Toshiyuki Uchikoshi

Expression of β-catenin in normal breast tissue and breast carcinoma : a comparative study with epithelial cadherin and α-catenin

Expression of β‐catenin was investigated in normal breast tissue and 66 breast carcinomas in conjunction with expression of epithelial cadherin (E‐CD) and α‐catenin. In normal mammary ducts and acini, intense β‐catenin immunoreactivity was present at the basolateral surfaces of luminal epithelium and weak immunoreactivity was observed at the lateral borders of myoepithelial cells. No β‐catenin was revealed at the myoepithelial basal surface. The intercellular expression of β‐catenin, as well as of E‐CD and α‐catenin, was also observed in carcinoma tissues with varying staining intensity. Almost all of 10 intraductal carcinomas and approximately 70% of 41 invasive ductal carcinomas expressed the three molecules at the same level as in normal glands, whereas approximately 80% of 13 invasive lobular carcinomas showed severe deficiency of them. Two lobular carcinomas in situ showed complete absence of all of the proteins. Some of these findings were confirmed biochemically by immunoblotting analysis. In invasive ductal carcinomas, α‐catenin was reduced more frequently in diffuse than in solid type tumours, whereas the level of expression of β‐catenin and E‐CD was unchanged between them. No correlation was present between reduced expression of the adhesion molecules and lymph node metastasis.

Genetic polymorphisms of interleukin-1β in association with the development of alcoholic liver disease in Japanese patients

OBJECTIVE:Cytokine interleukin-1β plays a central role in the inflammation process. Serum levels of IL-1β are elevated in patients with alcoholic liver disease (ALD), especially in those with cirrhosis and alcoholic hepatitis. Recently, the presence of genetic polymorphisms of this cytokine was confirmed. The aim of this study was to determine whether IL-1β polymorphisms are associated with the development of ALD.METHODS:We examined the frequency of two polymorphisms in the IL-1β gene located in promoter −511 and exon 5 +3953 locus by restriction fragment length polymorphisms in 142 male patients with ALD, 30 heavy drinkers without ALD, and 218 healthy controls.RESULTS:The carriers of −511 IL-1β allele 2 were present significantly more often in patients with alcoholic cirrhosis than in those with noncirrhotic ALD (p = 0.026), heavy drinkers without ALD (p = 0.001), and healthy controls (p = 0.032). The frequencies of allele 2 and heterozygotes of +3953 polymorphism were both significantly higher in heavy drinkers without ALD than in patients with ALD (allele, p = 0.030; genotype, p = 0.027) and healthy controls (allele, p = 0.047; genotype, p = 0.043). The haplotype, IL-1β−511 allele 2/+3953 allele 1 was associated with the development of alcoholic cirrhosis (p < 0.05).CONCLUSIONS:These results suggest that IL-1β polymorphisms may be related to the development of ALD in Japanese alcoholics.

Immunohistochemical analysis of TrkA neurotrophin receptor expression in human non-neuronal carcinomas

The Trk family of tyrosine protein kinase receptors plays a significant role in the development and maintenance of neural tissues. It has been recently shown that Trk receptors are also expressed by a wide range of normal non‐neuronal tissues in humans in a cell type‐specific manner. In the present study, the expression patterns of TrkA in 337 non‐neuronal invasive carcinomas of 15 different human tissues were investigated immunohistochemically. Overall, 133 (39%), 101 (30%) and 103 (31%) tumors exhibited strong, moderate and no TrkA Immunoreactivity, respectively. Esophageal and thyroid carcinomas expressed high levels of TrkA, whereas the levels in gastric and colon cancers were low. TrkA expression was detected not only in carcinomas originating from TrkA‐positive normal counterpart tissues, Including the esophagus, breast, lung and uterus, but also in those from TrkA‐negative tissues/cells of the thyroid, liver and ovary. Immunostaining for nerve growth factor‐β, the specific ligand for TrkA, in esophageal and breast carcinomas demonstrated its immunoreactivity in stromal fibroblasts and some TrkA‐expressing tumor cells. These results suggest that paracrine/autocrine regulation via stromal/tumoral NGF‐tumoral TrkA interaction may be involved In the growth of certain non‐neuronal carcinomas.

Demonstration of apoptosis in neuroblastoma and its relationship to tumour regression.

The in vivo occurrence of apoptosis in neuroblastomas was investigated. Histologically, a number of tumour cells showed typical apoptotic changes, including cell shrinkage, condensed and fragmented nuclei, eosinophilic cytoplasm, and absence of the inflammatory response. These cells coincided closely with the so-called karyorrhectic cells. An electrophoretic DNA ladder, a functional hallmark of apoptosis, was demonstrated in four of six tumours, and DNA fragmentation was detected in situ by terminal deoxytransferase-mediated nick end-labelling in 26 of 35 tumour specimens (74%). The labelled cell counts ranged from 5 to 62 per 5000 tumour cells (mean±SD: 15.0±14.5). Immunoperoxidase staining revealed that an apoptosis-suppressing protein, bcl-2, was expressed abundantly in advanced-stage tumours, whereas it was absent from karyorrhectic-apoptotic cells. Several tumours with the potential for spontaneous regression were bcl-2-deficient. Immunostaining of the Fas receptor for apoptosis demonstrated that the tumour cells expressed this molecule on their cell surfaces. Our results provide evidence of apoptosis in neuroblastomas and suggest that bcl-2 and the Fas receptor may play a role in its regulatory mechanisms.

Increased serum levels and sinusoidal expression of thrombomodulin in acute liver damage

Thrombomodulin (TM) is a surface glycoprotein of endothelial cells involved in both anticoagulation and antifibrinolysis. In this study, we assessed the clinical significance of TM in acute liver damage by using a rat model induced by intraperitoneal injection of D-galactosamine (Gal-N). Serum TM levels were measured with enzyme immunoassay utilizing rabbit anti-rat TM antibody. Simultaneously, immunohistochemical examination was performed using the same antibody. Serum TM levels increased significantly after the injection of Gal-N compared with preinjection levels, peaking from 48 to 72 hours after injection and normalizing by 168 hours. Changes in parenchymal damage were synchronized with changes of TM, and changes of TM levels mirrored changes of liver weight. In immunohistochemical examination, TM immunoreactivity was observed only on the endothelial surfaces of both the artery and portal vein within Glissons sheath in controls. After injection of Gal-N, TM immunoreactivity was gradually intensified, especially around the necrotic area and the central veins. These findings disappeared with improvement of parenchymal damage. Both the increase of serum TM levels and intensified TM immunoreactivity in the liver were synchronized with acute liver parenchymal damage induced by Gal-N. These findings on TM are related to endothelial damage with parenchymal necrosis and liver regeneration interacting with both homeostasis of microcirculation and healing of parenchymal damage.

Apoptosis in favourable neuroblastomas is not dependent on Fas (CD95/APO-1) expression but on activated caspase 3 (CPP32).

The mechanisms of apoptosis in neuroblastomas have been investigated by examining the expression profiles of Fas, Fas ligand (FasL), and caspase 3 in 42 primary tumour tissues. Immunohistochemically, no or weak Fas expression was detected in 25 out of 29 neuroblastomas (86 per cent), whereas high levels of expression of FasL and pro‐caspase 3 were noted in 30 and 29 of 42 tumours, respectively (∼70 per cent). Overexpression of pro‐caspase 3, but not FasL, correlated significantly with a younger age and low tumour stage. Western blot analysis of ten neuroblastomas confirmed the lack of Fas expression and the presence of strong FasL expression in all samples and pro‐caspase 3 expression in five tumours, of which four belonged to the favourable type. These favourable tumours also showed vigorous Asp‐Glu‐Val‐Asp (DEVD) hydrolytic, or caspase 3‐like activities, while the unfavourable tumour lacked such activity. Moreover, immunostaining for the p17 subunit of the caspase 3 heterodimer showed that active caspase 3 was mainly localized in apoptotic tumour cells. Combined together, our results suggest that caspase 3, activated via a Fas‐independent pathway, may play important roles in apoptosis, suppression of growth, and, in some cases, regression of favourable neuroblastomas. Copyright

Plasma thrombomodulin in liver diseases

Thrombomodulin (TM) is an endothelial cell surface membrane protein that binds to thrombin and acts as both a cofactor for protein C activation and an inhibitor of fibrinogenolysis (I). Since TM was discovered in plasma (2), the clinical significance of plasma TM (p-TM) has lately attracted attention. However, alteration of p-TM in liver disease has not yet been reported. Levels of p-TM in liver diseases were measured using a newly developed EIA (3). The levels of p-TM were significantly higher in patients with fulminant hepatitis, decompensated liver cirrhosis, hepatocellular carcinoma (p<0.01) and chronic active hepatitis (p/0.05) than healthy controls, while there was no significant increase in acute hepatitis and compensated liver cirrhosis. (Fig.) Thus the levels of p-TM might be considered to increase as a progression of liver damage, whereas they could be correlated statistically with neither liver function tests nor conventional coagulation-fibrinolytic parameters. The metabolism of p-TMremains obscure. Recently it has been suggested that the levels of p-TMmay increase in patients with collagen disease which involve destruction of vascular endothelial cells. The levels of p-TMare also said to increase in cases of chronic renal failure, in which p-TM is correlated with serum creatinin level. In liver disease, however, no correlation was found between the levels of p-TM and serum creatinin levels. These results indicate that a possible mechanism of increase in p-TM is present in liver disease, which may vascular endothelial cells TM (ng/ml} in damaged liver. ,20

Immunohistochemical expression of manganese superoxide dismutase in hepatocellular carcinoma, using a specific monoclonal antibody

The expression of manganese superoxide dismutase (Mn−SOD) was studied immunohistochemically, using a specific monoclonal antibody, in surgically resected hepatocellular carcinoma (HCC) and noncancerous tissues from 47 patients (2 with well-differentiated HCC, 36 with moderately differentiated HCC, 8 with poorly differentiated HCC, and 1 with undifferentiated carcinoma). Cancer cells in 44 patients (93.6%) were positive for Mn−SOD. The staining pattern of cancer cells was mostly homogeneous in well-differentiated HCC, whereas it was heterogeneous in poorly differentiated HCC. Moreover, strongly positive immunoreactivity was observed in noncancerous liver tissues in all patients, especially in normal hepatocytes surrounding HCC, regenerative small hepatocytes in the tumor boundary, and mononuclear inflammatory cells in the necroinflammatory lesions. The positive immunoreactivity for Mn−SOD in patients with HCC appears to reflect increased production of the enzyme protein.

HISTOPATHOLOGICAL INVESTIGATION OF DMF‐INDUCED HEPATOTOXICITY

N, N‐Dimethylformamide (DMF) has been implicated in the production of hepatotoxicity in male and female F344 rats. Repeated administration of dosages of 0.75 and 1.0 ml/kg DMF for up to 12 weeks produced massive liver necrosis associated with decreased body weight gain. Macroscopically, areas of necrotic change were well pronounced in every hepatic lobe, being yellowish‐red in coloration, irregular in shape, and varying in size, but were most striking immediately adjacent to the porta hepatis. Among the lobes, those which were relatively small were most markedly affected, and occasionally an entire lobe was involved. Light and electron microscopic studies revealed the hepatic architecture to be occupied by massive fibrosis. There were, however, sharp lines of demarcation between surviving normal and necrotic areas. Hemosiderosis involving macrophages was accompanied by proliferative bile ductules and a number of multinucleated giant cells. The distribution and quality of the hepatic lesions produced by DMF were discussed.

Hepatic veno-occlusive lesions in severe alcoholic hepatitis and alcoholic liver cirrhosis: a comparative histopathological study in autopsy cases.

Clinicopathological features of veno-occlusive lesions in hepatic veins were studied in autopsy cases of severe alcoholic hepatitis (15 cases) and alcoholic liver cirrhosis (15 cases). All the cases were heavy drinkers and died of liver failure or variceal rupture. The frequency and degree of veno-occlusive lesions, and the diameter and number of hepatic veins were studied from stained sections of liver blocks from each case. The hepatic veins observed ranged from 60 to 3000 μm in diameter. The veno-occlusive lesions were found in hepatic veins mainly 60 to 1200 μm in diameter. These findings were recognized in the majority of severe alcoholic hepatitis cases and alcoholic liver cirrhosis cases. Furthermore, more severe veno-occlusive lesions were noted in severe alcoholic hepatitis, compared with alcoholic liver cirrhosis. In the cases with obstruction in hepatic veins of <400 μm, a decrease in the number of hepatic veins and zonal necrosis were noted. In addition, some of the veno-occlusive lesions were recognized focally in the same cases. Clinical findings also indicated that ascites increased with the progression of the veno-occlusive lesions. We conclude that investigation of veno-occlusive lesions in severe alcoholic liver disease has clinicopathological significance.