Epidermal keratinocyte-derived basophil promoting activity. Role of interleukin 3 and soluble CD23.

Human epidermal keratinocytes (EK) secrete factors able to sustain the proliferation of early myeloid cells and, in particular, the generation of basophils. This activity was previously attributed to IL-3, although no definitive in situ demonstration of this cytokine was provided. In regard to the possible physiological relevance of these data, we investigated herein the nature of EK-derived factors responsible for basophil promotion. Our data show that EK-derived supernatants (EK-sup) contain IL-3 as well as soluble CD23 (sCD23), both known for their colony stimulating activity. Messenger RNA for IL-3 and CD23 were also detected in EK. Blocking experiments using specific neutralizing monoclonal antibodies (mAb) further indicate that EK-derived basophil promoting activity is mainly due to the presence of IL-3 and sCD23 in EK-sup. Furthermore, by contrast to IL-3, sCD23 secretion by EK is cortisone sensitive and highly enhanced by IL-4, suggesting distinct regulatory mechanisms for their production.

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.

Monoclonal antibodies to the human immunodeficiency virus p18 protein cross-react with normal human tissues.

The immunohistochemical reactivity of four monoclonal antibodies (MAbs): CVK, 49-5, 49-6 and 63-FH2, raised against the p18 protein of HIV-1 was assessed in tissues obtained from HIV-infected and uninfected individuals. As already reported, all the MAbs specifically labelled follicular dendritic cells (FDC) in lymph nodes from HIV-infected patients with lymphadenopathy, and cells of microglial nodules in the brain from patients with AIDS-related encephalopathy. However, cross-reactivity with normal uninfected tissues was also observed: epithelial cells of the skin, the thymus and tonsils with CVK, and astrocytes in the brain of 49-6 and 63-FH2. Such cross-reactivities suggest that 'molecular mimicry' could exist between p18 of HIV and normal constituents of human cells. This phenomenon could be relevant for the diagnostic use of anti-p18 MAbs on pathological specimens, and it could be of importance in the pathophysiology of HIV infection.

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.

[Determination of the prothrombin time and of the activated cephalin time wit semi-automatic Electra 600 apparatus (author's transl)]. / Détermination du temps de Quick et du temps de céphaline activée avec l' appareillage semi-automatique Electra 600.

This work studies the quality of the response of the Electra 600 for the determination of the prothrombin time (PT) and the activated cephalin time (ACT). The intra-serial precision was good for the PT and the ACT with normal or pathological plasmas (CV 2%). The precision was excellent for the PT (r = 0.98; Electra 600-fibrometer). The correlation coefficient varies between 0.85 and 0.95 for the ACT depending on the nature of the activator chosen. Lactescent plasmas having a protein concentration greater than 85 g/l should be treated manually for measurement of the PT. No interference was noted with other biological substances: eg glucose, bilirubin, hemoglobin. The increasing addition of heparin indicates a correct sensitivity of the response with Electra 600 both for short and long ACT. Autoanalysis renders the determinations of PT and ACT independant of the manipulator, that of the PT being much more rapid than with the manual method

Differential effect of D-penicillamine on the cell kinetic parameters of various normal and transformed cellular types.

The cell-growth-inhibitory and phase-specific effects of D-penicillamine on cell-cycle progression were investigated using cell-proliferation patterns, quantitative cell-cycle analysis by flow cytometry, and determination of the mitotic index and binucleate cell fraction of normal (rabbit articular chondrocytes, L 809, rabbit fibroblasts) and transformed (HeLa, L 929) cells. D-penicillamine treatment resulted in an inhibition of growth within a dose range of 5 x 10(-4) M to 7.5 x 10(-3) M. Examination of DNA by flow cytometric analysis revealed that rabbit articular chondrocytes were preferentially arrested in the G0/1 phase of the cell cycle, whereas the other cell lines were blocked in the G2 + M phase; the increase in the proportion of cells with G2 + M DNA content was partially due to an enhancement of binucleate cells, resulting in a cytokinesis perturbation for HeLa and L 929 cells. These results showed that D-penicillamine affects cell proliferation through different events according to cell type.

The effects of sodium butyrate on transcription are mediated through activation of a protein phosphatase.

In this study we have investigated the molecular mechanism by which sodium butyrate modulates gene expression when added to cultured cells. As a model system we used hepatoma tissue culture cells in which sodium butyrate treatment increases histone H1(0) mRNA level and decreases c-myc mRNA level. Because we observed that stimulation of histone H1(0) gene expression could take place in the absence of protein neosynthesis, we hypothesized that sodium butyrate induced a post-translational modification of a factor involved in the transcription process. Using different types of well known kinase and phosphatase inhibitors, we studied the implication of kinase or phosphatase activity in this pathway. Interestingly, cell treatment with potent serine-threonine-phosphatase inhibitors, calyculin A or okadaic acid, prevented the regulation of both histone H1(0) and c-myc gene expressions by sodium butyrate. On the other hand, the tyrosine phosphatase inhibitor, vanadate, or the protein kinase C inhibitor, staurosporine, did not significantly modify sodium butyrate effects. Using protein phosphatase 1 and 2A for in vitro assays, we found a 45% increase of phosphatase activity after cell treatment by sodium butyrate, possibly due to a protein phosphatase 1-type protein phosphatase. These data strongly suggest that signaling pathway(s) triggered by sodium butyrate to modulate gene expression involve(s) a serine-threonine-phosphatase activity.

[A study of the specificity of amylases. Measurement of pancreatic isoamylases (author's transl)]. / Spécificité des amylases. Détermination des isoamylases pancréatiques.

Serum lipase, pancreatic isoamylases and serum inhibitory activity on proteases were measured in 29 patients, 17 of whom had pancreatic disorders. The new test used to measure pancreatic isoamylases is rapid and useful, as it is relatively specific of pancreatic lesions. In acute abdominal syndromes, it undoubtedly enhances the value of biochemical investigations by confirming or excluding pancreatic lesions or involvement.

Effects of dexamethasone on the growth of cultured rabbit articular chondrocytes:relation with the nuclear glucocorticoid-receptor complex.

This study reports that dexamethasone at a high dose (10(-4) mol/l) induced slowing of the in vitro proliferation of rabbit articular chondrocytes in both monolayer and clonal culture. This effect is consistent with an inhibition of DNA and RNA synthesis and was characterised by an accumulation of cells in the G0G1 phase of the cell cycle, as shown by flow cytometric analysis. Therefore we determined the extent of nuclear localisation of dexamethasone-receptor complexes. The results showed a discrepancy between 50% growth inhibitory dose (10(-4) mol/l) and the apparent affinity, KD (1.4 (SD 0.2) X 10(-9) mol/l). Thus the growth inhibition of rabbit articular chondrocytes by dexamethasone did not seem to be related exclusively to an interaction with the glucocorticoid-receptor complexes.

Interleukin-1 beta-mediated glucose uptake by chondrocytes. Inhibition by cortisol.

The aim of this study was to investigate the in vitro effects of interleukin-1 beta (IL-1 beta) on cultured human articular chondrocytes from patients with osteoarthritis, by the evaluation of glucose uptake. We also investigated the inhibitory effect of cortisol on IL-1 beta-mediated glucose uptake. Experiments were performed by using 2-deoxy-D-[1-3H]glucose (2-DOG) and confluent monolayer cells at first passage. Confluent cells were also treated for 24 h with different concentrations of cortisol (10(-5), 10(-6) and 10(-7) mol/l). IL-1 beta (100 pg/ml) was added 6 h before glucose uptake studies. Glucose uptake stimulation was observed 3 h after the addition of 100 pg/ml IL-1 beta (+70%) and increased up to 24 h (+145%). The sensitivity and responsiveness of chondrocytes to IL-1 beta, studied after a 6 h association time, appeared to be dose-dependent from 0.1 pg/ml IL-1 beta (+50%) to 100 pg/ml (+130%) over basal values. The effect of the cytokine was protein synthesis-dependent, as demonstrated by using cycloheximide. Cortisol inhibited the action of IL-1 beta on glucose uptake because it reduced stimulating effects by 28% at concentrations as weak as 10(-6) mol/l. Results appeared similar when IL-1 beta and cortisol were added simultaneously 6 h before 2-DOG uptake. The rapid effect of cortisol was protein-synthesis dependent, as indicated by inhibition by cycloheximide. These results suggest that IL-1 beta stimulates chondrocyte metabolic activity. The inhibition of IL-1 beta-mediated glucose uptake is suggested for studying the anti-IL-1 effect of other anti-rheumatic drugs.

Characteristics of homogeneously small keratinocytes from newborn rat skin: possible epidermal stem cells.

The aims of the present study were to characterize the phenotype, growth kinetics, and proliferative activation in culture of a population of poorly differentiated homogeneously small (HS) keratinocytes. These slow-cycling cells were separated by unit gravity sedimentation from a population of actively proliferating basal keratinocytes in newborn rat skin. This population (approximately 1% of the total basal keratinocytes) consisted of extremely small cells with little cytoplasm or RNA. Their positive KL4 staining demonstrates that they were keratinocytes. HS keratinocytes did not, however, contain epidermal calcium binding protein. Acridine orange, bivariate Hoechst, and ethidium bromide flow cytometry of in vitro bromodeoxyuridine-labeled cells as well as Ki67 staining showed that HS keratinocytes were in the G0 stage of the cell cycle and did not actively proliferate in vivo. [3H]thymidine label-retaining cells were found only in the HS cell population, showing that HS cells may originate from a central position in the epidermal proliferative unit. Growth of HS cells in vitro was characterized by a delayed but progressive increase in RNA before entry into the cell cycle. The clonogenic efficiency of HS cells in primary culture was much less than that of larger cells. Subclones of HS cell colonies exceeded primary colonies in their cloning efficiency and proliferative potential, suggesting that HS cells, although normally prevented from dividing, retain a high self-renewal capacity. They also maintain the ability to differentiate. The results are consistent with the concept that HS cell population may represent the epidermal-specific progenitor cells which act as stem cells in this tissue.

DNA flow cytometric analysis of monogranulocytic colony forming cells from mouse bone marrow in vitro.

An adaptation of a previously reported flow cytometric technique is described. This technique was applied to study the DNA distribution of mouse granulocyte-macrophage colony forming cells (GM-CFc) grown in a methyl cellulose culture system. This method involved the collection of cell clusters and colonies from the methyl cellulose culture medium for the subsequent determination of DNA content. The DNA content of the homogeneous GM-CFc suspensions was determined by applying a modification of the propidium iodide staining procedure described by Crissman et al. The relative percentage of cells in G0 + G1, S, and G2 + M stages of the cell cycle was calculated by applying mathematical analysis to the resulting DNA histograms. This method could be useful in studying the effects of drugs on GM-CFc kinetics.

Proliferation kinetics of rabbit articular chondrocytes in primary culture and at the first passage.

The in-vitro proliferation kinetics of young rabbit articular chondrocytes were compared in primary culture and at the first passage. The growth curves labelling and mitotic indices, percentage labelled mitosis (PLM) curves and DNA content distributions by flow-microfluorometric analysis during a 7-day growth period were determined in both cases. The length of the cell cycle and the doubling time calculated from the exponential part of the growth curve were quite similar: Tc = 19 hr and Td = 20 hr for the primary culture, Tc = 17 X 3 hr and Td = 20 hr for the first passage. However, the growth curve and the DNA distribution during the 7-day period showed some differences. The duration of the lag period studied by the growth curve was longer in the primary culture than at the first passage. This phenomenon was also observed using the FCM analysis. The growth fraction determination on the second day of culture was in accordance with the lower proliferation capacity of the cells in primary culture. These data suggest that it would be better to study growth kinetics and drug modifications in articular chondrocytes at the first passage than in primary culture.

Preliminary clinical studies of a biological skin equivalent in burned patients.

The possibility of covering large areas of full thickness skin loss with 'living skin equivalent' produced by a modification of Bell's method was studied. Living skin equivalents, composed of a dermal equivalent (fibroblasts plus collagen) covered by epithelial cells were grafted, meshed or non-meshed, onto granulation tissue and, in one patient, onto fascia. Eight patients with full skin thickness burn wounds covering over 15 per cent of the body surface area were thus partially covered. The graft 'take' was evaluated every 48 h. In every patient grafted, an extensive lysis (60-90 per cent) of the skin equivalent graft was observed at the first dressing (48 h). In one patient only, a significant percentage of 'take' (40 per cent) was observed 14 days after grafting. These disappointing results were probably related to the presence of collagenases or proteases produced on the wound bed either by bacteria or by surrounding human cells. It appears that at the present time the biochemical nature of the dermal equivalent used is not yet completely appropriate to serve routinely as a substitute for human skin.