The skin of hairless (Ng/Bln) mice topically treated with dimethylbenzanthracene (DMBA) was investigated by light microscopy, histochemistry, electron microscopy and autoradiography in order to gain some insight into the mechanisms by which the DMBA-induced cutaneous hyperpigmentation is mediated. The results of the present study indicate that this phenomenon is due to the DMBA-induced stimulation of both the tyrosinase system and the mitotic activity of dopa-inactive dormant melanocytes.
Isozyme patterns of alkaline phosphatase (ALP) were electrophoretically examined in human cell lines derived from one hepatoblastoma, five hepatocellular carcinomas (HCCs) and two cholangiocellular carcinomas. Most of the cell lines tested had a liver-type ALP isozyme. In addition, an abnormal ALP isozyme, which was similar to variant ALP, was detected in one hepatoblastoma and two HCC cell lines. One HCC cell line of these variant-like ALP-positive cell lines was alpha-fetoprotein (AFP)-negative. These findings suggest that variant-like ALP may be useful for the identification of human hepatoma cell lines, especially in AFP or albumin-negative cell lines.
A primary culture of cortical neurons was established from a 17-day-old rat embryo. Twenty-four hours after plating (day 0), the medium was changed to a chemically defined one. Hypoxia was exerted for 4 h by placing cultures in an air-tight chamber connected to a vacuum pump and a cylinder filled with 95% N2 and 5% CO2. Hypoxic stress was exerted on one of the consecutive days from day 0 to day 3. Hypoxia on days 0, 1 or 2 reduced the number of surviving neurons immediately following exposure. However, hypoxia on day 3 resulted in delayed reduction. Anit-synaptophysin antibody was used immunocytochemically to examine whether the difference is related to teh synaptic formation of cultured neurons. Neurons which had more and longer neurites were clearly stained for synaptophysin. We suspect that neurons with synaptic formation release substances from their presynaptic vesicles, and that these substances might account for the delay reduction.
The balance of phagocytic function among Kupffer cells, hepatic endothelial cells and splenic macrophages in the chronically ethanol-fed rats has been investigated. Clearance of latex particles in the blood was measured to estimate the function of the reticuloendothelial system. Phagocytosis of latex particles by Kupffer cells, hepatic endothelial cells or splenic macrophages in vivo was measured by counting the number of ingested particles in a cell after isolation of hepatic nonparenchymal cells or spleen cells following injection of different amounts of latex particles. Latex particle clearance was suppressed in the ethanol-fed rats, demonstrating a decreased phagocytic capacity of the reticuloendothelial system. Markedly decreased phagocytic function was found in 40% of Kupffer cells of the chronically ethanol-fed rats. In contrast, the number of latex particles in hepatic endothelial cells and in splenic macrophages was increased after injection of a triggering dose of latex particles. From these results it may be concluded that an increased phagocytosis of hepatic endothelial cells and splenic macrophages could compensate for the decreased phagocytic function of Kupffer cells.
Annulus fibrosus of intervertebral discs from diabetic and non-diabetic sand rats were examined by microspectrophotometry for fibronectin content. This was higher in the diabetic animals both in the dorsal and ventral parts and in the outer and inner lamellae of the annulus. It is suggested that diabetes-related changes in fibronectin are similar to changes in annular collagen observed in species other than sand rats.
Pulmonary intravascular macrophages (PIMs) have been recognized as the site of substantial uptake of blood-borne particles in the lungs of a number of domestic animal species. Concomitantly, there is a pronounced lung susceptibility to endotoxin in calves, goats, sheep, pigs, and cats. Hemodynamic changes and initial lung injury after endotoxin administration are mediated by arachidonic acid metabolites from a pulmonary source. A significant role of PIMs in regulating pulmonary hemodynamics is implicated.
Urinary extracts from patients with aplastic anemia are known to promote murine megakaryocytopoiesis. In this report, we show a simple method for the partial purification of megakaryocyte colony-stimulating factor from human urine. A four-step purification procedure, which included ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-Sepharose chromatography and high-resolution hydroxyapatite chromatography, resulted in an about 430- to 630-fold increase of specific activity. The final fractions were still contaminated with erythropoietin, but the contaminated content of erythropoietin was not enough to stimulate mouse megakaryocytopoiesis in our culture system. We also demonstrate that human urinary extracts stimulated human megakaryocyte colony formation.
Laminin, a basement membrane glycoprotein, has been implicated in a number of stages in tumour invasion and metastasis. In addition to its roles in cell adhesion and migration, laminin may be important in mediating interactions of tumour cells with the immune system and have more subtle roles in controlling metastatic behaviour. Recent work on the prognostic significance of laminin and the possible role of the molecule in antimetastasis therapy will also be discussed.
Electrically regulated morphological and cytoskeletal changes of HeLa cells were studied on an optically transparent electrode (OTE), on which potential-applied surface cellular behavior and morphogenesis were easily observed. Upon application of a potential, HeLa cells in an OTE exhibited remarkable morphological changes above +0.5 V (vs. Ag/AgCl) and below 0 V. At each potential in this potential range, change in F-actin distribution was observed using a fluorescent probe (rhodamine phalloidin). These results suggest that an electrical field induces a subcellular cytoskeletal change. Electrostimulation of cells with OTE can be a valuable strategy for the manipulation of cultured human cells.
Human hematopoietic survival and stem cell growth factor (SCGF), derived from the KPB-M15 myeloid cells, is a heat- and pH-stable protein. Chemical modification with various denaturing agents and proteolytic enzymes abrogated SCGF activity. The granulocyte-macrophage colony-potentiating and erythroid burst-promoting activities of SCGF were proportional to the density of the bone marrow (BM) cells cultured, the optimal BM cell density for delta granulocyte- and delta burst-promoting activities being 5 to 10 X 10(5)/ml. These data could be important in enabling the use of SCGF to induce proliferation of human hematopoietic stem cells in vitro.
Thirty-one samples representing Hodgkin's and non-Hodgkin's lymphomas, angioimmunoblastic lymphadenopathy (AILD), and benign follicular hyperplasia in HIV infections were examined for rearrangements of the immunoglobulin (Ig) and T cell receptor (TcR) beta-chain gene loci. In 11 of 12 non-Hodgkin's lymphomas (classified as Burkitt lymphoma (2), centrocytic lymphoma (1), centrocytic-centroblastic lymphoma (5), centroblastic lymphoma (3], only rearranged Ig genes could be detected. The exceptional case was an unclassified high-grade lymphoma, which represented a rearrangement of the TcR beta-chain. We also examined DNA from lymphoid neoplasms in which the lineage of the malignant cell was still controversial. Rearrangement of the TcR could exclusively be demonstrated in all 3 cases of AILD. One Ig gene rearrangement and 4 TcR beta-chain rearrangements were found in 13 samples of Hodgkin's lymphomas (11 lymph nodes, 1 pleura effusion and 1 bone biopsy with proven infiltration). Examination of 3 cases of benign follicular hyperplasia in HIV infection represented one Ig rearrangement.
The stratum corneum can be dissociated into single squames by several extraction methods: mechanically, with organic solvents, and with detergents. We have performed studied of reaggregation from single squames prepared by these methods. Mechanically dispersed corneocytes recombined into a lamellar-like structure closely resembling intact stratum corneum. Squames obtained by dissociation in ether formed a multilayered structure which differed from the intact tissue in the collapse of the bilayered lamellar structure into a single lucent band with osmiophilic deposits. Squames obtained after extraction with detergent failed to reaggregate.
Embryonic chick retinal pigmented epithelium (RPE) has been grown on glass derivatized with covalently bound proteins of basement membrane and treated with transforming growth factor-beta (TGF-beta). In the present paper we show that over the concentration range tested (0.1-10 ng/ml) TGF-beta has no effect on RPE cell proliferation either in the presence or the absence of serum, cell motility and the organization of cytoskeleton-extracellular matrix linkage complexes with respect to their structure and presence of actin, vinculin, talin, integrin and fibronectin. The protein profiles of total cell/ECM extracts of cells grown in the presence or the absence of TGF-beta are similar although some stimulation of protein synthesis and of production of fibronectin-containing extracellular matrix has been detected.
Thirty-two femoral heads from autopsy material with arthrotic changes and 12 surgical specimens of femoral heads with different diseases have been examined with respect to their ability to form a 'double contour'. An additional bony plate within the cartilage, giving rise to the double contour, occurred only in the non-pressure zone of the articular cartilage. There were no major differences between autopsy and surgical material and these changes could also not be found in normal femoral heads. The origin of a double contour could be traced to tissue elements penetrating the subchondral bone from the bone marrow, and was the result of a gradual transformation with chondroid tissue as the starting material. We observed a similar transformation leading to lamellar bone in association with pannus.
Bone marrow fibroblasts from normal and leukemic patients were used to investigate the relationship between serial subcultivation and changes in collagen synthesis. A regime was established to generate subcultures up to 35 cumulative population doublings (CPDs) in normal cells and to 9 CPDs in leukemic cells. In both types of cells, collagen synthesis decreased as subcultivation progressed. In normal cells, collagen synthesis was reduced to 10% of the original levels at 18 CPDs and in leukemic cells at 8 CPDs. In normal fibroblasts, collagen synthesis was more profoundly affected than overall protein synthesis by subcultivation. In acute lymphoblastic leukemia-derived fibroblasts, the decrease in collagen synthesis paralleled that of total protein.
In order to study the angiogenic activity, entire ovaries of immature or adult golden hamsters in different functional stages were implanted into the chick chorioallantoic membrane (CAM). Ovaries or other organs (heart, lung, liver, spleen, kidney, adrenal gland, testis) were applied during days 9-14 of incubation. Histological serial sections and ultrastructural studies displayed vascularization from the host to the ovary. Vascularization was marked for unstimulated immature ovaries compared to superstimulated immature ovaries or adult ovaries obtained during the period of proestrus-estrus. When no vascularization developed, grafted ovaries of immature or adult animals induced a mild erosive reaction of the CAM. In contrast, few other implanted organs obtained from immature animals caused a response with vascularization which was absent in grafted organs from adult hamsters. The erosive reaction of the CAM was rare, but the ulcerative response accompanied by infiltrating eosinophils was frequently seen both in immature and adult animals. These results may indicate that angiogenetic activity of the ovary is not the sole cause for vascularization of grafts. Other factors such as tissue death or a low histocompatibility barrier are likely to be involved.
Cytotoxic effect(s) of N6-cycloalkylated adenine and adenosine derivatives, upon the viability of mouse hepatoma cells, were studied in vitro. N6-Cyclopropyl- and N6-cyclobutyladenine and adenosine derivatives (33 micrograms/ml; 24-48 h) exerted significant cytotoxic effects upon the cells. N6-Cyclopentyl- and N6-cyclohexyladenines exerted similar effects under different experimental conditions (133-166 micrograms/ml; 48-72 h), while no significant cytotoxic effect(s) were observed with the corresponding adenosine derivatives under these conditions. Observed physical changes in the treated cells included cell elongation, short stubby filaments, wide intracellular spaces and ruptured cell membranes. N6-Cycloalkylated nucleosides were usually more cytotoxic than the cycloalkylated bases.
The present study describes the behavior of in vitro grown normal human oral mucosal epithelial cells and that of a tumorigenic epithelial cell line following subcutaneous inoculation into nude mice. A successful recovery of viable human epithelial cell inocula was seen in 25-90% of mice and there was no improvement in recovery rates after addition of fibroblasts. These inocula resulted in cyst formation lined by a 2-6 cell layer unkeratinized squamous epithelium without rete ridges. There was no increase in recovery rate or size of cysts when coinoculated with fibroblasts. The tumorigenic cell inocula were successfully recovered in all cases. Tumors established from these inocula had a low grade of differentiation and were without signs of metastasis. Inocula of tumorigenic cells showed an increased size after addition of fibroblasts to the inocula. The model may be useful in studies of interactions between inoculations of heterologous normal and pathologic cells as well as in studies of differentiation of carcinogen-treated epithelial cells.
Expression of type 1 and type 2 chain Lewis antigens was studied in 32 rectal adenocarcinoma specimens; the results were correlated with the patients' Lewis phenotype and secretor status. In addition, the pattern of expression of these antigens was analyzed in adjacent and distant normal mucosa. We used an indirect immunofluorescence technique with p-phenylenediamine counterstaining (Oriol technique) and a panel of monoclonal antibodies directed against the different antigenic specificities. Normal distal colonic mucosa only expresses monofucosylated structures (Lea and X) arising from activity of the alpha 1-3,4-fucosyltransferase coded by the Le gene. Rectal adenocarcinomas also show Lea and X, but also reexpress blood group antigens ABH and exhibit difucosylated determinants (Leb and Y). The accumulation of mono- and difucosylated type 2 chain in neoplastic processes, independently of the Le and Se genes, could be due to the enzymes coded by reactivation of the H and X genes. Blood group antigens form a complex signal code, genetically regulated, which intervenes in differentiation, growth and cellular recognition processes, and which may undergo important modifications during malignant transformation. These alterations could be useful in the diagnosis and prognosis of some types of carcinoma.
