Functional canine dendritic cells can be generated in vitro from peripheral blood mononuclear cells and contain a cytoplasmic ultrastructural marker.

For physiological and practical reasons the dog is a large animal model used increasingly to study the pathogenesis of human diseases and new therapeutic approaches, in particular for immune disorders. However, some immunological resources are lacking in this model, especially concerning dendritic cells. The aim of our study was to develop an efficient method to generate dendritic cells (DC) in vitro from dog peripheral blood mononuclear cells (PBMC) and to characterize their functional, structural and ultrastructural properties. PBMC were cultured in vitro with IL-4 and GM-CSF. After 1 week of culture, a great proportion of non-adherent cells displayed typical cytoplasmic processes, as evidenced both by optical and electron microscopy. Cytometric analysis revealed the presence of 41.7+/-24.6% CD14+ cells expressing both CD11c and MHC class II molecules. Allogeneic mixed lymphocyte reactions confirmed the ability of these cultures to stimulate the proliferation of allogeneic lymphocytes as already reported as a characteristic of DC in other species. In addition, we describe for the first time the presence in canine DC of cytoplasmic periodic microstructures (PMS) that could represent ultrastructural markers of canine DC. In conclusion, our study provides an easy method to generate DC from PBMC in sufficient numbers for immunological in vitro investigations in dogs, a pre-clinical model for many human diseases.

Constitutive presence of cytochrome c in the cytosol of a chemoresistant leukemic cell line.

The release of holocytochrome c (cyt c) from mitochondria into the cytosol is reportedly a landmark of the execution phase of apoptosis. As shown here, the P-glycoprotein- (P-gp) expressing K562/ADR cell line (but not the parental K562 cell line) exhibits both cytosolic and mitochondrial cyt c in the absence of any signs of apoptosis. K562/ADR cells were found to be relatively resistant to a variety of different inducers of apoptosis, and blocking the P-gp did not reverse this resistance. The release of cyt c in non-apoptotic K562/ADR cells was not accompanied by that of any other mitochondrial apoptogenic protein, such as AIF or Smac/DIABLO, and was inhibited by Bcl-2 over expression. In addition, using a cell-free system, we show that mitochondria isolated from K562/ADR cells spontaneously released cyt c. These data suggest that cyt c release may be compatible with the preservation of mitochondrial integrity and function, as well as cell proliferation.

Hepatocyte-mediated transport to the bile of AMI-HS, a particulate contrast agent.

RATIONALE AND OBJECTIVES: The elimination of hepatocyte-directed particulate contrast agents has not been studied in the same detail as particles eliminated mainly by the mononuclear phagocyte system. The aim of the present study was to elucidate the fate of these particles by a multidisciplinary approach. METHODS: After intravenous injection of AMI-HS particles directed to the hepatocytes, rats were killed and cytological studies, by both electron microscopy and histochemistry, and spectroscopic studies of the bile were performed. The data were compared with a dynamic magnetic resonance study of the heart and liver. RESULTS: The particles were rapidly cleared from the blood by Kupffer cells and hepatocytes and then found first in the vascular and later in the biliary pole of the hepatocytes. After 24 hours, a relaxometric characterization of the bile showed the presence of unchanged particles in the bile. CONCLUSIONS: These results show the capacity of the liver to excrete unchanged AMI-HS particles directly into the bile.

Only fibres promoting a stable butyrate producing colonic ecosystem decrease the rate of aberrant crypt foci in rats.

BACKGROUND: Dietary fibres have been proposed as protective agents against colon cancer but results of both epidemiological and experimental studies are inconclusive. AIMS: Hypothesising that protection against colon cancer may be restricted to butyrate producing fibres, we investigated the factors needed for long term stable butyrate production and its relation to susceptibility to colon cancer. METHODS: A two part randomised blinded study in rats, mimicking a prospective study in humans, was performed using a low fibre control diet (CD) and three high fibre diets: starch free wheat bran (WB), type III resistant starch (RS), and short chain fructo-oligosaccharides (FOS). Using a randomised block design, 96 inbred rats were fed for two, 16, 30, or 44 days to determine the period of adaptation to the diets, fermentation profiles, and effects on the colon, including mucosal proliferation on day 44. Subsequently, 36 rats fed the same diets for 44 days were injected with azoxymethane and checked for aberrant crypt foci 30 days later. RESULTS: After fermentation had stabilised (44 days), only RS and FOS produced large amounts of butyrate, with a trophic effect in the large intestine. No difference in mucosal proliferation between the diets was noted at this time. In the subsequent experiment one month later, fewer aberrant crypt foci were present in rats fed high butyrate producing diets (RS, p=0.022; FOS, p=0.043). CONCLUSION: A stable butyrate producing colonic ecosystem related to selected fibres appears to be less conducive to colon carcinogenesis.

Differentiation of mucous neck cells into parietal cells: a new concept of mitochondrial biogenesis.

Parietal cells of the gastric fundic mucosa are small and contain only a few tiny mitochondria when they begin to differentiate from mucous neck cells. The canalicular ATPase activity characteristic of mature parietal cells is discrete in these young cells, whereas areas of very high activity are apparent in the Golgi complex, reticulum, nuclear envelope, mitochondrial wall, and plasma membrane. Close relations and contacts occur between mitochondria and these organelles, and the size and number of mitochondria increase progressively. These relations, as well as mitochondrial ATPase activity (a true differentiation marker), cease once the mitochondria become as numerous and large as those of a mature parietal cell. Our observations suggest that a secondary form of mitochondrial biogenesis, involving the massive participation of other organelles and independent of the classical mechanisms inherent in mitosis, occurs in parietal cells at the beginning of G1 phase during the 6 days of their maturation.

Production and characterisation of a monoclonal antibody specific for apoptotic bodies derived from several tumour cell lines.

We have recently shown that apoptotic bodies (apobodies) derived from rat colon carcinoma cell lines (PROb) after sodium butyrate (NaB) treatment were able to cure rats with induced peritoneal carcinomatosis ( [BOISTEAU] ). A specific immune response was assumed to be involved since the serum of cured rats contained antibodies specific for apobodies. In the present study, a mAb (clone 6E8) produced by immunisation of rats with apobodies strongly recognized apobodies but had little reactivity with parental tumour cell lines, as demonstrated by enzyme-linked immunosorbent assay (ELISA), immunostaining and flow cytometry. Immunoelectron microscopy showed that 6E8 mAb mainly stained the hyaloplasm or cytosol of apobodies. A protein was detected at 67 kDa by immunoprecipitation of apobodies with mAb, followed by immunoblotting, using serum of rats immunised with apobodies. The 6E8 mAb recognized apobodies derived from several rat or human colon cancer cell lines and a rat glioma cell line, regardless of the apoptosis stimulus used (NaB, staurosporine or UV). Our results clearly show that 6E8 mAb defines an epitope specifically generated during apoptosis, which suggests that the protein recognized may be involved in the molecular cascade of apoptotic cell death.

Cellular mechanisms of calcium phosphate ceramic degradation.

Calcium phosphate (CaP) ceramics are widely used for bone substitution in orthopedic, maxillofacial and dental surgery. Many environmental factors are involved in the gradual degradation of calcium phosphate ceramic after implantation, including physiocochemical processes (dissolution-precipitation) and the effects of various cell types. Several of these cell types degrade ceramics by phagocytotic mechanisms (fibroblasts, osteoblasts, monocytes/macrophages) or by an acidic mechanism with a proton pump to reduce the pH of the microenvironment and resorb these synthetic substrates (osteoclasts). Various mesenchymal cells located at the implantation sites can induce the solubilization of CaP ceramics. Crystal-cell contacts were required to induce such crystal dissolution. Mesenchymal cells such as fibroblastic cells are also actively involved in the ceramic degradation process. In this context, CaP crystals underwent dissolution into the phagosome. If osteoclasts resorb CaP ceramics similarly to the natural bone, they possess a phagocytic capability. This phagocytosis mechanism consisted of three steps: crystal phagocytosis, disappearance of the endophagosome envelope membrane and fragmentation of phagocytosed crystals within the cytoplasm. Similar phenomenons have been observed during the phagocytic mechanism induced by monocytes/macrophages. The cellular mechanisms of CaP ceramic degradation are modulated by various parameters, such as the properties of the ceramic itself, the implantation sites and the presence of various proteins (cytokines, hormones, vitamins, ions, etc.). The cells involved in these mechanisms could intervene directly or indirectly through their cytokine/growth factor secretions and their sensitivity to the same molecules. This article reviews recent knowledge on the cellular mechanisms of calcium phosphate ceramic degradation.

Electron microscopy study of intrahepatic ultrasmall superparamagnetic iron oxide kinetics in the rat. Relation with magnetic resonance imaging.

Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents for use in magnetic resonance imaging (MRI) are currently undergoing clinical evaluation. However, the images observed and the kinetic profiles obtained differ from one agent to another. In this study, BD IX rats received an intravenous penis injection of the USPIO contrast agents AMI-HS and AMI-227. A cytologic study of the liver was performed, and the data obtained were compared with those of MRI. Images acquired in light microscopy, transmission electron microscopy, microanalysis and electron diffraction provided data on the cell categories involved in the processing of these contrast agents, the importance and modalities of each category relative to this processing, and the modalities of agent elimination. AMI-HS was rapidly removed from the bloodstream by Kupffer's cells and hepatocytes and then eliminated through bile ducts. AMI-227 remained much longer in the blood compartment since it was processed very slowly by endothelial and Kuppfer's cells in the near absence of hepatocytic participation and thus of elimination by the bile ducts. These results allowed us to base our interpretation of MRI sequences on cytologic observations.

Ultrastructural study of degradation of calcium phosphate ceramic by human monocytes and modulation of this activity by HILDA/LIF cytokine.

Biodegradation of ceramics in vivo is achieved essentially by monocytes and multinuclear cells (osteoclasts). Monocytes are the key element in this process because they intervene first at the biomaterial implantation site during inflammatory reaction. In this work, in vitro studies were conducted on an ultrastructural scale to determine the specific behavior of these cells with regard to a calcium phosphate (CaP) ceramic. Two types of phagocytosis were observed when cells came into contact with the biomaterial: either CaP crystals were taken up alone and then dissolved in the cytoplasm after disappearance of the phagosome membrane or they were incorporated together with large quantities of culture medium, in which case dissolution occurred after the formation of heterophagosomes. Phagocytosis of CaP coincided with autophagy and the accumulation of residual bodies in the cells. Addition of HILDA/LIF factor to these cultures induced a very marked decrease in phagocytotic activity directed at the capture of CaP crystals and culture medium. Autophagy was reduced, and residual bodies were rare or absent. This study specifies the role of monocytes in CaP biodegradation and demonstrates for the first time that HILDA/LIF has a biological effect on this cell line.

Effects of interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha) association in intraperitoneal radioimmunotherapy: studies on an in vitro micrometastasis model.

We considered the usefulness of an association of two cytokines interferon gamma (INF gamma) and tumor necrosis factor alpha (TNF alpha) in optimizing intraperitoneal (i.p.) radioimmunotherapy of ovarian cancer. Studies were performed in conditions similar to those observed in vivo, using tumor multicell spheroids of the SHIN-3 ovarian adenocarcinoma line which expresses CA125 and O3 antigens. Light and electron microscopy showed that this cytokine association caused considerable modification of the three-dimensional morphology of the spheroids and the cells composing them. The uptake and retention kinetics of 125I-labeled F(ab')2 fragments of OC125 and OVTL-3 antibodies indicated that the presence of the cytokines led to a 1.5-fold increase in the quantity of O3 antigen at the spheroid surface. These results were confirmed by autoradiographs showing that the INF gamma-TNF alpha association produced extensive direct antitumor action, with better penetration and uptake of OVTL-3 antibody. Thus, i.p. radioimmunotherapy of micrometastases could be optimized by an initial injection of the IFN gamma-TNF alpha combination.

Ultrastructural and cytochemical analysis of Na+, K+, ATPase and H+, K+, ATPase in parietal cells of gastric mucosa in the rabbit.

Rabbit gastric secretion has the physiological peculiarity of being continuous and uninfluenced by food intake. In this respect, ultrastructural analysis of rabbit parietal cells has revealed morphofunctional features situated between states of rest and very active acid secretion. Our cytochemical study shows that Mg2+ ATPase and ADPase activities vary from cell to cell and can even be totally absent. These activities concern either microcanaliculi or laterobasal folds or both, but never tubulovesicles. Application of the technique of Mayahara to K+ pNPP, associated or not with inhibitors (ouabain, vanadate, N-ethyl-maleimide, sodium fluoride), enabled us to confirm the coexistence of H+, K+, ATPase and Na+, K+, ATPase activities in the rabbit and to determine that these activities concern basolateral folds, microcanaliculi, hyaloplasm and tubulovesicles. The global activity of K+, pNPPase varied considerably in intensity. The results of using inhibitors suggest that proton transport ceases completely in certain cells. The signs of functional alternation found in this study are in agreement with physiological data relative to this animal.

Study of in vitro drug sensitivity on a newly established cell line from a primary bronchial epidermoid carcinoma of human origin (NSCLCN6).

We studied a cell line established from a primary non-small-cell lung cancer (non-SCLC) of human origin and characterized by midly differentiated epidermoid carcinoma, a human karyotype and keratin expression. Doubling time was about 48 h in vitro and 12 days when transplanted into nude mice. In vitro, this cell line was mainly sensitive to dactinomycin and mitotic poisons such as Vinca alkaloids. Most chemotherapeutic drugs proved ineffective. Our findings are comparable to previous results in patients who showed 30% objective response and less than 5% complete response regardless of the therapeutic associations used against non-SCLC. Our line would also seem to provide a good model for studying new potentially antitumor substances.

Three-dimensional analysis demonstrates the presence of exocrine cytoplasmic vela between endocrine cells and basal lamina in the stomach of mammals.

Three-dimensional analysis demonstrated the presence of cytoplasmic vela extending from exocrine cells into the space between endocrine cells and basal lamina in the gastrointestinal epithelium of the rabbit; these structures were also observed in various other mammals. The following techniques were used to determine the morphologic characteristics of these vela and to study their significance: preparation of semiserial thin sections, three-dimensional reconstruction in plexiglass and lanthanum staining of pericellular spaces. It was found that these fine vela, devoid of major differentiated cell-constituents, sometimes form a pseudocircular crown at the base of endocrine cells. If the zone of basal apposition of the plasma membrane is referred to as ZBA and the zones of lateral apposition as ZLA, the presence of this velum makes it possible to distinguish a zone of immediate apposition without interposition (ZIA) and a mediate zone of apposition with interposition (ZMA) within the ZBA. Exocrine cell processes can also penetrate within endocrine cells in invaginations, and the depth of these invaginations can be demonstrated by lanthanum staining. Adjacent to the membrane zones defined above, other cytoplasmic microdomains-M(ZLA) and M(ZBA), as well as M(ZIA) and M(ZMA) of different morphofunctional significance may also be envisaged.

Process for analysis of an area delimited by a nonparametric curve and divided into microdomains, and for localization of possible constituents: exploitation by "digisector" software.

The process presented here was designed and developed for study of various cell parameters in electron microscopy photographs and analysis of the location of certain differentiated cell-constituents relative to plasma membrane. It can also be used for many other applications, for example, in metallurgy, petrography and geostrategy. A photograph is analyzed using a plotting table interfaced with a microcomputer.

Effects of bistramide A on a non-small-cell bronchial carcinoma line.

The antiproliferative effects of bistramide A, a nitrogenous dilactam polyether from Lissoclinum bistratum Sluiter (Urochordata), were studied at the level of the cell cycle in asynchronous cells of the NSCLCN6-L16 line. Bistramide A has a dual mechanism that induces blockade in the G1 phase (compatible with differentiation properties reported elsewhere) and causes polyploidy that is suggestive of inaptitude for cytokinesis. These effects confirm the results of cytomorphology studies in electron microscopy.

Ultrastructural identification of round-granule EC cells in rabbit fundic mucosa.

In endocrine (EC) cells of rabbit fundic mucosa, it is practically impossible to obtain unequivocal ultrastructural identification of all cells found in order to perform morphometric analysis. In addition to classic EC cells with pleomorphic granules, a cell type with entirely round granules is encountered which can be confused with non-EC cells. To solve this problem, all EC cells in our study were first identified by their 5-HT (immunocytochemistry) and argentaffinity. Examination of the fine structures of reactive cells then revealed that the round granules of EC cells were differentiated from those of non-EC cells by the existence of a dense core surrounded by a less dense halo, a feature providing unequivocal ultrastructural identification. EC cells with round granules showed less argentaffinity and less immunoreactivity to 5-HT as compared with classic EC cells. After labelling with [3H]L-dopa, EC cells with round-granules displayed an overall staining index higher than that of classic EC cells and comparable with that of D cells; however, the nuclear staining index was higher than that of D cells.

Quantitative assessment of the glandular activity of endocrine cells of gastrointestinal mucosae by computer-assisted analysis of ultrastructural images: methodology.

A method is described for studying the morphological features of endocrine cells of gastrointestinal mucosae in man and some animal species by semi-automatic analysis of ultrastructural images. The method enables possible ultrastructural variations in gastrointestinal endocrine glandular cells of different types to be determined with an acceptable margin of error. Various causes of error are investigated. It is found that the main cause is the choice of microscope magnification, despite the corrections made. The factor of inconsistency in the reproducibility of measurements performed by several operators is also calculated.

A new technique for staining sections and recovering celloidin film in high resolution autoradiography.

Ultrathin sections are stained immediately after cutting by placing them in contact with staining solution and then placed on a slide covered by a celloidin film. This method largely avoids precipitates of heavy metals. The recovering of celloidin film is improved using a stainless steel basket. This technique is far more reliable than that involving use of a filter paper.

[Resorption of drugs through the vaginal wall]. / La résorption des médicaments par voie vaginale.

Drug absorption during vaginal administration. A method is described (systems approach to vaginal delivery of drugs and development of in situ vaginal drug absorption procedure) and is able to show vaginal absorption of drugs. Some drugs are rapidly and largely absorbed through vaginal epithelium: Metronidazole (Flagyl), Prostaglandins, Estrogens, Polyvidone-iodine (Betadine), Chloroquinadol (Gynotherax), d-Methadone, Hexachlorophene (Phisohex et Ultralan), Insulin and Triclosan. Other drugs are poorly absorbed: Amphotericin B (Fungizone, Amphocycline), Econazole (Pevaryl) and Trimethoprim. At least, some drugs are not absorbed: Nystatine (Mycostatine) and Furazolidone (Tricofuron).

Quantitative autoradiography, electron microscopy, and granulometry of endocrine cells in the rabbit colon.

A high-resolution autoradiographic study was conducted on 268 thin sections of endocrine cells in the rabbit colon following injection of (3H) L-dopa. Using quantitative autoradiography, silver-proteinate impregnation, granulometry, and statistical analysis of cell populations by the Falck procedure, three cell types were identified--EC, L; and H--as well as a single cell with distinct ultrastructural characteristics. For the first time, a fine quantitative estimation of the handling ability of amine precursor related to the cross-sectional areas of each cell type was obtained, using a Kontron Digiplan image analyzer. All cells studied showed the ability to take up the precursor. Labeling indices were 6.63, 2.15, and 2.14 for EC, L, and H, respectively. These data and silver-proteinate impregnations provide good criteria for differentiating EC from H cells despite morphological similarities. After critical analysis of granule diameters, L cells were considered to be pluripotential in secretory activity.