Activation mechanism of anticoagulant protein C in large blood vessels involving the endothelial cell protein C receptor.

Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has been identified as an essential component for protein C activation. Although protein C can be activated directly by the thrombin-TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In this study, we demonstrate the constitutive, endothelial cell-specific expression of EPCR in vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity for protein C activation mediated by the thrombin-TM complex. By these mechanisms, EPCR appears to enable significant levels of protein C activation in large vessels. These results suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.

Incidence of adult T-cell leukemia/lymphoma among human T-lymphotropic virus type I carriers in Saga, Japan.

Using a population-based cancer registry, we tabulated 69 definite adult T-cell leukemia/lymphoma cases (36 males and 33 females) and 2.20 expected cases (0.95 for males and 1.25 for females) diagnosed from 1981 to 1983 in Saga, Japan. The number of human T-lymphotropic virus type I carriers was computed by applying sex- and age-specific anti-human T-lymphotropic virus type I antibody positive rates among blood donors at the blood center in 1986 to the whole population of Saga Prefecture in 1982. The age-specific incidence rates among male human T-lymphotropic virus type I carriers from 40 to 79 yr of age per 100,000 were significantly higher than those of female carriers (P less than 0.05), and the rates from 60 to 69 yr of age were the highest in both sexes. The annual crude incidence rates among carriers were 115.9 for males and 66.4 for females. The summary incidence rates with 95% confidence intervals were 115.9 (58.4 to 193.0) for males and 65.9 (30.0 to 115.9) for females. The cumulative risks were 4.5% (0.8 to 11.0) for males and 2.6% (0.3 to 7.0) for females. These morbidity figures were assumed to be underestimated partly due to the newly proposed clinical entity of adult T-cell leukemia/lymphoma.

Physical mapping of the genome and sequence analysis at the inverted terminal repetition of adenovirus type 4 DNA.

The genome of adenovirus type 4 (Ad4), the unique member of Ad group E, has been mapped with nine restriction endonucleases. Comparison of the occurrence of restriction endonuclease cleavage sites on Ad2, Ad7, Ad12 and Ad4 indicates that there is very little homology between these serotypes. Sequence analysis at the ITR of Ad4 showed that the "CAT" box which is present in all the ITRs of Ad's so far sequenced is absent in Ad4. The length of 116 bp for the ITR of Ad4 is also different from that of other Ad subgroups.

In situ demonstration of the HTLV-I genome in the spinal cord of a patient with HTLV-I-associated myelopathy.

Using polymerase chain reaction (PCR) and in situ hybridization, we investigated the HTLV-I genome in the CNS of an HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patient with a 20-year disease duration. Neuropathologically, there was severe white matter degeneration throughout the spinal cord, but lymphocytic infiltrates were not evident in any lesion. PCR amplification of the pX region of HTLV-I DNA detected its sequence in the spinal cord and all extra-CNS tissue samples. In situ hybridization using probes complementary to the pX and gag regions detected the HTLV-I genome in the cytoplasm and nucleus of cells in the thoracic cord. The findings indicate a direct involvement of HTLV-I in the neurodegeneration of HAM/TSP.

Localized lymphoid hyperplasia (pseudolymphoma) of the pancreas presenting with obstructive jaundice.

A case of hyperplastic lymphoid lesion of the pancreatic head manifested by obstructive jaundice is presented. A 57-year-old woman who complained of malaise and icterus underwent pancreatoduodenectomy under the clinical diagnosis of pancreatic carcinoma. The lesion was diagnosed as localized lymphoid hyperplasia (pseudolymphoma) based on the presence of hyperplastic follicles with germinal center and mixed infiltration of plasma cells and mature lymphocytes with no significant cytologic atypia. The immunostaining revealed polyclonal origin of the lymphoplasmocytic component. Localized lymphoid hyperplasia occurs in a wide variety of sites; however, to our knowledge, its occurrence in the pancreas has not been documented previously.

Multinucleated variant endothelial cells (MVECs) of human aorta: expression of tumor suppressor gene p53 and relationship to atherosclerosis and aging.

Multinucleated variant endothelial cells (MVECs) generally exist in atherosclerotic human aorta and even in nonatherosclerotic aorta. Because the number of nuclei is increased in every MVEC, and because DNA instability was suspected, a series of oncogene expressions was conducted to clarify the nature of nuclear abnormality. The tumor suppressor gene p53 was found to be specifically expressed in the multinuclei of MVECs, while double nuclei were sometimes positive, and mononuclear typical endothelial cells were always negative for p53. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) revealed extra bands in exons 5 and 7 of the p53 gene, but no additional band in exons 6 and 8. In a BCL family, BCL-2 was coexpressed in one or two nuclei in the perinuclear space of the multinuclei of MVECs, whereas MCL-1, BCL-XS/L, and BAX were all negative, indicating that the BCL-2 coding gene is expressed only in the corresponding one or two nuclei of the multinuclei. Another oncogene, c-MET (hepatocyte growth factor receptor), was universally expressed in either type of endothelial cells, but other oncogenes, k-RAS and c-ERBB2, were not expressed in either type. MVECs were derived from human aorta and therefore non-tumorous somatic cells. No morphologic evidence of apoptosis was found. Although it is unclear that the extra bands came from the MVECs or just from ECs associated with atherosclerosis, combined immunocytological studies and PCR analysis suggest that MVECs express mutant type p53.

Phlegmonous colitis: a specific and severe complication of chronic hepatic disease.

Phlegmonous colitis (PC) is an acute infectious entity caused by bacteria. In this study, we reviewed 8,822 autopsy cases and found 13 cases of PC (0.15%). PC affected 2.43% of patients with hepatic cirrhosis or subacute liver atrophy, both of which were considered to be due to hepatitis viral infection. Before autopsy, none of the cases studied was suspected to involve PC, irrespective of the immediate cause of patient death. Thirteen autopsy cases showed some or all of the following pathohistologic characteristics: (1) involvement of the cecum (9 cases, 76.9%), (2) phlegmonous inflammatory changes and edema in the submucosa (100%), (3) bacterial infection (100%), (4) no microscopically detectable mucosal injuries (12 cases, 92.3%), and (5) acute serositis (peritonitis) (2 cases, 15.4%). These results suggest that PC is an unrecognized, but fatal complication of patients with some hepatic diseases and that PC has pathohistologic characteristics in common with previously reported spontaneous bacterial peritonitis in animal models. PC probably arises due to spontaneous infection in patients with hepatic cirrhosis.

Endothelial cell heterogeneity in experimentally-induced rabbit atherosclerosis. Demonstration of multinucleated giant endothelial cells by scanning electron microscopy and cell culture.

We investigated the aortic endothelial cells of cholesterol-fed rabbits, using scanning electron microscopy and a cell culture technique. Rabbits were given a 1% cholesterol diet intermittently for up to 40 weeks. In these animals, the area of endothelial cells was increased and the cells showed polymorphism in relation to the progression of atherosclerosis. In animals fed the cholesterol diet for 12, 28 and 40 weeks, the average area of the endothelial cells was 436 +/- 15, 762 +/- 153, and 836 +/- 165 microns2, respectively. In the cholesterol-fed 40-week group, in particular, giant endothelial cells, measuring more than 1200 microns2, accounted for 14% of the population. In animals fed a standard diet there was no significant difference in endothelial cell morphology between control 0-week and control 40-week groups; in both, the luminal surface of the thoracic aorta formed a homogeneous sheet covered by small rhomboidal endothelial cells, the area of most being less than 400 microns2. Primary cultured endothelial cells harvested from those control groups were mononuclear typical small cells with a centrally located nucleus; the proportion of binucleated cells was less than 2% and no multinucleated giant cells with three or more nuclei were detected. Endothelial cells from the cholesterol-fed groups, however, contained larger numbers of binucleated cells, with the number increasing in proportion to the duration of cholesterol feeding. The major distinguishing feature of the endothelial cells in the cholesterol-fed groups was the presence of multinucleated giant cells with three or more nuclei; these accounted for 2.3% and 3.3% of the total cell population in the cholesterol-fed 28- and 40-week groups, respectively. No bromodeoxyuridine uptake was found in the nuclei of the cultured multinucleated giant cells. Heterogeneity of endothelial cells, with the concomitant appearance of multinucleated giant cells, emerges with the progression of diet-induced atherosclerosis. The morphological alterations of endothelial cells observed in the present study intimately reflect changes in their function associated with the progression of atherosclerotic lesions.

Expression of c-Met in laryngeal carcinoma.

Expression of c-Met, a gene for the hepatocyte growth factor/scatter factor (HGF/SF) receptor, is known to be associated with tumour development in several human carcinomas. The expression of c-Met was examined using immunohistochemistry in 82 cases of primary laryngeal carcinoma to evaluate the tissue distribution of c-Met and the clinicopathological significance of c-Met expression. In normal larynx, c-Met expression was observed only in some minor salivary glands. Positive reaction for c-Met in neoplastic epithelium was noted in 45 out of 82 (54.9%) cases. In 44 cases, structures adjacent to the carcinoma (noncancerous squamous epithelium, some stromal fibroblastic cells, and endothelial cells) showed positive reaction for c-Met. c-Met expression in cancerous epithelium was significantly correlated with lymph node status (P<0.04) and proliferating activity expressed by the Ki-67 labelling index (P<0.02). There was no correlation between c-Met expression and age, sex, histological type, T category, distant metastasis or clinical stage. The data suggest that overexpression of c-Met in laryngeal carcinomas represents a growth advantage for cancer cells, which may be conferred by the mitogenic effect of HGF/SF. Simultaneous c-Met expression in noncancerous components of the larynx may represent a paracrine modification of c-Met.

Primary intestinal T-cell lymphoma resembling lymphomatous polyposis: report of a case.

We report an interesting case of primary intestinal T-cell lymphoma (ITL) resembling lymphomatous polyposis (LP) in a 24-year-old man. The neoplasm macroscopically showed numerous small polyps throughout the colon and microscopically showed diffuse proliferation of small-sized tumor cells with occasionally cleaved or irregularly shaped nuclei. The tumor cells were immunohistochemically positive for CD3, CD8, TIA-1, and CD56, and a polymerase chain reaction study showed a single band, indicating monoclonal rearrangement of the T-cell receptor beta gene. The phenotypic features in the current case are consistent with those of ITL derived from cytotoxic CD56+ CD8+ intraepithelial lymphocytes. This is the second documented case of primary ITL with a morphologic pattern of LP.

Association of vascular endothelial growth factor and mast cells with angiogenesis in laryngeal squamous cell carcinoma.

We investigated the expression of vascular endothelial growth factor (VEGF) and microvascular density in 54 cases of invasive laryngeal squamous cell carcinoma (SCC) and in ten samples of normal laryngeal tissue using immunohistochemistry methods. The study also focused on the distribution of mast cells in and around the SCCs. The microvascular density in laryngeal carcinoma tissue was higher than that in normal tissue (P = 0.02). VEGF was localized in SCCs, stromal cells, endothelial cells, minor salivary glands, and non-cancer epithelium adjacent to the tumor. VEGF expression in the tumor cells was found in 13 of 54 cases (24.1%), whereas mast cells around the carcinomas were VEGF positive in all 54 cases. Staining of VEGF in SCCs was strong in the area of high microvascular density (P = 0.0002). Using a multi-labeling subtraction immunostaining method, VEGF-positive stromal cells were classified mostly as mast cells and, in a few instances, as macrophages. VEGF staining in SCCs was associated with the mast cell count (P = 0.0001). There was no distinct correlation between VEGF expression and pTNM stage of an SCC. In conclusion, the results suggest that VEGF might be an important angiogenic factor in cancer invasion. Laryngeal cancer cells and mast cells may control the angiogenic response by releasing VEGF.

HTLV-I associated and non-associated primary T-cell lymphoma of gastrointestinal tract.

The gastrointestinal (GI) tract is the common extranodal site for non-Hodgkin's lymphoma (NHL), and primary lymphoma of GI tract are mostly of B-cell origin. We have treated 16 patients with primary lymphoma of GI tract between 1981 and 1991, of whom 10 (62%) were of B-cell origin, while 6 (38%) were of T-cell origin. The incidence of T-cell phenotype in our hospital was considered to be much higher than that of previous reports and these 6 patients with primary T-cell lymphoma of GI tract were carefully studied. The primary sites were stomach in 4, ileocecum in 1, and duodenum in 1 case. Their T-cell nature was confirmed by immunohistochemical methods. All were peripheral T-cell lymphomas; one was CD 3+ 4- 8- and the other 5 were CD 3+ 4+ 8-. The antibody against human T-cell leukemia virus type I (HTLV-I) was positive in 3 cases (HTLV-I associated), but negative in 3 (HTLV-I non-associated). The integration of HTLV-I proviral DNA in HTLV-I associated patients was demonstrated by Southern blot analysis after DNA amplification by means of polymerase chain reaction (PCR). The clinical features of the HTLV-I associated and HTLV-I non-associated primary T-cell lymphoma of the GI tract were quite different. HTLV-I associated patients showed leukemic manifestations and tumor involvement of the skin at a later stage of the disease. These observations indicated that HTLV-I can play an important role in the occurrence of primary T-cell lymphoma of GI tract.

Angiogenesis and mast cells in non-Hodgkin's lymphoma: a strong correlation in angioimmunoblastic T-cell lymphoma.

Mast cells are likely to play a role in angiogenesis under pathological conditions. Solid tumor growth is dependent on angiogenesis, but the influence of mast cells on angiogenesis in non-Hodgkin's lymphoma, (NHL) is not clear. We investigated mast cell number and vessel count in 61 cases of NHL. We also evaluated expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), both important cytokines for angiogenesis. The number of mast cells was greater in T-cell lymphomas than in B-cell lymphomas. Of the T-cell lymphomas, the greatest number of mast cells was observed in the angioimmunoblastic T-cell lymphoma (AIL). In all NHLs, significant correlation was found between vessel count and the number of mast cells (p < 0.0001) and between vessel count and the number of VEGF-expressing cells (p < 0.05) but not between vessel count and bFGF-expressing cells. Strong correlation was detected between the number of mast cells and the number of VEGF-expressing cells (p < 0.0001) in all NHLs. Double fluorescence staining of VEGF mRNA and mast cell tryptase revealed that mast cells expressed VEGF mRNA. Our data suggest that mast cells play a very important role in angiogenesis by expressing VEGF in NHL, especially in AIL.

Differences in Prognostic Factors between Diffuse Non-Hodgkin's Lymphoma and Lymphoma Type of Adult T-Cell Leukemia.

Sixty-four Japanese patients with diffuse non-Hodgkin's lymphoma (NHL) were classified into human T-cell leukemia virus type I (HTLV-I) non-related diffuse NHL (41 cases) and HTLV-I related diffuse NHL, i.e. lymphoma type of adult T-cell leukemia (ATL-lymphoma, 23 cases) on the bases of serological detection of HTLV-I antibody and the detection of HTLV-I proviral DNA in malignant cells. The survival curves between diffuse NHL and ATL-lymphoma were quite different. Therefore, it is important to know the differences in prognostic factors, if any, affecting the survival in two groups: diffuse NHL and ATL-lymphoma cases. In diffuse NHL patients, factors found to be important were age, performance status, stage, organ involvement, hemoglobin concentrations and platelet count in the peripheral blood, serum lactic dehydrogenase (LDH) and calcium levels. However, none of these factors were useful for ATL-lymphoma patients, except for the serum calcium level. Thus, prognostic factors should be considered separately for diffuse NHL and ATL-lymphoma.

Occurrence of primary biliary cirrhosis, CREST syndrome and Sjögren's syndrome in a patient with HTLV-I-associated myelopathy.

We report the occurrence of three autoimmune diseases (primary biliary cirrhosis, CREST syndrome and Sjögren's syndrome) in a patient with HTLV-I-associated myelopathy (HAM). The patient had the depressed cell-mediated immune responses but the infiltration of CD8-T cells was found in the cerebrospinal fluid and liver. The clinical and immunological features of this case are similar to those of chronic graft-versus-host disease.

Polypoid adenomyoma of the gallbladder.

We describe a distinctive polypoid lesion of the gallbladder (16 mm in diameter) at the fundus, associated with a granulomatous mass in the liver adjacent to the gallbladder fossa, in a 64-year-old Japanese woman. Preoperatively, the lesion was diagnosed as advanced gallbladder cancer infiltrating the liver, and hepatopancreato-duodenectomy was performed. In the resected specimen, the polyp was round and pedunculate in shape, and, microscopically, it consisted of a mixture of small glandular and surrounding muscular elements, but atypia was not noted. The constituent elements were identical with those of adenomyoma, but the polypoid appearance was unusual. The hepatic lesion proved to be a foreign body granuloma containing multiple barium fragments and giant cells. A deep gastric ulcer, which penetrated into the gallbladder fossa, was also noted, near the granuloma. The histologic features indicate that the polypoid appearance of the tumor was due to a secondary modification of pre-existing adenomyoma by hepatic granuloma. To our knowledge, this is the second reported case of an adenomyomatous lesion of the gallbladder with a polypoid appearance.

Subacute necrotizing encephalomyelopathy (Leigh disease): report of a case with Lennox-Gastaut syndrome.

More than 100 cases of Leigh disease have been reported. None have shown Lennox-Gastaut syndrome. We report here the first known case of Leigh disease with Lennox-Gastaut syndrome, and discuss the clinical course in detail. A 3 1/2-year-old-boy was admitted with multiple symptoms and despite various therapies he died at age of ten years. Serial CT showed marked cerebral atrophy, ventricular dilatation, and an arachnoid cyst in the posterior fossa. Histopathological findings on autopsy of the brain and spinal cord were consistent with those characteristic of Leigh disease. In the chronic course of Leigh disease, the cerebral cortex was also involved and the Lennox-Gastaut syndrome might be complicated.

Experimental cerebral atherosclerosis in the rabbit. Scanning electron microscopic study of the initial lesion site.

The development and initial lesion sites of cerebral atherosclerosis were studied in hypertensive rabbits fed 0.5 g/day cholesterol in their diet. The earliest lesions developed at remarkably localized areas of the basilar artery-posterior cerebral artery Y-bifurcation (area A) and vertebral arteries-basilar artery confluence (area B). These findings were obtained from a thorough scanning electron microscopic survey of the dorsal surface of the cerebral artery segment covering from the vertebral arteries to the posterior cerebral arteries. By light microscopy intimal lesions were mainly composed of accumulations of foam cells and smooth muscle cells. Electron microscopically foam cells accumulated in the intima resembled those of a monocyte-macrophage lineage. Early lesions involving only a few endothelial cells with adherent leukocytes occurred at the dividing and confluent portions of the endothelial arrays formed in areas A and B, respectively. The results indicate that hypertension coupled with hypercholesterolemia induces atherosclerosis in particular vulnerable regions of the cerebral arteries.

Critical role of type IV collagens in the growth of bile duct carcinoma. In vivo and in vitro studies.

Most extrahepatic bile duct carcinomas (EBDC) are characterized by a striking stromal response (desmoplasia). Our previous studies showed deposition of type IV collagen in the desmoplastic stroma beyond the basement membrane. Although type IV collagen is expressed in EBDC, little is known about the pattern of deposition in tumor stroma and how this matrix component influences the behavior of tumor cells. With the progression of desmoplasia in EBDC, different changes occurred in the quantity and localization of type IV collagen from that of type I collagen. Type I collagen was diffusely distributed in the stroma and appeared to be concentrated in the center of the tumors. In contrast, type IV collagen was deposited in the interstitium alongside carcinoma cells at the tumors' periphery. Weak or no type IV collagen deposition was detected in the more central portion of the tumors containing sclerotic collagens. To investigate the role of stromal type IV collagen in tumor cell proliferation, EBDC cell lines were cultured in a three-dimensional matrix containing varying compositions of type I collagen and type IV collagen. They were also assayed for cell adhesion and migration using in vitro models. Type IV collagen more extensively stimulated tumor cell proliferation, adhesion and migration in a dose-dependent manner than did type I collagen. All of these results suggest that modified tumor stroma with the presence of type IV collagen in EBDC provides a better environment for tumor growth and invasion.

Ultrastructural demonstration of mast cells in varicose veins of lower limbs: presence of mast cell-mediated mechanism.

OBJECTIVE: Mast cells are suggested to play an essential role during development of varices in the lower limbs. EXPERIMENTAL DESIGN: Investigation of the ultrastructure of mast cells in varicose lesions. SETTING: Saga Medical School, Yamamoto Surgical Hospital. PATIENTS: Eighteen varicose veins of 17 patients and 9 normal saphenous veins of 9 patients were examined. Patients who had undergone sclerotherapy for varices were excluded. INTERVENTIONS: Radical stripping surgery was performed on all varicose veins. MEASURES: Ultrastructural observations. RESULTS: In normal saphenous veins, mast cells usually singly embedded in dense collagen bundles as resident cells. They have characteristic crystalline granules of storage type. In varicose veins, mast cells show different features such as an increase of altered granules of discharging type, degranulation and intimate relationship with fibroblasts and lymphocytes. CONCLUSIONS: The observations suggest the presence of mast cell-mediated mechanism by releasing some mediators in the development of varices.