[After the COVID crisis, what solutions for the future nursing home?] / Rapport 22-02. Après la crise COVID, quelles solutions pour l'EHPAD de demain ?

The ageing of the population induces situations of large vulnerability and dependence. Home care usually remains the best response to comply with the person's wish, the family's desire, and the civil society's interest. However, there are circumstances where patient management in a nursing home (EHPAD) is the only solution. The present pandemic of coronavirus COVID-19 has highlighted the issue of EHPAD and their limitations to provide high quality care. To analyze the current position of EHPAD into the care chain and to understand difficulties to their functioning, it seems essential to seek out accelerated changes in the EHPAD since their establishment in 1999 and then in the light of the current crisis, propose possible solutions with a positive view of the role which each EHPAD will have to ensure for future.

Effect of sodium cis-beta-4-methoxybenzoyl-beta-bromacrylate (Cytembena) on HeLa cell kinetics.

The treatment of HeLa cells with various concentrations of sodium beta-4-methoxybenzoyl-beta-bromacrylate (Cytembena) results in inhibition of growth and modification of cell cycle distribution. These phenomena were observed at concentrations between 7.5 x 10(-5) and 2.5 x 10(-5) M. The estimation of DNA content by flow cytometry showed an important shift in the distribution of cycling cells with a relative decrease of G0 + G1 cells and a striking accumulation of G2 + M cells. According to our experimental conditions, the blocking up in G2 + M is irreversible at 7.5 and 5 x 10(-5) M.

Flow cytometric analysis of pentakis(aziridino)thiatriazadiphosphorine oxide (SOAz)-induced changes in cell cycle progression of HeLa and HL-60 cells.

The treatment of HeLa and HL-60 cells with various concentrations of pentakis(arizidino)thiatriazadiphosphorine oxide results in inhibition of growth and modification of cell cycle distribution. These phenomena were observed at 10(-4) M and 5 X 10(-5) M for HeLa cells and 10(-5) M and 5 X 10(-6) M for HL-60 cells. The estimation of DNA content by flow cytometry showed an important shift in the distribution of cycling cells with a striking arrest in G2 for both cell lines with a concomitant late S-phase accumulation for HeLa cells. Incubation of cells in drug-free medium 3 days after treatment did not show any change in DNA distribution, suggesting the irreversibility of drug action.

Type II transglutaminase expression in rabbit articular chondrocytes in culture: relation with cell differentiation, cell growth, cell adhesion and cell apoptosis.

Depending on the cell type studied, the involvement of type II transglutaminase (TGase) has been proposed in almost any event of the cell life such as differentiation, apoptosis, growth, aging, cell morphology and adhesion, metastatic capacity or extracellular matrix stabilization. In order to define the field(s) where this enzyme may be implicated in chondrocytes, type II TGase expression was studied in chondrocytes at different passages which differentiated state was modulated by retinoic acid, dihydrocytochalasin B or staurosporin. Results showed that (i) type II TGase expression is not incompatible with type II collagen expression, a main marker of chondrocyte differentiation (ii) type II TGase expression is higher when cells are in the exponential phase of growth than when growth arrested (iii) a high type II TGase expression does not imply that cells are apoptotic although cell apoptosis correlates with increased type II TGase expression (iv) non-adherent cells do not express type II TGase whereas adherent cells do whatever their differentiation state as assessed by type II collagen synthesis. These results suggest that, in articular chondrocytes, type II TGase is specifically implicated in the cell adhesion capacity.

Transglutaminase activity in rabbit articular chondrocytes in culture.

In vivo, articular chondrocytes produce an important amount of extracellular matrix (cartilage) whose quality is impaired upon inflammation or aging leading to arthritis or arthrosis. Transglutaminases (EC 2.3.2.13) are a family of enzymes which have been shown to be involved in extracellular matrix stabilization, cell differentiation and possibly in initiation and propagation of inflammatory diseases. It is therefore of interest to study transglutaminase activity in chondrocytes. Transglutaminase activity was studied in rabbit articular chondrocytes in primary culture, where cells are in a well-differentiated state as assessed by collagen-type synthesis, as well as in subculture and in retinoic acid-treated cells, where cells are in a dedifferentiated state. Results showed that two different TGases activities are expressed in chondrocytes. One, down-regulated upon retinoic acid treatment of cells, preferentially membrane bound and strongly activated upon trypsin treatment of cell lysates, is expressed at a high level in primary culture. The other one is up-regulated upon retinoic acid treatment, preferentially cytosolic and inactivated upon trypsin treatment of cell lysates. The rate of expression of the TGase down-regulated by RA seems to correlate with the differentiation state of the chondrocyte. This suggests that this TGase activity may have a physiological role in cartilage and merits further study.

[Pharmacology, an innovative factor in therapeutic research]. / La pharmacologie, facteur d'innovation en recherche thérapeutique.

As members of the pharmacology training group set up by the committee of pharmacological science of the French Academy of Pharmacy, we examine the situation of pharmacology in drug discovery. Today, it is obvious that by integrating genome sequencing, cellular and molecular biology, and bioinformatics, pharmacology has become a cross-disciplinary science. Pharmacologists must become knowledgeable in a wide range of domains, using the major points in each to direct them towards the discovery and development of new therapeutic agents. It is also clear that pharmacology remains a major factor in the different steps of drug discovery, from the molecular and cellular stages, to clinical and pharmaceutical developments.

Effects of oxazaphosphorine cytostatics on granuloid progenitor cell (CFUc) proliferation in mice.

The effects of oxazaphosphorine cytostatics were studied on granulocytic/monocytic colony forming cells from mice bone marrow in methylcellulose culture. Cyclophosphamide, ifosfamide, trofosfamide and two secondary metabolites show a weak activity in vitro (Inhibitory Dose--50% (ID50) between 5 X 10(-4) M and 5 X 10(-5) M). By contrast, a high cytostatic activity was observed with phosphoramide mustard and especially with hydroperoxycyclophosphamide (ID50: 2.5 X 10(-6) and 4 X 10(-7) M). These results suggest that these metabolites are active. The differentiation of the three types of colonies (granulocytic, monocytic and mixed) showed no specific effect of the drugs for a given series. After in vivo treatment, cyclophosphamide, ifosfamide and trofosfamide induce an important decrease of nucleated bone marrow cells. This decrease is maximum on the third day and regresses when the treatment is interrupted. On the first day of the culture an inhibition of the proliferation of granulocytic/monocytic progenitor cells is observed. An important statistically significant stimulation of these same progenitor cells is however noted later.

Studies on in vitro colony formation by mouse bone marrow cells using different inflammatory pleural exudates. Relation to colony stimulating factor (CSF).

An inflammatory exudate obtained in Swiss mice 3 hours after intrapleural injection of dextran was able to increase the number of cultivated peritoneal macrophages in S phase. This exudate was also able to stimulate the formation of colonies from mice bone marrow progenitors of macrophages and granulocytes in methylcellulose culture. The stimulating activity of this acute inflammatory exudate was compared with that of colony stimulating factor (GM.CSF). The qualitative and quantitative results showed that the biological activity of the mitogenic factor of this inflammatory exudate, inflammatory mitogenic factor (IMF) was very close to GM.CSF at optimal concentration, but when used at the same concentration, the exudate was less active than GM.CSF. A stimulating activity was also found with another inflammatory pleural exudate induced by calcium pyrophosphate. The comparative kinetics of the action of the two exudates on CFUc appeared alike but the rise was earlier with calcium pyrophosphate. These results suggest the release of a growth factor for monocyte-macrophage in different acute inflammatory exudates.

Effect of in vitro treatment with indomethacin on mouse granulocyte-macrophage colony-forming cells in culture (CFUC). Possible role of prostaglandins.

The effect of indomethacin (6 mg/kg daily orally for 2, 3 or 4 days) on myelopoiesis was studied in mice by estimating 1) differential cell counts of bone marrow, 2) proliferation of the CFUC in presence of GM-CSF, 3) proliferative state of CFUC after tritiated thymidine suicide. After 4 days of treatment, a rise in the recognizable myeloid precursors, an increase of both the colony forming capacity and the number of CFUC in S-phase were observed. These data suggest that indomethacin produced an hyperplasia of the committed stem cell compartment. The decrease of PGE2 amounts in bone marrow cells following treatment with indomethacin could explain the hyperplasia observed. These in vivo results are in accordance with the in vitro data which show that PG could control the proliferation and differentiation of myeloid progenitor cells.

Production of insulin-like growth factors and their binding proteins by rabbit articular chondrocytes: relationships with cell multiplication.

Articular chondrocytes from 2- to 3-month-old rabbits were cultured in serum-free medium supplemented with fibroblast growth factor. The effects were studied of GH, insulin-like growth factors (IGFs), and insulin on the production of IGF-I, IGF-II, and their binding proteins (BPs) and on cell multiplication. In the control culture medium, IGF-I levels were about one fifth those of IGF-II. Western blot analysis of the BPs revealed a predominant 30K form and 24K and 20K forms which appeared inconsistently and in small quantities. Ten to 100 ng/ml human GH had no mitogenic effect, and even had a slightly inhibitory effect. IGF-I at 10 ng/ml stimulated cell multiplication above the control level by 41% and at 50 ng/ml by 74%, whereas the mean increase obtained with IGF-II (10 and 50 ng/ml) was only 19%. At the same doses, insulin had no effect, but at 5 micrograms/ml it stimulated cell multiplication by a mean of 67%. There was a positive correlation between cell number and release into the medium of both IGF-I (r = 0.86) and IGF-II (r = 0.77). Neither IGF-I nor IGF-II production was affected by GH. Insulin (5 micrograms/ml) increased IGF-I production by a factor of 2.6, but increased IGF-II production by a factor of only 1.4. Under the various conditions of culture with different doses of GH and insulin, cell multiplication, relative to the control value was positively correlated to the IGF-I/IGF-II production ratio (r = 0.77). It would, therefore, seem that IGF-I secreted by the chondrocytes may stimulate their own proliferation. When IGFs or insulin were added to the culture medium, changes in the electrophoretic profiles of the BPs included an increase in the 30K form and an increase in or the appearance of the 24K and 20K forms. Ten and 50 ng/ml IGF-I or IGF-II had effects equal to or greater than those induced by 5 micrograms/ml insulin. These results indicate that the syntheses of BPs and IGFs are coordinated and that IGFs may be implicated in the control of the synthesis of their BPs.

Age-related changes in rabbit articular chondrocytes.

Cell culture techniques have been used extensively in the study of the aging process at the cellular level. The "senescent" articular chondrocyte seems to be a good model to examine the responses to aging in osteoarthritis, one of the most frequent diseases of old age. Thus in vitro chondrocyte "senescence", established by weekly subculture was characterized by a declining proliferation rate during late passages, from a rapid growth rate in early subculture to a complete loss of the proliferation capacity after 8 +/- 1 passages. Flow cytometric analysis show a time course decrease in the fraction S and G2 + M during the subculture, and a concomitant enhancement in protein content related to the increase of cell size. The immunocytochemistry assays revealed an appearance of a rigid cytoarchitecture with an increase in the number, and organization, of three cytoskeletal components: actin, tubulin and vimentin. The cultured chondrocytes therefore undergo in vitro aging analogous to that described for diploid fibroblasts, and could constitute a cellular model for pharmacological and toxicological assays.

Effects of donor's age on growth kinetics of rabbit articular chondrocytes in culture.

The in vitro proliferative capacity of articular chondrocytes derived from young and old rabbits was investigated to examine if the modifications incurred can be related to the in vivo aging. Determinations were made of the cartilage cell density, cell volume, cell number at confluency, plating efficiency, growth curve and DNA content distributions. The old donor cells were characterized by a decline in all the parameters of cartilage growth studied: cell number at confluency, cell replication rate (from 20 h to 45 h) as well as an increase in cell volume. The mean cycle time in vitro increased from 17.5 h compared to 27 h during in vivo aging, essentially because of an elongation of the G1 phase. Chondrocytes derived from young and old donors may be an appropriate model system for studying the in vitro effects of drugs on rheumatoid diseases as a function of in vivo aging.

Partial characterization of intracellular and secreted glycosidases from rabbit articular chondrocytes in culture.

N-Acetyl-beta-hexosaminidase, beta-galactosidase and beta-glucuronidase activities were shown to be present in cultured rabbit articular chondrocytes. Secretion of enzyme activity seems to preferentially result in the accumulation of N-acetyl-beta-hexosaminidase. Three days after seeding, the amount of N-acetyl-beta-hexosaminidase activity found in the medium accounts for about 140% of the total N-acetyl-beta-hexosaminidase activity after complete disruption of the cell pellet. Optimal conditions of incubation time, cell numbers, substrate concentration, and pH for glycosidase activities were determined in 0.1% Triton X-100. Intracellular and secreted glycosidases have shown similar elution profiles by chromatofocusing. N-acetyl-beta-hexosaminidase exhibits two major forms which may play a role in the catabolism of glycosaminoglycans.

A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.

A simple and sensitive cell-cell adhesion microplate assay was established using the cytoplasmic fluorescent dye, calcein AM. The procedure involves three steps: the labeling of lymphocytes with an adequate concentration of calcein AM (20 microM) during a short incubation period (30 min); the adhesion of 2 x 10(5) labeled lymphocytes per well to confluent keratinocyte or fibroblast monolayers grown in microtiter plates for 90 min; and, finally, measurement of the fluorescent signal utilizing a new system of cold-light microfluorimetry (Rat, 1993). During the adhesion assay, the release of calcein from labeled lymphocytes is low and the method permits the detection of as few as 1000 adherent cells. This non-radioactive procedure takes less than 4 h to perform and has proven to be as accurate and reliable as the common method using radioactive isotopes. In addition to its simplicity, the use of a fluorescent molecular probe in conjunction with cold-light microfluorimetry (CLF) offers many advantages of safety and economy, and can readily be adapted to the different cell types that participate in cell-cell adhesion.

Sodium butyrate-induced changes in cultured rabbit articular chondrocyte transmembrane electrical potentials. Relationship between these changes and the proliferative response.

Sodium butyrate at 5mM reversibly induced a significant increase of transmembrane potentials (Em) in normal chondrocytes (24 hours after seeding) and arrested their proliferation. This increase in Em levels, which could be temporarily abolished by Tetra-ethyl Ammonium (TEA 5mM), was related to an increase in membrane permeability to K+. This hyperpolarization was correlated with the reversible inhibition of growth in G1 induced by the sodium butyrate.

Rabbit articular chondrocytes: an in vitro model for studying the effect of sodium aurothiopropanol sulfonate on proliferation kinetics, type II collagen phenotype and mitochondrial activity.

Despite the benefits of chrysotherapy the responsible mechanism of action of gold compounds remains unclear. At a concentration of 5 x 10(-4) M, sodium aurothiopropanol sulfonate (SAS) modified the in vitro proliferation kinetics of articular chondrocytes by reducing growth, viability and plating efficiency. Flow cytometry analysis, using propidium iodide DNA staining, revealed slight but significant cell arrest in G2+M which, in fact, represents an increase in the proportion of binucleate cells. SAS did not induce any variations in chondrocyte phenotype stability as far as the biosynthesis of type II collagen was concerned, and no appreciable changes in overall mitochondrial activity reflected by rhodamine 123 incorporation.

In vivo effects of indomethacin and cyclophosphamide on CFU-GM proliferation and cyclic nucleotide levels in bone marrow of mice.

The effects of indomethacin (6 mg/kg daily, orally, for 4 days) or cyclophosphamide (150 mg/kg, a single dose, intraperitoneally) on myelopoiesis were studied in mice. A hyperplasia of the committed stem cell compartment (rise in the recognizable myeloid precursors, increase of both colony forming capacity and number of CFU-GM in S-phase) associated to an increase of cyclic AMP and GMP amounts in bone marrow cells of treated mice were observed. These results suggest a possible relationship between the in vitro enhancement of proliferative activity of CFU-GM and the in vivo increase of both cyclic nucleotide levels in medullary environment.

Cytotoxicity of sulphur mustard on a human keratinocyte cell line: direct effects compared to conditioned-medium effects.

Direct toxic effects of sulphur mustard (SM) were compared to SM-conditioned medium effects in a human keratinocyte cell line. Cytotoxicity was evaluated by measurement of cell viability with the Neutral Red uptake assay. Culture directly exposed to SM exhibited cytotoxic dose-response curves after 24, 48 or 72 h. Inhibitory concentration 50 (IC50) appeared to be more than 10 times lower after 72 h than the corresponding value after 24 h. In contrast, no cytotoxic activity was observed in media which were collected 4 h or 24 h after SM exposure. Cell viability was unchanged even if observation was extended to 48 h. Results suggested that SM cytotoxicity was not due to potential mediators secreted by human keratinocytes.

Monolayer, tridimensional and immortalized cell line cultures as models in pharmacology and toxicology.

The articular cartilage located in movable joints can be divided into cellular and intercellular components. This latter category, also called the extracellular matrix, is responsible for the mechanical properties of cartilage and results from the functional activity of the cartilaginous cells, the chondrocytes. Through changes in their metabolism, chondrocytes are able to modify the composition of cartilage in response to physiological, pathological and pharmacotoxicological stimuli. The culture of articular chondrocytes is therefore important in biological research and various methods have been proposed. Models in vitro of normal and pathological chondrocytes are now described. Normal culture can be studied in organ explants, monolayers and various types of tridimensional culture. Chondrocytes immortalized by gene transfection will provide a further model of normal cells when progress has been made in the maintenance of differentiation. Models of pathological cells involve normal cultured chondrocytes treated with cytokines or oxygen free radicals, or rendered senescent by successive passages. Such models are useful in pharmacological and toxicological research. The advantages and limitations of these different models of cultured articular chondrocytes are discussed.

Comparative assessment of in vitro toxicity of xenobiotics using flow cytometry and spectrophotometry.

Cell viability and cell proliferation are endpoints that can be used to identify cytotoxic effects. In a study of the cytotoxicity of four biomaterials and drugs, these two criteria were determined by different techniques. There were notable similarities and differences among the different methods used. Cell viability, which was determined by the trypan blue exclusion test, spectrophotometric microtitration (neutral red) and flow cytometry (fluorescein diacetate) gave similar results. However, the neutral red assay was found to be the most sensitive method for determining the cytotoxicity of these biomaterials and drugs. Cell proliferation measurement, by cell counts and quantitative protein estimation (coomassie blue), revealed important variations between the two methods and indicated poor sensitivity for the protein assay. A slight variability in the determination of the inhibitory concentration 50 (IC(50)) for the two drugs was observed for all the techniques.