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

Using monoclonal antibodies against proliferating cell nuclear antigen or PCNA (PC10) and the Ki-67 antigen (MIB1), an immunohistochemical and morphometric study was performed on routinely processed splenic tissue from ten patients with primary (idiopathic) osteomyelofibrosis (OMF). To determine the proliferation capacity of erythroid precursors and the endoreduplicative activity of megakaryocytes, corresponding antibodies (Ret40f and CD61) were applied in combination with the cell-cycle markers (sequential double-immunostaining). Morphometric analysis revealed no significant differences in PCNA or Ki-67 reactivity in either cell lineages. In comparison with previous studies on normal bone marrow, in splenic tissue showing myeloid metaplasia, the numbers of PCNA-labelled proerythroblasts, erythroblasts and megakaryocytes were conspicuously increased. Considering the ineffective erythropoiesis in OMF, there seemed to be a disproportional enhancement in PCNA and Ki-67 immunostaining of the red cell lineage. Similarly, the small size of megakaryocytes in advanced, OMF-associated myeloid metaplasia was in keeping with an impairment of endoreduplicative activity. In addition to various other contributory factors, anaemia in OMF may be partially caused by secondary folate (haematinic) deficiency. From experimental studies this defect is known to cause an abnormal arrest in the S-phase of the cell-cycle, comparable to that characterising pernicious anaemia. As a sequel of this pathomechanism, an undue overexpression of PCNA and Ki-67 has to be assumed, that is not necessarily associated with DNA synthesis or cell cycling.

Type X collagen is a short chain, non-fibril-forming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic alpha 1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, alpha 1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.

The distribution of the interstitial collagens I, II and III was analyzed immunohistochemically in cartilage and bone samples from 32 patients with degenerative osteoarthrosis at various morphological stages. The alterations observed showed a very patchy, focal distribution demonstrating significant heterogeneity in the tissue reaction. In minor osteoarthrotic lesions a focal pericellular deposition of collagens III and I was seen, while the majority of the interterritorial matrix reacted exclusively with collagen II antibodies. These changes were first seen in the superficial cartilage layer. At the more advanced stages of osteoarthrosis, particularly when osteophytic bone spur formation was present, extensive changes in the expression of collagen types in the pericellular matrix was revealed with extensive and overlapping localization of collagens I, II and III in the whole cartilage. These observations support the suggestion that degenerative cartilage shows a collagen type "switch". In addition, it was demonstrated that the interterritorial cartilage matrix was still mainly composed of collagen II even in advanced lesions. These observations may explain some of the previous discrepancies reported.

The presence of glutamate/aspartate-like immunoreactivity was studied in normal human skin and in skin with gold-induced inflammation. In normal skin all epithelial cells were glutamate and, apparently more weakly, aspartate immunoreactive. Both glutamate and aspartate immunoreactivities were also found in macrophage-like, HLA-DR positive cells in the dermis and in the epidermis. The intensity of glutamate and especially aspartate-like immunoreactivities seemed to be increased in the epidermis and dermis of the inflamed as compared to the normal skin, and this increase was particularly pronounced in the HLA-DR positive (dendritic) cells in the epidermis. Numerous cells, often of the mononuclear type, in the superficial dermis expressed glutamate- and aspartate-like immunoreactivities in the inflamed skin and many of these were HLA-DR positive. The functional role of glutamate and aspartate in normal skin, and the significance of the increase in the levels of these amino acids in several cell populations in the inflammatory skin is not known, but modulatory or protective roles may be considered. High concentrations of these amino acids could also induce cell damage. Moreover, the macrophage-like cells in the human skin may have a role in the processing of glutamate and aspartate on a recycling basis.

Frozen samples of minimal change glomerulopathy (MCG), and of membranous, segmental and diffuse lupus glomerulonephritis (MGN, SGN, DLGN) were studied to assess the distribution of tenascin (Ten), and the extradomains A and B (EDA- and EDB-) and oncofetal (Onc-) isoforms of cellular fibronectin (cFn). Cryosections were immunostained by the ABC method with specific monoclonal antibodies. In MCG, mesangial Ten and EDA-cFn reactions were increased. In MGN, mesangial Ten and EDA-cFn staining was enhanced except in segmental scars; convincing reactions were seen in cases with membranous transformation; spikes stained strongly. In SGN, variably intense staining for Ten and all cFn isoforms was seen in glomerular necrosis, proliferation and crescents; parietal epithelium EDA-cFn staining was noted. In DLGN, strong and extensive mesangial Ten and EDA-cFn staining was seen as were focal EDB- and Onc-cFn reactions. Parietal cells with and without crescents stained variably with all Mabs. Obsolete glomeruli were unreactive save for rare periglomerular Ten rims. Interstitial inflammation and fibrosis in MGN, SGN and DLGN had moderate to strong Ten and EDA-cFn staining with rare traces of EDB- and Onc-cFn. We conclude that enhanced Ten and EDA-cFn is a potentially reversible response to glomerular injury whereas the expression of EDB- and Onc-cFn apparently result from necrosis and/or cellular proliferation which lead to scarring. And, while mesangial cells are the major source of these molecules, epithelial cells might also partake in their synthesis.

The ultrastructure of hepatic granulomas induced by Cryptococcus (C.) neoformans was studied by a quick-freezing and deep-etching (QF-DE) method. Viable yeast cells were inoculated intravenously into rats and the livers were prepared for QF-DE replicas. Two cytoskeletal components were identified in the cytoplasm of macrophages composing the cryptococcal granulomas. These were: intermediate filaments, mainly located in the perinuclear region, and actin filaments, which were extensively decorated with myosin subfragment 1 (S1) and formed networks in the peripheral portion of the cytoplasm. In addition, two types of macrophage pseudopodia were observed in the granulomas. These were cobble stone-like pseudopodia at the yeast-macrophages contract areas, and thin, long and occasionally interdigitating pseudopodia in which actin filaments were consistently observed. Dense networks of actin filaments were also seen in pseudopodia protruding into the tight structure of the capsule of C. neoformans. These results suggest a role for actin filaments as one of the main factors in the force generating system of the phagocytic process.

The expression of cytokeratins, desmoplakin and vimentin has been studied immunohistochemically in the rat lung injured by x-irradiation using 14 well characterized monoclonal antibodies. A time-dependent relationship between the cytokeratin expression pattern and the morphological alterations observed was apparent. A cytokeratin 8 and 18 expression in normally cytokeratectable even at 3-6 h after irradiation. Between 14 days and 2 months, a remarkable heterogeneity in the epithelial cell cytokeratin pattern and an increasing immunoreaction for desmoplakin was found. In terminal bronchial epithelial cells, a heterogeneous CK8, 18 and 19 staining and a neoexpression of cytokeratins 4 and 7 was detected. Finally, peribronchiolar and vascular smooth muscle cells were cytokeratin-positive. At 6 months after irradiation, cytokeratin 13 and vimentin were focally present in bronchial epithelial cells and atypical type I and II pneumocytes as well as scattered epithelioid cell complexes were noted. During the course of injury, a loss of type III alveolar epithelial cells was found, which was characterized in the rat by a specific globular cytokeratin pattern and restricted immunoreactivity with cytokeratin-specific antibodies. These results show that the expression pattern of cytokeratins is a sensitive marker in monitoring epithelial alterations during lung injury.

The most prominent abnormality of ras proto-oncogenes in human lung tumours has involved point mutations at codon 12 of the Ki-ras gene. We have analysed 35 tumour samples of neuroendocrine lung neoplasms (ten carcinoid tumours, ten well-differentiated neuroendocrine carcinomas, and 15 intermediate/small cell neuroendocrine carcinomas) for a point mutation at this site. For this purpose, formalin-fixed and paraffin-embedded tissue sections were microdissected to remove non-tumours areas. DNA in the remaining tumour tissue was amplified in vitro by the polymerase chain reaction (PCR) and double-stranded PCR products were subjected to sequence analysis. Neither point mutations at codon 12 nor additional structural alterations at codons 1-32 were detected in Ki-ras gene. Our results suggest that point mutations at codon 12 of the Ki-ras gene do not seem to be involved in the pathogenesis of pulmonary neuroendocrine neoplasms.

In situ hybridization was performed to detect albumin mRNA in normal liver, liver cirrhosis, primary liver tumors and secondary liver neoplasms. In areas of normal liver, and liver cirrhosis, signals for albumin mRNA were present in hepatocytes, whereas no signals were seen in other cells such as endothelial and Küpffer cells, bile duct epithelium and smooth muscle cells. In 53 of 56 hepatocellular carcinomas signals were present in tumor cells but in eight cholangiocarcinomas and 14 metastatic adenocarcinomas from large bowel or pancreas, carcinoma cells were negative for albumin mRNA. In three metastatic tumors (from two neuroendocrine carcinomas and one gastric leiomyosarcoma), tumor cells contained no signals, while the surrounding hepatocytes showed diffuse grains. In 15 of the 84 specimens examined in situ hybridization was applied to routine formalin-fixed and paraffin-embedded blocks and strong signals were obtained for albumin mRNA. We conclude that in situ hybridization of human albumin is a valid tool in the differential diagnosis of hepatocellular carcinoma from cholangiocarcinomas and tumors metastatic to the liver.

The interaction between multinucleate giant cells (MGCs) and the fungus Aspergillus flavus as seen by transmission electron microscopy (TEM) is described in paranasal granulomas occurring in a Saudi patient dying from chronic aspergillosis. Two morphologically different types of MGCs were recognized; these were: a) 'Unhealthy looking' type I cells, rich in well organized organelles and containing few, partially degenerated and necrotic fungal elements. b) 'Healthy looking' type II cells that contained scanty, randomly dispersed cell organelles and normal, or partially degenerated fungal hyphae. The fungal elements had very thick and multilayered cell walls, and were found either in close contact to the host cell cytoplasm, or enclosed within phagosomes. The mechanism of the fungus destruction by the host MGCs is described and compared with that previous reports of MGCs involved in the elimination of extracellular microorganisms. The morphology and the various physiological activities of MGCs seems to depend mainly on whether the pathogen is extra- or intracellular. However, this study showed that MGCs are the cells best suited for killing pathogenic fungi.