390 AUTOANTIBODIES TO ALKALINE PHOSPHATASE IN AUTOIMMUNE LIVER DISEASES

V.D. Sch611horn~ R. Sumazaki~ P.A. Berg Medizinische Klinik, Lab. Prof. Berg, Otfried-MOller-Str., 7400 TObingen, W-Germany

In former studies we could observe severe changes in the metabolism of liver cells (be- ginning with a marked inhibition in the early phase of incubation and ending in a strong stimulation of DNA and protein synthesis) when they were incubated with antibodies specific for intracellular antigens (like mitochondria). Even though we never were able to detect binding of such antibodies to their targets in the cytoplasm of living cells. Therefore we postulated a crossreactivity with determinants on the surface of the tested cells. Because there are various molecules known to be expressed simultaneously on intracellular and on the cell membrane we chose alkaline phosphatase (ALP) among others as a possible target antigen. Sera from 51 PBC, 17 CAH patients and 20 controls were screened by ELISA for the occurrence of anti ALP antibodies. In 6 sera from the PBC group anti-ALP antibodies were present while none of the CAH or control group showed positive reactions. The six positive sera and nega- tive controls out of each group were further characterized in the immunoblot using affinity purified ALP. All six proved to be positive showing bands around 70 kD (suggesting that these antibodies recognized a part of the ALP) whereas the negative sera showed no bands. ALP co- valently coupled to CNBr-Sepharose was used to affinity purify anti ALP-positive and -nega- tive patients' sera. After elution only the anti ALP-positive sera showed positive stainmg in the immunofluorescence on fixed cells where they reeongized mltochondrial (strong I ) and cel l membrane (weak!) antigens. This study does not a11ow to draw conclusions about the meaning of this anLibodies in the etiopathology of PBC. However these resulta indicate that a pathogenetical role of outoanti- bodies to introeel lular antigens ~hould always be considered in autoimmune diseases.

391 CELLULAR EXPRESSION OF ALBUMIN AND ALPHA-FOETOPROTEIN GENES DURING }'-METHYL-DAB INDUCED HEPAIOCARCINOGENESIS IN THE RAI. J.Y. Scoazec~ D. Bernuau; A. Moreau~ G. Feldmann Laboratoire de Biologie Cellulaire and Inserm uz% ~aeulte de M~decine Xavier Biehat, Paris, France.

During hepatocarcinoqenesis, liver albumin (ALB) synthesis is frequently decreased. while large amounts of alpha-foetoprotein (AFP) may be syntheslzed. The hypothesis that these changes are reciprocally regulated has been raised. Thus, this work was desiqned to study at the cellular l@v@l ALB and AFP gene expressions during cRemical hepatocarcinogenesis in ord@r (i) to compare their ehanqes durinq the various staqes of the neoplastic process and (2) to gain an insiqht into their Factors of control. 32 male Fischer rats, fed a diet containing 0.06~ 3"-methyl-DAB, were sequentially sacrificed between the 4th and the 22nd week of intoxication. AdJacent sections from paraffin-embedded tissue were used for the detection of gene transcripts by in situ hybridization with ALB and AFP 35S-labeled cDNA probes, and for the detection of the correspondinq proteins by immunoperoxidase. The r@sults are presented according to the sequence of DAB-induced hepatoearcinogenesis. (i) At the preneoplastic stage, proliferating oval cells expressed high amounts of ALB and AFP mRNAs and both corresponding proteins. Hyperplastic nodules consisted of a mixture of cytologically abnormal hepatocytes. Most of these cells expressed decreased amounts of ALB mRNAs but only a few ones displayed increased AFP mRNAs. The so-called hyperbasophilic cells had a striking phenotype, characterized by the presence of high amounts of both ALB and AFP mRNAs with expression of the corresponding proteins= (2) At the neoplastic stage, ALB and AFP showed distinct expression patterns. All the hepatomas expressed variable amounts of ALB mRNAs, while AFP expression was inconstant. At the cellular level, co-expression of ALB and AFP mRNAs in the same neoplastic cell was possible. The level of ALB transcript detection was correlated with the degree of morpholoqie differentiation, while the distribution of cells containing AFP mRNAs was highly 5eterogeneous. In certain neoplastic cells where high levels of AFP mRNAs were present, no protein could be detected, whereas such a discrepancy was not observed for ALB. ]n conclusion, this work shows the absence of reciprocal ehanqes between ALB and AFP gene expressions in DAB-induced hepatocarcinogenesis and suggests different modulating factors. Hepatocyte differentiation seems to influence ALB but not AFP gene expression. Expressions of both genes mainly depend on transcript availability but, for AFP, the hypothesis of an additional post-transcriptional control may be raised.

$201