Comparative evaluation by semiquantitative reverse transcriptase polymerase chain reaction of MDR1, MRP and GSTp gene expression in breast carcinomas.

Identification and quantitative evaluation of drug resistance markers are essential to assess the impact of multidrug resistance (MDR) in clinical oncology. The MDR1 gene confers pleiotropic drug resistance in tumour cells, but other molecular mechanisms are also involved in drug resistance. In particular, the clinical pattern of expression of the other MDR-related genes is unclear and their interrelationships are still unknown. Here, we report standardization of the procedures used to determine a reliable method of semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) using a standard series of drug-sensitive and increasingly resistant cell lines to evaluate the expression of three MDR-related genes, i.e. MDR1 (multidrug resistance gene 1), MRP (multidrug resistance related protein) and GSTp (glutathione-S-transferase p), reported to be endogenous standard genes for normalization of mRNAs. A total of 74 breast cancer surgical biopsies, obtained before any treatment, were evaluated by this method. When compared with classical clinical and laboratory findings, GSTp mRNA level was higher in diploid tumours. However, the main finding of our study suggests a clear relationship between two of these MDR-related gene expressions, namely GSTp and MRP. This finding provides new insight into human breast tumours, which may possibly be linked to the glutathione conjugate carrier function of MRP. Well defined semiquantitative RT-PCR procedures can therefore constitute a powerful tool to investigate MDR phenotype at mRNA levels of different related genes in small and precious tumour biopsy specimens.

Nasal polyposis pathogenesis: a flow cytometric and immunohistochemical study of epithelial cell proliferation.

In nasal polyps, constantly associated with chronic inflammation, frequent epithelial morphological changes (squamous metaplasia, secretory hyperplasia) suggest a dysregulation of epithelial cell proliferation. Cell proliferation in nasal respiratory epithelium was therefore evaluated in nasal polyposis. In 20 patients, we compared cell proliferation in mucosa from the inferior turbinate to these in nasal polyps using two methods: Flow cytometry analyzing first the ploidy and the percentage of S-phase cells (propidium iodide DNA labeling), secondly the percentage of Ki-67-labeled cells and the green fluorescent index (fluorescein-conjugated anti-human Ki-67 antigen labeling, and thirdly the percentage of Ki-67-labeled cells being in S-phase. Immunohistochemistry, quantifying the expression of Ki-67 antigen in the epithelium permitting to calculate a Ki-67 index. All cell-populations studied were diploid. Percentages of S-phase cells, Ki-67-labeled cells, Ki-67 labeled cells being in the S-phase and green fluorescence index was significantly higher in nasal polyps than in mucosa Ki-67 index were significantly higher in nasal polyps than in mucosa in the epithelium. Epithelial cell proliferation which is therefore increased in nasal polyp could play an important role in nasal polyposis pathogenesis and its relationships with inflammation can be suggested.

Cisplatin-induced apoptosis and p53 gene status in a cisplatin-resistant human ovarian carcinoma cell line.

Cisplatin-induced apoptosis and p53 gene status were analyzed in human ovarian carcinoma using a parental IGR-OV1 line and a derived cisplatin-resistant IGR-OV1/DDP subline. Compared with parental cells, cisplatin-resistant cells exhibited a 5-fold higher resistance index and a 2-fold longer doubling time. Cisplatin induced apoptosis in both cell lines, as assessed by cell morphology and the presence of a DNA ladder. However, high concentrations were necessary to induce apoptosis in resistant cells. These cells elicited a 5-fold decrease in the number of platinum atoms bound per nucleotide. IGR-OV1/DDP cells also exhibited enhanced drug efflux and a higher glutathione content. Our data suggest that the levels of cisplatin-DNA lesions are critical for drug sensitivity and apoptosis induction in this in vitro ovarian carcinoma model. Comparative analysis of the p53 gene in sensitive and resistant cells revealed the presence of the same heterozygous mutation in exon 5. A 2-fold increase in p53 mRNA and protein amounts was observed in resistant cells as assessed by Northern and Western blots, respectively. Immunocytochemical staining revealed a higher percentage of p53 stained nuclei in resistant cells. RT-PCR analysis of p53 transcripts showed that both wild-type and mutated alleles were transcribed in sensitive as well as in resistant cells. However, mutated transcripts were 1.5-fold more abundant than wild-type transcripts in sensitive cells, whereas they were 2-fold higher in resistant cells. In addition, mdm-2 protein was over-expressed in resistant cells. Our results address the question of the functionality of p53 protein and its possible role in apoptosis induction in this model. In resistant cells, p53 protein might be inactivated by 2 mechanisms: mutation and complexation with mdm-2 protein. Therefore, the presence of non-functional p53 in resistant cells might be involved in the relative failure of cisplatin-induced apoptosis in these cells.

Increased epithelial cell proliferation in nasal polyps.

OBJECTIVE: To detect, quantify, and compare respiratory epithelial cell proliferation in nasal mucosa and polyps from patients with nasal polyposis. DESIGN: Cohort study. SETTING: Patients and samples were selected at the Hôpital Intercommunal de Créteil (France). Flow cytofluorometry and immunohistochemistry were performed at Hôpitaux Tenon and Mondor (Université Paris [France] VI et XII). PATIENTS: Twenty-one patients undergoing endoscopic ethmoidectomy for treatment of nasal polyposis. METHODS: In 10 cases, epithelial cells were removed from frozen inferior turbinate mucosa and polyps by mechanical disaggregation and were then analyzed by flow cytofluorometry, providing the cell DNA content (propidium iodide labeling) and the percentage of S-phase cells. In 11 cases, inferior turbinate mucosa and polyps were fixed in formaldehyde and embedded in paraffin. Proliferating cell nuclear antigen expression in the epithelium was quantified by immunohistochemistry; a proliferating cell nuclear antigen index was calculated for each sample in the basal area, suprabasal area, and full height of the epithelium. RESULTS: All cell populations studied were diploid, and percentages of S-phrase cells were significantly higher in nasal polyps than in mucosa. Proliferating cell nuclear antigen indexes were significantly higher in nasal polyps than in the suprabasal area and full height of the mucosal epithelium. CONCLUSION: Cell proliferation is increased in epithelium from nasal polyps. Epithelial damage caused by inflammatory mediators could induce this increased cell proliferation via epithelial repair processes. Inflammatory cells could up-regulate epithelial cell proliferation by secreting growth factors.

[Flow cytometry in the study of meningiomas. Preliminary results and attempt at clinical correlation]. / Apport de la cytométrie en flux dans l'étude des méningiomes. Résultats préliminaires et essai de corrélation clinique.

Meningiomas are meningeal primitive tumors. These benign neoplasms can recur but the rate of recurrence is unknown as there is no reliable factor of predictibility. The aim of this study was to test Flow Cytometry based on clinical data and follow up in a series of meningiomas operated on in a neurosurgical department. This method allows the study of DNA content matched with the study of the cellular cycle. S cellular phase was chosen to be tested related to immunostaining with 2 proliferating markers, Ki67 and PCNA. This prospective study was carried out on neurosurgical samples, immediately frozen. On the one hand, results confirm well known discrepancies between Ki67 and PCNA immunostainings. On the other hand, two recurrent meningiomas belong to the diploid group. This is unexpected as aneuploid tumors are known to be biologically more agressive than diploid tumors.

[Cholestyramine adsorbs Escherichia coli and Vibrio cholerae toxins by way of ion exchange mechanism]. / La cholestyramine fixe les toxines d'Escherichia coli et de Vibrio cholerae par une liaison ionique.

The use of cholestyramine in the treatment of enterocolitis is justified by its ability to fix the LT toxin of Vibrio cholerae and ETEC, the ST toxin of ETEC and the verotoxin of EHEC at the pH of intestinal fluid. To elucidate the nature of the ion-exchange mechanism in the binding of the toxins on the resin, we try to eluate the toxins with 0.1 M to 0.9 M NaCl solution at pH 7. Irrespective of their presence or absence in the different fractions, the biological activity of the toxins was assessed in cell culture and in the new-born mouse test. Desorption of the LT toxin from Vibrio cholerae is obtained with 0.6 M to 0.8 M of NaCl, with 0.3 M to 0.5 M of NaCl for the LT, 0.4 M to 0.5 M for the VT and 0.6 M for the TS toxins. The eluated toxins are biologically active; these facts demonstrate that the adsorption of toxin on cholestyramine result of a ion-exchange mechanism.

[Adsorption potency of 2 clays, smectite and kaolin on bacterial enterotoxins. In vitro study in cell culture and in the intestine of newborn mice]. / Pouvoir d'adsorption de deux argiles, la smectite et le kaolin sur des entérotoxines bactériennes. Etude in vitro sur culture cellulaire et sur intestin de souriceau nouveau-né.

The use of clays in the treatment of enterocolitis is justified by their ability to adsorb viruses, biliary acids and bacterial toxins secreted into the intestinal lumen. We have studied the in vitro inactivation of the LT toxins of Vibrio cholerae and E. coli, the ST toxin of ETEC and the verotoxin of EHEC. These various toxins were incubated with two types of clays, smectite and kaolin, to investigate the influence of dose, pH variations and the duration of contact of the clays with the toxins. Irrespective of their presence or absence in the supernatant, the biological activity of the toxins was assessed in cell culture and in the newborn mouse test. Both clays inactivated the LT toxin. Smectite was more efficient than kaolin as it was active immediately especially at the pH of intestinal chyme. The LT toxins were adsorbed on the clays by hydrogen bonding. This permitted the segregation of the toxins and prevented them from being fixed to the membrane receptors on the cells. The two clays were ineffective against the verotoxin of EHEC when the pH was alkaline although they were more efficient at acid pH. ST toxin of ETEC was slightly adsorbed by smectite and kaolin.

[Ability of cholestyramine to bind Escherichia coli and Vibrio cholerae toxins]. / Etude de l'aptitude de la cholestyramine à fixer les toxines d'Escherichia coli et de Vibrio cholerae.

Cholestyramine treatment of new born's infectious diarrhea has been shown to be effective. The study of in vitro binding of bacterial toxins from Vibrio Cholerae and from three strains of Escherichia coli suggest an ionic adsorption of the four toxins to this anion-exchange resin. This immediate binding is effective at the pH of intestinal fluid, therefore the protection of the enterocytes from the biological action of the toxins result of the toxins sequestering effect of the Cholestyramine.

[Experimental study in the rabbit of the effect of cholestyramine in the treatment of infectious diarrhea caused by cholera]. / Etude expérimentale chez le lapin de l'effet de la cholestyramine dans le traitement des diarrhées infectieuses d'origine cholérique.

Cholestyramine is a non-absorbable anion exchange resin. Cholestyramine treatment of new born's infectious diarrhea has been shown to be effective. Cholera toxin induces, in the adult rabbit ileal loop, histological, water electrolytes and enzymatic modifications similar to that observed in patients with acute infectious diarrhea. Using this experimental model, we try to exhibit the reduction of the pathologic secretory action of the toxin on intestinal loop, after contact with the resin. A prolonged contact of the cholera toxin with the resin does not abolish completely the activation of adenyl-cyclase induced by the toxin alone. However significant reduction of goblet cell degranulation, and of the secretory effect allowed to reduce the loss of water and electrolytes.

[Histo-autoradiographic study of the secretion of gastric mucus in the rat stomach. II. Role of feeding and its combination with an antacid]. / Etude histo-autoradiographique de la sécreétion du mucus gastrique del l'estomac du rat. II. Rôle de l'alimentation et de son avec l'administration d'une anti-acide.

The authors use the technique of histoautoradiography after injection of glucose 3H to study the production and excretion of acid mucus in the fundus mucosa of the rat stomach. They demonstrate the effect of fasting, of eating and of eating combined with a topical agent consisting of 3 active principles: beidellitic montmorillonite, aluminum hydroxide and magnesium hydroxide. Eating stimulates the excretion of mucus during the digestive phase. Gelox combined with food allows for the late creation (6 to 12 hours after the meal) of an abundant layer of protective mucus. This topical action on the secretion of acid mucus favourises the physiological protection of the gastric mucosa.

Experimental study, in the rat, on the protection of the gastric mucosa by the combination of an antacid and a montmorrillonite.

The stomach wall is protected by its own secretion of mucus. The superficial layer consist of a parietal film of acid mucus, adhering to the epithelium. The acid mucus reach its maximum viscosity at pH 5. We investigate, in the rat, the interaction between this mucus with a montmorillonite, a kind of clay, known to have a mucoprotective effect, and with the association of montmorillonite and an antiacid drug. Three hours after ingestion the clay particles saturated the parietal mucus, then, up to the sixth hour their abondance decrease. The parietal film of mucus is abundant until the sixth hour, this data confirm the protection of the gastric mucosa by this drugs association.

[Infectious diarrhea: weakening of mucosal protection caused by a pathogenic Escherichia coli in ileal loops in the rabbit]. / Diarrhées infectieuses : affaiblissement de la protection muqueuse induite par un Escherichia coli pathogène au niveau des anses iléales de lapin.

The histological, enzymatic and water-electrolyte modifications induced by a toxinogenic strain of E. coli (O128B12) were studied. The observed water loss was due to the action of the bacterial toxins on the ionic pumps of the epithelial cells. After 4 hours of contact with the bacterial strain, one-third of the goblet cells of the intestinal epithelium were totally degranulated. This indicated an increase in the destruction of the mucus and therefore the weakening of mucosa protection through action of bacterial enzymes and toxins.

Infectious diarrhoeas: weakening of mucosa protection induced in rabbit ileal loops by a pathogenic Escherichia coli.

The histological, enzymatic, and electrolyte changes induced by a toxigenic strain of E. coli (0128B12) were studied in the rabbit. After 4 hours of contact with the bacteria, one-third of the goblet cells of the intestinal epithelium were totally degranulated, indicating an increase in the destruction of mucus and hence a weakening of mucosal protection through action of bacterial enzymes. The good histological conservation of the ileal mucosa at the end of the study period was confirmed by the stability of the disaccharidase of the glycocalyx and of the luminal surface membrane enzymes, alkaline phosphatase and leucine amino-peptidase. The observed water, sodium and bicarbonate losses were principally due to the action of the bacterial toxins on the ionic pumps of the epithelial cells.

A histological, enzymatic and water-electrolyte study of the action of smectite, a mucoprotective clay, on experimental infectious diarrhoea in the rabbit.

The histological, enzymatic and water-electrolyte modifications induced by the administration of smectite, a type of clay reputed to be mucoprotective, have been studied in the rabbit ileal mucosa during infection by saprophytic bacteria and toxigenic E. coli 0128B12. Smectite diminished the bacterial mucolysis and the destruction of the luminal surface membranes of the intestinal epithelium by pathogenic bacteria, as evidenced by the elevation of the disaccharidase and alkaline phosphatase levels. As a result of these effects, the net ion fluxes and net fluid changes favour absorption. These results could account for the mechanism of action of smectite in infectious human diarrhoea.

[Ultrastructure of the oocyte follicles in the crustacean amphipod Orchestia gammarellus (Pallas)]. / Etude ultrastructurale des follicules ovocytaires chez le Crustacé Amphipode Orchestia gammarellus (Pallas).

The follicle cells of the Crustacean Amphipoda Orchestia gammarellus produce syntheses at the level of the ergastoplasm and the Golgi body. No secretary cycle is observed. A complex but permeable network of intercellular spaces provides a path for possible transfollicular penetration for the exogenous fraction of the vitellus.