Gut 1995; 36: A949-A952

British Association for the Study of the Liver

The 1995 Spring Meeting of the British Association for the Study of the liver was held at King's College, Chelsea 23-24February 1995. The 19 abstracts selected for presentation by the Programme Committee are printed below.

Fulminant hepatitis on withdrawal ofchemotherapy in chronic carriers ofhepatitis C virus (HCV)

S VENTO, L GUELLA, F MIRANDOLA, L COSCO,T FERRARO, E CONCIA (Department ofInfectiousDiseases, University of Verona; InfectiousDisease Unit, 'Pugliese' Hospital, Catanzaro,Italy) Acute liver failure following withdrawalof chemotherapy has been described inpatients with HBV related chronic liver dis-ease; in contrast, no cases have been reportedso far in patients with HCV infection. Wedescribe two such cases.

Case 1. Forty year old man with a two yearhistory of chronic hepatitis C (genotype lb)and non-Hodgkin's lymphoma treated for 10weeks with adriamycin, cyclophosphamide,vincristine, bleomycin, etoposide, pred-nisolone. Two weeks after starting pred-nisolone reduction from 50 to 10 mg daily,fulminant hepatitis developed (ALT 6030IU/L, AST 3575, bilirubin 17-2 mg/dl, pro-thrombin 15% of normal, grade III coma).Serum HCV-RNA was positive (102 genomeequivalents/ml); HBsAg, IgM anti-HBc,HDV, HAV, CMV, EBV, HIV antibodies,ANA, AMA, SMA, LKM were negative. Thepatient died six days later; necropsy showedmassive hepatocyte necrosis.

Case 2. Thirty one year old man with a sixyear history of chronic hepatitis C (genotype2a) and Hodgkin's lymphoma treated withfour courses of doxorubicin, bleomycin,vinblastine, dacarbazine. At the end of treat-ment HCV-RNA was very high (108/ml);ALT was 98 IUlL, AST 64. Twenty four daysafter stopping treatment fulminant hepatitisdeveloped (ALT 3870, AST 3220, bilirubin11-8 mg/dl, prothrombin 22%, grade IIcoma). Serum HCV-RNA was reduced to102/ml. Autoantibodies, HIV abs, HBsAg,IgM anti-HBc, HDV, HAV, CMV, EBV werenegative. The patient recovered in 50 daysbut HCV-RNA remained positive.

Massive liver cell necrosis can occur inchronic carriers of HCV on withdrawal ofchemotherapy, suggesting an immune medi-ated mechanism of hepatocyte damage.Monitoring of aminotransferases is manda-tory in HCV infected patients receivingcytotoxic treatments.

Longitudinal genotype analysis andquantification ofhepatitis C virus (HCV)in haemophilic patients undergoing a-interferon therapy

H L DEVEREUX, P T TELFER, D BROWN,A MORRIS, G M DUSHEIKO, V C EMERY, C A LEE(The Royal Free Hospital and School ofMedicine, Pond Street, London NW3 2QG)Six major HCV genotypes have beendescribed so far and 1-3 are common in theindigenous UK population. The aim of thisstudy was to assess the changes in viral loadand composition of the HCV quasispeciesin five haemophilic patients receiving a-interferon therapy using the four methodscurrently available for HCV genotyping -RFLP analysis, type specific primers,

serotyping, and DNA sequencing. Patients 1and 2 had low levels of viraemia, genotypes 1and 3 respectively, and had a complete ALTand PCR response to therapy. Patient 3 had ahigher level of viraemia, genotype 2, and hada complete ALT response. Patient 4 had thehighest level of viraemia, genotypes 1+2, andhad a partial ALT response. Patient 5 had asimilar level of viraemia to patient 3, genotype3, and had no response to therapy. The mostconsistent genotype results were obtainedusing RFLP analysis when compared with theDNA sequence analysis, and showed that allpatients had mixed infections and that geno-type changes occur frequently in patientsundergoing a-interferon therapy. This studyshows the difficulties in studying patients withmixed HCV genotype infections, and thatfrequent sampling is necessary to monitorresponse to a-interferon therapy.

The use of hepatitis C virus (HCV)quantitation in patients treated withinterferon

M M AHMED, B A B MARTIN, E ELIAS,R HARRISON, K O'DONNELL, J SHAW,D J MUTIMER (Liver Unit, Queen ElizabethHospital, Birmingham, UK) We havedeveloped a method for quantitating HCV bycoamplifying a constant amount of viralcDNA with varying amounts of a mutantcDNA fragment (containing the lac operatorsequence) using multiple nested PCRs. Themutant PCR products are detectedcolorimetrically following capture ontostreptavidin coated microtitre wells. TargetcDNA levels are derived from a quantitationcurve.Twenty two patients with chronic HCV

(RIBA positive) were treated with interferon(IFN): 3 MU, three times a week for up to 12months. Biochemical response to IFN wasdivided into three types: longterm response(LTR) with >6 months follow up, completeresponse with relapse after treatment (CR/R),and no/partial response (NR). Pretreatmentliver biopsy specimens were graded accordingto a modified Knodell histological activityindex (HAI, range 0-13, 13 is worst).Pretreatment serum samples were quantitatedfor HCV. In seven patients, sequential serumsamples (once before treatment, four timesduring treatment, and once after treatment)were quantitated for HCV to monitorvirological response.No correlation was found between pre-

treatment HCV cDNA levels and initial ALT(r=-0 095), initial HAI (r=-0 44) orpattem of biochemical response to IFN(p=0-57, Kruskal-Wallis test). SequentialHCV levels measured in seven patients(3 LTR, 2 CR/R, and 2 NR) complementedchanges in ALT.

In conclusion, competitive PCR using amutant cDNA fragment and colorimetricdetection of the PCR products provides arapid, reproducible, and non-radioactivemethod for HCV cDNA quantitation. HCVcDNA levels may be used to monitorresponse to IFN especially in patients who

have normal pretreatment ALT. We wereunable to show a relation between initial viralload and biochemical response.

Incidence, character, and clinicalrelevance of cryoglobulinaemia inchronic hepatitis C virus (HCV) infection

V S WONG, W EGNER, T ELSIE, D BROWN,G J M ALEXANDER (Departments of Histo-pathology, Community Health & Radiology,and University Department of Medicine,Addenbrooke's NHS Trust, Cambridge) Onehundred and thirteen consecutive patientswith chronic HCV and varied histologicalfeatures were evaluated prospectively to deter-mine the incidence, character, and clinicalrelevance of mixed cryoglobulinaemia.Twenty one of 113 (18.6%) patients had

cryoglobulinaemia at a concentration of 623mg/l (controls <55 mg/l). Thirteen of 15patients characterised had type III cryoglobu-linaemia and two type II. IgGl was present incryoglobulins in 14/15, IgG3 in 11/15, IgG27/15, and IgG4 in 3/15 with warm IgMrheumatoid factor present in 9/15.Autoantibodies and rheumatoid factor wererarely detected in serum and there was noevidence of complement depletion.

Only two patients (with type II disease)had evidence of systemic disease and renalinvolvement, both had classic pathwaycomplement activation. There was noevidence of renal disease or vasculitis orcomplement consumption in any of theremaining patients with cryoglobulinaemia.Thus although cryoglobulinaemia is

common in chronic HCV infection and pre-dominantly type III, evidence of systemic orrenal disease is rare. The cryoglobulinscomprised mixed IgG/IgM antibody, but it isnot certain whether they have anti-HCVspecificity.

Liver biopsy is essential in the assess-ment of patients with chronic hepatitis Cvirus (HCV)

V S WONG, S CARONIA, D G D WIGHT,P D BRITTON, C PALMER, G J M ALEXANDER(Departments of Histopathology, CommunityHealth & Radiology, and University Departmentof Medicine, Addenbrooke's NHS Trust,Cambridge) Chronic HCV is an important,common cause of cirrhosis. To assess the roleof liver biopsy a prospective study of 140patients seropositive for anti-HCV wasundertaken. In addition to a full history,demographic, clinical, biochemical, andultrasonographic details were collated.Fibrosis was scored 0-5. Each parameterwas determined by linear regression or x2univariate analysis and by multiple regressionfrom among age, sex, estimated duration ofinfection, clinical findings, ALT, andultrasound features and alcohol intake.

Median age was 36-5 (range 15-77) with106 men. Risk factors were: intravenous druguse in 87 (62%), blood transfusion in 37(24%), haemophilia in four (3%), and

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unknown in 16 (11 %). In 114 the meanmaximum estimated duration of infection was11 years (range 1-33). Excessive alcoholintake was found in 60 (42%). Most (75%)were asymptomatic, but only 10% werewithout fibrosis; grade 1, 11%; grade 2, 41%;grade 3, 21%; grade 4, 10%; grade 5, 7%.

Univariate analysis showed increasing ageand abnormal ultrasound features wereweakly associated with grade 4 or 5 fibrosis(p<0.05). However, by multiple regressionanalysis we could not identify any factorswhich could predict the degree of fibrosis.The inability to determine severity by

conventional clinical assessment, bio-chemistry or ultrasound shows that biopsy iscritical for patient management; this policyhas particular implications for a haemophiliacpopulation.

DNA haplotypes of CA dinucleotiderepeats associated with the Wilson'sdisease gene

P A MORRIS, D CURTIS*, 0 W QUARRELL*,M S TANNER (University Department ofPaediatrics and *Centre for Human Genetics,Children's Hospital, Sheffield S10 2TH)The gene for Wilson's disease (WD) hasrecently been sequenced and to date 29mutations identified. A number of micro-satellite markers around the WD locus showlinkage disequilibrium. We have used threehighly polymorphic markers (D13S314,D13S301, D13S296) with non-random dis-tribution on chromosomes carrying a WDmutation in other populations. To correlatehaplotypes with mutations in the Britishpopulation we have established allelefrequencies and haplotypes in 20 informationfamilies.Frequencies differ from data in the more

D13S314 D13S301 D13S296

Allele Frequency Allele Frequency Allele Frequency

8 0.01 2 0-14 1 0-019 0-06 3 0.04 2 0-0210 0-20 4 0.37 3 0.0710a* 0-14 5 0-14 4 0.3511 0-14 6 0-18 5 0-111la* 0-13 7 0.05 6 0-1112 0-25 8 0-06 7 0-1812a* 0-06 10 0-01 8 0-1313* 0-01 13* 0-01 10* 0-01

13* 0-01

*Alleles not previously identified in other populations.

heterogeneous North American populations.' 2Most common haplotypes in our popula-

tion are 12-4-7, 12a-4-7, and 10-4-4,accounting for 25% of total. Locus D 13S314shows a different allele frequency to publisheddata and not the expected haplotype distribu-tion. Haplotypes of British WD alleles are

being analysed at present, which means thatin future asymptomatic affected siblings couldbe diagnosed and penicillamine therapystarted earlier to reduce liver damage bycopper accumulation.

1 Petrukin, et al. Nature Genet 1993; 5: 338-43.2 Thomas, et ad. Am Hum Genet 1994; 54: 71-8.

IgG Subclass distribution of anti-neutrophil cytoplasmic antibodies inprimary sclerosing cholangitis andulcerative colitis

D S BANSI, R W CHAPMAN, K A FLEMING*

(Department of Gastroenterology and *NuffieldDepartment ofPathology & Bacteriology, Oxford

Radcliffe Hospital, Headington, Oxford OX39DU) Perinuclear antineutrophil cytoplasmicantibodies (ANCA) are found in primarysclerosing cholangitis (PSC) and ulcerativecolitis (UC) and are predominantly of IgGclass. The aim was to study IgG subclassdistribution of ANCA in PSC and UC.Patients: 33 PSC (23 male, mean age 56) and35 UC (15 males, mean age 51) were studied.Twenty eight PSC had UC, four had Crohn'scolitis, and one had PSC only. The IgGsubclass distribution was determined by analkaline phosphatase method using mono-clonal antibodies, HP 6001 for IgGI, HP6002 for IgG2, HP 6050 for IgG3, and SK 44for IgG4 (Sigma Immunochemicals).Four PSC and 3 UC tested over time showed

IgGI IgG2 IgG3 IgG4

UC (n=35) 91% 3% 14% 0%PSC only (n= 1) 0% 0% 100% 0%PSC+Crohn's (n=4) 100% 25% 50% 25%PSC+UC (n=28) 96% 14% 25% 11%All PSC (n=33) 94% 15% 30% 12%

no modification ofANCA IgG subclass.In conclusion the ANCA IgG subclass dis-

tribution in PSC and UC differs from that invasculitides, in which IgGI and IgG4 pre-dominate. Compared with UC, PSC serashow an increase in the IgG2, IgG3, andIgG4 subclasses. This suggests there may bedifferences in immune regulation and possiblya different antigenic species.

T-celi responses to mycobacterialextract (tuberculin-PPD) in patientswith primary biliary cirrhosis (PBC)

D E J JONES, 0 F W JAMES, A G DIAMOND,M F BASSENDINE (Department of Medicine,University of Newcastle upon Tyne) It has beensuggested that mycobacteria may play apotential pathogenic part in PBC.1 Toexamine this hypothesis we have undertaken astudy to compare the T-cell proliferativeresponses to tuberculin-PPD (prepared fromheat treated products of growth and lysis ofmycobacteria) in 60 PBC patients and 58controls (33 chronic liver disease and 25normal).Mean stimulation index (SI) in response to

PPD was 21-9±26-9 for PBC patients,46-5±89 for normal controls, and 26-8+50for chronic liver disease controls (NS).Positive responses (SI>2-95=mean control+2 SD) were seen in 44/60 patients, 17/25normal controls, and 27/33 chronic liverdisease controls (p=NS).

There is no difference in the frequency andmagnitude of proliferative responses to PPDin PBC patients and controls. These data failto confirm previous work suggestinggeneralised impairment of T-cell responses inPBC. In addition, it makes it highly unlikelythat chronic infection with mycobacteriaplays any part in the aetiology of PBC.1 J'Hepatol 1994; 21: 673-7.

The role of radiological intervention asthe first line treatment ofhepatic outflowobstruction

A E A MAHMOOD, S OLIFF, A MENDOZA, R WEST,J BUCKELS, E ELIAS (The Liver & HepatobiliaryUnit, Queen Elizabeth Hospital, Edgbaston,Birmingham, UK) Re-establishment of con-ventional hepatic venous outflow is the mostphysiological outcome of all therapeutic

modalities for treating hepatic outflowobstruction (HOO). We have recently usedradiological intervention (RI) in an attempt toachieve this in all new patients presentingwith HOO. Thirteen patients with HOO weretreated with balloon angioplasty or stentplacement. The mean age at presentation was38-6 years (range 21-65). Hepaticvenography showed IVC block in four, allhepatic veins blocked in five, and single ortwo hepatic veins block in four patients. MeanLFTs values; AST 328, bilirubin 54 9, alka-line phosphatase 401, and albumin 31. Twopatients were treated with stent placement,nine patients with balloon angioplasties(range 2-3/patient), and two patients weretreated with both. This was achieved by acaval approach in 10 but transhepatic percu-taneous puncture was necessary to gain accessto a segment of hepatic vein for recanalisationand subsequent dilatation in three patients.Clinical and biochemical improvement wasseen in nine patients (70%). Mean follow upwas 2-8 years (range 0.5-8). Radiologicalintervention was unsuccessful in relievinghepatic outflow obstruction in four patients.

Radiological intervention can be success-fully used as the sole treatment of HOOwith a favourable longterm prognosis.Furthermore, it provides a more physiologicalmethod of relieving hepatic outflowobstruction compared with other therapeuticmodalities.

Multicentre trial of octreotide versusinjection sclerotherapy (IS) for acutevariceal haemorrhage

S A JENKINS1, R SHIELDS1, R SUTTON1,A N KINGSNORTH1, M DAVIES2, E ELIAS2,A J TURNBULL3, M F BASSENDINE3, 0 F WJAMES3, J P IREDALE4, S K VYAS4, M J P ARTHUR4(1Royal Liverpool University Hospital, 2QueenElizabeth Hospital, 3Freeman Hospital,4Southampton General Hospital) The role ofoctreotide in the treatment of varicealhaemorrhage remains controversial. Weconducted a multicentre trial to assess the useof 50 pg/h intravenous octreotide for 48 hoursto control variceal haemorrhage. Consecutivepatients with endoscopically confirmedvariceal haemorrhage were randomised toeither octreotide (n=73) or emergency IS(n=77). Overall control of bleeding was notsignificantly different between octreotide(85%) and IS (82%) over the 48 hour trialperiod (relative risk of rebleeding 0-83; 95%CI 0-38, 1-82). Octreotide was as effective asIS irrespective of the severity of the liverdisease, and in those actively bleeding atendoscopy. Mortality during the 48 hour trialperiod was identical in the two groups, butmore patients died in the octreotide groupduring 60 days of follow up, although this didnot reach statistical significance (relative riskof dying at 60 days 1.91; 95% CI 0.97, 3-78;p=006). The results of this study show thatintravenous octreotide is as effective as IS inthe control of acute variceal haemorrhage.However, in view of the trend towards anincreased 60 day mortality in the octreotidegroup, further trials are necessary to evaluateits safety in variceal bleeding.

Functional assessment of liver allograftsusing CT perfusion imaging

R SINNATAMBY, K A MILES, M P HAYBALL, C JWATSON, G J M ALEXANDER (Department ofRadiology, Medical Physics, Surgery and

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University Department of Medicine,Addenbrooke's NHS Trust, Cambridge) Toquantitate arterial and portal perfusion ofliver allografts, CT perfusion imaging studieswere performed in 16 subjects. Hepaticarterial and portal perfusion were calculatedand functional perfusion images constructed.Perfusion was correlated with outcome,measured as survival of patient or allograft,chronic rejection (confirmed histologically),anastomotic failure, and liver biochemistry.On four occasions renal perfusion wasevaluated.

Combined arterial and portal perfusiongreater than 0-6 ml/min/ml was associatedwith good allograft function in six (of six)patients. All nine patients with reduced perfu-sion had abnormal liver function. Of these,three subsequently developed chronic rejec-tion and two suffered biliary anastomoticfailure. Functional images showed regionalvariations in perfusion. Significant reductionin renal cortical perfusion was associated withcyclosporin toxicity.CT perfusion imaging enables non-

invasive measurement of hepatic arterial andportal perfusion, combined with high spatialresolution. Measured perfusion is shown tocorrelate with allograft function and clinicaloutcome. Functional images show regionalperfusion abnormalities. This technique mayallow early detection of chronic rejection.Perfusion CT should become a valuable toolin functional assessment of liver transplantpatients.

The plasminogen activating system inhepatic lipocytes and its modulation byretinoic acid

H LEYLAND, R C BENYON, S K VYAS, M J PARTHUR (University Medicine, University ofSouthampton) Urokinase plasminogenactivator (uPA) generates plasmin, a processinhibited by plasminogen activator inhibitor(PAI-I). Plasmin degrades extracellularmatrix directly and by activation of metallo-proteinases MMP-1 and MMP-3. Aslipocytes are important in matrix homeo-stasis in the liver, their contribution toplasmin generation was assessed. Lipocytes(>98% pure) were prepared from rat liverand cultured on plastic. Northern blotanalysis showed that mRNA for PAI-I(3-2 kb), uPA (2-6 kb), and uPA recep-tor (uPAR) (1-4 kb) were expressedconstitutively by lipocytes cultured for 3, 7,14, or 21 days (n=3). PAI-I (55 kD) anduPA (48 kD) protein were both secreted bycultured lipocytes as shown by zymographicanalysis of media. Chromogenic assay ofuPA in media gave the following results(uPA pg/ml/gg DNA, n=3): 3 daysundetectable, 7 days 165±36; 14 days30±17; 21 days 6±9. As lipocytes containretinoids and release them on activation, theeffect of exogenous retinoic acid (RAc) onuPA secretion was assessed. Secretion ofuPA by 14 day old lipocytes incubated with1 gum RAc for 24 hours was significantlygreater than untreated controls (controls185 ±78 pg/mr uPA, treated 477 ± 124 pg/mluPA, p<Om.01, n=7). We conclude thathepatic lipocytes express mRNA for uPAR,uPA, and PAI-I and synthesise uPA andPAI-I during primary culture on plastic.Upregulation of uPA synthesis by retinoicacid may have implications in matrixremodelling in sites of lipocyte activationwhere high concentrations of retinoids maybe achieved.

Changes in tissue hepatocyte growthfactor concentration in liver disease

G W CURRY1 2, A MARKER3, K J HILLAN2 4A D BEAITIE1 ( 1 Southern General Hospital and2Department of Pathology, Western Infirmary,Glasgow; 3Department of Histopathology,Kings College, London; 4Genentech Inc, S SanFrancisco, CA) Hepatocyte growth factor(HGF) is thought to play a central part in liverregeneration. Serum HGF concentrations areincreased in many liver diseases. HGF is alsofound sequestered in the extracellular matrix.Concentrations of HGF within tissue duringinjury may more accurately reflect its contri-bution to cell proliferation and tissue repair invivo.

Liver tissue from 27 livers (six normalcontrols, five paracetamol overdose (POD),eight cirrhosis, eight hepatocellular carcinoma(HCC)) removed at, or before, transplanta-tion was homogenised and analysed by sand-wich type ELISA specific for human HGF toquantify total tissue HGF concentration.

HGF (SD)Liver sample (ng/mg protein) 95% CI Significance

Normnalcontrols 1-045 (0.79) 0-222 to 1-869

POD 5-760 (3.70) 1-170 to 10-360 p=0-0081tCirrhosis 3.300 (3.02) 0.770 to 5 830 p=001 18tHCC 1-063 (1-18) 0-076 to 2-051 p=0-6514t

p=0.02391

tCompared with normal liver; tcompared with cirrhotic liver.

Hepatic HGF concentrations increase indisease states, which are accompanied by cellproliferation, supporting the view that HGFis important for liver regeneration in vivo.These results mirror those obtained for serumHGF concentrations, but there are significantdifferences. Normal liver contains significantconcentrations of tissue HGF despite low cellturnover. HCCs show a biphasic spread oftissue HGF concentrations, with a subset ofHCCs having very low values of tissue HGF,which may point to a role for HGF inhepatocarcinogenesis.

Increased liver regeneration afterKupffer cell depletion

R A BOULTON, M R ALISON*, M GOLDING*,C SELDEN, S PEREIRA, M J HUDSON*, H J FHODGSON (Department of Gastroenterology and*Histopathology, Royal Postgraduate MedicalSchool, London W12 ONN) Kupffer cells havebeen implicated as both positive and negativemodulators of hepatic growth but are func-tionally impaired in liver failure. In thisexperiment we have depleted Kupffer cells invivo using liposome mediated delivery ofdichloromethylene bisphosphate (CI2MBP).Hepatocytes remain undamaged and thusthe role of Kupffer cells in modulatinghepatocyte proliferation can be assessed.Male rats received PBS-liposomes orCI2MBP liposomes and 24 hours later under-went partial hepatectomy (PHx).Bromodeoxyuridine (BRdU) was given everyfive hours until death at 24 hours post-PHx.Liver tissue was immunostained using anti-ED2 (to detect Kupffer cells) and anti-BRdUantibodies. Sections were assessed and scoredby a single blinded observer. Two thousandhepatocyte nuclei were counted and scored inrandom fields centred on terminal portaltracts. Kupffer cells were eliminated in theCI2MBP liposome treated animals as judgedby ED2 staining. At 24 hours post-PHx themitotic index and BRdU labelling in Kupffercell depleted animals were higher than in PBS

treated control. Results expressed as mean(SEM): Mitotic index 6-4 (1-7) v 3-2 (0.8):BRdU labelling index 63-3 (6.7) v 50-6 (4.5)p=0037. In conclusion, it was found thatKupffer cell depletion accelerates liverregeneration.

Immunoglobulin A response to a 140 kDagut protein in alcoholic liver disease

A C DOUDS, A G LIM, J D M LEWIS,*T A POULTON, J D MAXWELL (Departments ofMedicine and *Immunology, St George's HospitalMedical School, London SW17 ORE) Immuno-globulin A is characteristically increased inalcoholic liver disease (ALD). It has been pos-tulated that gut bacterial antigens or changedhepatocyte membrane proteins may accountfor this humoral immune response. We haveinvestigated the frequency and specificity ofthis IgA response against putative gut and liverantigens using immunoblotting.Human liver and gut aspirates were disag-

gregated to yield protein fractions. Thesewere resolved using 7-5% SDS polyacry-lamide gel electrophoresis and transferredonto nitrocellulose. Blots containing liver orgut proteins were then probed for IgAresponses using serum samples from 10normal controls, 22 ALD, and 10 primary bil-iary cirrhosis (PBC) patients. Reactivitieswere detected using an enhanced chemilumi-nescence system.

Seventeen ALD patients (77%, p<0-0001)showed an IgA response to a 140 kDa gutprotein. No other significant IgA responseswere detected to gut or liver proteins in ALDor PBC.

In conclusion, IgA antibodies are frequentlydetected to a 140 kDa gut protein, a responsespecific to ALD. The IgA response against gutantigens may account for the hypergamma-globulinaemia characteristic of ALD.

Identification of an acetaldehyde modi-fied 200 kD antigen in liver tissue ofpatients with alcoholic liver disease

L A RAMSAY, J G KENNA, J KOSKINAS,M E ALLISON, G J M ALEXANDER (St Mary'sHospital, London, Hippokration GeneralHospital, Athens and University Department ofMedicine, Addenbrooke's NHS Trust,Cambridge) Acetaldehyde, a metabolite ofalcohol, binds to the e-amino groups of lysineresidue of proteins to form highly immuno-genic adducts. It has been shown that serumsamples from most patients with alcoholicliver disease contains IgA that binds to a 200kD cytosolic liver protein modified byacetaldhyde in vitro.To discover if acetaldehyde modified

proteins might be present in liver tissue, wehave analysed liver tissue from 39 patientsundergoing diagnostic liver biopsy. Twelveproved to have alcoholic liver disease. Livertissue was western blotted using rabbit poly-clonal antisera raised against acetaldehydemodified proteins. In particular, a 200 kDantigen was detected in 10/12 (83%) of thosewith alcoholic liver disease in contrast with5/27 (18%) without histological features ofalcoholic liver disease (p<0-001).

Circulating IgA antibody was detected in5/7 (7 1%) patients with alcoholic liver diseaseand 3/22 (14%) controls (p<0 05). All fivepatients positive for both antigen and anti-body had alcoholic liver disease; in contrast1/17 (6%) was negative for both and hadalcoholic liver disease.

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These data support the hypothesis thatacetaldehyde modified proteins are immuno-genic and are present in liver tissue of patientswith alcoholic liver disease.

Hepatotoxicity induced by non-steroidalanti-inflammatory drugs

L T WADE, J G KENNA, J CALDWELL (Departmentof Pharmacology and Toxicology, St Mary'sHospital Medical School, London) Certain non-steroidal anti-inflammatory drugs (NSAIDs)cause very rare but severe hepatotoxicity. Ithas been suggested that covalent modificationof endogenous proteins by NSAIDs or theirmetabolites, or both, is responsible for thistoxicity. In view of this, we have used animmunochemical approach to investigatemodification of hepatic proteins by theNSAIDs diclofenac, sulindac (which maycause hepatotoxicity), and ibuprofen (whichis not hepatotoxic). Specific antisera wereproduced by immunisation of rabbits withKLH conjugates of the drugs. Subcellularfractions prepared from livers of mice dosedintraperitoneally with these drugs (diclofenac100, 200, 300 mg/kg, ibuprofen 50, 100, 200mg/kg or sulindac 25, 50, 100 mg/kg) wereanalysed for expression of NSAID modifiedproteins using SDS PAGE and western blot-ting. A novel polypeptide antigen of 1 10 kDawas expressed in livers of mice dosed withsulindac or diclofenac. In contrast, a 60 kDapolypeptide antigen was expressed in livers ofmice dosed with ibuprofen. The antigenswere concentrated primarily in the plasmamembrane fraction of liver homogenates.The identification and functional characteri-sation of these target polypeptides is underinvestigation currently.

Acknowledgements: This work wassupported by the Wellcome Trust.

Earlier identification of patients at riskfrom paracetamol induced liver failure

I MITCHELL, J WENDON, D BIHARI, R CHANG,R WILLIAMS (Institute of Liver Studies, King'sCollege Hospital, London SE5 and Departmentof Intensive Care, Guy's Hospital, London SE1)Prognostic indicators for paracetamolinduced liver failure used in specialist centresare unfamiliar to others. We have assessed theAPACHE II system, used routinely in generalintensive care in estimating their initial deathrisk, and compared it with the O'Gradycriteria.

Variables for APACHE II were collecteddaily until death or discharge from1 November 1993 to 31 October 1994 withhospital outcome as the endpoint. Of the 105patients studied (mean age 30± 11 years, meanICU admission APACHE II score 10±7-5),18 died (mortality 17%) and eight underwentliver transplantation, ofwhom six survived.

As the estimated APACHE II risk of deathwas 1.64% we derived a new coefficient fordrug overdose using our cohort of patientsgiving an estimated risk of death of 17-14%.Using the new coefficient, 23 patients wereidentified on the day of admission with a riskof death >25%; 15 died and eight survivedconferring a sensitivity of 83.3%, a specificityof 90-8%, and a false positive rate of 34-8%.The O'Grady criteria identified 16 patientson the day of admission, 12 died, and foursurvived giving a sensitivity of 66-7%, aspecificity of 95.4%, and a false positiverate of 25%. With further validation therecalibrated APACHE II system may prove to

be a useful tool in identifying patients for earlytransfer and transplantation.

Effect of temporary liver support withthe extracorporeal liver assist device(ELAD) on plasma hepatocyte growthfactor in acute liver failure

Y MIWA, A J ELLIS, R D HUGHES, P G LANGLEY,J A WENDON, ROGER WILLIAMS (Institute ofLiver Studies, King's College School ofMedicineand Dentistry, London SE5 91J) The HepatixELAD is currently under evaluation in thetreatment of patients with acute liver failure.The aim of this study was to investigate theeffects of ELAD treatment on plasmahepatocyte growth factor (HGF), the mostpotent stimulator of liver regeneration.Plasma HGF, as measured by ELISA, wasalready increased in both 12 ELAD treatedpatients (7*61 ±SE 1-72 ng/ml) and nine con-trol patients (9-82±4-09 ng/ml, normal range0-0.5 ng/ml). After six hours of treatmentwith ELAD, plasma HGF increased greatly(30 5_6-19 ng/ml, p=0-001) and thendecreased towards the initial values by 48hours (11-4±2-41 ng/ml). In the controlgroup, no significant increase in plasma HGFwas seen at six hours (6-82+2-02 ng/ml) orsubsequently during the 48 hour period. NoHGF was detected in the filtrate from the cellcompartment of the bioreactor, showing thatthe patient rather than the device was thesource ofHGF. There was a significant corre-lation between plasma HGF and plasmathrombin-antithrombin complexes, suggest-ing a link to activation of coagulation. HGF isa heparin binding growth factor and it is likelythat heparin given as an anticoagulant forELAD perfusion is involved in the effectsobserved.

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