April 1995 AASLD A l 1 1 9

O PBC-LIKE LESION IS INDUCED BY IMMUNIZATION WITH RECOMBINANT PDC-E2 BCOADH-E2 HYBRID MOLECULE AND LIPOPOLYSACCHARIDE INJECTION IN NEONATALLY THYMECI~MIZED MICE. T. Masanaga, Y. Watanabe, P.S.C. Lcung, M. Kamiyasu, E. Sanada, T. Nakanishi, G. Kajiyarna, M.E. Gershwin. First Department of Internal Medicine, Hirnshlma University School of Medicine., Internal Medicine, University of California at Davis.

<Introduction> Primary bitiary cirrhosis is characterized by the presence of autoantibodies to mltochondria with specific reactivity to the t!2 subunits of ~he pyruvate dehydrngenase complex and the branched chaIn 2-oxo-acid dehydrogeoase complex as the major and immunodominant autoantigens. It has been reported that some monoclonal antibodies to PDC-E2 reacted strongly with the luminal aspca of billary epithelial ceils of patients with PBC.

This study was designed to test the hypothesis that autoimmunity to PDC-E2 or BCOADH-E2 itself can induce the pathology of PBC. We took advantage of recombinant PDC-E2 and BCOADH-E2 hybrid molecule as major immunodominant autoantigens, and used nconatally thymectomized A/J mice as autoimmune prone animals.

< Methods> Neonatal thymeetomy was performed 2 to 3 days after birth. The mice in group A were i m m u ~ with a mixture of recombinant PDC-E2, BCOADH-E2 hybrid molecule (150/zg) and FCA aRer neonatal thymectomy. The mien in group B were immunized with a mixture of recombinant PDC-E2, BCOADH-E2 hybrid molecule (150,ug) and FCA without thymectomy. The mice in group C were immunized with a mixture of recombinant PDC-E2, BCOADH-E2 hybrid molecule (150/zg) and FCA after neonatal thyraectomy and LPS (0.5/~g) was injected intravenously 2 days after each immunization.

<Results> 6 of 7 mice (86%) in group C showed nonsuppurative cholangitis (grade C_,3 cholangitis) which is characterized by the mononuelear cell infiltration around the biliary epithelium and degeneration and destruction of biliary epithelial cells. One of 10 mice (10%) in group A showed grade C3 eholangitis. None of 6 mice in group B showed grade C3 cholangitis.

<Conclusion> PBC-Iike lesion is induced by immunization with PDC-E2 or BCOADH-E2 proteins, and endotoxin such as lipopolysaecharide may augment the immunopathology of PBC.

HISTOPAPHOLOGICAL CHANGES IN HEPATOCELLULAR CARCINOMA TREATED WITH PROTON IRRADIATION: ALTERED CELL PROLIFERATIVE ACTIVITY AND INDUCED APOPTOSIS. Y Matsuzaki ~, YSaito ~, T Ikegami 1, M Doy 1, T Chiba 2, M Abel , ' ~ Araki ~, J Shoda 1, N Tanaka ~, Y hal ~, T Osuga ~ and H Tsujii 2. ~lnstitute of Clinical Medicine, 2Proton Medical Research Center, University of Tsukuba.

We have reported that proton irradiation is a safe and effective therapeutic option in cases of hepatocellular carcinoma (HCC) (Gastroenterology,106(4), 1032, 1994). Excellent long-term local tumor control is achieved in most cases. However, the histologic features of the HCC treated with proton beam is not known. [Aim] VV~e aimed to Clarify the effect of proton irradiation on histopathology of HCC. Also, if proton irradiation alter cell proliferative activity or induce apoptosis of HCC was studied. [Materials and Methods] Tissue biopsy of the irradiated tumors was performed using a fine-diameter needle under ultrasonography before and 3 weeks after completing the proton therapy (mean 75 Gy) in 14 irradiated lesions. Formalin-fixed, paraffin-embedded tisuue sections were analyzed. As an index of cell proliferative activity, the expression of Ki-67 antigen was examined by an immunohistochemical method using MIB-I monoclonal antibody. MIB-1 labeling index(M) was determined by the percentage of positive nuclei. Apostosis was examined by immunohistochemical expressions of LeYantigen using BM-1/IIMRO antibody and also by Nick end labeling(TUNEL) using Gavrieli's methods. LSAB method was used for visualization. BM-I and TUNEL positivity W~R s evaluated by brown staining in cytoplasm and in nuclei, respectively.

esults[ 1): All 14 irradiated lesions showed excellent local tumor control after 2 yr completing theproton therapy. 2): Conventional histologic examination of biopsy samples of these HCC after treatment showed variable features; 4 of 14 lesmns became necrotic, 6 turned into degeneration, and 4 had viable cancer cells. 3): Cell proliferative activity of HCC decreased uniformly in ALL 14 lesions; MIB-1 LI decreased significantly from 4.2- ± 1.3%(M ± SE) before treatment to 1.1 ± 0.5 after treatment. 4): Apoptosis was induced in some but not all HCC following irradiation In 13 lesions with negative BM-1 staining before therapy, only 3 changed positive after irradiation(21.4%). In 6 lesions with negative TUNEL before therapy, 4 changed positive after teatment(66.7%). The other 4 showed positive TUNEL before and after the treatment.

[Conclusions] Histopathological examination following 3 weeks after completion of the proton irradiation revealed that not all HCC became necrotic despite excellent long-term local tumor control. It is suggested that altered cell proliferative activity Using MIB-I LI is related to the successful effect of the proton irradiation.

LONG-TERM EFFECTS OF PROTON IRRADIATION FOR HEPATOCELLULAR CARCINOMA. Y Matsuzaki/, S Yoshiga ~, T Chiba z, YSaito ~, M Abel ~, T Ikegami !, M Araki ', J Shoda ~, N Tanaka ~, T Osuga ~, Y Itai ~ and H Tsujii 2. ~ Institute of Clinical Medicine, 2Proton Medical Research Center, University of Tsukuba.

We have reported that proton irradiation is a safe and effective therapeutic option io cases of hepatocellular carcinoma (HCC) (Gastroenterology,106(4):1032,1994). However, the precise role of the proton irradiation for HCC is not known due to the lack of long-term results. [Aim] We here report the iong-term(4years) effects of proton irradiati0n for HCC, in comparison with that of Lipiodol-targeted cbemotherapy(I-TAl), in order to clarify the role of this new therapy for the treatment of HCC. [Materials and Methods] Informed consent was obtained from 48 HCC patients. 75 HCC lesions in the 48 patients which .were treated with either proton irradiation alone(Mono: 46 lesions) or with Lipiodol-targeted chemotherapy(l-TAD plus proton irradiation(Combi: 29 lesions) were included in the analysis. Proton beam was provided by the cynclotoron of the National Laboratory for High Energy Physics(KEK). Total amount of 75Gy (mean) of the proton beam was targeted to HCC, which ranged from 1 to 12cm in size. The results from these patients were compared with those from 42 patients(65 HCC lesions) treated with I-TAt alone(l-TAl group), whose background did not differ from the proton groups. All tumor was followed up by CT scan and ultrasonography. [Results] 1): After 1 yr, the reduction of tumor size was observed in 40 out of 46 lesions (87%) in Mono, 21 out of 29 lesions(72%) in Combi, compared with 30 of 65 lesions(46%) in I-TAI group. After 2 yrs, size reduction was seen in 11 of 12 (92%) in Mono, 10 o f l 0 (100%) in Combi and 10 of 16(63%) in I-TAI group. After 4 yrs, size reduction was seen in 1 of 1 (100%) in Mono, 3 o f 3 (100%) in Combi and 2 of 3(67%) in I-TAI group. 2): Local tumor control rate (no local recurrence rate) was significantly higher in the proton groups(Mono and Combi) than in the I-TAI group. By Kaplan and Meier's methods, cumulative local tumor control rates, at the end of ls tyr: 97% for both Mono and Combi, 70% for I-TAI group, at the end of4th yr: 60% for Mono, 90% for Combi, 30% for I-TAI. 3):Survival rate was also significantly higher in the proton groups than in the 1-TAI group during follow-up period. Cumulative survival rates at the end o f 2nd and 4thyrs, were 87%, 74% for the proton groups, and 74%, 45% for the I-TAI group, respectively. 4): Excellent QOL was maintained in all cases treated with proton therapy during observation period. [Conclusions] HCC patients treated with proton irradiation showed more excellent local tumor control as well as better survival and QOL, in comparison with I-TAI, which lasted for at least 4 yrs.

CHARACTERISTICS OF SERUM DEFENSE PROTEINS (DP) IN PATIENTS WITH CHRONIC HEPATOPATHY - A. A. Mattes, C. T. Both, J. F. C. Almeida, C. A. Marroni - Gastroenterology Service of Santa Casa Hospital - Porto Alegre,RS - BRAZIL

This paper intends to determine the serum levels of DP in cirrhot!c patients with aseites (ASC-CI), as they have propensity to desenvolve bacteremia with consequent infection in different sites. METHODS - Fifty patients with ASC - CI, 17 with aseites of other etiologies (AS C - OT) and 18 without hepatopathy or aseites (NO - ASC) were prospectively studied. It was determined the serum concentrations of C3, C4, lgG, IgA, lgM (mg/dl) and total hemolytic complement (CH50). In the statistical analysis, the significance level adopted was 1%. RESULTS -

ASC - CI ASC - OT NO - ASC ............................... ~.......£SD ....). ............... ~.......~S.9.._....) .......... .x ...LS. D.......~ ........................ C3' 80.4 ( 3 8 . 8 ) 135.4 (36.7) 83.6 (17.5) C4" 31.2 ( 2 3 . 4 ) 61.1 (21.7) 36.8 (10.4) lgG* 3157.1 (1976.1) 2437.5 (874.4) 1375.3 (290.9) lgA* 1130.6 (874.2) 587.6 (500.9) 295.6 (86.4) IgM* 465.1 (417.7) 251.1 ( 9 7 . 5 ) 208.8 (106.7) CHS0* 51.7 ( 1 2 . 4 . ) 7 9 . 8 (21.4). * P< 0,001 The serum determination of CHS0 was done only in the ASC - CI and ASC - OT group. The levels of C3, C4, and CH50 were smaller, and those of IgG, lgA e IgM were greater in the ASC _ CI group than in the controls. CONCLUSION ~ These results demonstrate that cirrhotic patients can present a great antigenic stimulus and complement consumption, probably in consequence to prolonged baeteremias.