Antineutrophil cytoplasmic antibodies in sera from colectomised ulcerative colitis patients and its relation to the presence of pouchitis.

Gut 1996; 38:894-898

Antineutrophil

cytoplasmic antibodies

in

sera

from

colectomised ulcerative

colitis patients and

its

relation

to

the

presence

of

pouchitis

M Esteve,J Mallolas,JKlaassen, A Abad-Lacruz, FGonzalez-Huix, ECabre, FFernandez-Bafiares, X Bertran, E Condom, J Marti-Rague, M AGassull

Departmentof Gastroenterology, Hospital Universitari 'Germans Trias i Pujol', Badalona, Catalonia, Spain MEsteve JMallolas JKlaassen ECabre FFemrndez-Bafiares XBertran M AGassull Department of Gastroenterology, Hospital de Sant Llorenq,Viladecans, Catalonia, Spain AAbad-Lacruz Department of Gastroenterology, Hospital 'Josep Trueta', Girona, Catalonia, Spain FGonzalez-Huix Departmentof Pathology,Ciutat Sanitaria Universitaria deBellvitge, L'Hospitalet, Catalonia, Spain ECondom Departmentof Surgery,Ciutat Sanitaria Universitaria deBellvitge, L'Hospitalet, Catalonia,Spain J Marti-Rague Correspondenceto: DrM AGassull,Hospital

Universitari 'Germans Triasi Pujol', CarreteradelCanyet

s/n,08916Badalona,Spain. Acceptedforpublication

13 December1995

Abstract

Background-Fewstudies have evaluated

the influence of colectomy on

anti-neutrophil cytoplasmic antibody (ANCA)

positivity in ulcerative colitis (UC). In small series ofpatients it has been

sug-gestedthat ANCApositivityin UCmight

bepredictivefordevelopment ofpouchitis

aftercolectomy.

Aimns-To

assess theprevalence ofANCA

inUC patients treatedby colectomy anda Brooke's ileostomy(UC-BI)orilealpouch

anal anastomosis (UC-IPAA), and the

relation between the presence ofANCA,

the type ofsurgery, and the presence of

pouchitis.

Subjects-63UCpatientstreatedby

colec-tomy (32 with UC-BI and 31 with

UC-IPAA), 54UC,and24controls.

Methods-Sampleswere obtainedatleast

two years after colectomy. ANCA were

detected by indirect immunofluorescent assay.

Results-There were no differences between patients with

(36/3%)

orwithout

pouchitis

(35O0%)

and between patients

with UC

(550/),

UC-BI

(40.6%),

and

UC-IPAA

(35.40/o).

However, ANCA preva-lence significantly decreases in the whole group ofoperated patients (38-0%)

com-pared with non-operated UC (p=0.044). Conclusions-Theprevalenceof ANCA in

operated patients was significantly lower than innon-operated UC, suggestingthat it mightbe related eitherto the presence

of inflamed or diseased tissue. ANCA

persistence is not related to the surgical procedure and itshould not be used as a marker forpredictingthe developmentof

pouchitis.

(Gut 1996;38:894-898)

Keywords: ulcerative colitis,proctocolectomy,ileal pouch anal anastomosis,antineutrophil cytoplasmic

antibodies,pouchitis.

Ulcerative colitis (UC) and associated condi-tions suchasprimarysclerosing cholangitisare

some of the diseases in which antineutrophil cytoplasmic antibodies (ANCA) have been

described.1-3 UC associated ANCA

(UC-ANCA) most commonly show an immuno-fluorescenceperinuclearpattern. Althoughnot

universally accepted,3 cytoplasmic and mixed ANCA

patterns

have been described associ-atedto

UC.±8

On theotherhand,becausethe

antigenic specificity of UC-ANCA is

unknown,'

3 910theyarereferredto asatypical ANCAorx-ANCA bysomeauthors.1-3

Few studies have specifically evaluated the influenceofcolectomyonANCA positivity in

UC.4

711-4 As well as only dealing with a small series ofpatients,noneof them includes

a large number of cases with Brooke's

ileostomy,whicharethe onlyonesthatmaybe

considered absolutely free of disease. On the otherhand, all these seriesreport ahigh preva-lence of ANCA in patients with pouchitis

(80-100%).4

711-13 Based on the results of thesestudies it has beensuggestedthatANCA assessmentinUCmaybeuseful in predicting the development of pouchitis in patients undergoingIPAA.2

Theaims of this studywere (a) toassessthe prevalence of ANCA in UC patients treated withcolectomy and eitheraBrooke'sileostomy or ileal pouch anal anastomosis, and (b) to determine the relation between thepresence of ANCA, thetypeofsurgeryperformed, and the presenceofpouchitis.

Methods

PATIENTS ANDCONTROLS

Ulcerativecolitispatients treated withcolectomy One hundred and forty eight patients with a

diagnosis of UC have had a colectomy since 1973 in three of four hospitals takingpart in thestudy. The diagnosishad been established

by the Lennard-Jones clinicopathological

criteria.15

Patientswithahistoryof indetermi-nate colitis were excluded from the study. Based onthe information available, 92 of114

patients livinginCatalonia (31 895

kM2)

could becontactedbyphoneandaskedtoparticipate

inthe study. Sixty three of them agreedto be included.ThirtytwohadaBrooke'sileostomy

(UC-BI) (15 male, 17female;43 years (range: 23-76)) and 31 hadanilealpouchanal anasto-mosis (UC-ANCA) (18 male, 13 female; 34 years (range: 24-61)). Thetechnique of IPAA includedmucosectomyofthe anorectal stump in five patients, whereas a staple ileal pouch analanastomosiswasperformedinthe remain-ingcases. In allpatients atwo loop J reservoir

wasmade.

Disease controls

Fifty four UC patients who had not had an

operation(35male,19female;40years(range:

Antineutrophil cytoplasmicantibodies inserafrom colectomisedulcerativecolitispatientsand its relationtothepresenceofpouchitis

TABLEI Clinical characteristics of the ulcerative colitis patients Patients Activity Inactive 21 Mild 21 Moderate 11 Severe 1 Extent Proctitis 16

Leftsided colitis 28

Pancolitis 10 Extraintestinalmanifestations 5 Treatment No treatment 12* Salicylates 37 Corticosteroids 8 Azathioprine 7 Corticosteroid refractory 13

*First episode. Time elapsed from diagnosis:4months (0-25).

19-74)), living in Catalonia, attending the

'Hospital Universitari Germans Trias i Pujol'

were included in the study. The diagnostic

criteriawerethesame asabove.

Healthy controls

Twenty four volunteers, living in the same geographical area (11 male, 13female;43years

(range: 23-72)) actedas ahealthycontrols.

CLINICALASSESSMENT

In all patients a complete clinical assessment wasperformed at the time of blood sampling

for ANCA detection. In non-operated UC

patients this included: activity and extent of the disease, current treatment, time from

diagnosis, extraintestinal manifestations, and outcome. The activity of the disease was assessed by means of the Truelove index.16

Table I shows the clinical characteristics of the non-operated UC patients. The assess-mentofUCpatients treated withacolectomy

included: time from diagnosis and surgery,

TABLE I Pouchitis diseaseactivityindex(PDAI)1415

Criteria Score

Clinical Stoolfrequency:

Usualpostoperative stoolfrequency 0 1-2Stools/day>postoperativeusual 1 3Ormorestools/day>postoperative usual 2

Rectalbleeding:

Noneorrare 0

Presentdaily 1

Faecal urgencyorabdominal cramps:

None 0 Occasional 1 Usual 2 Fever(temperature >37.8°C: Absent 0 Present 1 Endoscopicinflammation Oedema 1 Granularity 1 Friability 1

Lossofvascularpattern 1

Mucous exudate 1

Ulceration 1

Acutehistological inflammation

Polymorphonuclear leucocyteinfiltration:

Mild 1

Moderate+cryptabscess 2

Severe+cryptabscess 3 Ulceration per low power field(mean):

<25% 1

25-50% 2

>50% 3

extraintestinal manifestations, previous and currenttreatment, andpostoperativeoutcome.

Eight patients

with UC-BI were

previous

UC-IPAA that had failed. Theprimaryreason

forpouchexcisionwaschronicpouchitis resis-tant to medical treatmentin twopatients and fistula and

pelvic sepsis

in sixpatients.Four of the secondgroup also had chronic continuous

pouchitis.

None of thesepatients had

indeter-minate or Crohn's colitis. In UC-IPAA

patients, the degree of pouch inflammation

was assessed by means of the modified

Moskowitzindex17 18(TableII).Pouchitiswas defined as a score equal or greater than 7. Eleven of 31 UC-IPAA patients fulfilled this criterionatthetime ofinclusion.

Previous history of confirmed pouchitis

was also recorded. Four of 11 patients with

pouchitisatthetime ofinclusion hadhistoryof chronic continuous pouchitis resistant to medical treatment. Table III shows clinical data of UCpatients treated witha

colectomy.

Some UC-IPAA patients were taking more

than one drug. None of thepatients included hadprimarysclerosingcholangitis.

ANTINEUTROPHIL ANTIBODIES DETECTION Serumwasobtained of allpatientsand controls. Inoperated patients, sampleswere obtained at least two years after colectomy. ANCA were detectedusinganindirectimmunofluorescence microscopy detection method. Commercially

available slides containing cytospin smears of

human ethanol fixed neutrophils (INOVA

Diagnostic, SanDiego, CA) were used as sub-strate. Diluted serum samples in phosphate

buffered saline (PBS) were incubated on the slides for 30minutes. Afterwashing in PBS, the slides were incubated again for 30 minutes with fluorescein conjugated rabbit

immuno-globulins to human immunoglobulin G

(DAKO, Copenhagen, Denmark) at a pre-determined optimal dilution of 1/100 in PBS. Slides were washed in PBS twice, mounted in

glycerol, and read immediately under fluores-cence microscopy at 400X (Axioplan, Zeiss, Germany).

Slides were read by two independent

observers who were unaware of the clinical

diagnosis. Control ANCA positive and nega-tive sera and PBS were included in each test batch. A serum was considered positive for ANCA when a clear perinuclear fluorescence at a 1:20 dilution was observed or when a

TABLEIII Clinicalcharacteristics oftheUC patients

treatedwithacolectomy

UC-IPAA UC-IPAA UC-BI pouchitis nopouchitis

Years fromdiagnosis 14(4-36) 6(2-34) 7(2-24)

Years fromsurgery 9(2-16) 4(2-9) 4(2-9) Clinicalscore* - 2(0-4) 0(0-3) Endoscopicscore* - 4(3-6) 0(0-3) Histologicalscore* - 2(1-5) 1(0-3) Global score* - 8(7-11) 3(1-6) Treatment (patients): No treatment 32 3 14 Salicylates 0 2 3 Local corticosteroids 0 3 0 Antibioticst 0 4 5

*Pouchitisdiseaseactivityindex;tmetronidazole/tetracyclines.

Esteve,Mallolas,Klaassen,Abad-Lacruz, Gonzdlez-Huix, Cabre,Ferndndez-Baniares, Bertrdn,Condom,Marti-Rague,Gassull cytoplasmic staining was observed at a 1:40

dilution. The second cut off dilution was chosen to eliminate unspecific cytoplasmic positivity yielded by some negative sera. All negative sera and those given as positive by onlyone observer were retested blindly. Serum samples given as positive by only one observer induplicate assay were considered as negative. Positive ANCA samples were titrated to a dilution of 1/1280.

To differentiate positive ANCA from anti-nuclear antibodies (ANA) that mimic ANCA, allpositive sera were tested with HEp-2 cells andrat liversections, which are highly sensitive for ANA detection. All ANA positive sera with stronger titres than that observed in neutrophil slides were considered as ANCA negative. This occurred in four patients (three non-operated and one operated) and one healthy

control.

All assays were performed at the Research Laboratories of the Hospital Universitary

'Germans Trias iPujol'.

STATISTICAL ANALYSIS

Comparison of the frequencies between

patients with or without pouchitis were

analysed usingthetwotailx2andYates's cor-rection.ThefrequenciesbetweenUC,UC-IB,

and UC-IPAAwerecompared usinga twotail

X2

and the

comparison

between

operated

and

non-operated UC patients was performed using aleftsided Fisherexact test.Titreswere

compared using the Kruskal-Wallis test. The

Spearmanrank correlation coefficientwasused to determine associations between titres and the time elapsed from surgery. The resultsare

expressedbymeansof median andfrequencies

with their range and 95% confidence intervals (CI), respectively. The statistical procedures

were performed using the programs of the Biomedical DataProcessing, BMDP (BMDP,

Statistical Software, Los Angeles, California,

1986).

The positivity of ANCA was significantly

higherin all groups studied compared with the healthy control group (p<0 0001).

Relationbetween ANCApositivity andthe presenceofpouchitis

In patients with UC-IPAA, there were no differences inANCA positivity between those with (36-3% (CI: 109 to 69.2)) or without

pouchitis (35.0% (CI: 15.5 to 59.2)) (p=0.720).

Two offour(CI: 6.7 to 93.2%) patients with chronic continuous pouchitis andfour of eight UC-IPAA failure (CI: 15.7 to 84.3%) were ANCApositive.

As the percentage of ANCA positive

patients was thesameasthose with orwithout pouchitis, theywere considered together and compared with both patients treated with a colectomy and non-operated UC patients to

determine the influence of a Brooke's

ileostomyuponANCApositivity.

Relation between ANCA positivity and thetype

ofsurgeryperformed

There were no differences inANCApositivity between patients with UC-BI (40-6% (CI: 23.7 to59.3)) andUC-IPAA (35.4% (CI: 19.2 to54.6)) andnon-operated UC patients (55% (CI: 41-4 to 69.1)) (p=0 154).

Because there were nosignificant differences between both groups ofoperatedpatients,they were considered together (both UC-BI and

UC-IPAA) and compared with UC

non-operated patients to determine if colectomy

decreases ANCA positivity. There was a

significant decrease in ANCA prevalence in operatedpatients-38.0%(CI: 26.1 to 512)

-compared with non-operated UC (55%)

(p=0044).

The Figure shows the titres ofANCA and

UC, UC-BI, and UC-IPAA. There were no

differences in ANCA titres between groups

ETHICALCONSIDERATIONS

The studywas performed in accordance with the 1975Declarationof Helsinki ethical

guide-lines and was approved by the Research and EthicalCommittees of theHospitals.

Results

Table IV shows the numberof ANCApositive patients and controls in the groups studied. TABLEIV Number and percentageofANCApositive patients

Positive Negative

Ulcerative colitis 30(55.5%)* 24

UC-BI 13(406%)t 19

UC-IPAA withpouchitis 4(36.3%)A 7

UC-IPAA withoutpouchitis 7(35/0%)§ 13

Healthycontrols 0 24

UC-BI=totalproctocolectomy plusBrookesileostomy,

UC-IPAA=proctocolectomyplusilealpouchanal anastomosis. *26p-ANCA; 1c-ANCA;3mixedpattern,t11p-ANCA;

2mixedpattern,t3 p-ANCA;1mixedpattern,§6 p-ANCA; 1mixedpattern. 1/2560r 1/1280H 1/640

h

CO a) . x 1/320H 1/160[ 1/80H 1/40

h

m a ma a a m m a a a ma am* *-ma* m a a a 1/20- a aaUaE ma* a aEa *-m aE m aa a a a a m UC UC-IPAA *-UC-BI

TitresofANCA innon-operatedukerativecolitispatients

(UC), patientstreated withacolectomy plusilealpouch

anal anastomosis(UC-IPAA)andpatientstreated witha

colectomy plusBrooke'sileostomy (UC-BI).

n

Antineutrophil cytoplasmic antibodiesin serafromcolectomised ulcerative colitis patients and its relationtothe presence ofpouchitis

(p=0755). There was also no correlation

betweenthe timeelapsed fromsurgeryand the titresof ANCA (r=-0098).

Discussion

There is no agreement about those immuno-fluorescence ANCA patterns that should be considered associated with UC. Some labora-tories haveconsidered that allUC-ANCAare of the perinuclear

type,9

19-21 whereas others also include those less frequentcases showing eitherdiffuse cytoplasmicormixed

pattern.4-8

Weshare thissecond viewas,until the specific antigen(s) are identified, it does not seem

reasonable to exclude any type of ANCA pattern when an unequivocal

immunofluores-cencestaining is found.

Oneof the aims of this studywastoassess if removal of the colon results in a decline of ANCA inUC patients andif there isarelation tothetype ofsurgeryperformed. The percent-ageof ANCApositivityinourUCnon-operated patients

(55%/o)

issimilarto otherreports,622 23 and is within the range described in published

reports(40-80%).4-. 19-25Whetherorno circu-lating ANCApersist longtermafter colectomy inUC patientsisnotwellestablished. Although

most studies have reported the persistence of

ANCA after

surgery,47

11-1325 there are some reports of postoperative ANCA

disappear-ance.14

26 However,moststudiesincludeasmall number ofpatientsandnoneof them includesa largenumberofcaseswith Brooke's ileostomy. Although there are no data about the time course of ANCA behaviour after colectomy, wethought thattwo years might be a reason-ableperiodof timeforassessing ANCA clear-ance, assuming that they are related to the presenceof diseased tissue. We havenotfound a significant difference in ANCA positivity

betweennon-operated UC patients,thosewith

UC-BI, and those with UC-IPAA, showing

that these antibodiespersistin the serum ina

high percentage of UC operated patients long term after colectomy. However, we found a

significant decrease of ANCApositivity when both groups of operated patients were con-sideredtogether (thatis,increasing twofold the number of operated patients). This finding

mightdisclose theexistence ofaweak relation between ANCA positivity and inflammation,

ashas been shown insome

studies.6

26 Infact,

our non-operated patients showed a

non-significant trend towardsa greaterpercentage of ANCApositivityin those with activeversus

inactive UC (63% v 43%; p=0.13), and in those with extensive disease versus proctitis

(6/3%

v31%; p=007).Analternative explana-tion would be that ANCA clearance after colectomy was a very slow process taking more than two years and a prospective and paired assessment before and aftersurgery in

15 UCpatients foundasignificantdecrease in ANCA titres a mean oftwo years after

colec-tomy.14

In our study, no differences between groups with respect to ANCA titres, or any relation between the titres of ANCA and the time elapsed from surgery were found. However,asthiswas not apaired comparison,

interindividual differences in the ability of antibody production may have prevented the discoveryof such differences.

Inthisserieswefoundasimilar frequency of ANCA positivity between those patients with and without pouchitis. In contrast, several studies have reported a higher prevalence of ANCA in patients with

pouchitis.4

711-13

However, most of them include a small

number of patients with pouchitis or it is not well defined, or

both.4

11-13 In thisregard, to

distinguish between relapsing and chronic continuous pouchitis may be relevant. In the largest reported series, in which all patients with pouchitis had chronic inflammation resistant or dependent on medical treatment, frequency of ANCA positivitywas

100%.7

The authors suggest that the strong relation

between ANCA and pouchitis would be

related to a specific disease pattern (chronic continuous pouchitis) ratherthan to themere presence of

inflammation.7

In the remaining studies, it is not stated if those patients with pouchitis had relapsing orchronic continuous disease. However, a recentstudy published as anabstract,showedthat thosepatientswithout pouch inflammationatinclusion, but with

pre-vious episodes of pouchitis, had a

33.30/o

of ANCA

positivity.12

Thisfigure issimilartothe percentagefound in ourpatientswith

pouchi-tis, mostof themhavingrelapsingdisease. On the other hand, in our series two of four chronic resistant pouchitis and four of eight IPAAfailures hadpositiveANCA.

Takenas awhole,all these data suggest that ANCA would notpredict the development of relapsing pouchitis, and thestrong association between ANCA positivity and chronic con-tinuous pouchitis deserves further confirma-tion. However, even if a 100% association

(sensitivity) could beconfirmed, the low

speci-ficity of ANCA for chronic continuous

pouchitis7

will make the use of this marker

doubtfulas apredictorof this condition. Although all these queries could be only answeredbymeansofalongitudinal study, its

practicabilityseems tobequestionable. Sucha

study would be very long lasting, taking into accountthatpouchitismayappear aslateas 10 years after

surgery.27

28 In the case of chronic continuous pouchitis, an event occurring in

only

5%/o

of the total

IPAA,28

thegreatnumber

ofpatients to be included would be an addi-tionaldifficulty.

Inconclusion, ANCApersistinahigh

pro-portion of UC patientsafter colectomy and its presence is not related to the type ofsurgery

performed. The lower percentage found in

operated patients suggests that ANCA

positivity might be related to the presence of eitherinflamedordiseasedtissue. On theother hand, ANCA positivity in UC should not be used as a marker for predicting the develop-ment ofpouchitis and, at present, it does not seemagoodparameterfordeciding thetypeof surgerytobeperformedinUCpatients.

Partof thisstudyhas beenpresentedasaposteratthe95th Annual Meeting of the American Gastroenterological

Association held in SanDiegoinMay 1995,andpublishedas anabstract inGastroenterology1995;108:A816.

898 Esteve, Mallolas,Klaassen, Abad-Lacruz, Gonzalez-Huix, Cabre,Femrnndez-Bafnares, Bertran, Condom,Marti-Ragu4,Gassull

1 GrossWL,SchmittWH,CsernokE.ANCAandassociated

diseases:immunodiagnostic and pathogenic aspects. Clin ExpImmunol1993;91:1-12.

2 Shanahan F. Neutrophil autoantibodies in inflammatory

bowel disease:aretheyimportant? Gastroenterology1994; 107: 586-9.

3 Gross WL, Hauschild S, Mistry N. The clinical relevance of ANCA invasculitis. ClinExpImmunol1993; 93 (suppl1):

7-11.

4 Patel RT,Stokes R, Birch D, Ibbotson J, KeighleyMRB. Influence of total colectomy on serum antineutrophil

cytoplasmic antibodiesininflammatory boweldisease. Br

J3Surg1994; 81: 724-6.

5 LamproyeA, BelaicheJ, Louis E, Salmon J, Mahieu PH. Anticorps anticytoplasme des polynucleaires neutrophiles (ANCA) dans les maladies inflammatoires du tube

digestif.ActaGastroenterolBelg1994;LVII:171-6. 6 Broekroelofs J, MulderAHL, NelisGF,Westerveld BD,

Cohen TervaertJW, Kallenberg CGM.Anti-neutrophil

cytoplasmicantibodies(ANCA) in sera from patients with

inflammatory bowel disease (IBD). Relation to disease pattern and diseaseactivity.DigDisSCi1994;39: 545-9. 7 Sandborn WJ, Landers (CJ, Tremaine WJ, Targan SR. Antineutrophil cytoplasmic antibody correlates with

chronic pouchitisafter ilealpouch-analanastomosis. Am JGastroenterol1995;90: 740-7.

8 Sung JY, Chan FKL,LawtonJ, LeungJCK, Liew CT,

Leung NWY, et al.Anti-neutrophilcytoplasmicantibodies

(ANCA)andinflammatory bowel diseasein chinese. Dig

Dis SCi 1994;39: 886-92.

9 Ellerbroek PM, Outkerk Pool M, Ridwan BU, Dolman KM, vonBlombergBME, vondem Borne AEGKr,etal.

Neutrophilcytoplasmic antibodies (pANCA)inulcerative colitis.JClin Pathol 1994;47:257-62.

10KanekoK, Suzuki Y,YamashiroY, Yabuta K. Isp-ANCA

in ulcerative colitis directed against b-glucoronidase? Lancet1993;341: 320.

11 Remaux D, Colombel JF, Heresbach D, Duclos B, Chaussade S,BelaicheJ,etal.Antineutrophilcytoplasmic auto-antibodiesin serafrompatientsafterproctocolectomy for ulcerative colitis.Gastroenterology1993;104: A769. 12 Remaux D, Colombel JF, Heresbach D, Duclos B,

Chaussade S, DuclosJ,et al. ANCA andpouchitis in

ulcerative colitis afterproctocolectomywithileoanal

anas-tomosis.Gut 1995; 37: A162.

13 Vecchi M, Gionchetti P, Bianchi MB, Belluzi A, MeucciG, Campieri M,etal.p-ANCA and development ofpouchitis

in ulcerative colitis patients after proctocolectomy and

ileoanalpouch anastomosis.Lancet1994;344:886-7. 14 AitolaP, Miettinen A,MattilaA, Matikainen M,SoppiE.

Theeffectofproctocolectomy on serum antineutrophil cytoplasmic antibodies in patients with ulcerative colitis. Gastroenterology1995; 108: A769.

15 Lennard-Jones JE. Classification of inflammatory bowel

disease.ScandJ7Gastroenterol 1989; 24(suppl 170):2-6. 16Truelove SC, Witts U. Cortisone in ulcerative colitis.

Reportontherapeutic trial.BMJ31955; 2:1041-6.

17 Moskowitz RL,Shepherd NA, Nicholls RJ.Anassessment of inflammation in the reservoir after restorative procto-colectomywith ileoanal ileal reservoir. Dig Dis Sci 1986; 1: 167-74.

18 Sandborn WJ, Tremaine WJ, Batts KP, Pemberton JH,

PhillipsSF.Pouchitisafterileal pouch-analanastomosis:

apouchitis disease activity index. MayoClinProc 1994; 69:409-15.

19 SaxonA, SanahanF, Landers C, Ganz T, Targan S. A dis-tinct subsetofantineutrophil cytoplasmic antibodies is

associatedwithinflammatorybowel disease.JAllergyClin Immunol 1990; 86: 202-10.

20 Oudkerk Pool M, Ellerbroek PM, Ridwan BU, Goldschmeding R,vonBlomberg BME, Pefia AS,etal.

Serum antineutrophil cytoplasmic autoantibodies in

inflammatory bowel disease are mainlyassociated with ulcerative colitis. Acorrelation studybetweenperinuclear antineutrophil cytoplasmic autoantibodies and clinical

parameters, medical and surgical treatment. Gut 1993; 34:46-50.

21 OudkerkPool M, RocaM,RemauxD,BoumaG,PeflaS, ColombelJF,etal.Thevalue ofp-ANCAas aserological marker for ulcerative colitisindifferentEuropeanregions. EurJGastroenterolHepatol1994; 6: 399-403.

22 LeeJCW, Lennard-Jones JE, Cambridge G.Antineutrophil

antibodies in familial inflammatory bowel disease. Gastroenterology 1995;108:428-33.

23 Duerr RH, Neigut DA. Molecularly defined HLA-DR2

allelesinulcerative colitisanantineutrophil cytoplasmic antibody-positive subgroup. Gastroenterology 1995; 108: 423-7.

24 DuerrRH, TarganSR, LandersCJ,LaRussoNF,Lindsay KL, Wiesner RH, et al. Neutrophil cytoplasmic

anti-bodies: alink betweenprimarysclerosing cholangitisand

ulcerative colitis. Gastroenterology 1991; 100: 1385-91. 25 Duerr RH, Targan SR, Landers CJ, Sutherland LLR, Shanahan F. Anti-neutrophil cytoplasmic antibodies in

ulcerative colitis. Comparison with othercolitides and

diarrheal illnesses. Gastroenterology 1991; 100: 1590-6. 26 Rump JA,Worner I, Roth M, Scholmerich J, Hansch M,

Peter HH.p-ANCA of undefinedspecificityinulcerative colitis: correlationtodiseaseactivity and therapy.AdvExp

Med Biol 1993; 336: 507-13.

27Luukkonen P, Jarvinen H, Tanskanen M, Kahri A. Pouchitis-recurrenceoftheinflammatory bowel disease?

Gut 1994; 35: 243-6.

28 SandbomWJ. Pouchitisfollowing ileal pouch-anal

anasto-mosis: definition, pathogenesis, and treatment.