gut, histological and

Gut, 1991, 32, 424-429

Histological and immunohistochemical study of thegall bladder lesion in primary sclerosing cholangitis

G P Jeffrey, W D Reed, S Carrello, K B Shilkin

Department of Medicine,The University ofWestern Australia, Perth,AustraliaG P JeffreyW D Reed

Department ofHistopathology, SirCharles GairdnerHospital, Perth,AustraliaS CarrelloK B ShilkinCorrespondence to:Dr G P Jeffrey, Departmentof Medicine, The Universityof Western Australia,Nedlands 6009, WesternAustralia.Accepted for publication29 May 1990

AbstractThe histological features and type of mono-nuclear cell infiltrate in gail bladders from sixpatients with primary sclerosing cholangitiswere studied using routine staining techniquesand immunohistochemistry. Control studieswere performed using the gall bladders from sixpatients (age and sex matched) with chroniccholecystitis and four with primary biliarycirrhosis. A range of histological abnormalitieswas present in gall bladders from patients withprimary sclerosing cholangitis including a mildto moderate degree of epithelial hyperplasia,pseudogland formation, and mononuclear cellinfiltrate of the epithelium; moderate to severechronic inflammatory cell infiltrate and fibrosisaffecting the superficial and deep layers of thegail bladder wail; and minimal smooth musclehypertrophy. These abnormalities were non-specific and were also present in gall bladdersfrom patients with chronic cholecystitis andprimary biliary cirrhosis. Vasculitis and granu-lomas were not present in the patients withprimary sclerosing cholangitis. Immuno-histochemistry showed that the superficial anddeep mononuclear cell infiltrate in primarysclerosing cholangitis gail bladders wascomposed predominantly of lymphocytes, incontrast to chronic cholecystitis where macro-phages were found in similar or greaternumbers. Moreover, T lymphocytes (activatedand resting) were present throughout the lym-phocytic infiltrate and were apposed to thebase and interdigitated between the biliaryepithelial cells in significantly greater numbersthan in chronic cholecystitis gall bladders. Blymphocytes were present only in lymphoidfollicles. Comparative studies using liverbiopsy specimens from three of the primarysclerosing cholangitis patients showed asimilar T lymphocyte portal tract infiltrate. Weconclude that a number ofnon-specific chronicinflammatory histological abnormalities werepresent in primary sclerosing cholangitis gallbladders. Immunohistochemistry found otherfeatures that were present in this disease - apredominantly lymphocytic mononuclear cellinfiltrate of the superficial and deep layers ofthe gall bladder wail and the presence of Tlymphocytes that infiltrated the biliary epi-thelial cells. These findings support thehypothesis that aberrant cell mediated immunemechanisms may play a role in the patho-genesis of both the intrahepatic and extra-hepatic lesions in primary sclerosingcholangitis.

Primary sclerosing cholangitis is an importantand increasingly recognised cause of the vanish-

ing bile duct syndrome.' The characteristiccholangiographic features of multiple beads andstrictures are a result of chronic inflammationand obliterative fibrosis that affects the wholebiliary tree.2 Although the aetiology remainsunknown, evidence suggests that humoral andcell mediated immunological abnormalities playa role in the pathogenesis ofthis disorder. Studieshave reported the presence of hyperglobu-linaemia and circulating organ non-specific auto-antibodies,23 high values of circulating immunecomplexes in blood and bile,4" activation of thecomplement pathway (C3),6 and impaired Fcreceptor mediated systemic clearance of circulat-ing immune complexes.7 Cell mediated immuno-logical abnormalities found in this disorderinclude peripheral blood lymphocytes that aresensitised to bile antigens,8 T lymphocyte infil-trate of portal tracts and a deficiency in circulat-ing suppressor/cytotoxic T cells,9 and defectivesuppressor T cell function.' In addition,aberrant expression ofHLA-DR antigens, whichare a prerequisite for antigen presentation andinitiation of the immune response, have beenshown on biliary epithelium in primarysclerosing cholangitis."The pathognomonic lesion of intrahepatic

primary sclerosing cholangitis is reported to beinflammatory and fibrous obliteration of theinterlobular, septal, and segmental ducts withthe development of saccular and tubular biliarydilatations.'2 However, the extrahepatic biliarylesion in this disease, which may occur in isola-tion, has been less well characterised. The aim ofthis study was to review the histology anddetermine the nature of the inflammatory cellinfiltrate within the extrahepatic biliary lesion inprimary sclerosing cholangitis.

Methods

PATIENTSThe case records of 26 patients with primarysclerosing cholangitis attending the Sir CharlesGairdner Hospital Gastroenterology/Liver Unitwere reviewed. The diagnosis was based on thepresence of typical cholangiographic features inpatients who had not undergone previous bileduct surgery, other than simple cholecystec-tomy.`' Fourteen of these patients (58%) hadassociated ulcerative colitis. Cholecystectomyhad been performed in eight and the gall bladderspecimens from six of these patients were avail-able for study. The clinical characteristics ofthese patients are shown in Table I. Only onepatient had ulcerative colitis. None of theremaining five had symptoms suggestive ofulcerative colitis and fibreoptic colonoscopy orsigmoidoscopy with colonic biopsy specimen

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Histological and immunohistochemical study ofthe gall bladder lesion in primary sclerosing cholangitis

TABLE I Clinical, biochemical, and histological data ofpatients with primary sclerosing cholangitis at the time ofcholecystectomny

PatientNo

1 2 3 4 5 6

Sex M M F M F FAge (years) 70 72 44 49 64 44Indication for Possible chole- Recurrent Recurrent Possible chole- Possible chole- Recurrent

cholecystectomy docholithiasis pancreatitis pancreatitis docholithiasis docholithiasis pancreatitisERCP changes IH+EH IH IH+EH IH IH IH+EHUlcerative colitis No No No No No YesBilirubin ([mol/l)* 14 13 24 156 8 7ALP (N 100-350 U/i) 716 489 169 1273 920 1179Liver biopsy staget ND ND I II III NDCholelithiasis No No Yes No No No

ERCP=endoscopic retrograde cholangiopancreatographic; IH=intrahepatic; EH=extrahepatic; ALP=serum alkaline phosphatase;ND=not done.*Normal range 0-17 Wmol/l.tStaged according to LaRusso et al."

was normal in four of these. One patient refusedto have this procedure performed. Gall bladderspecimens from six patients (age and sexmatched) with chronic cholecystitis and fromfour with primary biliary cirrhosis were obtainedas controls. Liver biopsy specimens from threeprimary sclerosing cholangitis and all fourprimary biliary cirrhosis patients were availablefor comparative studies. All specimens had beenfixed in formalin and embedded in paraffin wax.

IMMUNOHISTOCHEMISTRYThe formalin fixed, paraffin embedded gallbladder and liver biopsy specimens were sec-tioned (5 [im), dewaxed, and blocked with 3%H202 in Tris buffered saline (TBS) (20mM Tris,500mM NaCl, pH 7T6) for five minutes and then20% normal swine serum in TBS for 20 minutes.The three stage immunoperoxidase techniquewas then performed to identify the type ofmononuclear cell infiltrate within the sections.Briefly, sections were incubated with optimallydiluted primary antibody overnight (Table II)

TABLE II Primary antibodies used in three stage immunoperoxidase technique

Antibody Reactivity Source Dilution

Monoclonal Strong; lymphocytes variable; Dakopatts 1:1002811+PD7/26 macrophages

Monoclonal L26 Strong; B cells Dakopatts 1:100Monoclonal UCHL1 Strong; activated T cells; some resting T Dakopatts 1:100

cellsPolyclonal muramidase Strong; monocytes/macrophages Dakopatts 1:400

TABLE iII Histological features ofgall bladders in primary sclerosing cholangitis (PSC) andchronic cholecystitis (CC)

Patients with PSC Patients with CC

Histological features 1 2 3 4 5 6 1 2 3 4 5 6

EpitheliumPapillary hyperplasia* 1 1 2 1 0 1 1 0 0 1 2 0Pseudoglands 1 1 1 1 1. 1 0 0 0 2 2 2Mononuclear cell infiltratet + + + + + + + + - + + -

InflammationSeverity 1 3 1 2 2 2 2 2 1 1 2 1Sitet S+D S SS S S S S D S D S+DFollicles - +.-+. + + + - - - - -

Smooth muscle hypertrophy 1 0 1 1 1 1 2 1 1 1 2 1Fibrosis

Severity 1 3 1 2 2 2 1 2 3 2 1 1Site S+D D D D D D D S+D D S D D

*0=absent, 1 =mild, 2=moderate, 3=severe.t+ =present, - =absent.fS=predominantly superficial, D=predominantly deep.

and washed in two changes of TBS for fiveminutes. Rabbit anti-mouse peroxidase con-jugated antibodies (Dakopatts) diluted 1/30 inTBS were added for 45 minutes, after which thesections were washed in TBS and then incubatedwith swine anti-rabbit peroxidase conjugatedantibodies (Dakopatts) diluted 1/30 in TBS for afurther 45 minutes. After washing in TBS thesections were covered with fresh diamino-benzidine (DAB) substrate (6 mg DAB in 10 mlTBS with 10 1l of 30% H202) for eight minutes,washed in TBS, counter stained with haema-toxylin, dehydrated, and mounted. Controllymph node sections and sections withoutprimary antibody were included in each assayrun.

EXAMINATION OF SECTIONSRoutine haematoxylin and eosin stained sectionswere reviewed blindly by two of us (KBS, GPJ).The presence, site, and severity (grade 0 to 3) ofthe following histological features were noted:biliary epithelial abnormalities (hyperplasia,pseudoglands, mononuclear cell infiltrate);inflammatory cell infiltrate (mononuclear cells,polymorphs); smooth muscle hypertrophy;fibrosis; vasculitis; and granuloma. Theimmunoperoxidase sections were also viewedblindly and the site, cell type, and severity of themononuclear cell infiltrate in each gall bladderwas estimated and graded using a scale from 0 to3 (0=absent, 1 =mild, 2=moderate, 3=severe).A semiquantitative method was used to estimatethe number of labelled cells that infiltrated thebiliary epithelium. The number of infiltratinglabelled cells in two high power fields wascounted and the mean cell number calculated.Those two areas of biliary epithelium that weremost severely affected by the inflammatory cellinfiltrate were chosen for each section.

STATISTICSStatistical analysis was performed using theMann-Whitney U test (two tailed) and X2 withYates's correction.

ResultsThe histological features present in the haem-atoxylin and eosin stained sections of gall blad-

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Figure 1: Haematoxylin and eosin staining ofa primary sclerosing cholangitis gall bladdershowing a dense mononuclear cell infiltrate in the superficial layers. (Original magnificationx250.)

ders from patients with primary sclerosingcholangitis compared with those from patientswith chronic cholecystitis are found in Table III.No distinguishing features characteristic of pri-mary sclerosing cholangitis were identified.Epithelial changes of papillary hyperplasia andpseudogland formation were present in bothdisorders. Although mononuclear cell infiltra-tion of the epithelium was present more fre-

B % sJ--F.Pis

Figure 2: Haematoxylzn and eosin staining ofa primary biliary cirrhosis gall bladdershowing: (A) superficial granulomas. (Original magnification x 100.) (B) Granulomainvolving the biliary epithelium. The arrow shows an epithelioid histiocyte. (Originalmagnification x400.)

quently in primary sclerosing cholangitis, it wasalso found in chronic cholecystitis. The site andseverity of inflammation within the gall bladderwall was also similar in both disorders, althoughfollicles were present more often in primarysclerosing cholangitis. However, the differencewas not significant (p>O 1). The cell infiltrate inprimary sclerosing cholangitis gall bladderswas predominantly mononuclear cells withoccasional polymorphonuclear cells (Fig 1).Smooth muscle hypertrophy and fibrosis werealso present to the same degree in primarysclerosing cholangitis and chronic cholecystitisgall bladders. The presence and severity ofhistological abnormalities in primary sclerosingcholangitis gall bladders did not correlate withthe presence of extrahepatic cholangiographicabnormalities. Vasculitis and granulomas werenot found in these gall bladders. A similar rangeof histological features was present in the fourprimary biliary cirrhosis gall bladders, althoughin one, multiple epithelioid granulomas werepresent in the superficial layers and affected thebiliary epithelium (Fig 2).Immunohistochemical staining of gall blad-

ders from patients with primary sclerosingcholangitis showed that the inflammatory cellinfiltrate was composed predominantly oflymphocytes (Table IV). Most of these cells wereT lymphocytes (active and resting), while Blymphocytes were present only in lymphoidfollicles (Fig 3). Comparative studies using liverbiopsy specimens from three primary sclerosingcholangitis patients found a similar T lympho-cyte infiltrate in portal tracts. In contrast, macro-phages were found in equal or greater numbersthan lymphocytes in the inflammatory cell infil-trate in chronic cholecystitis gall bladders. Gallbladders from patients with primary biliarycirrhosis had less severe inflammation than thosefrom patients with primary sclerosingcholangitis, although lymphocytes were the pre-dominant cell type. All four liver biopsy speci-mens from patients with primary biliary cirrhosishad a T lymphocyte infiltrate within the portaltracts which was similar to the findings inprimary sclerosing cholangitis.T lymphocytes were frequently found infiltrat-

ing the biliary epithelium of primary sclerosingcholangitis gall bladders, whereas B lymphocytesand macrophages were not present. T lympho-cytes were apposed to the base and interdigitatedbetween the epithelial cells (Fig 4). The numberofT lymphocytes per high power field infiltratingthe biliary epithelium in primary sclerosingcholangitis gall bladders (mean 21 5) was signifi-cantly more than in chronic cholecystitis gallbladders (mean 1-2; p<001) (Fig 5). Primarybiliary cirrhosis gall bladders also had fewer Tlymphocytes per high power field infiltrating thebiliary epithelium (mean 6), but the differencewas outside the range of significance (p=0052).This result was due mainly to the primary biliarycirrhosis patient with multiple superficial granu-lomas who had a mean of 14 T lymphocytes perhigh power field.

DiscussionGall bladder involvement in primary sclerosing

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Histological and immunohistochemical study of the gall bladder lesion in primary sclerosing cholangitis

TABLE IV Immunohistochemical typing ofgall bladder mononuclear cell infiltrate

Lymphocytes T lymphocytes B lymphocytes Macrophages

Patients Site* Severityt Site Severity Site Severity Site Severity

Primary sclerosing cholangitis1 S+D 1 S+D 1 NP 0 D 12 S+D 3 S+D 3 CF 1 D 23 S 1 S 1 NP 0 NP 04 S 2 S 1 CF 1 S 05 S 2 S 2 CF 1 S+D 16 S 2 S 2 CF 1 S+D 1

Chronic cholecystitis1 S 2 S 2 CF 1 S 32 S 2 S 1 CF 1 S 23 D 1 D 1 D 1 NP 04 D 1 D 1 NP 0 S+D 25 S+D 1 S+D 1 NP 0 S+D 16 S+D 1 S+D 1 S 1 S+D 0

Primary biliary cirrhosis1 S+D 1 S+D 1 CF 1 D 12 S+D 1 S+D 1 NP 0 D 13 D 1 D 1 NP 0 S+D 14 S 2 S 2 NP 0 S 3

*S=predominantly superficial, D=predominantly deep, CF=centre of follicle, NP=not present.t°=absent, 1 =mild, 2=moderate, 3=severe.

cholangitis has been recognised since the firstreports of this disorder were published,'4 but fewdetailed histological studies have been per-formed. An early study of gall bladders inprimary sclerosing cholangitis found that thestriking feature of this lesion was the presence oflymphoid aggregates and abundant plasma cellswithin the lamina propria.'5 A case reportdescribed the presence of mucosal ulceration,

Figure 3: Immunoperoxidase staining ofa primary sclerosing cholangitis gall bladder showinga predominant T lymphocyte mononuclear cell infiltrate: (A) Positive T lymphocyte stainingusing UCHL 1. (B) Negative B lymphocyte staining using L26. (Original magnificationx400.)

granulomatous inflammation, and fibrosis withinthe gall bladder wall,'6 and another found elon-gated mucosal papillary folds, pseudoglandularinvaginations, and noticeable chronic inflamma-tory fibrosis, principally involving the serosa. 7 Amore recent study of gall bladder disease inprimary sclerosing cholangitis patients foundthat 26% had gall stones, 15% had probableprimary sclerosing cholangitis affecting the gallbladder, and 4% had neoplasms (benign andmalignant).'8 Detailed histological features werenot reported, however, and no pathognomonicfeatures of the disease were identified.Only one of the six primary sclerosing

cholangitis patients in this study had ulcerativecolitis, although asymptomatic disease may havebeen present in one other patient who refusedfibreoptic sigmoidoscopy. This finding is notrepresentative of the proportion of patients withulcerative colitis in the total primary sclerosingcholangitis population, which was 58%. As aconsequence, those patients without ulcerativecolitis included in this study may be somewhatatypical, although the ulcerative colitis in thisdisorder is usually only mildly symptomatic andhas prolonged periods ofremission.2 In this studyno histological abnormality present in routinehaematoxylin and eosin stained sections ofprimary sclerosing cholangitis gall bladders wasidentified as being specific for this disorder. Theseverity and type of inflammation, fibrosis,smooth muscle hypertrophy, and epithelialabnormalities in primary sclerosing cholangitiswere similar to those present in chroniccholecystitis and primary biliary cirrhosis. Thepresence of lymphoid follicles associated withmoderately severe chronic inflammation was theonly feature that tended to occur more frequentlyin primary sclerosing cholangitis gall bladders,similar to the findings of Thorpe et al. '5Granulomatous inflammation was not present inany primary sclerosing cholangitis gall bladder incontrast with a previous report.'6 One primarybiliary cirrhosis gall bladder, however, hadmultiple granuloma within the submucosa andepithelial cell layer. Extrahepatic granuloma inthis disease have previously been reported inliver hilar lymph nodes, omentum, and lung."'The presence of gall bladder granulomas in

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Figure 4: Immunoperoxidase staining ofa primary sclerosing cholangitis gall bladder showingpositive staining ofT lymphocytes: (A) Numerous T lymphocytes apposed to the base ofbiliaryepithelial cells. (Original magnification x 100.) (B) High powerfield ofT lymphocytesinfiltrating the biliary epithelium. The arrow shows a T lymphocyte interdigitating betweenepithelial cells. (Original magnification x400.)

primary biliary cirrhosis suggests that lympho-cytes may also be sensitised to extrahepaticbiliary epithelium as well as intrahepatic bileducts and this finding requires further investiga-tion.Damage to the hepatic artery and peribiliary

vascular plexus has been associated withthe development of cholangiographic featurescharacteristic of primary sclerosing cholangitis.Hepatic artery infusion with 5-fluorodeoxy-uridine in patients with hepatic metastasesfrom colorectal adenocarcinoma has resulted inmultiple biliary strictures typical of primarysclerosing cholangitis.2` The infusion is thoughtto produce toxic hepatic arterial damage andnarrowing which results in ischaemic stricturesof the intrahepatic and extrahepatic bile ducts.Hepatic artery embolisation with alcohol in ananimal model has produced similar biliarylesions.2' Moreover, a patient with polyarteritisnodosa that affected the hepatic artery exten-sively was reported to have the cholangiographicfeatures of primary sclerosing cholangitis.22 Itwas postulated that vasculitis of the peribiliaryvascular plexus resulted in multiple biliary stric-tures and dilatations. Severe occlusive phlebitishas also been described in a primary sclerosingcholangitis gall bladder.'7 This lesion was

thought to be a specific feature of the disease and

l

Primary Chronic Primaryscierosing cholecystitis biliarycholangitis cirrhosis

Figure 5: The number ofT lymphocytes per high powerfieldinfiltrating the biliary epithelium in pnrmary sclerosingcholangitis, chronic cholecystitis, and primary biliarycirrhosis gall bladders. indicates mean number.

was implicated in its pathogenesis. Vasculitis wasnot found in any primary sclerosing cholangitisgall bladder in the present study, however, andthis suggests that while hepatic artery damagemay produce secondary ischaemic biliary stric-tures, it is unlikely to play a role in the patho-genesis of the disorder.The portal tract mononuclear cell infiltrate

present in both precirrhotic and cirrhoticpatients with primary sclerosing cholangitis haspreviously been shown to consist predominantlyof T lymphocytes.923 The ratio of T4 (helper/inducer) to T8 (suppressor/cytotoxic) cells with-in this infiltrate was similar to that found inprimary biliary cirrhosis patients. T lymphocyteshave also been shown to infiltrate the epitheliumof large and medium sized intrahepatic bile ductsin primary sclerosing cholangitis.2923 Similar Tlymphocyte infiltration occurs in other diseaseswhich result in the vanishing bile duct syndrome;namely primary biliary cirrhosis,24 chronic graftrejection,25 and grafts versus host disease.26 It hasbeen postulated that in all these disorders bileduct antigens co-expressed with either class 1 or 2major histocompatibility antigens induce animmune response directed at bile ducts or are atarget for immune effector cells, or both. In thisstudy, infiltrating T lymphocytes (activated andresting) were found within the extrahepaticbiliary epithelium as well as the portal tracts inpatients with primary sclerosing cholangitis.This suggests that lymphocytes in this disease aresensitised to a biliary antigen that is present inboth the extrahepatic and intrahepatic bile ductsand is consistent with the distribution of thebiliary lesions in this disorder. Moreover, infil-trating T lymphocytes were present in the gallbladder wall and biliary epithelium of threeprimary sclerosing cholangitis patients withcholangiographic abnormalities that were limitedto the intrahepatic biliary tree. This also suggeststhat cholangiography may not always detect thepresence of extrahepatic disease, especially if theextrahepatic lesion consists predominantly of

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Histological and immunohistochemical study ofthe gall bladder lesion in primary sclerosing cholangitis 429

inflammatory changes without any appreciablefibrosis and stricturing.

In conclusion, this study has shown that a widerange of non-specific histological features ofchronic inflammation and fibrosis were presentin routine haematoxylin and eosin stainedsections of primary sclerosing cholangitis gallbladders. Immunohistochemistry found otherfeatures that were present in the disease, andthese consisted of a lymphocytic mononuclearcell infiltrate that affected all layers of the gallbladder wall and the presence ofT lymphocytes(activated and resting) which infiltrated thebiliary epithelium. These findings support thehypothesis that lymphocytes in primary scleros-ing cholangitis are sensitised to a biliary antigenand may play a role in the pathogenesis of boththe intrahepatic and extrahepatic lesions inthis disorder. Further studies investigating Tlymphocyte subtypes and biliary epithelialexpression of class 1 and 2 major histocom-patibility antigens in optimally fixed primarysclerosing cholangitis gall bladders are planned.

Gary Jeffrey holds a NH&MRC Medical Postgraduate ResearchScholarship and the study was supported by a grant from the SirCharles Gairdner Hospital Research Foundation. We thank FranGeste for typing the manuscript.

1 Sherlock S. The syndrome of disappearing intrahepatic bileducts. Lancet 1987; ii: 493-6.

2 Chapman RW, Marborgh BA, Rhodes JM, et al. Primarysclerosing cholangitis: a review of its clinical features,cholangiography and hepatic histology. Gut 1980; 21:870-7.

3 Jeffrey GP, Reed WD, Laurence BH, Shilkin KB. Primarysclerosing cholangitis - a clinical and immunopathologicalreview of 21 cases. J Gastroenterol Hepatol 1990; 5: 135-40.

4 Bodenheimer HC, LaRusso NF, Thayer WR, Charland C,Staples P, Ludwig J. Elevated circulating immune com-plexes in primary sclerosing cholangitis. Hepatology 1983; 3:150-4.

5 Alberti-Flor JJ, Medina M, Jeffers L, Schultz DR, Schiff ER.Elevated levels of inumunoglobulins and immune complexesin the bile of patients with primary sclerosing cholangitis.AmJ Gastroenterol 1986; 81: 325-8.

6 Brinch L, Triesberg P, Schrumpf E, Akesson I. The in vivometabolism of C3 in hepatobiliary disease associated withulcerative colitis. ScandJ Gastroenterol 1982; 17: 523-7.

7 Minuk GY, Angus M, Brickman CM, et al. Abnormalclearance of immune complexes from the circulation ofpatients with primary sclerosing cholangitis. Gastroenterol-ogy 1985; 88: 166-70.

8 MacFarlane IG, Wojcicka BM, Tsantoulas DC, Portmann BL,Eddleston ALWF, Williams R. Leukocyte migration inhibi-tion in response to biliary antigens in primary biliarycirrhosis, sclerosing cholangitis and other chronic liverdiseases. Gastroenterology 1979; 76: 1333-40.

9 Whiteside TL, Lasky S, Si L, Van Thiel DH. Immunologicalanalysis of mononuclear cells in liver tissue and blood ofpatients with primary sclerosing cholangitis. Hepatology1985; 5:468-74.

10 Magrin S, Nouri-Aria KT, Donaldson PT, et al. The relation-ship between HLA-DR3 and T-cell regulation of immuno-globulin production in primary sclerosing cholangitis. ClinImmunol Immunopathol 1989; 50: 205-12.

11 Chapman RW, Kelly PMA, Heryet A, Jewell DP, FlemingKA. Expression of HLA-DR antigens on bile duct epithe-lium in primary sclerosing cholangitis. Gut 1988; 29: 422-7.

12 Ludwig J, MacCarty RL, LaRusso NF, Krom RAF, WiesnerRH. Intrahepatic cholangiectasis and large duct obstructionin primary sclerosing cholangitis. Hepatology 1986; 6: 560-8.

13 LaRusso NF, Wiesner RH, Ludwig J, MacCarty RL. Primarysclerosing cholangitis. N Engl J Med 1984; 310: 899-903.

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16 Goldgraber MB, Kirsner JB. Chronic granulomatous chole-cystitis and chronic fibrosing choledochitis associated withchronic ulcerative colitis. A case report. Gastroenterology1960; 38: 821-8.

17 Case records of the Massachusetts General Hospital. N EnglJMed 1982; 306: 349-58.

18 Brandt DJ, MacCarty RL, Charboneau JW, LaRusso NF,Wiesner RH, Ludwig J. Gallbladder disease in patients withprimary sclerosing cholangitis. AJR 1988; 150: 571-4.

19 Fox RA, James DG, Scheuer PJ, Sharma 0, Sherlock S.Impaired delayed hypersensitivity in primary biliary cir-rhosis. Lancet 1969; i: 959-62.

20 Kemeny MM, Battifora H, Blayney DM, et al. Sclerosingcholangitis after continuous hepatic artery infusions ofFUDR. Ann Surg 1985; 202: 176-81.

21 Doppman JL, Girton ME. Bile duct scarring following ethanolembolization of the hepatic artery. An experimental study inmonkeys. Radiolog 1984; 152: 621-6.

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24 Scheuer PJ. Liver biopsy interpretation. 3rd ed. London:Bailliere Tindall, 1980: 36-59.

25 Vierling JM, Fennell RH. Histopathology of early and latehuman hepatic allograft rejection: evidence of progressivedestruction of interlobular bile ducts. Hepatology 1985; 5:1076-82.

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