128A A A S L D A B S T R A C T S HEPATOLOGY O c t o b e r 1995

8 5 COMPLEMENT C1Q SYNTHESIS I N RAT KUPFFER CELLS, PERITONEAL MACROPHAGES BUT NOT IN BLOOD MONOCY- TES. MODULATION BY DEXAMETHASONE. Th. Armbrust. G. Ramadori Dept. of Internal Medicine, University of G6ttingen, FRG.

Phagocytosis of particulate material arriving from the gut is one of the important functions of Kupffer cells (KC). As the complement system could be crucial for this function, we investigated synthesis and secreti- on and modulation of the initiating factor C lq by KC and related pha- gocytes. Methods: KC, peritoneal macrophages (PM) and blood mo- nocytes (MC) of the rat were isolated by standard techniques and kept in culture. Freshly isolated and cultured cells were characterized immu- nocytochemically with the moabs ED1 and ED2. C lq synthesis of cultu- res was studied by endogeneous labeling of newly synthesized proteins with 35-S-methionine, immunoprecipitation, and SDS-PAGE. RNA was extracted from freshly isolated cells and cultures and analyzed by Nort- hern blotting and hybridization with a 32-P-labeled cDNA specific for C 1 q B-chain. For comparison, C 1-inhibitor (C 1-INH) synthesis was stu- died in parallel. Results: Freshly isolated KC constitutively expressed Clq and CI-INH mRNA. A decrease in the steady state of C lq mRNA was observed at dl of culture and a strong increase at d 5. Spontaneous C lq A-, B-, and C-chain synthesis and secretion was detected with an increase from dl to d5 of culture, while CI-INH synthesis remained constant. However, the amount of both proteins synthesized by PM was below that of KC. C lq and CI-INH gene expression were not detected in MC during the time of culture. Treatment of cultures with increasing concentrations of dexamettiasone (Dex) strongly increased C lq gene expression in KC and PM, but not in MC. In contrast, CI-INH synthesis was not affected by Dex. Conclusions: a) Beside CI-INH, constitutive C lq synthesis seems to be a further differentiation marker of rat liver macrophages, b) Modulation of C 1 q by Dex could indicate the important role of the complement system as one of the defense mechanisms of the body particularly in glucocorticoid-induced immunosuppression.

86 SPECIFIC DIAGNOSIS OF AMEBIC LIVER ABSCESS USING PRESERVED E.. histolyaca ANTIGENS WITHOUT ENZYMATIC INHIB1TORS1 . MS Flores- Castafieda*~ GL Menu, F Castafieda and S Said-Fernandez**. *Centro Regional de Control de Enfermedades Infecciosas, Fac. De Medicina, UANL, Monterrcy, N.L M6xico. **CIBN, IMSS. Monterrey N.L. M6xico

lnvasive amebiasis, as colitis and amebic liver abscess (ALA) is one of the three top causes of death by parasites through the world. The identification and characterization of E. histolytica antigens of diagnostic value which are recognized by specific antibodies induced during invasive liver infection, are of main importance. A major impediment to achieve this goal is the amebic extract high enzymatic activity that promotes a random degradation of the amebic antigen rending difficult to standardize a fine method for antigenicity analysis, even using enzymatic inhibitors.

We present a reproducibly method to: I.- Preserve amebic antigens without use enzymatic inhibitors, 2.- identify an amebic liver disease pattern. By this method we can, without doubt, to differentiate a non invasive amebiasis from an ALA disease. METHODS. The 'c rude freeze-dried trophozoite was treated with Chloroform-Methanol and heat to obtain the insolubl e fraction (IC:MC). SDS-PAGE electrophoresis and Western-Blot (EITB) were performed on IC:MC for 36 ALA patients, 32 non invasive amebiasis cases and 15 controls without amebiasis infection. Antiamebic antibodies titles were obtained by a commercial IHA kit. RESULTS. We obtain, by the IC:MC treatment, a 100% proteolytic activity reduction using azocasein as substrate and by EI"rB we obtain more antigenic bands than those obtained using the trophozoite extract treated with proteolytic inhibitors. We could identify 8 amebic molecules recognized exclusively by antibodies from ALA patients. The positive EITB ALA patterns was obtained even before the patients get a high Ilia titles level. In 8 more recent cases clinical diagnosis were inaccuracy because they were accepted as liver cancer or others liver pathologies, we could establish that they were ALA, and all the cases responded quite well to the antiamebic treatment CONCLUSIONS. l.-The method effectively preserve the antigenicity of the amebic molecules. 2.- The E1TB pattern can be effectively used for early diagnosis and to differentiate ALA from others liver pathologies.

Patent pending

8 7 BILIARY ATRESIA - AN AUTOIMMUNE MEDIATED DISORDER? EA Vasilianskas. SR Tartan. L Cobb. A Viddch. P Ro~nthal*. IBD Center, Cedars-Sinai Medical Center, Los Angeles, CA, and *Division of Pediatric Hepatology, UCSF Medical Center, San Francisco, CA.

Biliary atresia (BA), the leading eause of pediatric liver transplantation, is charaeterized by acute & chronic inflammation, resulting in scarring, fibrosis & ultimately obliteration of the biUary tree. The etiology is unknown. We have identified specific HLA associations in BA (Cw4/7 locus), implying genetic susceptibility & potentially an immune-mediated mechanism of disease. In adults, the expression of IgG autoantibodies, including antineutrophil cytoplasmic antibodies (ANCA), has been associated with hepatic disorders such as.PSC & AIH. Recently, we reported a high incidence of ANCA of the IgG isotype in sera of BA patients. Because BA occurs in the newborn period, the presence of IgM-ANCA would imply intrinsic immune dysregulation. Aim: To determine if IgM- ANCA is present in the sera o f BA patients. Methods : Sera from t l surgically proven BA patients (mean age 1.8y; range 6wk-7.Sy) & controls (see table) were analyzed for the presence oflgG- & IgM- ANCA. ANCA was determined by the fixed neutrophil ELISA assay at a 1:100 serum dilution, & antibody levels were expressed as percent of positive standard binding (positive >2SD above mean of control), Results:

Mean IgG ANCA+ IfrM ANCA+ Group n Age # positive ELISA # positive ELISA

(~s) (%) Mean (%) Mean BA 11 1.8 10(91) 19 10(91) 63 Neonatal Hepatitis (Nil) 3 1.2 3 (100) 40 3 (100) 42 HCV 2 12.5 2 (100) 10 1 (50) 18 HBV l0 10 .2 6(60) 17 8(80) 30 PSC/AIH 2 14.8 2 (100) 75 2 (11)(3) 30 Other Pediattic Liver Disease 5 10.1 0(0) 8 4(80) 19 Milk/Soy Allergy 3 0.2 2 (67) 12 0 (0) 7.5 CorOBlooa 24 0 t 0) 8 t (4) 9 Normal Adult Controls 20 Adult 1 (5) 8 14 (70) 28 Adult UC ANCA+ Controls 5 Adull 5 (100) 100 5 (100) 20

Conclusions: Both lgG- & IgM- ANCA isotypes are expressed in BA. The mean level of IgM-ANCA is higher in BA compared to other pt~.diatfie liver diseases (p < 0.01) and cord blood (p < 0.001). There is a higher level of IgM vs IgG ANCA in BA compared to other conditions. The combination of HLA associations & autoanribody production in BA provides indirect evidence for an autoimmune-mediated process. The strong IgM response in BA suggests that perinatal initiation of inflammation is then perpetuated by the child's own immune dysregulation.

88 GENETIC SUSCEPTIBILITY TO AUTOIMMUNE IIF~ATITIS (AIH). MDJ Strettell. A Czaia*. PT Donaldson. Roger Williams~ Imtimte of Liver Studies, King's College Hospital, London, UK, and *Mayo Clinic, Rochaster, MN, USA.

The HLA genes are essential elements in determining smceptibility and resistance to AIH. Previous studies at this Institute and elsewhere have identified associations with the DRBl*0301 (DR3), DRBI*0401 (DR4) and DRB3*0101 (DR52a) alleles in European white patients though whether these alleles predispose to different subsets of disease or whether there is a common shared susceptibility determinant remains to be ascertained. AIM: To confirm previous observations by PCR genotyping the HLA DRB1/3/4/5, DQAI, DQBI and DPBI genes and to investigate the nature of the DRBI*04 association by subtyping the DRBl*04 and DRB4 genes, in an independent, albeit racially similar, group of 114 patients with AIH from the Mayo Clinic. RESULTS: The analysis confirms the high frequency of the DRBI*0301 and DRBI*04 alleles (47% and 41% of patients respectively). Of those with DRBI*04, 70% carry 1"0401 and 21% carry 1"0404. In total, 80% of patients have either DRBI*0301 and/or an allele of DRBI*04. Of the 74% of patients carrying the DRB3 gene, 73% have DRB3*0101, 32% DRB3*0202 and 7% encode DRB3*0301. Similarly, of the 58% of patients carrying the DRB4 gene, 20% have 4*01011, 3% 4"01012N and 86% encode 4"0103. DRB3*0201 and DRB4*0102 were not represented. In total, 87% of patients have either DRB3*0101 and/or DRB4*0103. The DQAI, DQB1 and DPB1 alleles were relatively evenly distributed except for the high prevalence of DQA !.0501 (54%), DQBI*0201 (56%) and DPBI*0401 (64%) which most likely reflects linkage disequilibrium with DRB3*0101- DRBI*0301. Excluding either the DRBI*0301 or DRB3*0101 positive patients reveals a high frequency of DRB 1"04 positive patients (62%, both), 73% having the DRBl*0401 allele. More interesting however, is the previously unreported high frequency of the DRIM*0103 allele which was present in all 47 patients (100%) expressing the DRBI*04 gene, regardless of allele and in 70% of the DRBI*0301 and 72% of the DRB3*0101 negative patients respectively. This suggests a stronger secondary association than with DRBl*0401. CONCLUSION: These date are in keeping with the strong predisposition to AIH in patients with the extended haplotype A1-B8- DRB3*0101-DRBl*0301-DQAl*0501-DQBI*0201 but suggest that the secondary association may lie closer to the DRB4 rather than the DRB1 gane.