Virchows Archiv. B, Cell pathology including molecular pathology最新文献

The ultrastructure of the cellular contacts and bile canaliculi was examined in cultured neonatal (day 5) rat hepatocytes to elucidate the development of cellular polarity. A new scanning electron microscopic technique for cultured hepatocytes allowed a view of cell-cell attachment and the entire cell surface, including the underside on plastic dishes. At 3 h after plating, neonatal hepatocytes were shown to be round, with loss of the preferential localization of cell organelles. After 6 h of culture, the cells had become oblong; they were aggregated in groups of several cells and the cellular contacts were not as rigid or as straight as those in adult hepatocytes. Transmission electron microscopy showed the biliary functional polarity to be like that in vivo. On the undersurfaces of adjacent neonatal hepatocytes a hemicanalicular structure lined with microvilli was found, which probably corresponds to the ultrastructure of bile canaliculi in vivo. However, no canaliculi or orifices of bile channels were found in adult hepatocytes. These results suggest that in neonatal rat hepatocytes the formation of tight rigid cellular contacts was suppressed. Modulation of cell membranes appeared on the undersurfaces of neonatal hepatocytes in early culture stages. The differences in the development of cellular polarity could be caused by the proliferating activity of neonatal hepatocytes.

Systemic amyloidosis of the amyloid A (AA) type, is occasionally associated with various neoplasms, but the cause is still unclear. We obtained interleukin 6 (IL-6)-producing cells designated YO from a primary culture of a malignant peritoneal mesothelioma of epithelial type obtained from a 62-year-old woman. Post mortem examination revealed that the patient had systemic amyloidosis of the AA type. The supernatant media of YO cells, as well as recombinant human IL-6, successfully induced nonneoplastic liver cells to produce serum AA (SAA). Our data suggest that IL-6 produced by the tumor cells may have played an important role in the paraneoplastic syndrome of AA amyloidosis in this patient.

Using a lysosome-enriched "light mitochondrial" fraction of a rat liver homogenate, the effects of the reactive oxygen species hydrogen peroxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme, N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was assayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membrane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of about 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-dependent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a lag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indicate that neither superoxide radicals nor hydrogen peroxide are by themselves damaging to lysosomes. Available catalytically active iron in Fe(II) form, however, allows reactions yielding powerful oxidative species--probably hydroxyl radicals formed via Fenton reactions--to take place inducing peroxidation of the lysosomal membranes resulting in dissipation of the proton-gradient and leakage of their enzyme contents.

The in-situ polymerase chain reaction (in-situ PCR) is a novel molecular technique that combines the extreme sensitivity of the PCR with the anatomical localization provided by in-situ hybridization. A number of groups have recently reported studies using in-situ PCR for the detection of specifically amplified single-copy nucleic acid sequences in single cell preparations or low copy DNA sequences in tissue sections. In this overview, we describe the principles of in-situ PCR, review the applications of this technique and discuss future aspects of in-situ PCR. We critically compare the different in-situ PCR protocols described in the literature. Emphasis is placed on the absolute requirement for controls to allow accurate interpretation of results and the possible problems and pitfalls of the in-situ PCR methods, including artefacts related to diffusion of PCR products and non-specific incorporation of labelled nucleotides into fragmented DNA undergoing repair. It is concluded that this technique will eventually play an important role in specialized diagnostic laboratories in the evaluation of viral diseases, haematological and other malignancies which have unique genetic markers.

The light microscopic and immunohistochemical features of a novel localized senile amyloidosis in the gastrointestinal tract of C57BL/Ka mice are described. Senile gastrointestinal amyloidosis was predominantly found in the lamina propria of the ileum, cecum and stomach and infrequently in other segments of the gastrointestinal tract. The Congo red affinity of the senile amyloid was sensitive to potassium permanganate pretreatment. The amyloid did not react with anti-AA and anti-immunoglobulin antisera, but stained positively for apoAII, a major apolipoprotein of high density lipoproteins. A similar type of amyloid, termed AApoAII, has recently been described in a systemic form of senile amyloidosis in mice. In the present study, we investigated the effect of long-term immunosuppressive treatment on the incidence of systemic AA-amyloidosis and gastrointestinal AApoAII-amyloidosis in aged C57BL/Ka mice. Gastrointestinal amyloidosis occurred in 60% of the control mice, but significantly less in mice of the immunosuppressed groups. In contrast, systemic AA-immunoreactive amyloidosis was only found in mice that were given immunosuppressive treatment. There was no codeposition of AA and AApoAII-amyloid. These findings indicate that immunosuppressive drugs have a profound effect on the incidence as well as the type of amyloidosis in C57BL/Ka mice.

The expression of cytokeratin (CK) 17 was studied in 28 primary transitional cell carcinomas (TCCs) of the human urinary tract using CK 17-specific monoclonal antibody E3. While CK 17 was not detectable at all or only present in some areas of basal cells in normal--appearing urothelium, a certain subpopulation of cells of all G1 and G1/G2 TCCs examined (9 cases) stained positive for CK 17. These latter cells were either restricted to the basal compartment or located also in suprabasal layers exhibiting a decreasing intensity of immunoreactivity. CK 17 was seen in practically all cells in G2 and G2/G3 tumors (7 cases). In contrast, G3 TCCs and anaplastic carcinomas showed a highly variable CK 17 staining pattern ranging from completely negative to completely positive with several intermediate phenotypes. Our results indicate that CK 17 could be a useful marker for the progression of urinary tumors.

Sympathetic nerve fiber distribution and vascular smooth muscle morphology were investigated in the ophthalmic artery of stroke-prone spontaneously hypertensive rats (SHRSP) and were compared with those of normotensive Wistar Kyoto (WKY) rats at the age of 120 days. The distribution of fluorescent noradrenergic (NA) nerve fibers was examined by the glyoxylic acid method. The ophthalmic artery was divided into two portions according to the size of the outer diameter, that is into a proximal portion (above 100 microns) and a distal portion (30-70 microns). The distribution densities of noradrenergic nerve fibers were measured by quantitative image analysis using the Interactive Bild-Analyse System (IBAS). The distribution densities of NA nerve fibers in both portions of the ophthalmic artery were significantly higher (p < 0.01) in SHRSP than that in WKY rats. The difference in the density of NA fibers of the ophthalmic arteries between SHRSP and WKY rats was 1.9 times in the proximal portion and 1.5 times in the distal portion. The vascular smooth muscle cells of the ophthalmic arteries in SHRSP were observed by scanning electron microscope to examine the trophic effect of NA nerve fibers on the vascular smooth muscle cells. The smooth muscle cells of both portions of the ophthalmic arteries in SHRSP showed a smooth surface texture and no necrosis, and were very similar to those of WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The three-dimensional ultrastructure of glomerular basement membrane (GBM) in streptozotocin (STZ)-induced diabetic rats was examined by quick-freezing and deep-etching method. In three layers of the GBM of control rats, the outer and inner layers were formed by files of perpendicular fibrils, which connected the epithelial or endothelial cell surfaces with meshwork structures of the middle layer. In the diabetic rats, the inner layer was diffusely enlarged and the meshwork structure of the middle layer became markedly irregular due to the rupture of fine fibrils and thickening of material adherent to the fibrils. These ultrastructural changes correspond to those of subendothelial oedema, lamellation of lamina densa and fluffy material in the GBM, as revealed on conventional ultra-thin sections. It is suggested that the initial morphological change of STZ-induced diabetic nephropathy is disruption of matrix fibrils in the GBM, seemingly indicating a disturbance of size and/or charge barriers.

The effect of lysosomal storage diseases on the ultrastructure of human mast cells has not previously been reported. Indeed, there has been little published evidence indicating that mast cells contain typical lysosomes. However, mast cell cytoplasmic granules contain hydrolases similar to those found in lysosomes, but which differ from lysosomal hydrolases in exhibiting optimal activity at higher pH. We therefore examined by transmission electron microscopy the dermal mast cells in 58 biopsies of patients exhibiting 1 of 29 different lysosomal storage diseases. We found mast cells containing abnormal lysosomes in 16 of these disorders. In 6 of these 16 diseases, the mast cells' cytoplasmic granules appeared normal. These observations indicate that human mast cells can contain lysosomes, and provide evidence that the enzymes affected by lysosomal storage diseases are active in mast cells.

Repeated injections of mitomycin C-treated T2 fibrosarcoma cells into tumor-sensitized mice cause regression of a secondary tumor graft and more than 90% of the mice are cured. In the data presented here, an enhancement of the cytolytic cell-mediated activities measured in vitro against the specific T2 targets is shown in lymph nodes draining the tumor and in the spleen during the process of tumor rejection. Histopathologic studies revealed a rapid and marked accumulation of mononuclear cells mostly at the periphery of the rejected tumor tissue. A significant increase of CD8-positive, asialo GM1-positive and acid phosphatase-positive cells was observed in the rejected tumors whereas CD4-positive cells were similarly detected in both progressing and rejected tumor tissue. As macrophages seemed to be the population presenting the most persistent variation after immunization, the production of TNF-alpha was studied within the tumor site and in the lymphoid tissues during the regression process. Firstly, the presence of TNF-alpha within the cytoplasm of most of the adherent cell fractions isolated from the spleen and the tumor of immune mice was demonstrated by immunocytochemistry. Next, TNF-alpha mRNA-containing cells were determined by in situ hybridization of frozen tumor sections and identified essentially as tumor infiltrating macrophages. Finally, the macrophage populations isolated from tumors and from the spleen of immune mice were able to produce in vitro large quantities of TNF-alpha without exogenous stimulation. These findings support the role of TNF-alpha in the effector mechanisms contributing to the tumor regression process.