of treatment (in percent) 3.1.2. Expressivity of symptoms at start of treatment 3.1.3. Severe expressivity of symptoms at start of treatment 3.1.4. Symptom score at start of treatment Symptom score at start of treatment 3.2. Detection of specific IgG against food

3 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.2.1. Total number of food reactions 3.2.2. Number of food reactions with severity 3 or 4 3.3. Symptom expressivity in the course 3.3.1. Expressivity of symptoms after 2 weeks (in percent) 3.3.2. Expressivity of symptoms after 4 weeks (in percent) 3.3.3. Expressivity of symptoms after 6 weeks (in percent) 3.3.4. Expressivity of symptoms after 8 weeks 3.3.4.1. Expressivity of symptoms after 8 weeks 3.3.5. Severe expressivity of symptoms during initial & final documentation 3.4. Improvement rates 3.4.1. Improvement rates of symptoms 3.4.2. Exemplary symptom scores in the course 3.5. Consistency during the dietary change in the course (compliance) 3.5.1. Consistency during the dietary change in the course 3.5.2. Retention of new eating habits 3.6. Weight change in the course 3.6.1. Weight after 8 weeks in comparison with initial weight 3.6.2. Absolute weight change after 8 weeks in comparison with initial weight 3.6.3. Relative weight change after 8 weeks 3.6.4. Development of body weight in the observation period 3.6.4.1. Weight development in the course 3.7. Change of general state of health 3.8. Further changes 3.9. Final assessment of efficacy 3.9.1. Recommendation

4. Discussion Medical importance of IgG antibodies against food Why is the importance of food intolerance only recognized now? Why conduct a test of all IgG subclasses? Reasons for an unsatisfactory result of an elimination diet according to David

5. Abstract 6. Conclusion 7. Literature 8. Acknowledgments 9. Curriculum vitae

4 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

1. Introduction

Functional and psychovegetative complaints of fibromyalgia patients (e.g. polytopic pain, stress intolerance, lassitude, migraines, depressions, sleep disorders, rapid fatigability, etc.) and especially irritable colon are similar to the complaints of patients with food allergies or food intolerances. Specific histological changes are absent up to now; cellular inflammatory signs cannot be detected. A deterioration of symptoms is frequently found with intermittent inflammatory illnesses. Spontaneous remissions are described, but this frequently concerns a chronic clinical picture. Physiotherapy procedures, heat applications, antidepressants, NSAID or corticoids will normally be employed for therapy.

The motive for initiation of this project was unsolicited feedback from fibromyalgia patients who reported a clear improvement of their symptoms due to a dietary change and studies which showed a positive influence of a dietary change in patients 15,25,27,43,48 with rheumatoid arthritis . The investigator was motivated through research 9,33,54 regarding chronic fatigue syndrome (CFS) and food intolerances and the works from Enestrom, who was able to prove an augmented IgG deposition in the skin of 19,20 fibromyalgia patients .

The greatest challenge in the handling of food allergies and food intolerances is the identification of the responsible foodstuff. The differentiation of allergic reactions and intolerances is difficult. A clarification with RAST and prick test is generally 7,21,23,34,47,50 insufficient . The aim was to find new therapeutic approaches in the treatment of fibromyalgia through differentiated investigative.

1.1. Definitions

The term “food intolerance” is not to be equated with any specific reaction and first of all signifies merely a reproducible adverse reaction to a special foodstuff or a substance contained in this foodstuff. Immunological reactions (in the narrow sense, food allergy), enzymatic defects, pharmacological reactions, irritations and toxic effects come into question as mechanisms of a food intolerance. The term “food allergy” is frequently misused and thus does not take the causes into consideration 6,17,36,46 .

5 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

1.1.1. Food allergy

An allergic reaction is a reproducible adverse reaction to a substance which is mediated through an immunological process. This can be mediated through antibodies, mast cells or circulatory immune complexes. A food allergy is accordingly such a response to foodstuff. The “American Academy of Allergy and Immunology Committee on Adverse Reactions of Food” defines a food allergy as “an 6 immunological reaction which is attributable to the intake of food or additives” . Food allergies can be found via all types, according to Coombs, from I to IV or in any combination. The term “food allergy” is traditionally equated with type I, whereas types II, III and IV are designated as immune-mediated intolerances. The IgG- mediated problem investigated here thus concerns an allergy predominantly ascribed to type III or an immune-mediated intolerance. The processes proceeding within the framework of the allergy occur via predominantly inflammatory paths. At the same time, the triggering allergenic substance can be ingested, injected, inhaled or otherwise come into contact with skin or mucosa.

The reaction occurs on a delayed basis within 8 to 72 hours after ingestion. Therefore an affected individual can only react with gastrointestinal problems on Friday to a foodstuff which he/she had already consumed on Wednesday. This is why, long-term, this form of food allergy is frequently neither recognized by the affected individuals nor the attending physicians.

1.1.2. Anaphylaxis or anaphylactic shock

In this case this concerns a life-threatening, rapid allergic reaction with circulatory collapse. The term “anaphylaxis” is still erroneously equated with allergic reactions which are mediated through IgE antibodies. At the same time, the milder forms of an IgE-mediated allergy such as pollenosis are erroneously equated with this severe reaction.

1.1.3. Food intolerance

The intolerance does not presuppose any specific type or mechanism, and is defined as a reproducible adverse reaction to a specific foodstuff or an ingredient.

1.1.4. Enzymatic food intolerance

Congenital or acquired metabolic defects on account of enzyme defects can interfere with the digestion and absorption of carbohydrates, proteins or fats. One example is

6 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

lactose intolerance through lactase deficiency in the intestinal mucosa.

1.1.5. Pharmacological food intolerance

Pharmacological substances in food can also trigger adverse reactions. Coffee is able to cause intestinal and cardiac side effects, whereas sodium nitrate can cause vascular dilatation with flush, headache and urticaria 8. Vasoactive amines (tyramine, serotonin, tryptamine, histamine and others) are contained in various foodstuffs as normal ingredients, e.g. tuna fish, sardines, bananas, cheese, yeast extract, chocolate, wine, spinach and tomatoes. Side effects can appear through high supply, bacteriological interactions, interactions with pharmaceuticals (MAO inhibitors), release from mast cells and other inflammatory mediators. The symptoms vary from flush, constriction of the smooth musculature of the intestine and the bronchia, tachycardia, headache, changes in blood pressure, etc.

1.1.6. Mucosal irritations

Some foodstuffs have a directly irritating effect on the mucosa of the mouth or intestine, such as coffee, curry or hot spices.

1.1.7. Toxic food intolerances

Many plants contain toxins in order to protect themselves against their predators, such as solanine in potatoes, cyanides in tapioca, mycotoxins in fungi and cereals, phototoxic furocoumarins in angelica, parsley and dill. The toxic reactions are manifold.

1.1.8. Food aversion

An aversion to certain foodstuffs on account of psychological reasons (e.g. bad taste, the desire to lose weight) is to be distinguished from allergies and intolerances.

1.1.9. Psychological intolerance

Here this concerns a physical reaction which is triggered through emotions, less through the foodstuff itself. The emotions can have a direct connection to the ingested foodstuff or also be independent of this.

7 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

1.1.10. Strain on the organism through food

Food

Individual Toxic (Food Poisoning)

Non-immunological Immunological hypersensitivity (e.g. enzyme defects such as lactose intolerance)

IgE-mediated IgG-mediated food allergy intolerance

Figure 1

1.2. Clinical symptoms of a food allergy Urticaria, angioedemas, rhinitis, atopic reactions, asthma, nausea, abdominal symptoms (pain, diarrhea, constipation, increased bowel activity, hemorrhoids, bloody stool), rheumatoid complaints, myalgias, migraines, etc.

1.2.1. Sequence of allergic reactions Most allergic reactions to foodstuffs occur within a few minutes after ingestion. Some occur on a quite delayed basis. Hill et al. were able to differentiate three types of reactions with cow’s milk allergy. The reaction times varied between 45 minutes and more than 20 hours 30.

8 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

1.3. Food quantity and allergic reaction

The quantity of allergen as trigger of a reaction varies. For instance, very sensitive people already react to 1 µg casein, others only with quantities greater than 200 g 30. A connection between the ingested quantity and the period up until appearance of symptoms is also described, whereby the test persons which required greater quantities of allergen for reaction was also greater than the period up until 30 appearance of complaints .

Other factors for allergic reactions: Allergens can require other cofactors in order to bring about complaints, e.g. physical 16,17 training or medicaments (aspirin). Conversely, the tolerability can be better in a different environment or climate.

1.4. Frequency

Oberritter (1991) indicates the frequency of allergic illnesses for the Federal Republic 46 of Germany (FRG) with 10-20% . The British Allergy Foundation estimates the number of food intolerances in Europe and the USA at 45%. Other sources act on 22 the assumption of 2-8% in the western world . The types of allergies and food intolerances described by me thereby mix, so that no precise details can be made with regard to the frequency for the type III allergies with increased IgG levels described by me. In his paper published in 2004, Accomando points out that the proportion of non-recognized food intolerances in the example of celiac disease worldwide is very high and distinguishes classic, sub-clinical, silent and potential 1 courses .

1.5. The importance of the elimination diet

In the treatment of food intolerances and particularly of food allergies, the question is posed as to whether it will ever be possible at any other point in time for the people to ingest the incompatible substance without reactions. A large number of children with intolerances appearing in the first year of life lost their intolerances 8,50 over the course of time . Whereas it is generally assumed that intolerances persist on a lifelong basis in adults, in a follow-up study it could be shown that one third of 48 the adults had lost an allergy after a year-long elimination diet . Fasting also represents one form of the elimination diet.

9 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

1.6. Pathomechanism of a type III food allergy

The special feature of the specific defense system (= acquired immunity: lifelong learning) consists in the fact that the antibodies constitute a structure, in which case it can only detect a very specific antigen (“key-lock principle”). So our immune system is able to form several thousand different antibodies which are all different in their structure and can each bind to a very specific antigen.

If an antigen infiltrates into the organism, the suitable antibody adheres to it. In this way the antigen is visible for all other cells of the immune system. Bonds between antigens and antibodies are described as an immune complex. Cells which detect these immune complexes release messenger substances (mediators) and attract other immune cells. An automatically proceeding cascade of reactions develops. The immune complex is ultimately destroyed by phagocytes (scavenger cells) at the end of the reaction.

This automatism also proceeds with a food intolerance. However, here the antibody adheres to a specific foodstuff on account of the corresponding structure. But as long as incompatible foodstuffs will be consumed, this automatism is unstoppable. The immunological reaction inevitably proceeds, even if the effects can be detrimental to the body. This is clear with autoimmune diseases. Here a malfunction of the immune system leads to formation of IgG antibodies against endogenous tissue. Endogenous tissue becomes an antigen, the immune complex “endogenous tissue antibody” is “combated” through the body’s own immune system. Severe illnesses can be the result.

10 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Goal of the paper

The following questions shall be clarified with the paper at hand:

1. Are IgG-mediated food allergies causally involved in the fibromyalgia syndrome? 2. Can complaints with fibromyalgia syndrome change through a dietary change? 3. Which complaints particularly improve under an elimination diet? 4. How large is the compliance with regard to the practical implementation of an immunologically optimized dietary change? 5. Does an elimination diet have an influence on body weight? 6. How do the patients themselves assess this therapy?

2. Patient collective and research method

In collaboration with self-help groups, men and women were sought who are afflicted with the fibromyalgia syndrome and were willing to document their experiences within the framework of such a dietary change with the help of a detailed questionnaire. The diagnosis “fibromyalgia” had to be made by a physician at least once in the anamnesis. The questionnaire encompassed 5 documentation points (start of treatment, controls after 2, 4, 6 and 8 weeks). Amongst other things, questions regarding severity of 50 specific symptoms, weight as well as dietary change (consequence, problems) were to be answered for each point in time.

The expressivity of the 50 predetermined symptoms should be assessed at all documentation points with the help of a 4-step scale ranging from 0=nonexistent via 1=less, 2=moderate to 3=severe.

Specific antibodies against 272 different substances found in foodstuffs were identified on a semi-quantitative basis and quantified in 4 classes with the help of a standardized sandwich ELISA method. Varieties of meat, fish products, yeasts and baking additives, fruit varieties, nuts and seeds, vegetable varieties, salad varieties, fungi, milk products, food additives, grain and carbohydrate-rich products, coffee and tea varieties, saccharated products, spices, eggs and also heavy metals (in terms of secondary findings) were examined in this connection.

11 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

The results of the ELISA test were discussed with the patients. The patients additionally received a detailed written interpretation of findings and a personal recipe book in which concrete cooking recipes for the ordinary routine were provided to them. The proposed cooking recipes contained only substances no specific IgG had been detected in the ELISA test.

All patients had the possibility to take advantage of unlimited nutritional advice per telephone throughout the entire examination period. The nutritional advice was strictly oriented towards the laboratory results.

In order to attain the cooperation of patients and as consistent dietary change as possible, importance was attached to an interpretation of findings understandable for the patients and concrete instructions for the implementation, and a standardized interpretation of findings with written nutritional recommendation based on this, including cooking recipes, was employed.

A total of 73 participants documented their complaints over the indicated period. The diagnosis ‘fibromyalgia syndrome’ was not checked off with 5 patients. Here this possibly concerned family members of fibromyalgia patients, in which the diagnosis is not confirmed. At any rate, these 5 patients were excluded from the evaluation, so that 68 patients ultimately form the basis of the evaluation at hand.

This patient group consisted of 60 women and 8 men aged between 20 and 69 years (mean value: 53 years). The mean body mass index (BMI) at the start of treatment amounted to 28.4 kg/m2, and more than two thirds of all patients were overweight (41.2%) or obese (29.4%).

On average, the patients suffered for nearly 10 years from fibromyalgia syndrome (minimum: 1 year; maximum: 30 years).

12 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

2.1. Sex

Of the total 68 documented patients, 60 (88.2%) were female and 8 (11.8%) were male.

Sex of patients

Sex Number %

Male 8 11.8 %

Female 60 88.2 %

Total 68 100 %

Table 1

2.2. Age

The average age of the patients recorded at the start of treatment amounted to 53.1 (± 8.8) years. The youngest female patient was 20 years, the oldest 69 years old.

Age of patients

Sex Mean Value Standard Minimum Maximum Valid Deviation Male 48.5 6.6 35.0 55.0 N=8 Female 53.7 8.9 20.0 69.0 N=6 Total 53.1 8.8 20.0 69.0 N=6 Table 2

Age of patients (categorical)

Sex Total Male Female Age (years) Number % Number % Number % Up to 30 years 0 0 % 1 1.7 % 1 1.5 % 31-40 years 1 12.5 % 5 8.3 % 6 8.8 % 41-50 years 4 50.0 % 9 15.0 % 3 19.1 % 51-60 years 3 37.5 % 33 55.0 % 36 52.9 % 61-70 years 0 0 % 12 20.0 % 12 17.6 % Total 8 100.0 % 60 100.0 % 68 100.0 % Table 3

13 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Age distribution of patient collective

% of patients Graphic 1

14 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

2.3. Height, weight and BMI during admission visit

The mean body mass index at the start of treatment amounted to 28.4 kg/m2 (median: 28.0 kg/m2)

Height according to sex

Height [cm] Sex Mean Value Standard Minimum Maximum Valid N Deviation Male 178.0 11.8 166.0 198.0 N=7 Female 165.2 6.8 145.0 182.0 N=59 Total 166.6 8.4 145.0 198.0 N=66 Table 4

Weight according to sex

Weight [cm] Sex Mean Value Standard Minimum Maximum Valid N Deviation Male 84.1 21.8 62.0 124.0 N=8 Female 77.7 16.1 51.0 115.0 N=60 Total 78.5 16.8 51.0 124.0 N=68 Table 5

Body Mass Index according to sex

Body Mass Index [kg/m2] Sex Mean Value Standard Minimum Maximum Valid N Deviation Male 27.3 4.4 22.1 35.1 N=7 Female 28.5 5.7 19.2 45.2 N=59 Total 28.4 5.6 19.2 45.2 N=66 Table 6

15 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

2.4. Classification of initial weight

The initial examination revealed the following picture for the established BMI values: Most patients were overweight (41.2%), whereas 29.4% were obese and 26.5% of normal weight. The height data was missing with 2 patients, and so the body mass index could not be calculated.

Classification of initial weight

Sex Total Male Female Age (years) Number % Number % Number % Normal 20 25.00 % 16 26.7 % 18 26.5 % Overweight 4 50.0 % 24 40.0 % 28 41.2 % Obese 1 12.5 % 19 31.7 % 20 29.4 % No data 1 12.5 % 1 1.7 % 2 2.9 % Total 8 100.0 % 60 100.0 % 68 100.0 % Table 7

Based on the details regarding weight and height, the BMI was established and the patients were accordingly classified according to the recommended WHO threshold values for overweight and obesity.

% of Patients Graphic 2

16 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

2.5. Diagnosis

All patients had indicated the diagnosis ‘fibromyalgia syndrome’. On average, the patients suffered for nearly 10 years from the illness (minimum: 1 year; maximum: 30 years; 7 patients without data). The diagnosis had been made by a physician at least once in the anamnesis.

Fibromyalgia syndrome – when did it appear?

Fibromyalgia since … [years] Sex Mean Value Standard Minimum Maximum Valid N Deviation Male 6.8 4.4 3.0 15.0 N=6 Female 10.0 7.0 1.0 30.0 N=55 Total 9.7 6.8 1.0 30.0 N=61 Table 8

17 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

2.6. General risk factors

Risk factors

Sex Number %

Smoker 11 16.2 %

Alcohol consumption 12 17.6 %

Oral contraceptives 4 5.9 %

Table 9

Percentages related to all patients – multiple mentions possible

Risk Factors

% of patients Graphic 3

18 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3. Results

3.1. Symptoms at start of treatment

The blood test for detection of type III food allergies revealed the following results: on average, the patients had 47 food reactions, 9 of them very severe (severity 3 and 4).

The expressivities of the 50 predetermined symptoms have been documented with the help of a 4-step scale (from 0=nonexistent to 3=severe).

The expressivity of 50 predetermined symptoms at the time of the start of treatment has been processed in the following tables and illustrations.

The expressivities are indicated as percentages (always in relation to 68 patients) in the first table (see also Table 10).

Graphic 5 elucidates which symptoms were ‘very severe’ at the start of treatment. In particular, ‘tender points’ (72.1% of patients with severe expressivity), muscle pains (66.2%) as well as ‘back pains’ and ‘poor night sleep’ (each with 63.2%) are to be mentioned here.

In the end, the symptom score for all symptoms was established for better comparability. This mean value of the respective symptom was calculated from the evaluations 0=nonexistent, 1=less, 2=moderate and 3=severe (Table 11).

19 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.1.1. Expressivity of symptoms at start of treatment (in percent)

Expressivity of concomitant symptoms at start of treatment [percentages] Nonexistent Less Moderate Severe No data Tender points 7.4 14.7 72.1 5.9 Muscle pains 2.9 29.4 66.2 1.5 Morning stiffness 5.9 36.8 57.4 Morning exhaustion 4.4 35.3 60.3 Joint pains 11.8 33.8 54.4 Headaches 11.8 29.4 44.1 13.2 1.5 Migraines 54.4 16.2 16.2 11.8 1.5 Poor night sleep 2.9 11.8 22.1 63.2 Depressions 22.1 36.8 27.9 13.2 Mood swings 8.8 19.1 50.0 22.1 Fatigue 1.5 7.4 27.9 60.3 2.9 Intolerance reactions 5.9 19.1 35.3 29.4 10.3 Frequent diarrhea 26.5 36.8 17.6 19.1 Irritable bladder 13.2 27.9 39.7 17.6 1.5 Painful menstruation 58.8 4.4 5.9 8.8 22.1 Prolonged menstruation 63.2 5.9 5.9 2.9 22.1 Visibly swollen hands/feet/face 14.7 22.1 33.8 26.5 2.9 Feeling as if hands swollen 11.8 19.1 33.8 35.3 Tingling, numbness of hands/feet 13.2 19.1 32.4 33.8 1.5 Hemorrhages under the skin 39.7 23.5 29.4 5.9 1.5 Back pains 1.5 2.9 32.4 63.2 Increased chill 33.8 11.8 29.4 23.5 1.5 Hearing problems/tinnitus 26.5 25.0 19.1 29.4 Dry mucosae 8.8 14.7 33.8 42.6 Spasmodic abdominal pains 35.3 25.0 19.1 17.6 2.9 Concentration problems 19.1 27.9 51.5 1.5 Lack of drive 1.5 7.4 47.1 42.6 1.5 Forgetfulness 2.9 20.6 41.2 35.3 Feelings of anxiety 20.6 25.0 35.3 17.6 1.5 Vertigo 13.2 32.4 42.6 10.3 1.5 Hair loss 36.8 19.1 27.9 16.2 Throat tickle/hoarseness 25.0 22.1 36.8 16.2 Inflamed pharyngeal mucosa 36.8 29.4 23.5 8.8 1.5 Noise-sensitive/overloud hearing 16.2 20.6 26.5 36.8 Increased sweat production 11.8 20.6 32.4 35.3 Constipation 26.5 20.6 23.5 29.4 Painful stool or urinary urgency 45.6 22.1 20.6 11.8 Urination against increased resistance 61.8 19.1 14.7 4.4 Meteoropathy 8.8 16.2 26.5 48.5 Intensified venous thrombosis 23.5 20.6 30.9 23.5 1.5 Trembling hands 47.1 32.4 16.2 4.4 Thromboses 80.9 10.3 5.9 1.5 1.5 Restless legs 27.9 23.5 33.8 13.2 1.5 Sensation of pain with skin contact 16.2 19.1 33.8 30.9 Pressure & tightness over the heart 29.4 25.0 29.4 16.2 Eczema/skin rash 38.2 22.1 29.4 8.8 1.5 Allergies 27.9 26.5 22.1 23.5 Burning skin 32.4 27.9 29.4 10.3 Amnesic aphasias 14.7 27.9 33.8 23.5 Water retentions 20.6 23.5 30.9 23.5 1.5

Table 10

20 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.1.2. Expressivity of symptoms at start of treatment

Indicated is the percentage of 68 patients in which the respective symptom at the time of the start of treatment was nonexistent, less, moderate or very severe. On account of the missing details (above all with questions regarding menstruation), the addition of patient details does not always result in 100%.

Graphic 4

21 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.1.3. Severe expressivity of symptoms at start of treatment

Indicated is the percentage of 68 patients in which the respective symptom at the time of the start of the treatment was very severe.

Graphic 5

22 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.1.4. Symptom score at start of treatment

Indicated is the mean value of the respective symptom, calculated from the evaluations 0=nonexistent, 1=less, 2=moderate and 3=severe. Symptom score during admission visit

Symptom Mean value Standard Dev. Median Valid N

Tender points 2.69 0.61 3 N=64 Muscle pains 2.64 0.54 3 N=67 Morning stiffness 2.51 0.61 3 N=68 Morning exhaustion 2.56 0.58 3 N=68 Joint pains 2.43 0.70 3 N=68 Headaches 1.60 0.87 2 N=67 Migraines 0.85 1.09 0 N=67 Poor night sleep 2.46 0.82 3 N=68 Depressions 1.32 0.97 1 N=68 Mood swings 1.85 0.87 2 N=68 Fatigue 2.52 0.71 3 N=66 Intolerance reactions 1.98 0.90 2 N=61 Frequent diarrhea 1.29 1.07 1 N=68 Irritable bladder 1.63 0.93 2 N=67 Painful menstruation 0.55 1.05 0 N=53 Prolonged menstruation 0.34 0.78 0 N=53 Visibly swollen hands/feet/face 1.74 1.03 2 N=66 Feeling as if hands swollen 1.93 1.01 2 N=68 Tingling. numbness of hands/feet 1.88 1.04 2 N=67 Hemorrhages under the skin 1.01 0.98 1 N=67 Back pains 2.57 0.63 3 N=68 Increased chill 1.43 1.20 2 N=67 Hearing problems/tinnitus 1.51 1.18 1 N=68 Dry mucosae 2.10 0.96 2 N=68 Spasmodic abdominal pains 1.20 1.13 1 N=66 Concentration problems 2.33 0.79 3 N=67 Lack of drive 2.33 0.68 2 N=67 Forgetfulness 2.09 0.82 2 N=68 Feelings of anxiety 1.51 1.02 2 N=67 Vertigo 1.51 0.86 2 N=67 Hair loss 1.24 1.12 1 N=68 Throat tickle/hoarseness 1.44 1.04 2 N=68 Inflamed pharyngeal mucosa 1.04 0.99 1 N=67 Noise-sensitive/overloud hearing 1.84 1.10 2 N=68 Increased sweat production 1.91 1.02 2 N=68 Constipation 1.56 1.18 2 N=68 Painful stool or urinary urgency 0.99 1.07 1 N=68 Urination against increased resistance 0.62 0.90 0 N=68 Meteoropathy 2.15 1.00 2 N=68 intensified venous thrombosis 1.55 1.10 2 N=67 Trembling hands 0.78 0.88 1 N=68 Thromboses 0.27 0.64 0 N=67 Restless legs 1.33 1.04 1 N=67 Sensation of pain with skin contact 1.79 1.06 2 N=68 Pressure & tightness over the heart 1.32 1.07 1 N=68 Eczema/skin rash 1.09 1.03 1 N=67 Allergies 1.41 1.14 1 N=68 Burning skin 1.18 1.01 1 N=68 Amnesic aphasias 1.66 1.00 2 N=68 Water retentions 1.58 1.08 2 N=67

Table 11

23 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Symptom score at start of treatment

Graphic 6

24 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.2. Detection of specific IgG against food

On average, the patients had 47.2 ± 11.4 food reactions (median: 50 reactions). Of these, an average of 9.3 ± 11.7 reactions was very severe (severity 3 and 4). It is conspicuous that the total number of reactions with the fibromyalgia patients evaluated here is approx. 20% higher than in patients with comparable age structure who participated in parallel implemented application observation with other indication emphases.

3.2.1. Total number of food reactions

Number of food reactions according to sex

Number of all food reactions

Sex Mean Standard Minimum Maximum 25th Median 75th Valid N Value Deviation percentile percentile Male 47.6 8.5 32.0 61.0 42.5 50.0 51.5 N=8 Femal 47.1 11.8 20.0 70.0 41.0 50.0 53.0 N=59 e Total 47.2 11.4 20.0 70.0 41.0 50.0 52.0 N=67

Table 12

3.2.2. Number of food reactions with severity 3 or 4

Number of food reactions with severity 3 or 4 according to sex

Number of all food reactions with severity 3 or 4

Sex Mean Standard Minimum Maximum 25th Median 75th Valid N Value Deviation percentile percentile Male 7.1 8.3 1.0 27.0 2.5 5.0 6.8 N=8 Femal 9.6 12.2 1.0 70.0 4.0 7.0 12.0 N=55 e Total 9.3 11.7 1.0 70.0 4.0 6.0 11.0 N=63

Table 13

25 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.3. Symptom expressivity in the course

The expressivity of 50 predetermined symptoms should be assessed at all documentation points with the help of a 4-step scale ranging from 0=nonexistent via 1=less, 2=moderate to 3=severe.

The expressivity of 50 predetermined symptoms in the course of the 8-week observation period has been processed in the following tables and illustrations.

In the first 4 tables, the distribution of expressivities is presented after 2, 4, 6 and 8 weeks for all symptoms. The results after 8 weeks have also been graphically depicted (Graphic 7). In comparison with tables and illustrations in Section 5 it becomes clear how severely the expressivity of the individual symptoms has diminished in the course of 8 weeks. For instance, Graphic 8 shows the percentage of patients in which the symptom ‘tender points’ was very severe dropped from 72.1% at the start of treatment to 33.8% after 8 weeks. A few other examples: ‘muscle pains’ from 66.2% to 25%, ‘poor night sleep’ from 63.2% to 22.1% and ‘joint pains’ from 54.4% to 29.4%.

26 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.3.1. Expressivity of symptoms after 2 weeks (in percent)

Expressivity of symptoms after 2 weeks [in percent]

Nonexistent Less Moderate Severe No data Tender points 1.5 10.3 39.7 45.6 2.9 Muscle pains 1.5 16.2 41.2 41.2 Morning stiffness 1.5 16.2 45.6 35.3 1.5 Morning exhaustion 4.4 17.6 36.8 41.2 Joint pains 2.9 26.5 32.4 38.2 Headaches 25.0 30.9 38.2 4.4 1.5 Migraines 72.1 7.4 13.2 5.9 1.5 Poor night sleep 1.5 26.5 38.2 33.8 Depressions 30.9 33.8 23.5 10.3 1.5 Mood swings 13.2 35.5 33.8 16.2 1.5 Fatigue 4.4 20.6 38.2 36.8 Intolerance reactions 23.5 35.3 36.5 11.8 2.9 Frequent diarrhea 52.9 20.6 16.2 10.3 Irritable bladder 32.4 20.6 36.8 10.3 Painful menstruation 63.2 4.4 4.4 2.9 25.0 Prolonged menstruation 64.7 4.4 2.9 1.5 26.5 Visibly swollen hands/feet/face 25.0 29.4 32.4 11.8 1.5 Feeling as if hands swollen 20.6 26.5 36.8 16.2 Tingling, numbness of hands/feet 20.6 25.0 30.9 22.1 1.5 Hemorrhages under the skin 47.1 20.6 26.5 1.5 4.4 Back pains 1.5 32.2 36.8 48.5 Increased chill 38.2 11.8 30.9 17.6 1.5 Hearing problems/tinnitus 27.9 33.8 25.0 13.2 Dry mucosae 13.2 25.0 35.3 26.5 Spasmodic abdominal pains 50.0 23.5 16.2 8.8 1.5 Concentration problems 1.5 23.5 38.2 36.8 Lack of drive 4.4 22.1 45.6 27.9 Forgetfulness 2.9 29.4 36.8 30.9 Feelings of anxiety 29.4 29.4 30.9 10.3 Vertigo 25.0 41.2 23.5 10.3 Hair loss 41.2 27.9 16.2 11.8 2.9 Throat tickle/hoarseness 35.3 29.4 26.5 8.8 Inflamed pharyngeal mucosa 52.9 22.1 17.6 7.4 Noise-sensitive/overloud hearing 22.1 20.6 29.4 26.5 1.5 Increased sweat production 19.1 20.6 29.4 29.4 1.5 Constipation 45.6 8.8 29.4 13.2 2.9 Painful stool or urinary urgency 54.4 30.9 8.8 5.9 Urination against increased resistance 61.8 23.5 11.8 1.5 1.5 Meteoropathy 13.2 19.1 36.8 30.9 Intensified venous thrombosis 25.0 27.9 25.0 20.6 1.5 Trembling hands 55.9 27.9 14.7 1.5 Thromboses 82.4 7.4 5.9 1.5 2.9 Restless legs 32.4 33.8 25.0 8.8 Sensation of pain with skin contact 22.1 26.5 32.4 19.1 Pressure & tightness over the heart 39.7 25.0 25.0 10.3 Eczema/skin rash 52.9 23.5 17.6 5.9 Allergies 39.7 25.0 22.1 11.8 1.5 Burning skin 47.1 26.5 22.1 4.4 Amnesic aphasias 17.6 26.5 32.4 23.5 Water retentions 29.4 25.0 36.8 8.8 Percentages values in relation to all 68 patients Table 14

27 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.3.2. Expressivity of symptoms after 4 weeks (in percent)

Expressivity of symptoms after 4 weeks [in percent]

Nonexistent Less Moderate Severe No data Tender points 2.9 19.1 33.8 41.2 2.9 Muscle pains 1.5 19.1 44.1 33.8 1.5 Morning stiffness 2.9 22.1 42.6 32.4 Morning exhaustion 10.3 22.1 33.8 32.4 1.5 Joint pains 4.4 29.4 35.3 29.4 1.5 Headaches 32.4 32.4 26.5 7.4 1.5 Migraines 72.1 10.3 10.3 5.9 1.5 Poor night sleep 4.4 23.5 47.1 25.0 Depressions 35.3 35.3 20.6 7.4 1.5 Mood swings 19.1 44.1 25.0 10.3 1.5 Fatigue 5.9 22.1 47.1 25.0 Intolerance reactions 22.1 47.1 16.2 11.8 2.9 Frequent diarrhea 52.9 20.6 16.2 10.3 Irritable bladder 39.7 25.0 27.9 7.4 Painful menstruation 63.2 4.4 1.5 2.9 27.9 Prolonged menstruation 66.2 1.5 2.9 1.5 27.9 Visibly swollen hands/feet/face 30.9 32.4 29.4 5.9 1.5 Feeling as if hands swollen 26.5 26.5 41.2 5.9 Tingling, numbness of hands/feet 29.4 26.5 27.9 16.2 Hemorrhages under the skin 51.5 26.5 17.6 2.9 1.5 Back pains 20.6 44.1 35.3 Increased chill 41.2 17.6 29.4 10.3 1.5 Hearing problems/tinnitus 30.9 30.9 26.5 11.8 Dry mucosae 14.7 23.5 35.3 26.5 Spasmodic abdominal pains 51.5 25.0 14.7 7.4 1.5 Concentration problems 2.9 33.8 33.8 29.4 Lack of drive 4.4 32.4 45.6 16.2 1.5 Forgetfulness 8.8 32.4 35.3 23.5 Feelings of anxiety 36.8 32.4 23.5 7.4 Vertigo 32.4 36.8 20.6 10.3 Hair loss 45.6 23.5 19.1 8.8 2.9 Throat tickle/hoarseness 41.2 32.4 17.6 8.8 Inflamed pharyngeal mucosa 57.4 14.7 16.2 10.3 1.5 Noise-sensitive/overloud hearing 22.1 23.5 26.5 27.9 Increased sweat production 22.1 25.0 30.9 22.1 Constipation 48.5 19.1 17.6 14.7 Painful stool or urinary urgency 60.3 25.0 5.9 8.8 Urination against increased resistance 73.5 17.6 5.9 2.9 Meteoropathy 17.6 23.5 32.4 26.5 Intensified venous thrombosis 29.4 29.4 23.5 16.2 1.5 Trembling hands 55.9 27.9 13.2 2.9 Thromboses 86.8 5.9 4.4 2.9 Restless legs 36.8 27.9 25.0 10.3 Sensation of pain with skin contact 30.9 23.5 30.9 14.7 Pressure & tightness over the heart 42.6 29.4 19.1 7.4 1.5 Eczema/skin rash 57.4 25.0 10.3 7.4 Allergies 45.6 33.8 13.2 7.4 Burning skin 48.5 32.4 16.2 2.9 Amnesic aphasias 22.1 23.5 32.4 22.1 Water retentions 30.9 41.2 23.5 4.4 Percentages values in relation to all 68 patients Table 15

28 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.3.3. Expressivity of symptoms after 6 weeks (in percent)

Expressivity of symptoms after 6 weeks [in percent]

Nonexistent Less Moderate Severe No data Tender points 2.9 20.6 39.7 33.8 2.9 Muscle pains 2.9 20.6 45.6 30.9 Morning stiffness 4.4 30.9 39.7 25.0 Morning exhaustion 5.9 33.8 32.4 27.9 Joint pains 4.4 27.9 36.8 29.4 1.5 Headaches 32.4 32.4 29.4 4.4 1.5 Migraines 73.5 14.7 5.9 1.5 4.4 Poor night sleep 7.4 30.9 32.4 27.9 1.5 Depressions 42.6 27.9 16.2 10.3 2.9 Mood swings 26.5 35.3 23.5 13.2 1.5 Fatigue 7.4 33.8 32.4 26.5 Intolerance reactions 26.5 44.1 16.2 8.8 4.4 Frequent diarrhea 57.4 20.6 13.2 8.8 Irritable bladder 47.1 22.1 27.9 2.9 Painful menstruation 67.6 2.9 2.9 26.5 Prolonged menstruation 67.6 4.4 1.5 26.5 Visibly swollen hands/feet/face 29.4 38.2 20.6 10.3 1.5 Feeling as if hands swollen 20.6 33.8 30.9 13.2 1.5 Tingling, numbness of hands/feet 29.4 30.9 29.4 10.3 Hemorrhages under the skin 50.0 32.4 11.8 5.9 Back pains 1.5 26.5 32.4 39.7 Increased chill 39.7 23.5 20.6 11.8 4.4 Hearing problems/tinnitus 36.8 26.5 20.6 16.2 Dry mucosae 16.2 30.9 32.4 20.6 Spasmodic abdominal pains 55.9 29.4 10.3 2.9 1.5 Concentration problems 8.8 29.4 32.4 29.4 Lack of drive 7.4 39.7 32.4 20.6 Forgetfulness 7.4 30.9 38.2 23.5 Feelings of anxiety 41.2 32.4 17.6 8.8 Vertigo 32.4 35.3 27.9 4.4 Hair loss 42.6 29.4 19.1 7.4 1.5 Throat tickle/hoarseness 48.5 26.5 17.6 7.4 Inflamed pharyngeal mucosa 55.9 23.5 8.8 10.3 1.5 Noise-sensitive/overloud hearing 25.0 26.5 23.5 25.0 Increased sweat production 25.0 25.0 25.0 25.0 Constipation 48.5 27.9 11.8 10.3 1.5 Painful stool or urinary urgency 66.2 22.1 7.4 4.4 Urination against increased resistance 76.5 17.6 2.9 2.9 Meteoropathy 13.2 33.8 26.5 26.5 Intensified venous thrombosis 30.9 23.5 29.4 13.2 2.9 Trembling hands 61.8 19.1 16.2 2.9 Thromboses 85.3 4.4 5.9 4.4 Restless legs 39.7 25.0 23.5 11.8 Sensation of pain with skin contact 30.9 30.9 29.4 8.8 Pressure & tightness over the heart 52.9 22.1 20.6 4.4 Eczema/skin rash 55.9 27.9 11.8 4.4 Allergies 52.9 26.5 17.6 2.9 Burning skin 51.5 35.3 10.3 2.9 Amnesic aphasias 20.6 30.9 32.4 16.2 Water retentions 30.9 38.2 27.9 2.9 Percentages values in relation to all 68 patients Table 16

29 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.3.4. Expressivity of symptoms after 8 weeks

Expressivity of symptoms after 8 weeks [in percent]

Nonexistent Less Moderate Severe No data Tender points 2.9 20.6 39.7 33.8 2.9 Muscle pains 1.5 29.4 44.1 25.0 Morning stiffness 7.4 39.7 26.5 26.5 Morning exhaustion 11.8 33.8 25.0 27.9 1.5 Joint pains 4.4 27.9 36.8 29.4 1.5 Headaches 30.9 38.2 25.0 4.4 1.5 Migraines 79.4 10.3 4.4 2.9 2.9 Poor night sleep 11.8 35.3 29.4 22.1 1.5 Depressions 47.1 30.9 16.2 4.4 1.5 Mood swings 29.4 38.2 23.5 7.4 1.5 Fatigue 10.3 38.2 27.9 22.1 1.5 Intolerance reactions 42.6 33.8 13.2 5.9 4.4 Frequent diarrhea 57.4 26.5 10.3 4.4 1.5 Irritable bladder 50.0 26.5 17.6 4.4 1.5 Painful menstruation 66.2 2.9 2.9 27.9 Prolonged menstruation 67.6 2.9 1.5 27.9 Visibly swollen hands/feet/face 32.4 36.8 14.7 14.7 1.5 Feeling as if hands swollen 22.1 41.2 23.5 11.8 1.5 Tingling, numbness of hands/feet 35.3 27.9 23.5 11.8 1.5 Hemorrhages under the skin 55.9 29.4 10.3 2.9 1.5 Back pains 2.9 29.4 30.9 35.3 1.5 Increased chill 42.6 26.5 16.2 11.8 2.9 Hearing problems/tinnitus 36.8 25.0 25.0 11.8 1.5 Dry mucosae 22.1 23.5 32.4 20.6 1.5 Spasmodic abdominal pains 61.8 23.5 7.4 4.4 2.9 Concentration problems 7.4 36.8 26.5 27.9 1.5 Lack of drive 11.8 38.2 30.9 17.6 1.5 Forgetfulness 7.4 32.4 38.2 20.6 1.5 Feelings of anxiety 42.6 29.4 19.1 7.4 1.5 Vertigo 32.4 36.8 22.1 5.9 2.9 Hair loss 47.1 25.0 19.1 5.9 2.9 Throat tickle/hoarseness 50.0 22.1 19.1 7.4 1.5 Inflamed pharyngeal mucosa 60.3 20.6 4.4 13.2 1.5 Noise-sensitive/overloud hearing 32.4 23.5 23.5 19.1 1.5 Increased sweat production 32.4 27.9 22.1 16.2 1.5 Constipation 47.1 29.4 14.7 7.4 1.5 Painful stool or urinary urgency 66.2 23.5 5.9 2.9 1.5 Urination against increased resistance 73.5 19.1 4.4 1.5 1.5 Meteoropathy 11.8 38.2 23.5 25.0 1.5 Intensified venous thrombosis 32.4 26.5 26.5 11.8 2.9 Trembling hands 60.3 22.1 11.8 4.4 1.5 Thromboses 85.3 5.9 5.9 2.9 Restless legs 38.2 27.9 19.1 13.2 1.5 Sensation of pain with skin contact 36.8 23.5 26.5 8.8 4.4 Pressure & tightness over the heart 52.9 23.5 17.6 4.4 1.5 Eczema/skin rash 60.3 20.6 10.3 7.4 1.5 Allergies 52.9 27.9 13.2 4.4 1.5 Burning skin 57.4 30.9 5.9 4.4 1.5 Amnesic aphasias 19.1 33.8 27.9 17.6 1.5 Water retentions 32.4 35.3 25.0 5.9 1.5 Percentages values in relation to all 68 patients Table 17

30 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.3.4.1. Expressivity of symptoms after 8 weeks Indicted is the expressivity of the respective symptom 8 weeks after start of treatment. The percentages always relate to all 68 patients. On account of the missing details (above all with questions regarding menstruation), the addition of patient details does not always result in 100%

Graphic 7

31 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.3.5. Severe expressivity of symptoms during initia l& final documentation Percentage representation of the respective very severe symptoms during initial & final documentation:

Graphic 8

32 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.4. Improvement rates

During the control documentations held after 2, 4, 6 and 8 weeks, the expressivity of all symptoms with most patients was much lower that at the start of treatment. For instance, the symptoms ‘migraines’ had improved with 86.2% and ‘intolerance reactions’ with 80.4% of the concerned patients. Amongst other things, further very high improvement rates were established for the symptoms ‘painful stool or urinary urgency’ (78.9%), ‘irritable bladder’ (73.7%), ‘spasmodic abdominal pains’ (72.7%), ‘constipation’ (72.5%), ‘burning skin’ (71.7%) and ‘allergies’ (70.8%). The expressivity of tender points had improved by at least 50% of the concerned patients.

With 63.2% of patients, the general state of health after an 8-week dietary change had improved in comparison to the admission visit, whereas the general state of health remained unchanged amongst 29.4% and it had deteriorated amongst 7.4%.

The presented results have been graphically depicted in the form of improvement data for better overview.

In this connection it is to be noted that only those patients have been included in which the respective symptom was at least ‘less expressive’ in at least one point in time. Patients with which the respective symptom was not expressive at all documentation points were therefore not utilized for Graphic 9.

33 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.4.1. Improvement rates of symptoms Depicted is the percentage of patients in which the expressivities of the respective symptoms have improved in the observation period. Patients in which the symptom was neither expressive at the start of treatment nor during the last documentation after 8 weeks have not been taken into account.

Graphic 9

34 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.4.2. Exemplary symptom scores in the course

In order to exemplarily illustrate the development of symptoms in the course of the observation period, the symptom scores (mean values calculated from 0=nonexistent, 1=less, 2=moderate and 3=very severe) have been calculated for some symptoms for each documentation point and graphically depicted. In this connection it is to be noted that only those patients have been included in which the respective symptom was at least ‘less expressive’ in at least one point in time. Patients with which the respective symptom was not expressive at all documentation points were therefore not utilized for the following tables and illustrations.

Only patients with which the symptom was expressive in at least one point in time; mean values calculated from 0=nonexistent, 1=less, 2=moderate, 3=severe

Development of symptom “tender points” Mean Value Standard Median Number Deviation Start 2.69 0.61 3.0 64 After 2 weeks 2.34 0.74 2.0 64 After 4 weeks 2.16 0.86 2.0 64 After 6 weeks 2.08 0.82 2.0 64 After 8 weeks 2.06 0.83 2.0 34 Table 18

Development of symptom “migraines” Mean Value Standard Median Number Deviation Start 1.90 0.80 2.0 30 After 2 weeks 1.17 1.12 1.0 30 After 4 weeks 1.07 1.11 1.0 30 After 6 weeks 0.71 0.85 0.5 30 After 8 weeks 0.66 0.94 0.0 30 Table 19

Development of symptom “poor night sleep” Mean Value Standard Median Number Deviation Start 2.53 0.71 3.0 66 After 2 weeks 2.09 0.78 2.0 66 After 4 weeks 1.97 0.78 2.0 66 After 6 weeks 1.86 0.92 2.0 66 After 8 weeks 1.68 0.94 2.0 66 Table 20

35 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Development of symptom “constipation” Mean Value Standard Median Number Deviation Start 2.08 0.87 2.0 51 After 2 weeks 1.49 1.10 2.0 51 After 4 weeks 1.31 1.12 1.0 51 After 6 weeks 1.12 1.02 1.0 51 After 8 weeks 1.08 0.96 1.0 51 Table 21

Development of some symptom scores Depiction of symptom scores (mean values calculated from 0=nonexistent, 1=less, 2=moderate and 3=very severe) for 4 exemplary symptoms in the course. In this connection it is to be noted that only those patients have been included in which the respective symptom was expressive in at least one point in time.

Graphic 10

36 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.5. Consistency during the dietary change in the course (compliance)

In addition to development of symptoms, questions regarding the experiences gained within the framework of dietary change should be documented: about two thirds of the 68 patients assessed their consistency during the dietary change after 2 weeks with ‘very good’ or ‘good’. The percentage of these patients remained constant after 4 weeks, then sagged after 6 weeks to under 60%, and then increased again to 63% up until the end after 8 weeks.

The percentage of patients which assessed their consistence as ‘poor’ or ‘very poor’ amounted to 10% in the beginning.

Two weeks after start of treatment, 42.6% of all patients documented that the immunologically-adapted dietary change was ‘difficult’ or ‘very difficult’ for them. After 4 weeks, only 27.9% of all patients indicated that the retention of new eating habits was ‘difficult’ or ‘very difficult’ for them. The percentage of these patients decreased after 6 weeks to 22.1%, and at the end of the observation period after 8 weeks amounted to 26.5%.

37 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.5.1. Consistency of dietary change in the course

How difficult is it today for you to retain your new eating habits?

Consistency during the dietary change

After 2 weeks After 4 weeks After 6 weeks After 8 weeks Very good 17.6 % 13.2 % 17.6 % 20.6 % Good 48.5 % 54.4 % 41.2 % 42.6 % Average 23.5 % 22.1 % 33.8 % 29.4 % Poor 10.3 % 10.3 % 5.9 % 4.4 % Very poor 0 % 0 % 1.5 % 1.5 % No data 0 % 0 % 0 % 1.5 % Total 100.0 % 100.0 % 100.0 % 100. 0 % Table 22

Graphic 11

38 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.5.2. Retention of new eating habits

Two weeks after start of treatment, 42.6% of all patients documented that the dietary change was ‘difficult’ or ‘very difficult’ for them. After 4 weeks, only 27.9% of all patients indicated that the retention of new eating habits was ‘difficult’ or ‘very difficult’ for them. The percentage of these patients decreased after 6 weeks to 22.1%, and at the end of the observation period after 8 weeks amounted to 26.5%.

Assessment for the last respective period up until the current documentation

Retention of eating habits

After 2 weeks After 4 weeks After 6 weeks After 8 weeks Very good 19.1 % 10.3 % 5.9 % 5.9 % Good 23.5 % 17.6 % 16.2 % 20.6 % Average 32.4 % 38.2 % 50.0 % 36.8 % Poor 25.0 % 30.9 % 25.0 % 33.8 % Very poor 0 % 1.5 % 1.5 % 2.9 % No data 0 % 1.5 % 1.5 % 1.5 % Total 100.0 % 100.0 % 100.0 % 100. 0 % Table 23

Graphic 12

39 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.6. Weight change in the course

In the course of the approximate 8-week dietary change, a weight decrease could be document in 86.8% of all patients. The average weight of patients at the start of treatment amounted to 78.5 kg, and continuously decreased via 77 kg (after 2 weeks), 76.0 kg (after 4 weeks) and 75.2 kg (after 6 weeks) to 74.9 kg. The mean value of weight loss amounted to 3.6 kg (maximum: 10.9 kg). If one considers the relative weight change in comparison to the initial weight, it turned out that the patients had lost 4.9% of their body weight on average in the course of the 8-week observation period. The maximum weight loss amounted to 15% of body weight.

3.6.1. Weight after 8 weeks in comparison with initial weight

Sex Total

Male Female

Age (years) Number % Number % Number % Higher 0 0 % 4 6.7 % 4 5.9 % Unchanged 0 0 % 5 8.3 % 5 7.4 % Lower 8 100.0 % 51 85.0 % 59 86.8 % Total 8 100.0 % 60 100.0 % 68 100.0 % Table 24

3.6.2. Absolute weight change after 8 weeks in comparison with initial weight

Weight change after 8 weeks [kg] Sex Mean Standard Min. Max. Percentile Median Percentile Valid N Value Deviation weight weight loss loss Male -4.9 2.3 -7.8 -1.0 -6.9 -5.3 -3.2 N=8

Female -3.4 2.8 -10.9 1.5 -5.3 -3.2 -1.1 N=60

Total -3.6 2.7 -10.9 1.5 -5.7 -3.5 -1.4 N=68

Table 25

40 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.6.3. Relative weight change after 8 weeks

Solely the absolute weight changes have only a limited expressivity. The consideration of relative weight change in comparison with initial weight is more helpful here.

On average, the patients lost 4.9% of their body weight (median: 4.7%) in the course of the 8-week observation period. The maximum weight loss amounted to 15% of body weight.

Weight change percentage after approx. 8 weeks in comparison with initial weight Sex Mean Standard Min. Max. Percentile Median Percentile Valid N Value Deviation weight weight loss loss Male -6.5 3.7 -14.4 -1.3 -7.6 -5.9 -5.1 N=8

Female -4.7 3.9 -15.0 2.1 -7.2 -4.2 -1.5 N=60

Total -4.9 3.9 -15.0 2.1 -7.2 -4.7 -2.1 N=68

Table 26

3.6.4. Development of body weight in the observation period

The weight should be documented at the start of treatment as well as during the controls after 2, 4, 6 and 8 weeks. The following table lists the mean values of weight details along with standard deviation and median for all documentation points. A graphic illustrates the development.

Development of body weight [kg]

Mean Value Standard Median Valid N Deviation Initial visit 78.5 16.8 76.3 N=68 After 2 weeks 77.0 16.6 74.2 N=68 After 4 weeks 76.0 16.5 73.0 N=68 After 6 weeks 75.2 16.6 72.0 N=68 After 8 weeks 74.9 16.3 72.0 N=68 Table 27

41 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.6.4.1. Weight development in the course

The mean value of body weight at the respective documentation point is always depicted.

Graphic 13

42 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.7. Change of general state of health

With 63.2% of patients, the general state of health after an 8-week dietary change had improved in comparison to the admission visit, whereas the general state of health remained unchanged amongst 29.4% and it had deteriorated amongst 7.4%.

Change of general state of health after 8 weeks

Sex Total

Male Female

Health Number % Number % Number % Improved 4 50.0 % 39 65.0 % 43 63.2 % Unchanged 3 37.5 % 17 28.3 % 20 29.7 % Deteriorated 1 12.5 % 4 6.7 % 5 7.4 % Total 8 100.0 % 60 100.0 % 68 100.0 % Table 28

Graphic 14

43 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.8. Further changes

In addition to the predetermined symptoms, so-called ‘further changes’ (between 1 and 9 mentions, 161 mentions altogether) had been indicated as clear texts with 63 patients. These statements were coded and summarized in groups. Most frequently mentioned were ‘weight loss’ (69.1% of patients), followed by ‘fitter’ (38.2% of patients), ‘more seldom tired’ (36.8%) and ‘better skin’ (26.5%).

Further changes

Changes Number % Better skin 18 26.5 Fitter 26 38.2 More seldom tired 25 36.8 Gastrointestinal complaints improved/gone 13 19.1 Weight loss 47 69.1 Happier, livelier, more balanced 8 11.8 Negative observations 10 14.7 Positive observations (other) 14 20.6 Table 29

44 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.9. Final assessment of efficacy

Nearly two thirds of the patients (64.7%) assessed the efficacy of dietary change with ‘very good’ or ‘good’, and altogether 82.4% of all patients would recommend this therapeutic approach.

Patient opinion of efficacy

Sex Total

Male Female

Efficacy Number % Number % Number % Very good 2 0 % 11 18.3 % 11 16.2 % Good 6 75.0 % 27 45.0 % 33 48.5 % Average 1 12.5 % 15 25.0 % 16 23.5 % Poor 1 12.5 % 5 8.3 % 6 8.8 % No data 0 0.0 % 2 3.3 % 2 2.9 % Total 8 100.0 % 60 100.0 % 68 100.0 % Table 30

Final patient assessment of efficacy

Graphic 15

45 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

3.9.1. Recommendation Altogether 82.4% of all patients would recommend the diagnostics and therapy.

Sex Total

Male Female

Recommendation Number % Number % Number % Yes 6 75.0 % 50 83.3 % 56 82.4 % No 1 12.5 % 7 11.7 % 8 45.8 % None 1 12.5 % 3 5.0 % 4 5.9 % Total 8 100.0 % 60 100.0 % 68 100.0 % Table 31

46 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

4. Discussion

The fibromyalgia syndrome presents a vast number of complaints and symptoms. Establishing a diagnosis is more difficult as a result. The pathogenesis of the illness has not been clearly clarified up to now. In addition to the classic medicamentous therapies with analgesics, muscle relaxants or antidepressants, there are a vast number of therapeutic approaches. Fibromyalgia patients deal with increase of 58,59 complaints with alternative medical therapeutic procedures . They equally seem 60 to benefit from the application of these procedures .

One of the most frequent approaches in alternative medicine is the application of dietetic measures. The number of patients who attempt a diet (omission attempt or 59,61 supplementation with foodstuffs or additives) is indicated with 26-40% . Similar numbers are found in related illnesses such as chronic fatigue syndrome. In this case, 54% of patients indicated having attempted a diet. Of these patients, 74% 62 were of the opinion that the diet attempt improved their illness . It is evident that dietetic measures are subjectively and objectively helpful in the treatment of 63,64,65,26,27,53 fibromyalgia .

The connection between allergic-inflammatory reactions of type III allergy to food and the frequent complaints with regard to fibromyalgia should be clarified with the paper at hand. This immunological approach in the diagnostics and therapy of fibromyalgia was not the emphasis of research in the previously known papers. Moreover, food allergies are frequently reduced to the type I allergy. The necessary IgG diagnostics are often not undertaken as a result.

The diagnosis of a food allergy requires a careful anamnesis and thorough clinical examination. The examination by means of prick test and RAST usually detects IgE antibodies. Since a number of allergies are not IgE mediated, we must inevitably reckon with erroneous negative findings. For instance, patients who react quickly to cow’s milk protein frequently have positive prick test results, whereas the delayed 30 reacting patients do not react . There is a slight correlation between food 7,34 provocation test and prick test . Mast cells of the gastrointestinal tract (GIT) differ from mast cells of the connective tissue with regard to their characteristics, amongst other things also with regard to their mediator substances of intracellular granules and the stimuli which leads to their release. Therefore a positive cutaneous reaction 4,29,40,47, does not have to be synonymous with a positive reaction in the GIT . RAST

47 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

and ELISA track down antibodies against food. A correlation between the laboratory result and the clinical findings could not always be established. For instance, IgE and 5,47 IgG antibodies against food are also found in complaint-free patients . This prompted the authors to postulate that this is an indication for the irrelevance of an IgG and IgE diagnosis. One could assume that the existence of IgG in asymptomatic individuals could be an indication that the organism always produces IgG on the presentation of a foodstuff over the course of time. It is to be countered that patients with a completely unbalanced diet also do not show any increased IgG level in a great number. Possibly the symptom-free patients with increased IgG levels at the time of examination simply have better compensation possibilities. Papers regarding celiac disease were able to show that there are asymptomatic gene 36,52 carriers which can never – or only after years – develop complaints .

At the same time, the long-term exposition seems to have a certain significance vis- 1,12 à-vis the potential allergen . In my opinion, this is also an indication of an up to now functioning compensation mechanism. Tolerance now becomes intolerance.

The connection between allergy and fibromyalgia or generally the complaints of patients is ideally verifiable through a placebo-controlled, double-blind study. This is possible in special cases. However, this approach is not ordinarily suitable in practical reality. The understandable desire to conduct a double-blind, placebo-controlled study in order to diagnose IgG-mediated food allergies and to implement a subsequent elimination diet encounters limits. David demonstrates these as follows:

Limits and difficulties of a double-blind, placebo-controlled study according to David 16:

• Unclear dose-effect relationship • Difficulties with concealing some substances • Altered means of intake • Difficulties taking a capsule • Danger of anaphylaxis • Additive effects of various foodstuffs

A double-blind, placebo-controlled study is thus an impracticable way to answer questions posed in this study.

Simpler testing methods which determine the incompatible foodstuff with greater precision are therefore required. At the same time, false positive reactions can be more accepted than false negative findings. A single false negative finding with one

48 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

basic foodstuff can call the effectiveness of measures into question. In allergological practice, with the type I allergy one generally relies on the detection of specific IgE. An oral provocation is reserved for severe or unclear cases. Up to now, no great importance has been attached to the detection of specific IgG. This is incomprehensible, since particularly IgG is recognized in the mediation of inflammatory reactions with infectious diseases. The role of the IgG antibody in the 67 treatment of celiac disease is already established . So it is astonishing that in August 2002 a review was published in The Lancet in which only the IgE-mediated food allergy is causally mentioned under the heading “food allergy”, whereas all 68 other reactions are subsumed as food intolerances .

The importance of IgG antibody in the pathogenesis of allergic-inflammatory illnesses is negated by several authors. They understand the formation of IgG as a normal 69,70,71,72 reaction of the body to the supplied nutritional allergens . However, studies on atopic children show that in contrast to healthy children, these children form 73,74 lastingly high IgG (and IgE) titer against milk and chicken protein . This corroborates a fundamental immunological disorder with involvement of IgG. This hypothesis is supported through proven increased IgG titer with respiratory allergies. So it has to be postulated that the immunological hypersensitivity (allergy) to food knows more than one reaction pathway. In intraindividual terms, this can dominate IgG or IgE formation.

The paper at hand shows that particularly with the fibromyalgia syndrome, the detection of specific IgG against foodstuff and an elimination diet building on this has a high success rate in the therapy.

These successes first of all pertain to the general state of health of the respondents. For instance, at least 63.2 % of all study participants indicate an improvement of the general state of health in this connection. Women (65%) seem to benefit more strongly than men (50%). The positive overall result through the very high percentage of the recommendations of the diagnostic and therapeutic approach undertaken in the work is emphasized. At least 82.4% are willing to recommend this procedure. Therefore it is clear that in addition to the amelioration of complaints, an additional benefit has emerged for the study participants as a result of the applied elimination diet.

All classic complaints of fibromyalgia can be significantly improved through an IgG-

49 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

adapted elimination diet during the treatment of individual symptoms. For instance, the tender points reduce amongst 50% of the patients, muscle pains amongst 55.2 %, morning feeling of exhaustion amongst 61.2%, sensation of pain with skin contact amongst 66.7%, burning skin amongst 71.7% and morning stiffness amongst 57.4%. Particularly high improvement rates are found with migraine-like headaches (86.2%), emotional complaints such as depressions (64.2%), mood swings (65.6%), poor sleep (63.1%) and fatigue (60.9%). Above-average improvement rates are also found with gastrointestinal symptoms such as painful stool or urinary urgency (78.9%), spasmodic abdominal pains (72.7%) and constipation (72.5%).

The submitted paper thus shows that in addition to a general improvement, the manifold pain syndromes and the emotional well-being of patients will also be improved through an elimination diet if this diet avoids foodstuffs in which an increased specific IgG level has been measured. This allows the conclusion that the type III allergy plays a noteworthy role in the development or at least with regard to the severity of fibromyalgia complaints.

A prerequisite for the expressivity of a food allergy is possibly that foodstuffs penetrate the intestine and will then be recognized as exogenous. A healthy intestine is lined by a mucous membrane, a strong, particularly stably structured defense system. The first pathogens are already repelled here. Another function of the intestine is the extensive separation of nourishment into component parts so that they can be absorbed in the blood. From there they reach the body cells, where they fulfill numerous tasks. Infections, stress, medicaments such as antibiotics and antiphlogistic agents as well as a disturbed intestinal flora destroy this equilibrium and bring about an increased permeability of the small intestine, whereby larger undigested or not completely digested nutritive elements can penetrate the blood. A permanently increased intestinal permeability leads to a continual increase of food intolerances — measurable with the concomitance of increased IgG antibodies 10,29,45,51 against this foodstuff .

50 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Medical importance of IgG antibodies against food

If IgG antibodies against food can be detected, this is a very reliable sign that constant contact between food and the immune system comes about. It could be proven that the concentrations of antibodies diminish and finally vanish if the corresponding foodstuffs will be avoided for a while.

The antibody binds to the foodstuff and an immune complex emerges. This activates the complement system – a part of the immune cascade – and thus attracts phagocytes (“scavenger cells” which “digest” the intruders and thus make them innocuous), which destroy the immune complex. At the same time, various signal substances of the immune system (interleukin, TNF-α) will release large quantities of oxygen radicals (O •) and proteases.

TNF-α has a predominantly phlogistic effect in the tissue, where it is formed in the course of immune response. Oxygen radicals can also attack and destroy endogenous cells. This happens with a surplus of oxygen radicals if in the absence of antioxidants the endogenous cells are no longer able to protect themselves against an oxidation (decomposition of the cell membrane’s components) through the oxygen radicals. Moreover, oxygen radicals can decompose (oxidize) the cell fatty substances (lipids) which normally keep the cell membranes smooth, with the result of cell destruction. Proteases are enzymes which decompose unspecific proteins and can thus also destroy surrounding body tissue. If the immune complexes are found in lesser number, this occurs without appreciable damages. On the other hand, in the event of intolerance, the immune complexes are available in a larger concentration. These affix to certain activated cells of the blood vessels, can leave the bloodstream and settle in the tissue. Immune complexes repeatedly settle on these activated cells and lead to chronic local inflammations. These are responsible for a vast number of chronic complaints. Since these inflammatory processes continually proceed, more and more mediators will be formed, which subsequently interfere in numerous metabolic processes such as with insulin resistance.

51 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Why is the importance of food intolerance only recognized now?

Food intolerances give rise to varying symptoms. They partially resemble the symptoms of an allergy. As a result, they have often been erroneously designated as food allergy, although the markers responsible for this are not detectable. The time delay between ingestion and onset of symptoms makes detection difficult. Astonishingly, the author was able to find very few scientific papers which address 15,18,25,27,28,37,53,55 the connection between food and myalgias or rheumatic illnesses . A connection between complaints and nutritional habits is evident. An immunological / 25 allergological approach is thereby the exception. Hafstrom et al. show a connection between gluten-free nutrition and rheumatoid arthritis. In accordance with the notion that especially with a type III allergy increased immune complexes form and these can be deposited in soft parts and joints, the subsequent inflammatory reaction explains the complaints of these patients. Myalgic complaints are generally known within the framework of IgE-mediated allergies, yet the type III allergy is particularly predisposed towards inflammatory reactions.

Enestrom et al. frequently found deposits of mast cells as well as IgG intradermal and in the vascular walls with fibromyalgia patients, and thus allow the hypothesis of 19,20 a neurogenic inflammation . In 1998, Baraniuk et al. found markers of an increased vascular permeability and augmented IgG secretion in the nasal secretion, but precluded a causal connection because there was also one group of affected 2 patients which did not these markers . But possibly this is to be assessed more as an indication of a multicausal phenomenon, since the results presented here point out that there is a connection between the complaints of fibromyalgia and the existence of specific IgG. At the same time, it cannot be said with certainty whether the type III allergy is the cause or whether it acts as an additional trigger. For the clinical practice, IgG diagnostics against specific foodstuffs offer a valuable approach for gaining substantial relief for the patients.

In the opinion of the author, it is erroneous if the IgE determination or a prick test will be utilized as a screening instrument in the consideration of food allergies, yet a specific IgG diagnosis is not undertaken. The results of this paper clearly show that there is a connection between increased IgG levels and complaints. Amongst other things, a repeated attack by free radicals leads to a further chronification, further damages in the long term, and possibly accelerates the progress of such illnesses as a result of the inflammatory activity situation which is more pronounced vis-à-vis the type I allergy.

52 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

It is already customary to determine IgG antibodies – e.g. against gluten with celiac 38 disease – with individual diseases . However, even an individual analysis is very expensive. But this rarely concerns monocausal sequences in which only one allergen is involved. The vast number of basic foodstuffs used in everyday life necessitates broad screening. For one thing, it is a matter of identifying the potential allergens in order to prevent the allergic-inflammatory events. For the clinical daily routine it is furthermore decisive to give the patients a recommendation regarding what they can eat. In my opinion, a broadly applied diagnosis is not only useful with regard to the aspiration of a diversified diet, it is virtually a prerequisite for a successful therapy. A diversified diet is also the basis for avoiding malnutrition.

In the past, the relatively high costs arising in this connection have certainly contributed towards refraining from this examination. Or dietary recommendations based on the testing of 10 or 15 foodstuffs were implemented. This usually did not render the desired success, since the recommendations also allowed incompatible foodstuffs which were not tested. On the other hand, fasting cures – the most stringent form of omission diet – are often successful, since all incompatible foodstuffs will also be automatically avoided.

If one considers that an undetected type III food allergy accelerates the progress of a chronic illness or leads to a deterioration of symptoms, follow-up costs will be caused because of this, which are not in any proportion to the financial expenditure of a specific IgG diagnosis.

The utilization of orthomolecular substances – such as vitamins, minerals, fatty acids, etc. – with chronic inflammatory illnesses and the reduction of free radicals is being increasingly discussed. With a dietary change as described here, the causal emergence of free radicals can be diminished. The basic inflammatory events will be reduced as a result. The study at hand was able to show that quite a few complaints improved under an elimination diet. This possibly results from the reduction of free radicals and inflammatory mediators.

53 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Why conduct a test of all IgG subclasses?

There are the following varying systems which are utilized for detection of food intolerance.

IgG test: detects only IgG antibodies; the test is unsuitable for diagnosing an 4 4 incompatibility in infants, since IgG is not transmitted on a transplacental basis. 4

ANT: is a nutrophil function test. The difference of neutrophils in the quiescent state and after exposure with foodstuffs is measured. Only antibodies which have opsonizing characteristics (i.e. antibodies of class IgG and IgG ) will be detected. 1 3

A great disadvantage of the method is that reproducible results are only to be anticipated with fresh blood or serum. Furthermore, disturbances can emerge upon existence of infections or through any existing antiphlogistic medicaments. Therefore it is only usable on a limited basis.

The granulocytic transformation tests do not detect any IgG , since this has 4 insufficient opsonizing capacities. But in order to cover Th as well as Th reactions, 1 2 all subtypes are necessary. Reproducible results are also only achievable here with fresh blood.

Activity IgG IgG IgG IgG 1 2 3 4 Neutralization ++ ++ ++ ++ Opsonization +++ * ++ (+) Sensitization of mast calls + - + - Complement activation ++ + +++ (+) Transport through the placenta +++ + ++ (+/-) Extravascular diffusion +++ +++ +++ +++ Th - Th Th Th Th + Th Th 1 2 2 1 1 2 2 reaction type Type 2 Type 4 Type 3 Type 1 mean concentration 9 3 1 0.5 Table 32 Only a test that records all characteristics of the IgG classes is able to actually depict all forms of intolerance and the related immunological reaction.

IgG antibodies are components of acquired, adaptive immunity. They bind to the specific antigen for them and thus initiate the immunological reaction for destruction of the antigen. While doing so, they act as an opsonin and make the antigen visible for the scavenger cells. The complement system is also activated, which attracts the phagocytes. They have a high persistence and are detectable in the serum.

54 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

This makes them popular markers in infection serology for any infection that has occurred. However, one cannot differentiate whether it was a longstanding contact or a recent contact. Since they bind the antigens and release for destruction, and thus prevent that these antigens propagate in the body, protective characteristics are attributed to them and apply as a standard for the immunity against a specific germ.

The behavior is similar, yet different, with foodstuffs. Since foodstuff cannot propagate in the body, the supposed protective factor does not play any role. Although one encounters an infectious antigen such as a hepatitis virus yet quite seldom, we still eat the same things every day, such as bread or milk products. If there is an incompatibility, the bread or milk antigens will be combated in the same way as the virus antigens, with the result of a chronic inflammation. The supposed protective characteristic of the IgG antibody has now turned out to be a rather burdening characteristic.

Within the framework of an elimination diet there are a series of variables which cannot always be precisely recorded, and the results can vary. These variables are depicted as follows:

16 Reasons for an unsatisfactory result of an elimination diet according to David : • The test person is not allergic to the foodstuff. • The omission period was too short. • The foodstuff was incompletely avoided (hidden ingredients, etc.). • The test person is allergic to other non-tested foodstuff. • The test person had an intercurrent illness. • The symptoms were inordinately indicated.

But during weighting of these variables, these mainly lead to a poorer result. So the results achieved here can possibly be improved even more if it is possible to control individual variables more precisely. However, an omission period that is too short or undetected (hidden) incompatible foodstuffs can worsen the result. The cooperation between patient and physician during anamnesis and therapy is a decisive factor for success.

55 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

If one compares the results of the paper at hand with other works which have examined medicamentous therapeutic approaches, they will discover that the IgG- adapted elimination diet provides at least equally good results. For instance, the general state of health (as status “overall”) improved by 63.2% within the observation period of 8 weeks. In particular, migraines and the gastrointestinal symptoms of the fibromyalgia patients even had much greater improvement rates (up to 86%). In subjective terms, the benefit of such a dietary change was recognized by 84.2%, which can be realized in the degree of recommendation.

The benefit of medicamentous therapy is indicated for amitriptyline with 74%, for 75 moclobemide with 54% and 49% with placebo . However, undesirable effects with this medication are also found with amitriptyline (77%) and with moclobemide (74%) 75 . Combinations such as tramadol/acetaminophen also do not reveal any better results and even lead to a high termination rate in the therapy 76. A sole dietetic attempt through an IgG-adapted elimination before a medicamentous therapy is at any rate justified during consideration of benefit and risks. But at the same time, a consideration of IgE-mediated food allergies and other intolerance is also recommended, since these set similar reaction chains in motion.

An association of fibromyalgia with autoimmune diseases of the thyroid gland is also found. This supports the thesis of an immunologically caused or impressionable fibromyalgia presented here. The therapeutic successes with thyroid hormones in the treatment of these cases is not by any means contradictory, since these hormones influence the metabolism in toto and also assume an immunologically pivotal role 77,78,79 .

The clinical picture of fibromyalgia as a complex illness is depicted under consideration of known literature and the results of this paper. Immunological aspects are evident. A step-by-step approach in diagnostics and therapy appears useful. Less invasive procedures which are in the realm of the patient are to be utilized as a priority. Suitable exercise and stress management is also included for this purpose. Analgesics and antidepressants are a consideration in severe or therapy-resistant cases. The clarification of food allergies is part of basic diagnostics. Not only IgE-mediated allergies are to be diagnosed in this connection, but particularly IgG-mediated allergies are also to be disclosed. Particularly on account of the partially very long latency up until appearance of symptoms they are clinically hard to detect and require immunological diagnosis. The statement of IgG diagnosis

56 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

is exactly sufficient to be able to give a therapeutic recommendation. Oral provocation tests are generally not necessary. In order to ensure a well-balanced and diversified nutrition, it is necessary to examine a large allergen pool. This has to include all foodstuffs eaten by the patient, otherwise the patient is to continue to incorporate only tested foodstuffs in the diet. Should the success of an IgG-adapted elimination be unsatisfactory, an additional endocrinological diagnosis for thyroid gland – but also for sexual and adrenal hormones – is useful. In addition to the known analgesic and antidepressive medicaments, all complementary techniques are also appropriate, since they have a noticeable benefit for the patients and give them more quality of life.

57 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

5. Abstract

The results show that nutritionally-specific IgG antibodies are involved in the emergence and/or the severity of a fibromyalgia. Alone the percentage of patients with increased IgG is about 20% higher than during a study conducted on a parallel basis with another collective.

This is emphasized through the high percentage of patients who indicated an amelioration of their complaints through an elimination diet. For instance, the number of very painful pressure points was reduced from 72.1% to 33.8% after an 8-week dietary change. Other symptoms also improved to a similar extent under elimination of foodstuff with increased IgG levels. A large number of test persons even benefited with a not very consistent change. A significant weight loss was shown as a positive side effect. An elimination diet which rests on the avoidance of IgG-positive foodstuffs significantly reduces the complaints of fibromyalgia patients.

A dietary change which avoids foodstuffs with increased IgG levels is successful in the treatment of fibromyalgia. It ameliorated all investigated complaints within 8 weeks by usually more than 50%. In particular, painful events – e.g. migraines, spasmodic abdominal pains, painful defecation, and hyperesthesias of the skin – are subjectively much improved.

In the first two weeks it was especially difficult for the test persons to change their previous eating habits, particularly since “favorite dishes” were frequently affected. In the aggregate, two thirds of the patience assessed their consistency as good to very good. The retention of the new eating habits was much easier for the test persons with continuation of the study. A continual accompaniment of the patients and a good presentation of findings are important for the therapeutic success.

Of the most overweight (41.2%) or obese (29.4%) patients, 86.8% lost an average of 4.7% of their body weight in the observed 8 weeks. An elimination diet which takes into consideration IgG-specific food allergies is successful in weight reduction.

The study participants assessed the success of dietary change as good to very good and were mainly satisfied with the result.

58 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

6. Conclusion

The greatest challenge in the handling of food allergies and food intolerances is the identification of responsible foodstuffs. Nutrition not only inspires the appetite, but also the emotions. The differentiation of allergic reactions and intolerances is difficult. A clarification with RAST and prick test is not sufficient. A differentiated approach is required. Delayed immunological sequences via IgG play a certain role as trigger of myalgias. An elimination diet which takes into consideration this IgG increase is successful in the treatment of fibromyalgia patients. Observations of other investigators reveal connections between complaints with rheumatic illnesses and 25,43,27 fibromyalgia to the ingestion of foodstuffs . A consistent elimination diet currently represents the only practical management in the treatment of food allergies and complaints triggered as a result. In addition to the classic oral provocation test, the IgG diagnosis is particularly indicated with delayed reactions and as a screening instrument can provide important references for which foodstuff it is worthwhile to make an omission attempt within the framework of an elimination diet. Moreover, it is to be implemented in a low-risk and patient-friendly manner. This is corroborated through the clinical results of the study.

59

IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

8. Acknowledgments

I would like to express my sincere thanks to Professor Dr. med. Herbert Kellner for the opportunity of dissertation and the very helpful support at all times.

My thanks also go to Mr. Ralf Schierl for the support with the data acquisition and the statistical processing.

I also owe thanks to Mr. Walther Werner for the graphic and linguistic review of the manuscript.

I would like to thank Mr. Gerd Michael Richter for the friendly support and his endless cooperativeness.

I would especially like to thank my wife Anna for the lasting motivation, her understanding and her patience, without which I could not have finished this paper.

61 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

9. Curriculum vitae

Mario Günther Krause Born on July 3, 1960 in Rotenburg a.d.Fulda (Germany)

School and University

1966 - 1970 Grundschule Lispenhausen [elementary school] 1970 - 1979 Jakob-Grimm-Gymnasium Rotenburg [academic high school] 1979 - 1980 Justus Liebig University Giessen 1980 - 1986 Georg August University of Göttingen

Medical internship in Krankenhaus am Bürgerpark [Red Cross hospital] , Bremerhaven

Clinical Activity

1986 - 1988 Surgery, Rotenburg Hospital 1988 - 1989 Gynecology, Sigmaringen Hospital 1989 - 1989 Cardiosurgery, Rotenburg Cardiovascular Center 1990 - 1990 General medicine practice, Dr. Grotehans, Bad Hersfeld 1990 - 2002 General practitioner with own practice in Rotenburg a.d. Fulda 2002 - 2003 Leading physician of the Medical Vital sector of the Medical One AG in Dortmund and Hanover Since November 2003 General medicine practice and leadership of the VIP³ MEDICAL CENTER in the Eilenriede Clinic Hanover

Main emphases of activity and special expertise

Acupuncture German Medical Association for Acupuncture (DÄGfA) diploma and member since 1990

62 IgG-mediated food allergy as trigger of fibromyalgia complaints and the influence of an elimination diet

Homeopathy Member in the Central Association of Physicians for Naturopathy (ZÄN) since 1990; additional qualification and diploma of the State of Hesse Medical Association: Homeopathy

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GASTROINTESTINAL INFLAMMATION Gastrointestinal Candida colonisation promotes sensitisation against food antigens by affecting the mucosal barrier in mice N Yamaguchi, R Sugita, A Miki, N Takemura, J Kawabata, J Watanabe, K Sonoyama ......

Gut 2006;55:954–960. doi: 10.1136/gut.2005.084954

Backgrounds and aims: Controversy still exists as to whether gastrointestinal colonisation by Candida albicans contributes to aggravation of atopic dermatitis. We hypothesised that Candida colonisation See end of article for authors’ affiliations promotes food allergy, which is known to contribute to a pathogenic response in atopic dermatitis. We ...... tested this using a recently established murine Candida colonisation model. Methods: Candida colonisation in the gastrointestinal tract was established by intragastric inoculation with Correspondence to: Dr K Sonoyama, C albicans in mice fed a synthetic diet. To investigate sensitisation against food antigen, mice were Laboratory of Food intragastrically administered with ovalbumin every other day for nine weeks, and antiovalbumin antibody Biochemistry, Graduate titres were measured weekly. To examine gastrointestinal permeation of food antigen, plasma School of Agriculture, Hokkaido University, Kita- concentrations of ovalbumin were measured following intragastric administration of ovalbumin. 9, Nishi-9, Kita-ku, Results: Ovalbumin specific IgG and IgE titres were higher in BALB/c mice with Candida colonisation than Sapporo-shi, 060-8589, in normal mice. Gastrointestinal permeation of ovalbumin was enhanced by colonisation in BALB/c mice. Japan; ksnym@ Histological examination showed that colonisation promoted infiltration and degranulation of mast cells. chem.agr.hokudai.ac.jp Candida colonisation did not enhance ovalbumin permeation in mast cell deficient W/Wv mice but did in v Revised version received congenic littermate control +/+ mice. Reconstitution of mast cells in W/W mice by transplantation of bone 25 December 2005 marrow derived mast cells restored the ability to increase ovalbumin permeation in response to Candida Accepted for publication colonisation. 31 December 2005 Published online first Conclusions: These results suggest that gastrointestinal Candida colonisation promotes sensitisation 19 January 2006 against food antigens, at least partly due to mast cell mediated hyperpermeability in the gastrointestinal ...... mucosa of mice.

andida albicans is part of the indigenous microbial flora However, given that C albicans is indigenous to the gastro- of the human gastrointestinal tract. In healthy indivi- intestinal tract of healthy humans, such methods of admin- duals, populations of this fungus pose no threat to the istration, particularly the immunosuppressive route, should C 1 host. However, in hosts receiving antibiotic treatment, be avoided so that an animal model that is typical of immunocompromised states,2–4 and occasionally in appar- gastrointestinal colonisation by C albicans can be developed ently healthy persons,5 elevated populations can pose a and the relationship between gastrointestinal Candida colon- significant risk.5 In addition, it has been hypothesised that isation and allergic responses can be studied further. We excessive colonisation by C albicans in the gastrointestinal recently reported a model of sustained gastrointestinal tract may constitute aggravating factors in atopic dermatitis Candida colonisation by a single intragastric inoculation of C (AD), but this remains controversial.467 AD is a chronic, albicans in healthy adult mice without administration of relapsing, highly pruritic inflammatory skin disease with antibiotics or immunosuppresants.25 This was achieved by multifactorial causes, such as susceptibility genes, conditions feeding mice a synthetic diet that resulted in reduced within the host environment, and immunological factors.8 To numbers of lactobacilli in the stomach. While animals show date, laboratory and clinical investigations have demon- a good healthy appearance in this model, high faecal recovery strated that IgE mediated food allergy plays a pathogenic role of C albicans is still observed at least 18 months after the in a subset of patients with AD.9–11 Some reports have shown single inoculation (unpublished data), suggesting asympto- 12–14 increased gastrointestinal permeability in AD patients. matic Candida colonisation in the gastrointestinal tract. Hyperpermeability of the gastrointestinal mucosal barrier Therefore, using this model it was possible to investigate results in enhanced transport of intact and degraded antigens the role of gastrointestinal colonisation by C albicans in the across the gastrointestinal mucosal barrier, which could sensitisation against food allergens. favour food protein sensitisation and food allergy in To test our hypothesis, we initially determined whether 15 susceptible individuals. We therefore hypothesised that sensitisation against food antigens was promoted in mice gastrointestinal colonisation by C albicans may be involved with gastrointestinal tracts that had been chronically in aggravation of AD by affecting the mucosal barrier in a colonised by C albicans. We then assessed the effect of manner that results in increased permeation of food allergens gastrointestinal Candida colonisation on permeation of food and subsequent manifestation of a food allergy. antigens. Finally, because the barrier and transport properties Most models of gastrointestinal colonisation by C albicans of the gastrointestinal epithelium are actively regulated by have used oral inoculation of C albicans in adult mice treated with antibiotics and immunosuppressive agents,16–21 22 23 Abbreviations: AD, atopic dermatitis; BMMC, bone marrow derived or in infant mice. These treatment regimens have been mast cells; BSA, bovine serum albumin; HRP, horseradish peroxidase; necessary because competitive indigenous bacterial flora and OVA, ovalbumin; PAS, periodic acid-Schiff; PBS, phosphate buffered the immune system prevent colonisation by C albicans.17 19 24 saline; TLR, toll-like receptor; TNF, tumour necrosis factor

www.gutjnl.com Candida promotes food allergy 955 mast cells which are activated in response to various embedded in OCT compound (Sakura Finetechnical, Tokyo, pathogen associated stimuli,26 27 we investigated whether Japan) for histological examination. mast cells were involved in the permeation of food antigens in mice with gastrointestinal Candida colonisation. ELISA For determination of OVA specific IgG titre in the sera of MATERIALS AND METHODS mice, 96 well microtitre plates (Corning, New York, USA) Animals were coated overnight at 4˚C with 200 mg/ml OVA in Specific pathogen free female five week old mice were used in 50 mmol/l carbonate buffer, pH 9.6. Plates were blocked all experiments. BALB/c mice were purchased from Charles with PBS containing 1% bovine serum albumin (BSA, River Japan (Yokohama, Japan). Mast cell deficient mice fraction V; Serologicals Proteins, Illinois, USA) (PBS-B) at (WBB6F1-W/Wv) and congenic littermate control mice 37˚C for one hour. Test sera serially diluted with PBS (WBB6F1-+/+) were purchased from Japan SLC containing 0.2% BSA and 0.02% Tween 20 (PBS-BT) were (Hamamatsu, Japan). All mice were housed in a temperature then added and incubated at 37˚C for two hours. After controlled (23¡2˚C) room with a dark period from 20:00 to incubation, HRP conjugated goat antimouse IgG (Zymed, 08:00 and allowed free access to water and a purified diet California, USA), diluted 1:2000 in PBS-BT, was added and prepared according to AIN-93G.28 The study was approved by incubated at 37˚C for one hour. Wells were washed five times the Hokkaido University Animal Use Committee, and with PBS containing 0.02% Tween 20 (PBS-T) between each animals were maintained in accordance with the guidelines step. Plates were developed at room temperature after for the care and use of laboratory animals of Hokkaido addition of o-phenylendiamine (0.4 mg/ml) and hydrogen University. peroxide (0.012%) in 7 mmol/l citrate buffer, pH 5.0. Finally, 1 mol/l H2SO4 was added, and absorbance was measured at 490 nm with a microplate reader (model 550; Bio-Rad, Inoculation and enumeration of Calbicans California, USA). Preimmunised serum was used as a C albicans (JCM 1542) was obtained from Japan Collection of negative control. The average extinction in negative control Microorganisms of the Institute of Physical and Chemical wells, to which three times the standard deviation was Research (Saitama, Japan) and maintained as previously added, provided the reference for determination of the titre in described.25 For inoculation, all mice were acclimatised to the the test sera. Antibody titres were expressed as the reciprocal purified diet for two weeks before being deprived of the diet of the last dilution yielding an extinction value higher than for 16 hours. Mice were then inoculated intragastrically with the reference value. 0.2 ml of saline containing 16108 cells of C albicans. Control For determination of the OVA specific IgE titre, 96 well mice were intragastrically administered 0.2 ml of the vehicle. microtitre plates were coated overnight at 4 C with 10 mg/ml Faecal specimens and tissue homogenates were quantita- ˚ antimouse IgE monoclonal antibody (LO-ME-2; Zymed) in tively cultured using a standard pour plate technique, as 50 mmol/l carbonate buffer, pH 9.6. Plates were blocked with previously described.25 PBS-B at 37˚C for one hour. Test sera serially diluted with PBS-BT were then added and incubated overnight at 4˚C. Oral immunisation experiment After incubation, 20 ng/ml OVA-DIG conjugate in PBS-BT At three weeks after inoculation, oral immunisation with was added and incubated at 37˚C for one hour. Coupling of ovalbumin (OVA) was started in BALB/c mice. Phosphate DIG to OVA was performed using a DIG protein labelling kit buffered saline (PBS 0.2 ml) containing 0.1 mg of OVA (Roche Diagnostics, Tokyo, Japan) according to the manu- (grade V; Sigma, Missouri, USA) was intragastrically facturer’s instructions. HRP conjugated sheep anti-DIG Fab administered every other day for nine weeks. Blood samples fragments (Roche Diagnostics), diluted 1:4000 in PBS-BT, were obtained from the tail vein at weekly intervals and was then added and incubated at 37˚C for one hour. Wells subjected to ELISA for measurement of OVA specific were washed five times with PBS-T between each step. Plates antibody titres, as described below. In addition, every week, were developed at room temperature after addition of faecal specimens were analysed for number of C albicans 3,39,5,59-tetramethylbenzidine (0.123 mg/ml; Sigma) and organisms, as described above. On the last day of the hydrogen peroxide (0.012%) in 2 mol/l citrate buffer, experiment, mice were anaesthetised with diethyl ether and pH 5.0. Finally, 1 mol/l H2SO4 was added, and absorbance sacrificed by exsanguination from the carotid artery. was measured at 450 nm with a microplate reader. Antibody Following laparotomy, the stomach, jejunum, ileum, and titres were determined as described above. colon were excised, homogenised in PBS, and the number of To measure plasma HRP concentrations, 96 well microtitre C albicans organisms were counted, as described above. plates were coated with goat anti-HRP antibody (Sigma) for two hours at 37˚C. All subsequent steps were performed at Gastrointestinal permeation experiment 37˚C with extensive washing between steps. Plates were Gastrointestinal permeability was determined in vivo by blocked with 1% BSA for two hours. Diluted plasma samples measuring the appearance in blood of horseradish peroxidase in PBS-BT were added to the wells and incubated for two (HRP) and OVA, administered by gavage according to Wang hours. Plate development and measurement were as and colleagues29 and Saitoh and colleagues,30 respectively, described for the OVA specific IgE. with some modifications. Plasma OVA concentration was measured by sandwich At four to five weeks after inoculation, mice were deprived ELISA using anti-OVA IgG (Chemicon, California, USA) and of the diet for 12 hours, after which 0.6 ml of PBS containing HRP conjugated rabbit anti-OVA IgG (Rockland, 0.6 mg of HRP (Sigma) or 0.2 ml of PBS containing 2 mg of Pennsylvania, USA) for capture and detection antibodies, 30 OVA were intragastrically administered. Blood samples were respectively, as described previously. then collected from the tail vein at 0, 30, 60, and 120 minutes after administration, and plasma HRP and OVA concentra- Histology tions were measured by ELISA, as described below. Cryostat sections (5 mm) of stomach were prepared and Thereafter, mice were anaesthetised with diethyl ether and stained with haematoxylin and periodic acid-Schiff (PAS) sacrificed by exsanguination from the carotid artery. reaction for detection of C albicans or with toluidine blue for Following laparotomy, the stomach was excised, opened identification of mast cells. The number of mast cells in the along the greater curvature, washed with ice cold saline, and stomach was counted using a high power field in a section

www.gutjnl.com 956 Yamaguchi, Sugita, Miki, et al

A Control A 8 Candida 20 Control Candida 7 15 *†

6 *† 10

CFU/g faeces) 5 Start OVA administration 10 Viable C albicans (log 4 5 Plasma HRP concentration (ng/ml) <3 024681012 0 03060 Time after inoculation (week) Time after administration (minutes)

B B 18 Control * 250 Control Candida *† 16 * Candida *

titre 200 2 14

150 12

Reciprocal log 10 100

8 50 <6.6

024 6 810 concentration (ng/ml) Plasma OVA Time after starting administration (week) 0 0 30 60 90 120 Time after administration (minutes) C * 6 Control Figure 2 Time course of changes in plasma concentration of intragastrically administered proteins in BALB/c mice with and without Candida gastrointestinal Candida colonisation. Concentrations of horseradish 5 peroxidase (HRP) (A) and ovalbumin (OVA) (B) were measured in titre

2 plasma samples of mice taken at the times indicated after intragastric administration. Values are means (SEM) of six mice per group. *p,0.05 compared with time point = 0; p,0.05 compared with control mice at 4 each time point.

Reciprocal log material dispersed diffusely was taken as evidence of 3 degranulated mast cell. All mast cell counts were performed by a single observer. <2 024 6 810 Reconstitution of mast cells in W/Wv mice Time after starting administration (week) Mast cell deficient W/Wv mice were reconstituted with in vitro bone marrow derived mast cells (BMMC) from congenic Figure 1 Time course of changes in recovery of organisms from faeces control +/+ mice according to Kung and colleagues.32 Bone and titres of serum antibodies specific to intragastrically administered ovalbumin (OVA) in BALB/c mice with and without gastrointestinal marrow from femurs was flushed out and seeded in Candida inoculation. The number of viable C albicans in faeces (A) was RPMI1640 medium (GIBCO-BRL, Tokyo, Japan) supplemen- determined weekly by quantitative culture method. Anti-OVA IgG ted with 10 ng/ml recombinant murine interleukin 3 (B) and IgE (C) titres were measured in sera taken at the times indicated (PeproTech EC, London, UK), 10% heat inactivated fetal calf after starting intragastric administration of OVA. Values are means serum (Biological Industries, Kibbutz Beit Haemek, Israel), * , (SEM) of six mice per group. p 0.05 compared with control mice 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mg/ml without Candida inoculation at each time point. gentamycin, at a concentration of 56105 nucleated cells/ml, and cultured at 37˚C in a humidified atmosphere containing 31 from each specimen. According to Nakajima and colleagues, 5% CO2. Culture medium was replaced every seven days. with some modifications, the number of mast cells in the After five weeks of culture, cells were harvested and gastric mucosa was counted in a high power field measuring suspended in PBS. Staining of cells with toluidine blue 0.65 mm2 using a light microscope equipped with a 106 indicated that nearly 99% of viable cells were mast cells. A objective and a 106eyepiece containing a reticule (Olympus, total of 16107 BMMC were injected intravenously into each Tokyo, Japan). The counting areas included stratified W/Wv mouse through the tail vein. Five weeks later, the squamous epithelium, lamina muscularis mucosa, and reconstituted mice were inoculated with C albicans,as submucosal layer in the forestomach, and gastric pits and described above, and then subjected to gastrointestinal fundic glands in the glandular stomach. The cell with stained permeation experiments.

www.gutjnl.com Candida promotes food allergy 957

Statistics simple to detect this macromolecule by ELISA. On the day of Results are presented as means (SEM). The unpaired or the permeation experiment, we confirmed high faecal paired t test or Tukey-Kramer’s test following one way recovery of C albicans (6.8 (0.2) log10 CFU/g faeces) in all analysis of variance was used to compare mean values. mice inoculated with C albicans. Figure 2A shows the time StatView for Macintosh (version 5.0, SAS institute Inc., course of changes in plasma HRP concentrations in BALB/c North Carolina, USA) was used for analysis. mice after intragastric administration of this molecule. In control mice without Candida colonisation, no significant increase in plasma HRP concentrations was observed after RESULTS administration. Conversely, plasma HRP concentrations in Sensitisation against orally administered OVA in mice with gastrointestinal Candida colonisation increased BALB/c mice with gastrointestinal Candida significantly, peaking at 30 minutes and remaining high for colonisation 60 minutes after administration. HRP concentrations were To study the effect of chronic colonisation of C albicans in the significantly higher in mice with Candida colonisation than gastrointestinal tract on sensitisation against orally adminis- control mice at 30 and 60 minutes. We next examined tered antigen, BALB/c mice were intragastrically inoculated permeation of a food antigen, OVA. As shown in fig 2B, with . By weekly counting of faecal specimens after C albicans compared with control mice, plasma OVA concentrations in inoculation, a high faecal recovery of C albicans was observed mice with gastrointestinal Candida colonisation increased in all mice throughout the experimental period (fig 1A). significantly 30 minutes after administration. Additionally, counting of organisms in gastrointestinal tissues by quantitative culture after euthanasia of animals revealed that colonisation occurred in the stomach, jejunum, Mast cell degranulation in gastrointestinal mucosa of ileum, and colon in all mice (5.5 (0.2), 1.8 (0.1), 2.3 (0.1), BALB/c mice with gastrointestinal Candida colonisation and 3.3 (0.0) log10 CFU/g tissue, respectively). No organism was detected in faeces or tissues of control mice without On histochemical examination of gastric mucosa in BALB/c inoculation of C albicans. These data suggest that chronic mice killed on the day of the permeation experiment, PAS colonisation of C albicans was established in the gastrointest- staining revealed colonisation of C albicans on the surface of inal tract of mice after single inoculation of C albicans. the forestomach in all mice inoculated with C albicans (data Figure 1B and 1C show the time course of changes in the not shown). Toluidine blue staining showed that a number of titre of serum antibodies against intragastrically adminis- mast cells were infiltrated in both the forestomach and tered OVA. In mice with gastrointestinal Candida colonisa- glandular stomach in Candida inoculated mice, and that tion, the anti-OVA IgG titre began increasing at three weeks degranulation was evident in the majority of cells. after starting administration of OVA and stabilised at six Quantitative examination of mast cells in histological weeks (fig 1B). The anti-OVA IgG titre tended to be higher in sections of stomach in BALB/c mice is shown in fig 3. In mice with Candida colonisation than in control mice without the forestomach, numbers of granulated cells were the same Candida colonisation throughout the experimental period, in mice with Candida colonisation and control mice, whereas and there was significant difference between the two groups numbers of degranulated and total cells (that is, granulated at 4, 5, and 7 weeks after starting OVA administration. Anti- plus degranulated) were significantly higher in mice with OVA IgE titre began increasing at five weeks after initiating Candida colonisation than in control mice. In the glandular OVA administration (fig 1C). As with the IgG titre, the anti- stomach, numbers of both granulated and degranulated cells OVA IgE titre tended to be higher in mice with Candida were higher in mice with Candida colonisation than in control colonisation than in control mice, with a significant mice. difference apparent between the groups nine weeks after initial OVA administration. Permeation of orally administered OVA in mast cell deficient mice with gastrointestinal Candida colonisation Permeation of orally administered proteins in BALB/c To elucidate the involvement of mast cells in increased mice with gastrointestinal Candida colonisation permeation of OVA in mice with gastrointestinal Candida We examined gastrointestinal permeation of HRP in BALB/c colonisation, we examined OVA permeation in mast cell mice with gastrointestinal Candida colonisation, because it is deficient W/Wv mice. As shown in fig 4A, W/Wv mice with gastrointestinal Candida colonisation showed no significant 250 * increase in plasma OVA concentrations after administration, 2 Control and levels were comparable with those in mice without 200 Candida Candida colonisation for the duration of the experimental period. Conversely, plasma OVA concentrations increased 150 significantly in congenic littermate control +/+ mice with gastrointestinal Candida colonisation. Concentrations peaked * * 100 at 30 minutes after administration and were significantly higher in mice with Candida colonisation than in those ** 50 without Candida colonisation (fig 4B). Quantitative culture of Mast cell numbers/mm C albicans isolated from pooled faeces for each Candida 0 inoculated group showed that the number of organisms Granulated Degranulated Total Granulated Degranulated Total v was higher in W/W mice (6.5 log10 CFU/g faeces) than in +/+ Forestomach Grandular stomach mice (5.5 log10 CFU/g faeces). No organism was detected in the faeces of control mice without inoculation of C albicans. Figure 3 Number of granulated and degranulated mast cells and total These results indicate that the increase in OVA permeation by number of mast cells in the forestomach and glandular stomach of mice gastrointestinal Candida colonisation requires mast cells. with and without Candida inoculation. The cryostat section of stomach To confirm this finding, we then determined whether was stained with toluidine blue and the number of mast cells in the reconstitution of mast cells enhances OVA permeation in stomach was counted under high magnification in a section from each v sample. Values are means (SEM) of six mice per group. *p,0.05; W/W mice with gastrointestinal Candida colonisation. As **p,0.01 compared with control mice without Candida inoculation. shown in fig 4C, BMMC transplanted W/Wv mice with

www.gutjnl.com 958 Yamaguchi, Sugita, Miki, et al

A OVA concentrations at 30 minutes tended to be higher than in W/Wv mice without BMMC transplantation and were 500 Control comparable with those in +/+ mice. Toluidine blue staining of Candida cryostat sections of forestomach revealed that mast cells were 400 almost absent in W/Wv mice and that there was no significant difference in the number of granulated and 300 degranulated mast cells between +/+ and BMMC trans- planted W/Wv mice (40 (8) v 42 (7) cells/mm2 for granulated cells and 44 (8) v 45 (8) cells/mm2 for degranulated cells). In 200 addition, quantitative culture of C albicans isolated from pooled faeces for each group on the day of the permeation 100 experiment showed that the number of organisms was higher in W/Wv mice and BMMC transplanted W/Wv mice (7.0 and Plasma OVA concentration (ng/ml) Plasma OVA 0 6.8 log10 CFU/g faeces, respectively) than in +/+ mice (5.8 0 30 60 90 120 log10 CFU/g faeces). Time after administration (minutes) DISCUSSION B The present study showed that gastrointestinal colonisation 500 with C albicans stimulated increased serum IgG and IgE Control specific to intragastrically administered OVA in BALB/c mice Candida (fig 1), indicating therefore that gastrointestinal Candida 400 colonisation promotes sensitisation against food antigen. *† Theoretically, increased permeation of food antigens in the 300 gastrointestinal tract would elicit such immunological responses as sensitisation. Indeed, intestinal inflammation, 200 associated with increased gut permeability, led to enhance- ment of sensitisation to ingested milk proteins in guinea pigs.33 In healthy humans, macromolecules such as food 100 antigens would be excluded by the gastrointestinal mucosal barrier to prevent food allergy. However, in individuals with Plasma OVA concentration (ng/ml) Plasma OVA 0 impaired gastrointestinal mucosal barriers, the likelihood of 0 30 60 90 120 permeation of food antigens in an intact form would be Time after administration (minutes) increased. This is an important prerequisite for the develop- ment of a food allergy and may explain the prevalence of food C allergy in infants and children with immature gastrointest- 200 inal mucosal barriers. In the present study, plasma concen- +/+ trations of intragastrically administered proteins (that is, W/W v HRP and OVA) were significantly increased in BALB/c mice v 150 W/W -BMMC with gastrointestinal Candida colonisation (fig 2). This suggests that gastrointestinal Candida colonisation impairs the gastrointestinal mucosal barrier, which in turn enhances *† 100 permeation of food antigens. Consequently, gastrointestinal colonisation by C albicans is likely to promote sensitisation against food antigens, at least in part, by affecting the * 50 gastrointestinal mucosal barrier and to increase the risk of food allergy. We investigated the mechanism by which gastrointestinal Plasma OVA concentration (ng/ml) Plasma OVA 0 Candida colonisation facilitates permeation of food antigens 0 30 60 90 120 through the mucosal barrier of the gastrointestinal tract. Time after administration (minutes) Mast cells have been reported to be involved in the regulation of the barrier and transport properties of gastrointestinal Figure 4 Time course of changes in plasma concentrations of 34 intragastrically administered ovalbumin (OVA) in WBB6F1 mice with epithelium via mediators such as rat mast cell protease II, and without Candida inoculation. OVA concentrations were measured tumour necrosis factor (TNF)-a,35 and interleukin 4.36 37 In in plasma samples obtained at the times indicated after intragastric the present study, mice with gastrointestinal Candida administration. (A, B) Results from mast cell deficient (W/Wv) and wild- colonisation showed an increased number of degranulated type (+/+) mice, respectively. (C) Data on +/+, W/Wv, and bone v mast cells in both the forestomach and glandular stomach marrow derived mast cell (BMMC) transplanted W/W mice inoculated (fig 3). Under these conditions, mast cells should secrete with C albicans. Values are means (SEM) of six mice per group. *p,0.05 compared with time = 0; p,0.05 compared with control mice mediators to increase the permeability of the gastric without Candida colonisation in (A) and (B) and W/Wv mice in (C), at epithelium. In fact, despite gastric colonisation with C each time point. albicans, mast cell deficient W/Wv mice showed no overt increase in the permeation of OVA (fig 4). In addition, reconstitution of mast cells in W/Wv mice with gastrointest- gastrointestinal Candida colonisation as well as +/+ mice inal Candida colonisation enhanced OVA permeation whose showed a significant increase in plasma OVA concentrations levels were comparable with those in +/+ mice with Candida 30 minutes after administration, although no such signifi- colonisation. The present results therefore suggest that mast cant increase was observed in W/Wv mice without BMMC cells mediate an increase in the permeation of food antigens transplantation. OVA concentrations at 30 minutes were in C albicans colonised gastrointestinal tracts. McDermott et al significantly higher in +/+ mice than in W/Wv mice without reported that mast cells play a critical role in increased BMMC transplantation. In BMMC transplanted W/Wv mice, intestinal permeability during enteric nematode infection via

www.gutjnl.com Candida promotes food allergy 959 release of mouse mast cell protease 1 and degradation of tight our preliminary experiments using this Candida colonisation junction proteins.38 This may also be the mechanism by model, however, denaturing gradient gel electrophoresis of which permeation of food antigens in C albicans colonised 16S rDNA amplicons showed that the bacterial composition gastrointestinal tract is increased. of the stomach and faecal specimens was the same in mice It remains unclear how C albicans colonisation stimulates with and without Candida colonisation (unpublished data). mast cells. Nosal et al reported that cell wall glycoproteins Therefore, the possibility described above could be ruled out. from C albicans induce the release of histamine in isolated rat In conclusion, we propose that gastrointestinal Candida mast cells.39 In addition, Jouault et al showed that phospho- colonisation promotes sensitisation against food antigens, at lipomannan in C albicans stimulated TNF-a production by the least partly due to mast cell mediated hyperpermeability in mouse macrophage-like cell line J774 via toll-like receptor 2 the gastrointestinal mucosa of mice. In contrast with this (TLR2).40 Because mast cells reportedly recognise pathogenic murine model however, it is not true to say that C albicans products via TLR2,41 a ligand for TLR2 such as phospholipo- colonises the gastrointestinal tract of a healthy human. C mannan in C albicans may elicit TNF-a release from mast albicans colonises the posterior dorsum of the tongue of 40– cells. We also observed that the water exudate of C albicans 60% of healthy humans. Yeast cells dislodge from the tongue, induces degranulation of rat basophilic leukaemia cells are swallowed, and survive transit through the gastrointest- RBL-2H3 in a concentration dependent fashion, suggesting inal tract, being detectable by culture of faeces. In addition, degranulation of mast cells by some water soluble constitu- the stomach of humans lacks a non-secretory epithelium ents of C albicans (unpublished data). Alternatively, anti-C which is the major site of Candida colonisation in the mouse albicans antibody may stimulate mast cells. We previously forestomach. Thus it remains unclear whether the transient observed that the titre of serum IgG1 specific to C albicans yeast cells in the human gut increase the permeability of the increases in BALB/c mice chronically colonised with C intestinal epithelium and/or affect oral immune tolerance in albicans.25 Similarly, increased anti-C albicans IgG1 levels in healthy humans. Further studies are required to prove this. the sera of mice were observed on the day of the gastrointestinal permeation experiment in the present study ACKNOWLEDGEMENTS (data not shown). Although IgE antibodies which bind to We are indebted to Dr Tatsuya Morita of Shizuoka University and Dr high affinity FceRI receptors on mast cells play a central role Taizo Nagura of Nippon Beet Sugar Mfg Co. Ltd. This work was in mediating type I allergic responses, non-IgE antibodies supported in part by a grant from Uehara Memorial Foundation. such as IgG1 could also mediate allergic responses via FccRI receptors on mast cells.42 Therefore, it seems possible that the ...... Candida antigen-IgG1 complex stimulates mast cells via FccRI Authors’ affiliations receptors, which in turn enhances gastrointestinal perme- N Yamaguchi, R Sugita, A Miki, N Takemura, J Kawabata, K Sonoyama, ability. Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, The present study did not elucidate the precise site at Sapporo, Japan which permeation of OVA occurred in mice with gastro- J Watanabe, Creative Research Initiative ‘‘Sousei’’, Hokkaido intestinal Candida colonisation. Although the small intestine University, Sapporo, Japan is usually considered the primary site of uptake of food Conflict of interest: None declared. antigens,43 previous reports suggest that the stomach is a site of uptake of food antigens under some physiological conditions. Hatz et al reported that antigen challenge in rats REFERENCES sensitised with dinitrophenol induces degranulation of mast 1 Verghese A, Prabhu K, Diamond RD, et al. Synchronous bacterial and fungal cells and results in increased permeation of a bystander septicemia. A marker for the critically ill surgical patient. Am Surg macromolecule OVA in the stomach.44 In addition, gastric 1988;54:276–83. Helicobacter infection has been reported to increase gastric 2 Bodey GP. Candidiasis in cancer patients. Am J Med 1984;77:13–19. 45 3 Eras P, Goldstein MJ, Sherlock P. Candida infection of the gastrointestinal permeability to macromolecules. Therefore, it is believed tract. 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