Antibodies to Saccharomyces cerevisiae in patients

Transcription

1 Gut 1992; 33: Gastoenteology Unit, M H Giaffe C D Holdswoth and Univesity of Sheffield Medical School, Depatment of Clinical and Expeimental Micobiology, Royal Hallamshie Hospital, Sheffield A Clak Coespondence to: D C D Holdswoth, Gastoenteology Unit, Wad J 1, Royal Hallamshie Hospital, Glossop Road, Sheffield S 1 2JF Accepted fo publication 1 1 Novembe 1991 Antibodies to Sacchaomyces ceevisiae in patients with Cohn's disease and thei possible pathogenic impotance M H Giaffe, A Clak, C D Holdswoth Abstact Sacchaomyces ceevisiae (bake's yeast) may play an impotant pat in the pathogenesis of Cohn's disease Because of this the levels of and IgA antibodies against thee S ceevisiae stains (NCYC 77, NCYC 79, and NCYC 118) wee assayed in 49 patients with Cohn's disease, 43 with ulceative colitis, 14 with coeliac disease, and 21 healthy contols Coded seum samples wee tested by ELISA Simila antibody pattens to all thee stains wee found and IgA antibody levels wee significantly aised in patients with Cohn's disease compaed with healthy contols (p<1 and p<1 espectively) and with ulceative colitis patients (p<1 and p<6 espectively) Raised IgA, but not, yeast antibody levels wee found in two patients with Cohn's disease who wee intoleant to yeast, but these values wee simila to those in othe patients without yeast intoleance In ulceative colitis, both and IgA levels wee simila to nomal contols Patients with small bowel Cohn's disease had significantly highe antibody levels than those with colonic disease (p<1) High levels of, but not IgA, antibody wee pesent in patients with coeliac disease, the antibody esponses being indistinguishable fom those found in Cohn's disease It is concluded that the pesence of antibody to S ceevisiae is chaacteistic but not specific to Cohn's disease Although aised IgA antibody levels ae moe fequently found in Cohn's disease, thei pathogenic impotance emains to be established (Gut 1992; 33: ) Thee is no fim epidemiological o expeimental evidence that diectly links diet and Cohn's disease, although the esults of dietay suveys' and the as yet unconfimed theapeutic value of 'exclusion diets'23 suggest that dietay factos may pecipitate o pepetuate acute exacebations in some patients with this disode A local intestinal immune esponse to as yet unidentified antigen(s) esulting in tissue damage and ganuloma fomation has been suggested as a possible mechanism of tissue injuy in Cohn's disease Both dietay and bacteial antigens have been implicated Thus ciculating antibodies to vaious dietay and bacteial antigens have been descibed in patients with inflammatoy bowel disease5-' as well as in coeliac disease ' A ecent epot suggested that milk may be an 'aetiologic facto' in a patient with Cohn's ileitis"` Main et al'2 showed that and IgA antibodies to Sacchaomyces ceevisiae (bake's yeast) ae pesent in high tites in patients with Cohn's disease but not in those with ulceative colitis o nomal contol subjects Thus, they claimed that yeasts may be aetiologically elevant in some patients with Cohn's disease In view of the unconfimed pathological impotance of yeast antibodies in Cohn's disease, this study was designed to examine the pevalence and possible pathogenic significance of and IgA antibodies specific to S ceevisiae in patients with Cohn's disease and ulceative colitis using patients with coeliac disease and healthy subjects as contols Patients and methods 171 PATIENTS Foty nine patients with Cohn's disease wee studied Thee wee 21 men and 28 women with a mean age of 45 yeas (ange 19-77) The diagnosis was based on established clinical, adiological, and histological citeia as peviously descibed'3 In 14 patients the disease was confined to the lage bowel; the emaining 35 had adiological evidence of small bowel disease with o without colonic involvement The disease was consideed clinically active in 18 patients and quiescent in the emaining 31 The disease was consideed active if the Cohn's disease activity index'4 was geate than 15 and the value of at least one laboatoy investigation (such as the eythocyte sedimentation ate, C eactive potein, ct acid glycopotein, platelet count, haemoglobin, and seum albumin) was abnomal " Foty thee patients had ulceative colitis - 18 men and 25 women with a mean age of 47 yeas (ange 22-81) Eighteen of these had active disease, confimed sigmoidoscopically and by histology, and the emaining 25 had quiescent disease None of the patients with inflammatoy bowel disease was eceiving steoids o othe immunosuppessive dugs at the time of the study Most patients with Cohn's colitis and all those with ulceative colitis wee taking sulphasalazine o othe 5-amino salicylic acid eleasing pepaations The contol goups included 2 healthy voluntees (eight men, 12 women with a mean age of 34 yeas (ange 19-48)) and 14 patients with established coeliac disease In the latte goup, 11 wee taking gluten fee diet; the othe thee had newly diagnosed disease and wee on a nomal unesticted diet Gut: fist published as 11136/gut on 1 August 1992 Downloaded fom on 25 August 218 by guest Potected by copyight

2 172 Giaffe, Clak, Holdswoth IMMUNOLOGICAL METHODS A 1 ml blood sample was collected fom each patient This was allowed to clot at oom tempeatue, centifuged, and the seum was sepaated into a steile containe The sample was coded and stoed at -2 C fo late testing Seial seum samples befoe and afte teatment with the elemental diet (Vivonex) wee obtained fom fou patients with clinically active Cohn's disease The enzyme linked immunosobent assay (ELISA) was applied to quantitate the specific and IgA antibody esponses to the yeast S ceevisiae Antigen pepaation Thee S ceevisiae stains wee used to pepae yeast antigens These included NCYC 77 and NCYC 79 (bake's yeast) and NCYC 118 (bewe's yeast) The stains wee kindly povided by D H McKenzie, Univesity of Abedeen, UK The yeasts wee cultued on sepaate Sabouaud aga slopes (Oxoid Ltd, Basingstoke, Hampshie) fo 48 hous at 37 C The gowth was havested and washed twice befoe being esuspended in 15 M steile saline so that a 1:1 dilution had an optical density of 25 at 54 nm The suspension was then boiled fo 6 minutes and stoed at 4 C A 1:1 dilution of the boiled antigen suspension in 5 M cabonate bicabonate buffe (ph 96) was used to coat the wells of the micotite plates Antibody assay Flat bottomed micotite plates (96 wells) (Alpha Laboatoies Ltd, UK) wee coated with 1 1d of antigen suspension pe well and incubated at 37 C fo 6 minutes and at 4 C fo 16 hous Each well was then washed fou times with 5 M phosphate buffeed saline, ph 73, to which 1% (v/v) Titon X-1 was added (PBS-T) Test sea wee diluted 1:1 using PBS-T to which 1% (w) bovine seum albumin was added (PBS-T-BSA) Each seum sample was tested in duplicate A 1 itl aliquot of seum was added to each antigen coated well In addition to test sea, each plate included a positive and a negative efeence seum and a negative contol whee no seum was added The plates wee then incubated at 37 C fo 6 minutes and washed fou times with PBS-T A 1,ul aliquot of affinity puified alkaline phosphatase conjugated antihuman o IgA (Sigma, Poole, Doset) diluted 1:4 in PBS-T-BSA was added to each well and the incubation and washing steps wee epeated One hunded 1d of a 2 mg/ml solution of P-nitophenyl phosphate (Sigma) in 5 M glycine-sodium hydoxide buffe ph 1-2, wee then added to each well The plates wee incubated at 37 C fo 3-4 minutes until the optical density of the positive efeence seum eached the pedetemined value of 1- at 41 nm, at which time the optical densities of the test sea wee ead (Micoelisa Minieade MR59, Guensey, Channel Islands, UK) Thus the quantities of and IgA anti-yeast antibodies wee expessed as optical density measuements in elation to the efeence positive seum STATISTICAL ANALYSIS Data wee expessed as mean (SEM) The Mann- Whitney U test was used to examine the significances in the diffeences in antibody levels between study goups Intecoelation between and IgA antibodies to vaious S ceevisiae stains wee tested using Speaman's equation Results Figue 1 (A, B, and C) shows the distibution of and IgA levels against baking (NCYC 77 and NCYC 79) and bewing (NCYC 118) S ceevisiae stains in all study goups In Cohn's disease, values against NCYC 77, NCYC 79, and NCYC 118 stains wee significantly highe than those ofhealthy contols (p<o1, p<-1, and p<*1 espectively) and ulceative colitis patients (p<-2, p<1, and p<q5 espectively) IgA tites wee geneally pesent at lowe seum concentations than But like, IgA tites against all thee yeast stains wee significantly aised in patients with Cohn's disease compaed with those with ulceative colitis and nomal contols (Figs 1A, B, and C) Patients with ulceative colitis, egadless of disease activity, had and IgA antibody values that wee not significantly diffeent fom those found in healthy contols Figues 2 and 3 show that the site of Cohn's disease influenced the antibody tites Patients with small bowel involvement had significantly highe antibody tites against NCYC 77 and NCYC 118 than those with colonic disease, whethe o not the small bowel was also affected (p<-1 and p<q4 espectively) Although thee was a diffeence between small and lage bowel disease in IgA antibody values, this diffeence was not statistically significant Othe factos such as age, sex, disease activity, o pevious intestinal esection had no effect on the antibody tites (data not shown) Twenty patients with Cohn's disease wee tested fo individual food intoleances,'5 16 and thee identified yeast as a possible tigge food Two of these had aised IgA but not tites, although the antibody values wee simila to those found in subjects without yeast intoleance The thid declined testing Seial measuements of and IgA wee made in fou patients befoe and fou weeks afte teatment with an elemental diet" This teatment had no effect on the o IgA tites Raised o IgA values against one S ceevisiae stain coelate well with o IgA values against the othe two stains (Table) Of the 26 patients with Cohn's disease who had aised against NCYC 77 stain, 24 (92%) and 21 (81%) also had aised against NCYC 79 and NCYC 118 espectively In the same goup, 2 (77%), 2 (77%), and 18 (69%) also had inceased IgA levels against NCYC 77, NCYC 79, and NCYC 118 espectively In patients with coeliac disease, but not IgA values against all thee S ceevisiae stains wee significantly aised compaed with those in ulceative colitis and nomal contols The aised antibody concentations in these patients wee compaable with those seen in Cohn's disease; high concentations wee equally Gut: fist published as 11136/gut on 1 August 1992 Downloaded fom on 25 August 218 by guest Potected by copyight

3 1 1 A; p c -4- _ O~g ' ) ^ ) c -7' z o X) -4 < n -2c ' C Co 8 > E -7 zo Z -6 ((X -5 OL c a a C -2-1 C 3 * 9 s en <x) ) C > s _ * - C) e" -, Jn 8o Q J I p z -1 il A C L m **4% e,e,< ID-4- IgA * * -=o ~ 4) 4) j ~ ~ Ig = to = CD 3 dl~~~~~~~~~ 1 -dn - 1~ i 7 C - pesent in teated and unteated patients with coeliac disease In contast, the mean IgA antibody tites in coeliac disease wee simila to those in nomal contols and patients with ulceative colitis (Figs IA, B, and C) Inceased IgA antibody values wee theefoe found mainly in patients with Cohn's disease * Discussion In this study, patients with established Cohn's disease had significantly aised and IgA values against baking and bewing yeasts (S ceevisiae) In contast, the ulceative colitis * patients had antibody values compaable with * healthy contols This confims pevious obsevations12 17 which also showed that the patients with Cohn's disease tended to have high levels of and IgA anti-yeast antibodies In maked U contast to these authos, howeve, we found that the aised levels against S ceevisiae depended upon disease site Patients with confimed small bowel involvement, whethe o not the lage bowel was also affected, had highe levels than those in whom the disease was confined to the colon Apat fom anatomical location, no othe patient o disease facto influenced the acquisition of high antibody levels The pesence of aised levels against all thee S ceevisiae stains in coeliac disease aises questions about the impotance of this antibody in Cohn's disease which was claimed by pevious wokes'2 17 In ageement with a ecent epot,'8 IgA antibody against S ceevisiae was moe specific to Cohn's disease, fo significantly highe levels wee found only in this condition Coeliac disease patients and those with ulceative colitis geneally had IgA levels that wee not diffeent fom healthy contols Futhemoe, aised IgA levels in Cohn's disease wee equally found in - patients with small and lage bowel disease Thus, the aised IgA levels could not be accounted fo on the basis of disease site Even so O n its pathogenic significance is not cetain fo the pesence of yeast antibodies did not coelate with a pevious histoy of yeast intoleance Two of ou patients who had documented intoleance to yeast had and IgA antibody levels that wee not diffeent fom those of othe patients without such a histoy It could be agued that antibody is usually of highe affinity than IgA and might mask an IgA esponse, but, if this was the case this would meely incease the significance of the aised IgA levels epoted hee It is accepted that seum IgA esponses must be intepeted as a eflection of the mucosal secetoy IgA esponse which could not be evaluated in this study It is possible that the aised tites against S ceevisiae eflect enhanced intestinal pemeability The highe levels of antibodies found in ou patients with small bowel Cohn's disease ae consistent with this hypothesis A mucosal pemeability defect has been shown Gut: fist published as 11136/gut on 1 August 1992 Downloaded fom on 25 August 218 by guest Potected by copyight M >o ~~ ( ) (D (*Jv- Figue 1: Levels ofseum and IgA antibodies to Sacchaomyces ceevisiae (A) NCYC 77, (B) NCYC 79, and (C) NCYC 118 in patients with inflammatoy bowel disease, coeliac disease, and healthy contols *Significantlv diffeentfom contols and ulceative colitis (p<-1) IgA

4 174 Giaffe, Clak, Holdswoth E *7 p < 48 Small bowel 46 o p Lagebowel optical -3 density at 41 nm) in Cohn's disease C2 QI) C 4-o p <175 NCYC "TMY 7 CC NCYC77 NCYC79 NCYC 118 Figue Fig'ue 2: Effect of disease site on the mean (SEM) seum IgA antibody levels (expessed as optical density at 41 nm) in Cohn's disease Spamnsceainmti o g n g gis l 4eSchmcscvsa Spanans oeltin atixofig adg Sm allbowllteschomeseevia s stinsncc7,nyc79,an Small bowel 2 21 Anioy I ggi oiaiega Ig/NY in patients with Cohn's disease and thei elatives,9 as well as in those with coeliac disease2 The abnomally pemeable mucosa may pemit the tansfe of lage quantities of luminal antigens acoss the intestinal wall This antigenic load may then initiate an immune esponse esulting in inceased levels of ciculating antibodies against the antigens This concept is suppoted by the fact that patients with Cohn's disease have high antibody tites against a wide vaiety of dietay antigens to which the patient is usually not clinically intoleant5-7 Hypesensitivity to gliadin is consideed to be an impotant aetiological featue in coeliac disease and is manifested by the pesence of aised antigliadin antibody levels Howeve, antibodies to othe dietay antigens have also been shown in coeliac disease patients without clinical evidence of intoleance 21 Since it is postulated that both conditions shae the same mucosal pemeability defect, the pesence of these antibodies may simply epesent a seconday phenomenon The effect of teatment on intestinal pemeability is not cetain In Cohn's disease, Logan et a122 claimed that elemental diet educes mucosal pemeability, although the data of Hollande et all9 suggest that the pemeability defect of Cohn's disease still exists in patients with quiescent disease Janasson et al'3 showed that the pemeability defect in coeliac disease pesists even afte teatment with gluten fee diet These obsevations indicate that in both conditions some degee of mucosal pemeability exists even when the disease is quiescent and this may theefoe explain the pesence of high and IgA tites in patients with inactive Cohn's disease and the failue of elemental diet teatment to educe antibody levels S ceevisiae has not been diectly implicated in the pathogenesis of Cohn's disease The oganism is a ubiquitous mico-oganism that aely causes significant human infections The immune esponse obseved to it descibed in this study may have esulted fom a eaction eithe to antigens on the S ceevisiae itself o to an as yet unidentified antigen(s) which coss eacts with S ceevisiae antigens This wok was suppoted by eseach gants fom the Tustees of the fome United Sheffield Hospitals and Cow & Gate Ltd 1 Heaton KW, Thonton JR, Emmett PM Teatment of Cohn's disease with an unefined cabohydate, fibe-ich diet BMJ 1979; 2: Alun-Jones V Compaison of total paenteal nutition and elemental diet in induction of emission of Cohn's disease Long-tem maintenance of emission by pesonalized food exclusion diets DigDis Sci 1987; 32: Alun-Jones V, Dickinson RJ, Wokman E, Wilson AJ, Feeman AH, Hunte JO Cohn's disease: maintenance of emission by diet Lancet 1985; ii: Kisne JB, Shote RB, eds Inflammatoy bowel disease Philadelphia: Lea & Febige, 1975: Falchuk KR, Isselbache KJ Ciculating antibodies to bovine albumin in ulceative colitis and Cohn's disease Gastoenteology 1976; 7: Knoflach P, Pak BH, Cunningham R, Weise MM, Albini B Seum antibodies to Cow's milk poteins in ulceative colitis and Cohn's disease Gastoenteology 1987; 92: Davidson IW, Lloyd RS, Whowell PJ, Wight R Antibodies to maize in patients with Cohn's disease, ulceative colitis and coeliac disease Clin Exp Immunol 1979; 35: Van de Mewe JP, Schmitz PIM, Wensinck F Antibodies to Eubacteium and Peptosteptococcus species and the estimated pobability of Cohn's disease JX Hyg 1981; 87: Tabaqchali 5, O'Donoghue DP, Bettelheim KA Escheichia coli antibodies in patients with inflammatoy bowel disease Gut 1978; 19: Kenick KG, Walke-Smith JA Immunoglobulins and Gut: fist published as 11136/gut on 1 August 1992 Downloaded fom on 25 August 218 by guest Potected by copyight

5 Antibodies to Sacchaomyces ceevisiae in patients with Cohn's disease and thei possible pathogenic impotance 175 dietay potein antibodies in childhood coeliac disease Gut 197; 11: Ginsbeg AL, Albet M Teatment of patients with sevee steoid-dependent Cohn's disease with nonelemental fomula diet Identification of possible etiologic dietav facto Dig Dis Sci 1989; 34: Main J, McKenzie H, Yeaman GR, Ke MA, Robson D, Pennington GR, et al Antibody to Sacchaomvces ceevisiae (bake's yeast) in Cohn's disease BMJ 1988; 297: Donaldson RM In: Sleisenge MH, Fotan JS, eds Gastointestinal disease Philadelphia: Saundes, 1989: Best WR, Becktel JM, Singleton JW, Ken F Development of a Cohn's disease activity index National Co-opeative Cohn's Disease Study Gastoenteology 1976; 7: Giaffe MH, Noth G, Holdswoth CD Contolled tial of polvmeic vesus elemental diet in teatment of active Cohn's disease Lancet 199; i: Giaffe MH, Cann P, Holdswoth CD Long tem effects of elemental and exclusion diets in Cohn's disease Aliment Phamacol Theap 1991; 5: McKenzie H, Main J, Pennington CR, Paatt D Antibody to selected stains of Sacchaomyces ceevisiae (bake's and bewe's yeast) and Candida albicans in Cohn's disease Gut 199; 31: Banes RMR, Allan S, Taylo, Robinson CH, Finn R, Johnson PM Seum antibodies eactive with Sacchaomyces ceevisiae in inflammatoy bowel disease: is IgA antibody a make fo Cohn's disease? Int Ach A llegy Appl Immunol 199; 92: Hollande D, Vadheim CM, Bettholz E, Peteson GM, Delhaunty T, Rotte JI Inceased intestinal pemeability in patients with Cohn's disease and thei elatives A possible etiologic facto Ann Inzten Med 1986; 15: Cobden I, Dickinson RJ, Rothwell J, Axon ATR Intestinal pemeability assessed by excetion atios of two molecules: esults in coeliac disease BM3' 1978; 2: Feguson A, Caswell F Pecipitins to dietay poteins in seum and uppe intestinal secetions of coeliac childen BM3 1972; 1: Logan FRA, Gillon J, Feington C, Feguson A Reduction of gastointestinal potein loss by elemental diet in Cohn's disease of the small bowel Gut 1981; 22: Janasson I, Petes TJ, Veal N A pesistent defect in intestinal pemeability in coeliac disease demonstated by 5 1Clabelled EDTA absoption test Lancet 1983; ii: Gut: fist published as 11136/gut on 1 August 1992 Downloaded fom on 25 August 218 by guest Potected by copyight