ULTRASTRUCTURE OF KIDNEY FROM THREE PATIENTS WITH HBeAg-ASSOCIATED NEPHROPATHY WITH SPECIAL REFERENCE TO VIRUS-LIKE PARTICLES IN THE GLOMERULAR TUFTS

The biopsied kidneys from three patients with hepatitis Be antigen (HBeAg)-associated nephropathy were observed by light microscopy, immunohistochemistry and electron microscopy. By an indirect technique utilizing horseradish peroxidase-conjugated antisera, HBeAg was found to be deposited in a diffuse granular fashion along the glomerular capillary wall. No deposition of hepatitis Bs or hepatitis Be antigen was detected. The three cases were diagnosed as HBeAg-associated nephropathy. Ultrastructurally, there were finely granular electron-dense deposits in the subendothelial area, basement membrane, mesangial area and subepithelial area of the glomerular tufts. In all three cases, virus-like particles between 30 and 70 nm in diameter were also found in such areas of the glomerular tufts, and rarely in the glomerular capillary lumen and space of Bowman. They occasionally formed clusters in the phagosomes of mesangial cells. In addition, tubulo-reticular structures were noted in the cytoplasm of endothelial cells in the glomerular capillaries. The presence of HBeAg both in the serum and in the kidney and of virus-like particles in the glomerular tufts suggests that HBeAg is causally related to the development of HBeAg-associated nephropathy.     

Malignant transformation of gastric hyperplastic polyps: alteration of phenotypes,proliferative activity,and p53 expression

The aim of this study was to clarify the mechanism of malignant transformation of gastric hyperplastic polyps, focusing on phenotypic expression, cell proliferation, and p53 overexpression. Twenty-two lesions of gastric hyperplastic polyps with neoplastic foci were selected for this study. The phenotypes were divided into 3 types (G, gastric; incomp I, incomplete intestinal; and comp I, complete intestinal), according to immunohistochemical stains (human gastric mucin [HGM], MUC2, and CD10). The cell proliferative activity by Ki-67 and overexpression of p53 protein were also examined. Eleven of these lesions contained carcinoma components (CA, category 5 by the Vienna classification), 6 of which were accompanied by low-grade dysplasia (LGD, category 3) and 4 of which were accompanied by high-grade dysplasia (HGD, category 4). Another 2 were composed only of HGD, and the remaining 9 were composed of both LGD and HGD components. As a result, 15 LGD, 15 HGD, and 11 CA components were recognized. The 15 LGD components were classified as 1 G type and 14 incomp I type. All hyperplastic components expressed HGM, 5 (22.7%) of which were accompanied by focal intestinal metaplasia demonstrated by MUC2 expression, whereas intestinalization frequently occurred in neoplastic components (93% of LGD, 53% of HGD, and 64% of CA components). The labeling index was 22.2% in hyperplastic, 42.2% in LGD, 55.7% in HGD, and 53.9% in CA components. p53 protein overexpression was recognized in none of hyperplastic, in 40% of the LGD, in 60% of the HGD, and in 45% of the CA components. These results suggest the importance of the dysplasia-carcinoma sequence in malignant transformation of hyperplastic polyps. Interestingly, intestinalization frequently occurs during neoplastic transformation, although it is not common in the surrounding hyperplastic components.     

Temporospatially regulated expression of subtilisin-like proprotein convertase PACE4 (SPC4) during development of the rat submandibular gland.

The temporospatial expression of PACE4, a member of the mammalian subtilisin-like proprotein convertase family involved in the activation of growth/differentiation factors, was investigated by in situ hybridization during the development of the rat submandibular gland (SMG). At the initiation stage (day 15.5 of gestation; E15), PACE4 was intensely expressed in the submandibular epithelium, but weakly expressed in the mesenchymal cells. At E16 when the branching morphogenesis becomes obvious, the expression of PACE4 in the mesenchyme was further decreased, although its level in the submandibular epithelium had not changed remarkably from that at E15. During the next stage of embryonic development (E17-E20), PACE4 was expressed in the cells derived from the submandibular epithelium, which include the proacinar, terminal tubular, and presumptive ductal cells. In the perinatal SMG, PACE4 was still expressed intensely in the terminal portion of the SMG containing the proacinar and terminal tubular cells, whereas its expression in the ductal cells was obviously decreased at the second postnatal day (P2) and at P6. Acinar cells expressing no PACE4 appeared, and their numbers increased following their development (P9-P20). At P30 when the PACE4 expression in the acinar cells was completely suppressed, its expression in the ductal cells became intense again. This temporospatially regulated expression of PACE4 suggests its apparent association with the proliferation, differentiation, and establishment of functional acinar and ductal cells of the SMG.     

Gastric carcinosarcoma (sarcomatoid carcinoma) with rhabdomyoblastic and osteoblastic differentiation

A rare case of carclnosarcoma (sarcomatoid carcinoma) with rhabdomyoblastic and osteoblastic differentlatlon occurrlng in the gastric remnant is reported. A 69-year-old Japanese man who had undergone a partial gastrectomy for a duodenal ulcer 30 years earlier, presented with anemla, eplgastralgia, and an abdominal mass. The diagnosls of gastric carcinosarcoma was made based on the findings of endoscoplc biopsies. The patient was thus scheduled to undergo a surgical operatlon, but he died of respiratory failure. At autopsy, a huge polypoid tumor measurlng 2Ox18times8 cm was located on the greater curvature of the gastric remnant. Microscopically, the tumor conslsted of Intimately mixed tubular adenocarcinoma and heterologous mesenchymal elements contalning rhabdomyosarcoma and osteosarcoma. Between these components, a morphological transition from the adenocarclnoma element to the sarcomatous element was observed. Ultrastructually, rhabdomyoblastlc dlfferentlation was conflrmed In the sarcomatous areas. lmmunocytochemlcal expresslons of epithelial markers includlng epithelial membrane antlgen and cytokeratlns (35bHll and 34bE12) were recognlzed not only in the carclnomatous cells but also In the sarcomatous cells. These findlngs suggest that carclnomatous cells appear to transform Into cells wlth sarcomatous features.     

Relationships of Differential Leukocyte and Lymphocyte Subpopulations with Carotid Atherosclerosis in Elderly Men

To examine the relationship between systemic immune status and carotid atherosclerosis in elderly men, differential leukocyte counts and lymphocyte subpopulations were measured in 557 apparently healthy Japanese men aged 60-75 years. Each individual also underwent high-resolution ultrasonography for measurement of intima-media thickness (IMT) of the common carotid arteries. The increased numbers of circulating lymphocyte subpopulations, including memory T cells (CD4+CD45RO+T cells) and late-phase activated B cells (CD19+CD80+B cells) correlated significantly and positively with the mean IMT of the common carotid artery after adjustment for age, smoking, and other cardiovascular risk factors. The positive associations of CD19+CD80+B and CD4+CD45RO+ T cell counts with mean IMT were more evident among nonsmokers, hypertensives, and men with lower HDL-cholesterol levels. The present epidemiological study provided the evidence that alterations in lymphocyte subpopulations, in particular memory T cells and late-phase activated B cells concur with carotid atherosclerosis among free-living elderly men.     

Lipopolysaccharide-induced elevation and secretion of interleukin-1beta in the submandibular gland of male mice

The intraperitoneal injection of lipopolysaccharide (LPS) (400 microg/kg body weight) induced the expression of mRNAs of inflammatory cytokines such as interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha in the submandibular gland (SMG) of C3H/HeN mice but not that of C3H/HeJ mice, a mutant strain for Toll-like receptor-4 (TLR-4(-) mutant). The mRNA levels of these cytokines in the SMG of the wild-type mice increased as early as 3 hr after injection, peaked at 3-6 hr, and had decreased again by 24 hr. In this study, we particularly focused on IL-1beta, and induction by this endotoxin was investigated in detail. Denervation of the superior cervical trunk and chorda tympani nerve did not diminish the LPS-induced elevation of IL-1beta mRNA in the SMG, indicating the irrelevance of the central nervous system in this induction. TLR-4 mRNA and protein were shown to be strongly expressed in the SMG, suggesting the direct action of LPS on this gland. IL-1beta proteins were localized in the secretory granules of granular convoluted tubular (GCT) cells, and their molecular weights in the gland were 17.5 and 20 kDa. IL-1beta of the same size appeared in the saliva 6 hr after LPS injection in C3H/HeN but not in C3H/HeJ mice. The present study thus suggests that IL-1beta, an inflammation cytokine, is induced and secreted into the saliva in response to endotoxin injected intraperitoneally.     

Detection and quantitation of phenotypically mixed viruses: Mixing of ecotropic and xenotropic murine leukemia viruses

Methods were devised for detection and titration of phenotypically mixed particles in harvests of mixed infections by ecotropic and xenotropic murine leukemia viruses. Such harvests contain, in addition to parental-type virions, particles which induce ecotropic virus infection of heterologous cells and particles which induce xenotropic virus infection of mouse cells. The phenotypically mixed particles show neutralization and interference specificities corresponding to the host range determinant. These findings, in conjunction with the relative proportions of infectious particles in mixed infection harvests, are compatible with the hypothesis that the restriction of mink cells for infection by ecotropic viruses is fully circumvented by phenotypic mixing, while the resistance of mouse cells to xenotropic virus infection is at two levels, one of which cannot be overcome by phenotypic mixing.     

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

Summary We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3 termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I fromEscherichia coli, which has 35 exonuclease and replacement activities as well as its main 53 polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.     

Age-dependent basal level and induction capacity of copper-zinc and manganese superoxide dismutase and other scavenging enzyme activities in leukocytes from young and elderly adults.

Several enzymes, including superoxide dismutase (SOD), catalase, glutathione peroxidase, and D-glucose-6-phosphate dehydrogenase are capable of scavenging reactive oxygen species in in vivo. We assessed both basal levels and the capacity of these enzyme activities to be induced in human leukocytes in response to a variety of agents. Basal activity of copper-zinc SOD, and manganese SOD showed little variation with age. In contrast, the basal activity of the three H2O2 scavenging enzymes, catalase, glutathione peroxidase, and D-glucose-6-phosphate dehydrogenase, was significantly higher in younger adults than in elderly individuals. Both manganese SOD and copper, zinc SOD activities were significantly induced by paraquat, interleukin-1, tumor necrosis factor, adriamycin, and bleomycin in lymphocytes and neutrophils from asymptomatic non-aged adults, whereas neither activity was induced in aged individuals. In contrast, glutathione peroxidase activity was significantly induced in both groups of subjects, whereas catalase and D-glucose-6-phosphate dehydrogenase were only slightly induced in either. Enzyme induction with paraquat, adriamycin, or bleomycin was inhibitable by neutralizing antibody to interleukin-1 and tumor necrosis factor, suggesting that the inductions observed with these three drugs are due to the distal mediators, interleukin-1 or tumor necrosis factor released from the cells. Finally, as observed in the regulation of genes in eukaryotes (Storz et al: Bacterial defenses against oxidative stress. Trends Genetics 1990, 6:363-368, ref. 1) O2- and H2O2 seem to differ in the rate of change with age in both basal levels and inducibility under oxygen stress.