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

The effect of in situ autolysis on cerebral mitochondrial structure and function has been investigated. Mice (n = 9) were sacrificed and stored for up to 24 h under unfavorable post-mortem conditions at 25 degrees C. At different time intervals groups of three animals were submitted to post-mortem dissection and tissue from different regions of the brain was used for the preparation of "free" and synaptosomal mitochondria. On electron microscopic examination, the post-mortem period had no significant influence on mitochondrial morphology and enzymatic activities of complexes I-V of the mitochondrial oxidative phosphorylation system were still present in all the mitochondrial preparations from different regions of the brain, albeit at a reduced levels. Degradation of mitochondrial DNA was virtually absent from mitochondrial preparations during the 24-h period of autolysis, as shown by the presence of intact DNA by Southern blot and PCR analysis. Based on these results, alterations in mitochondrial DNA and deficiencies of mitochondrial respiratory complexes I-V can be recognized in cerebral tissue even after 24 h of unfavorable post-mortem storage conditions.

This study concerns the cytogenetics of 23 gastric carcinomas, classified histologically as intestinal or diffuse types. In carcinomas of the diffuse type, the only numerical changes observed were Y chromosome loss associated with X-chromosome disomy in four of seven male patients. A 46, XX karyotype without recognizable alterations was observed in three of five female patients, and rare structural changes in diffuse carcinomas involved chromosomes 1 and 18. In contrast, intestinal type tumors were exclusively aneuploid, with chromosome modes ranging from 48 to 84. The most consistent change was trisomy 20 in seven of 11 patients, each of which displayed a number of both single and clonal structural aberrations. Frequent structural changes were translocations involving chromosome 13 (including a putative isochromosome 13q in three of 11 patients), and alterations in chromosomes 1, 6 and 12. This study therefore suggests that diffuse and intestinal types of gastric carcinomas do not share a common sequence of genetic changes. The tumor with the worse prognosis (diffuse type) is surprisingly diploid, with uniform X-disomy in both males and females. The clinically less aggressive tumors (intestinal type) show multiple changes, both numerical and structural, of which some are reminiscent of changes seen in tumors of the lower gastrointestinal tract. Cytogenetics may thus be a valuable adjunct in establishing the diagnosis, classification, and prognosis of gastric carcinomas.

The object of the present investigation was to extend fundamental understanding of the composition, function and origin of bronchoalveolar lavage (BAL) cells, and to reduce the considerable gaps in our existing knowledge. BAL cells from the B10.D2 mouse were identified by morphological, enzyme cytochemical and immunocytochemical methods and a further functional characterisation of macrophages was undertaken in vitro using latex phagocytosis. The possible bone marrow origin of BAL cells was investigated with a radiation chimera. The bone marrow from H2k-positive F1 mice (B10.BR x B10.D2) was transplanted into the irradiated H2k-negative parent animals. At various time intervals after transplantation, BAL cells were tested immunocytochemically for the presence of H2k-positive cells. The principal finding was that BAL cells are functionally heterogeneous. It was possible by morphological, enzyme cytochemical and immunocytochemical techniques to subdivide the total BAL cell count into 89% macrophages, 8.5% lymphocytes and 2.5% granulocytes. Only 14.3% of the macrophages expressed the corresponding epitope, and this small population could be further subdivided immunocytochemically into mature and immature (7:1) macrophages. The ability to phagocytose, which is typical of active macrophages, could be induced in vitro in only one-third of all BAL macrophages. Three-quarters of the macrophages carried the H2d antigen and 9.5% of the BAL cells were natural killer cells showing a macrophage phenotype. Subdivision of the lymphocytes showed a significant majority of T-cells (75%) to B-cells (20%), and 5% Asialo GM1-positive cells. The CD4/CD8 ratio amounted to 1.3:1 and 38% of the lymphocytes expressed on the surface the H2d antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

The death of a cell results in a large amount of membrane lipid, predominantly phospholipids and cholesterol, that must be eliminated. In this study, we have examined what happens to phospholipids in dying rat platelets. Rat platelets were incubated for up to three days following their activation with thrombin. Platelet death occurred during the first day of incubation. This was indicated by a complete loss of platelet lactate dehydrogenase into the incubation medium. The platelets progressively lost over one-half of their phospholipid content during the three days of incubation. Cholesterol and sphingomyelin (the phospholipid with the highest affinity for cholesterol) were not lost during the same period. Our findings suggest that significant degradation of cellular non-sphingomyelin phospholipid can be triggered by cell death. The preservation of sphingomyelin in dying platelets, may be an adaptive response to maintain cholesterol in a solubilized state within dying cells.

Hepatic granulomas induced by a single or several subcutaneous injections of carbon tetrachloride (CCl4) in Balb/c mice were examined electronmicroscopically and immunocytochemically. Stellate cells (fat-storing cells; lipocytes; Ito cells) were identified by the detection of cytoplasmic desmin, while T-lymphocytes and monocytes/macrophages were identified with monoclonal antibodies Thy 1.2 and MOMA-2, respectively. Following pericentral necrosis induced with CCl4, clear foci containing lymphocytes, monocytes, macrophages and perisinusoidal stellate cells occurred in the surrounding hepatic parenchyma on day 5. These clear foci developed to granulomas with increasing numbers of macrophages and stellate cells. Mitotic and apoptotic figures in randomly distributed macrophages, and direct contacts between macrophages and stellate cells were frequently seen within the granulomas. The stellate cells were characterized by a well-developed granular endoplasmic reticulum and Golgi complex. Collagen fibrils were closely applied to the stellate cells and connective tissue septa extended between neighboring granulomas and/or the pericentral necrotic areas after several injections of CCl4. CCl4-induced hepatic granulomas provide a model for investigating paracrine and/or autocrine modulation within a well-organized microenvironment during progressive hepatic inflammation and fibrosis.

Incubation of the human renal carcinoma cell line CaKi-1 with tumour necrosis factor alpha (TNF alpha) or the phorbol ester phorbol-12-myristate 13 acetate (PMA) strongly enhanced the immunocytochemical staining of the intercellular adhesion molecule ICAM-1, in a non-linear manner. Since PMA is capable of activating Ca2+/phospholipid-dependent protein kinase C (PKC), we investigated the role of this kinase during TNF alpha signal transduction. Calcium ionophore A23187 significantly enhanced PMA, but not TNF alpha-induced ICAM-1 staining. The PKC inhibitors H7, staurosporine and sphingosine abrogated the action of PMA, while TNF alpha was unaffected. Simultaneous incubation with TNF alpha and PMA resulted in maximal ICAM-1 staining significantly above values obtained when cultures were treated with either agent alone. Finally, chronic PMA treatment with subsequent TNF alpha stimulation enhanced ICAM-1 staining above values from cultures where TNF alpha was omitted. Our findings suggest that the immunocytochemical staining of ICAM-1 in CaKi-1 cells can be induced by TNF alpha through mainly PKC-independent mechanisms or by PMA through PKC-dependent mechanisms. The two agents may work synergistically in this respect.

Amplification of genomic DNA encoding oncogenes such as HER-2 (syn.c-erbB2/c-neu) may be substantially involved in the initiation and progression of breast cancer. In order to refine and facilitate the quantitative analysis of HER-2 amplification in breast cancer, differential polymerase chain reaction (PCR) was performed on DNA derived from single cryosections of tumor tissue. This technique is based on the simultaneous amplification of a potentially amplified oncogene (HER-2) and a reference gene (IFN-gamma). Differential PCR yielded reproducible results that were in agreement with gene copy quantification using the dot blot technique. Thus we suggest differential PCR to be a reliable and rapid method for determining relative gene dosage in a minute amount of tumour tissue.

We have recently shown that interferon-gamma (IFN-gamma) stimulated immunocytochemical staining of the intercellular adhesion molecule ICAM-1 may be dependent on inositol phosphate formation in the human renal carcinoma cell line CaKi-1. In the present study we investigated the possible role of GTP-binding proteins (G-proteins) during IFN-gamma signalling. Preincubation of CaKi-1 cells for 24 h with increasing amounts of pertussis toxin (PT) or cholera toxin (CT), two regulators of G-protein activity, inhibited IFN-gamma induced ICAM-1 staining. Preincubation with PT or CT for 24 h also inhibited IFN-gamma induced inositol 1-monophosphate (Ins 1-P), inositol 1,4 bisphosphate (Ins 1,4-P2) and inositol 1,4,5 trisphosphate (Ins 1,4,5-P3) formation. Our findings suggest that IFN-gamma induced ICAM-1 staining and inositol phosphate formation in CaKi-1 cells is dependent on a PT and CT sensitive signalling pathway. This may reflect a role for G-proteins in the coupling of IFN-gamma receptor activation and phospholipase C catalyzed phosphoinositide hydrolysis.

Morphologically atypical cells were first detected in the adjacent connective tissue 98 days after implanting a paraffin pill containing 2 mg of 7,12-dimethylbenz[a]anthracene (DMBA) into the subcutaneous tissues of rats. These cells subsequently formed groups and finally produced gross malignant fibrous histiocytomas (MFH). Early atypical cells were located between proliferating fibroblasts and histiocytes in the center of a fibrous capsule surrounding the DMBA pill. They exhibited a smooth cell surface, dilated rough endoplasmic reticulum, multiple Golgi complexes, and were often associated with newly formed collagen. These cells incorporated [3H]thymidine and [3H]proline intensively, and showed weak acid phosphatase activity but no features diagnostic of macrophages (microvilli, numerous lysosomes, high acid phosphatase and non-specific esterase activities, antigens recognized by monoclonal antibodies ED1 and OX-42 and vital staining with trypan blue). There was no evidence that atypical cells differentiated into muscle cells (no expression of desmin or the alpha-sarcomeric form of actin) or Schwann cells (no expression of S-100 protein). No point mutation in the neu gene at nucleotide 2007, specific for N-ethyl-N-nitrosourea- and DMBA-induced malignant rat schwannomas, was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analyses. These results support the view that malignant fibrous histiocytoma is derived from immature fibroblasts exhibiting pronounced phenotypic diversity during the later stages of carcinogenesis.

We have examined the effects of human epidermal growth factor (hEGF), a growth-promoting factor for hepatocytes, on the cytoskeletal structure and intercellular contacts in neonatal hepatocytes in primary culture. The ultrastructural characteristics of cellular contacts and bile canaliculi (BC) were examined using a newly developed technique of scanning electron microscopy for cultured hepatocytes. The neonatal hepatocytes incubated with hEGF plus insulin for 3 h showed more variety in shape than controls, and their cellular contacts were very loose with many long fibers. At this time, there were no changes in the distribution of microtubules or microfilaments. After treatment with hEGF plus insulin for 21 h, the distribution of microfilaments was altered. Actin filaments no longer surrounded BC, but were observed near the cell periphery; in addition, DNA synthesis was increased to 3.9 times the rate in controls. Treatment with dexamethasone for 3 h caused tight straight cellular contacts, and after 21 h actin filaments appeared around slightly dilated BC, but there was no increase in DNA synthesis. Transmission electron microscopy showed that the transport and secretion of horseradish peroxidase in BC was inhibited after 3-h incubation with hEGF. These results suggested that hEGF first affected the cellular contacts of hepatocytes necessary for the development of cellular polarity, and then affected the distribution of actin filaments, the development of functioning BC being suppressed.