Evaluation of transcatheter arterial embolization with epirubicin-lipiodol emulsion for hepatocellular carcinoma.

A total of 18 patients with hepatocellular carcinoma (HCC) were treated by transcatheter arterial embolization (TAE) with a 4'-epi-doxorubicin (EDX)-lipiodol emulsion. Infusion of the EDX-lipiodol emulsion (EDX-L) via the hepatic artery was followed by the injection of gelatin sponge in 12 cases. The response and survival of these 12 patients following EDX-L treatment were compared with those of 42 subjects treated with a doxorubicin-lipiodol emulsion (DX-L) and those of 23 patients treated by TAE with gelatin sponge (GS) only. In the group treated with EDX-L, nine cases were AFP-positive in sera and four showed a decrease in serum AFP values to less than 10% of the pretreatment level. Seven cases showed a partial response, and nine cases showed no change in the size of the tumor. In the group treated with EDX-L, nine cases are alive, and the oldest has survived for more than 431 days since the treatment. The half-year survival value was 57%, and the 1-year survival value was 49%. These values did not differ significantly from those calculated for the group treated with DX-L. The 1-year survival value determined for patients treated with a lipiodol emulsion (EDX-L or DX-L) followed by GS was 65%, and the 2-year survival value was 39%. These results rates are significantly better than those obtained in patients treated with GS only (1-year survival, 39%; 2-year survival, 13%.

Subcellular abnormalities of liver sinusoidal lesions in human hepatocellular carcinoma.

Liver sinusoidal lesions in 20 cases of human hepatocellular carcinoma (HCC) were examined with the electron microscope. Kupffer cells existed in the compact type of HCC. In area with pseudoglandular and trabecular cell patterns, Kupffer cells could not be observed. Only a few macrophages were distributed in the tumor tissues. Endothelial cells were altered and showed a poly-layered arrangement and were attached to each other with desmosome-like junctional complexes. There was a general loss of endothelial fenestrae. Endocytotic vesicles could be recognized in these endothelial cells. Poly-layered endothelial cells and accompanying layers of basal laminae were variably arranged between sinusoids and tumor cells with pseudoglandular, trabecular, or compact types of tumor cell patterns. Atypical cells containing numerous lysosomes, together with altered fat-storing (Ito) cells, were located in the tissue space bordering the capillaries. Moreover, tumor cells possessed flattened sinusoidal surfaces. These alterations of the sinusoidal wall suggest that capillarization of liver sinusoids in HCC took place, by loss of fenestrations, formation of basal laminae, and loss of microvilli on the surface of tumor cells. These architectural alterations are thought to completely change the physiological pattern of exchange of metabolites between tumor cells and the sinusoidal lumen. The absence of large numbers of Kupffer cells suggests that at this stage of tumor development, local cellular defense mechanisms were inoperative.

Collagen production in fat-storing cells after carbon tetrachloride intoxication in the rat. Immunoelectron microscopic observation of type I, type III collagens, and prolyl hydroxylase.

Monospecific antibodies directed against type I, type III collagens, and prolyl hydroxylase were used to clarify the process of liver fibrosis after CCl4 intoxication in rats by the direct immunoperoxidase method. In acute CCl4 intoxication, fat-storing cells (FSCs) were increased in number in the areas of necrosis around the central veins. These FSCs exhibited intense positive stainings for type I, type III collagens, and prolyl hydroxylase in well-developed rough endoplasmic reticula and Golgi apparatus. This was the direct evidence that the collagens formed after CCl4 intoxication are produced by FSCs. In chronic CCl4 intoxication, increased FSCs in and around the fibers also contained strong immunoreactive materials of both collagens and prolyl hydroxylase mainly in the rough endoplasmic reticula. These collagens were also present in the Golgi apparatus and vesicles close to the cytoplasmic membrane, demonstrating the exocytic process of collagen formation of FSCs. In contrast, faint immunoreactions of both collagens were found in the rough endoplasmic reticula and Golgi apparatus of hepatocytes during the process of fibrosis. These findings indicate that FSCs play an important role in fibrogenesis after acute and chronic CCl4 intoxication in the rat.

Ultrastructural localization of type IV collagen, laminin and prolyl hydroxylase in biliary epithelial cells of rat liver following ligation of the common bile duct.

Type IV collagen and laminin are major components of basement membrane (BM), whereas prolyl hydroxylase (PH) is a key enzyme in the hydroxylation of proline to hydroxyproline in collagen synthesis. In order to elucidate the exact mechanism of the formation of BM, immune electron microscopic observation of type IV collagen, laminin and PH was made in rat liver with marked proliferation of bile ducts following ligation of the common bile duct. Extracellular localization of type IV collagen was found in the BM of bile ducts and blood vessels and in the space of Disse in both normal rat liver and the liver of rats undergoing operation. Type IV collagen was localized in lamina rara and lamina densa. Laminin was codistributed with type IV collagen in BM but rarely in the space of Disse even in the liver of rats undergoing operation. Immunostaining of laminin was diffusely spread in lamina densa, but sparsely in lamina rara. Though no reaction products of type IV collagen and laminin were detected in the cytoplasm of normal biliary epithelial cells, they were found in rough endoplasmic reticulum (rER) and the vesicles close to the basal surfaces of the plasma membrane of the proliferating biliary epithelial cells. No evident localization of these components in Golgi apparatus was found. PH was found in rER of the biliary epithelial cells, hepatocytes, endothelial cells of vessels, fibroblasts and perisinusoidal cells except for Kupffer cells in normal rat liver. More intense and diffuse staining of PH was observed in rER in the proliferating biliary epithelial cells of the liver of rats undergoing operation in concomitance with the evident localization of type IV collagen in this organelle. These findings suggest that the major components of BM, such as type IV collagen and laminin in the proliferating biliary epithelial cells, are produced in rER and secreted by vesicles to the basal extracellular spaces, thus forming new BM in these circumstances.

Extracellular matrix formation in piecemeal necrosis: immunoelectron microscopic study.

Immunolocalization of Type I, Type III and Type IV collagens, laminin and prolyl hydroxylase (PH), a key enzyme in collagen synthesis, was examined to clarify the fibrotic process in chronic, active liver disease. In piecemeal necrosis of chronic, active hepatitis (CAH) and active liver cirrhosis (LC), fat-storing cells (FSCs) and transitional cells (TSCs), containing abundant rough endoplasmic reticulum (RER), were increased in number and stained intensely for PH. Immunodeposits of extracellular matrix (ECM) components were found in the RER, Golgi apparatus (GA) and vesicles of these cells, especially in cases with marked inflammation. On the other hand, in the periportal areas of chronic, persistent hepatitis (CPH) or inactive LC, immunoreaction of ECM components was seldom found in the RER of FSCs and TSCs. In the portal tract, immunodeposits of ECM components were seldom found in the organelles of fibroblasts, although ECM was increased there. These findings indicate that FSCs and TSCs in piecemeal necrosis might play a role in the production of ECM components in the progression of fibrosis during the development of chronic active liver disease. In addition, ECM component production by FSCs and TSCs is associated with marked inflammation.

Hepatocellular carcinoma in 13 patients with hepatitis C virus-associated chronic hepatitis.

Thirteen of 81 patients with chronic hepatitis and positive hepatitis C virus (HCV) antibody developed hepatocellular carcinoma (HCC) during a follow-up period of 54 +/- 38 months. The histopathological findings in HCC-bearing liver in these patients included six cases of chronic persistent hepatitis [CPH; mean hepatitis activity index (HAI) score: 5.8] and seven cases of chronic aggressive hepatitis (CAH) 2A, or 2B (HAI) score: 13.6). Multiple biopsies of the liver in six cases revealed that five cases, including four with CPH at the time of HCC diagnosis, previously had histopathological findings identical to CAH 2A, and another case constantly had CPH during the 8-year follow-up. These findings suggest that HCV-associated HCC can occur even in patients with HCV antibody positivity and inactive or mild chronic hepatitis. This is of interest in the pathogenetic mechanisms of HCV-associated HCC.

Etiology and prognosis of cryptogenic liver cirrhosis: possible contribution of hepatitis B virus.

Three female patients without type B or type C viral hepatitis, alcoholic, metabolic or autoimmune liver disease, were selected from 250 cases with histologically proven liver cirrhosis (M:F = 183:67). All three cases showed at least one positive aspect among three parameters of serum anti-HBc (RPHA, x1), HBV-DNA (gene S, nested PCR) and liver HBs and/or pre-S2 antigen (immunoperoxidase methods). Two cases may suggest a spontaneous disappearance of HBV from sera. Another case may suggest a contribution of mutant HBV which can not be detected by the routine tests. These HBV-related cirrhotic patients have done well clinically and have not been associated with hepatocellular carcinoma during the period from 6 to 12 years of follow-up when compared with 59.6% and 65.4% prevalence of hepatocarcinogenesis in type B and type C hepatitis-associated cirrhosis during the observation period of six and seven years on average, respectively.

Analyses of proliferating cell nuclear antigen-positive cells in hepatocellular carcinoma: comparisons with clinical findings.

Proliferating tumour cells in 92 patients with hepatocellular carcinoma (HCC) were identified by an immunohistochemical method using a monoclonal antibody against proliferating cell nuclear antigen (PCNA). The rate of PCNA-positive cells in HCC tissues was positively correlated with histological grade and the tumour size and T factor of the tumour. In order to analyse the relationship between prognostic factors and cumulative survival rate after obtaining tumour specimens, 49 patients whose clinical courses could be followed after needle biopsy were selected for evaluation. These patients were treated by medical therapy alone. Analyses of prognostic factors by Cox's proportional hazard model revealed that the patient's prognosis was significantly correlated with PCNA-positive rates as well as the tumour size and mode of therapy. Moreover, the cumulative survival rates were significantly (P < 0.001) higher in patients with rates of PCNA-positive cells < 15% than in those with > or = 15%, even when tumour sizes were under 50 mm or tumours demonstrated the same degree of histological differentiation. These findings indicate that the PCNA-positive rate in biopsied tissues provides useful prognostic information in patients with HCC treated only by medical therapy.

Effects of an infusion of branched-chain amino acids on neurophysiological and psychometric testings in cirrhotic patients with mild hepatic encephalopathy.

Psychotropic action of a branched-chain-enriched amino acid solution (Aminoleban) was quantitatively and visually examined in six cirrhotic patients with mild hepatic encephalopathy (grades I and II) using electrophysiological and psychometric methods. Neurophysiological effects of the amino acid solution were observed by comparing topographic spectrum analyses of electroencephalography (EEG) before and immediately after an intravenous 3 h infusion of the solution. The delta wave in the frontal region diminished from 61 +/- 13 to 12 +/- 4% (P < 0.01) and the alpha wave in the occipital region increased from 11 +/- 3 to 51 +/- 11% (P < 0.01). Latencies of the P3 wave in visual evoked potentials, which were topographically recorded in the occipital region, shortened from 220 +/- 32 to 148 +/- 19 ms (P < 0.01). Latencies of the P300 wave in event-related potentials, which were topographically recorded in the centro-temporal region, shortened from 493 +/- 81 to 360 +/- 93 ms (P < 0.05). Topographic reaction pattern of P300 was irregular toward the occipital or parietal region in cirrhotic patients. The EEG frequency power spectrum, illustrated by the colour density spectral array of computer-aided polysomnography analysis, clearly showed a gradual increase of the alpha wave spectrum and a gradual decrease of the delta wave spectrum after initiation of the infusion. These immediate neurophysiological changes were confirmed by improvement of quantitative psychometric tests including number connection test, reaction time to sound, and digit symbol and block design tests of Wechsler Adult Intelligence Scale.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased expression of matrix metalloproteinase-II in experimental liver fibrosis in rats.

Matrix metalloproteinase-II (MMP-II, 72-kd type IV collagenase, or gelatinase) is one of the gene families of zinc enzymes capable of degrading extracellular matrix molecules, and specifically of degrading type IV and V collagens, gelatin, fibronectin, and elastin. In this study, we used both the liver fibrosis model and the reversibility model of experimental cirrhosis to clarify how MMP-II participates in liver fibrosis of rats. To produce fibrosis model, rats received subcutaneous injections of CCl4 twice weekly for 7, 9, or 14 weeks. For the reversibility model, rats were treated with CCl4 three times a week for 8 weeks and killed at 3, 7, 14, 28, or 42 days after discontinuation of treatment. MMP-II gene expression was studied by Northern hybridization technique, and gelatinase activity of MMP-II was examined by zymography using gelatin substrate. At the same time, an immunohistochemical study using anti-type IV collagen antibody was carried out. In liver fibrosis model, nodule formation was established at 14 weeks. Immunodeposit of type IV collagen was increased in wide fibrous septa and was clearly observed along sinusoidal wall. Gene expression of MMP-II increased up to 7 to 12 times compared with that of controls, with the expression rate being maximum at an intermediate stage of fibrosis. Zymography showed the expressions of both 65-kd latent MMP-II, which is confirmed to be activated by adding p-aminophenylmercuric acetate, and 62-kd active MMP-II during fibrosis. The expression of both forms increased 13 to 28 times as the fibrosis progressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural localization of type IV collagen and laminin in the Disse space of rat liver with carbon tetrachloride induced fibrosis.

Monospecific antibodies, directed against type IV collagen and laminin, were used to clarify the process of sinusoidal capillarization in rats after carbon tetrachloride (CCl4) intoxication by the direct immunoperoxidase method. After acute intoxication, both type IV collagen and laminin were increased in the area of hepatic necrosis, adjacent to the central veins; however, sinusoidal capillarization was not found. During chronic intoxication, deposition of laminin was co-distributed with that of type IV collagen, but deposition proceeded more slowly than that of the type IV collagen. Deposition of laminin was increased in the Disse space. Sinusoidal capillarization was noted as thick deposition of both antigens by light microscopy. Immunoelectron microscopy showed that both components were continuously present in the Disse space. Intracellularly, both antigens were found in the rough endoplasmic reticulum (RER) of fat-storing cells (FSC) and endothelial cells, and these cells showed morphological changes, becoming slender and flattened. In contrast, few immunoreactive products of the two components were observed in the hepatocytes. These findings suggest that type IV collagen and laminin are indispensable for the establishment of sinusoidal capillarization, and that FSC play an important role in the production of both components.