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1.
Gastroenterology ; 121(6): 1451-9, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11729124

ABSTRACT

BACKGROUND & AIMS: Acute liver failure (ALF) of viral origin results from massive hepatocyte apoptosis induced by the interaction between Fas expressed on hepatocytes and Fas ligand on activated T lymphocytes. Because Fas-induced apoptosis of hepatocytes involves mitochondrial damages and potential reactive oxygen species (ROS) overproduction, we investigated whether manganese III tetrakis (5,10,15,20 benzoic acid) (MnTBAP), a nonpeptidyl mimic of superoxide dismutase (SOD), can inhibit Fas-induced ALF. METHODS: An agonist anti-Fas monoclonal antibody was used to induce hepatocyte apoptosis in vitro and ALF in vivo. RESULTS: Preventive and curative treatments by MnTBAP significantly increased survival rates and significantly reduced aspartate aminotransferase levels and parenchymal lesions. ROS generation was suggested by those beneficial effects and significant increases in SOD and Gpx activities after anti-Fas injection. Flow cytometry of isolated hepatocytes incubated with anti-Fas monoclonal antibody showed that ROS production was associated with the collapse of transmembrane potential and loss of cardiolipin content. After injection of anti-Fas monoclonal antibody, mitochondrial Bcl-2 was decreased, cytochrome c released, and caspase-3 activated. Mitochondrial alterations and their consequences were abrogated by MnTBAP. CONCLUSIONS: ROS are key executioners in Fas-induced hepatocyte apoptosis. This finding explains why a nonpeptidyl mimic of SOD can cure ALF in a model of viral hepatitis, pointing out the potential interest of this molecule in humans.


Subject(s)
Liver Failure/chemically induced , Liver Failure/prevention & control , Metalloporphyrins/pharmacology , fas Receptor , Acute Disease , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis , Cell Separation , Female , Glutathione Peroxidase/metabolism , Hepatocytes/drug effects , Hepatocytes/physiology , Liver/drug effects , Liver/pathology , Mice , Mice, Inbred BALB C , Mitochondria, Liver/pathology , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism , Superoxide Dismutase/pharmacology , fas Receptor/immunology
2.
Hepatology ; 33(5): 1173-80, 2001 May.
Article in English | MEDLINE | ID: mdl-11343246

ABSTRACT

Drug-induced acute liver failure (ALF) is a devastating and often fatal disease mainly caused by poisoning by acetaminophen (APAP). The toxic metabolite, N-acetyl-p-benzoquinone-imine (NAPQI), that leads to gluthatione depletion has been suspected to be the main effector of hepatocyte apoptosis during APAP-induced ALF. We have investigated whether reactive oxygen species (ROS) also play a role in APAP-induced ALF, and whether manganese III tetrakis (5,10,15,20 benzoic acid) (MnTBAP), a mimic of superoxide dismutase (SOD) with catalase-like activity, can treat the disease in mice. The effects of MnTBAP were tested on APAP-intoxicated mice and on isolated hepatocytes incubated with APAP. MnTBAP preventively and curatively administered significantly improved survival times, and dramatically reduced serum transaminase activity levels and parenchymal lesions in APAP-intoxicated mice. Whereas pretreatment with N-acetyl-L-cysteine (NAC) prevented ALF in a dose-dependent manner, the molecule was ineffective when curatively administered. The significant increase in glutathione peroxidase (Gpx) activity following APAP administration, and the beneficial effects of MnTBAP suggested that ROS were produced during APAP-induced ALF. A direct evidence of ROS generation was provided by flow cytometry of isolated hepatocytes incubated with APAP. In vitro, ROS production was associated with mitochondrial damage characterized by the collapse of transmembrane potential and the loss of cardiolipin content. In livers of intoxicated mice, ALF was associated with cytochrome c release that led to the activation of caspases-9 and -3. The capacity of MnTBAP to abrogate all those alterations suggests that ROS play a role in APAP-induced apoptosis of hepatocytes, and explains the beneficial effects of MnTBAP, which could be of interest in APAP-induced ALF in humans.


Subject(s)
Acetaminophen , Free Radical Scavengers/antagonists & inhibitors , Free Radical Scavengers/pharmacology , Liver Failure/chemically induced , Liver Failure/drug therapy , Metalloporphyrins/pharmacology , Acetaminophen/pharmacokinetics , Acute Disease , Animals , Caspases/metabolism , Cytochrome c Group/metabolism , Enzyme Activation/drug effects , Female , Free Radical Scavengers/chemistry , Glutathione Peroxidase/metabolism , Inactivation, Metabolic , Liver/drug effects , Liver/pathology , Mice , Mice, Inbred BALB C , Mitochondria, Liver/drug effects , Mitochondria, Liver/pathology , Oxidation-Reduction/drug effects , Superoxide Dismutase/chemistry , Superoxide Dismutase/metabolism , Survival Analysis , Transaminases/blood
3.
Hepatology ; 33(1): 10-5, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11124814

ABSTRACT

Stable gene transfer into hepatocytes has been proposed to compensate for genetic deficiencies that affect liver function, or to deliver diffusible factors into the circulation. This strategy can be achieved using retroviral vectors; however, cell division must occur. We describe a simple and reproductive method that enables the induction of hepatocyte replication in a controlled fashion, thus allowing an efficient in vivo retroviral liver transduction that is applicable to mouse models of human genetic disorders. The approach is based on liver susceptibility to apoptosis via the Fas/CD95 pathway. We show that, 4 days following a single Fas agonist antibody (JO2) injection, hepatocyte replication occurs, the intensity of which is correlated with the level of the induced hepatic cytolysis. This treatment enables in vivo liver transduction, and its efficiency also correlates with the level of hepatic cytolysis. When recombinant retroviral vectors were infused intravenously during the period of hepatocyte replication, 15.4% +/- 1.7% of the hepatocytes were transduced, reaching up to 32.5%.


Subject(s)
Gene Transfer Techniques , Genetic Vectors , Liver Regeneration/physiology , Retroviridae/genetics , fas Receptor/physiology , Alanine Transaminase/blood , Animals , Antibodies, Monoclonal/pharmacology , Cell Division/drug effects , Dose-Response Relationship, Drug , Female , Hepatocytes/cytology , Injections , Mice , Mice, Inbred C57BL , Transduction, Genetic , Ultrafiltration , fas Receptor/immunology
5.
J Biol Chem ; 272(39): 24148-53, 1997 Sep 26.
Article in English | MEDLINE | ID: mdl-9305863

ABSTRACT

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.


Subject(s)
Butyrates/pharmacology , Phosphoprotein Phosphatases/metabolism , Transcription, Genetic/drug effects , Butyric Acid , Enzyme Activation , Enzyme Inhibitors/pharmacology , Genes, myc , Histones/genetics , Marine Toxins , Oxazoles/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Protein Phosphatase 1 , Protein Processing, Post-Translational , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Cells, Cultured
6.
J Clin Immunol ; 17(3): 228-33, 1997 May.
Article in English | MEDLINE | ID: mdl-9168403

ABSTRACT

Autoantibodies characteristic of autoimmune bullous skin diseases (AIBDs) can be detected by immunoblotting on epidermal, dermal, or bovine muzzle extracts. However, none of those substrates contain all the autoantigens involved in AIBDs, and the diagnosis requires the use of various substrates. Human keratinocytes were cultured under such conditions that they expressed the major autoantigens associated with AIBDs. Forty-two sera with antiepidermal antibodies were immunoblotted on the keratinocyte extract. Bands corresponding to desmoglein III, desmoglein I, BPAg2, BPAg1, and type VII collagen were found in 38 sera. Desmoplakins I and II were revealed by specific monoclonal antibodies. A review of the patients' charts showed a perfect correlation between the blots and the diagnoses of pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, and epidermolysis bullosa acquisita. Four sera revealing no band typical of AIBD were from patients with no autoimmune skin disease. Therefore, a single extract of keratinocytes can be used for the differential diagnosis of AIBDs.


Subject(s)
Autoantigens/immunology , Autoantigens/isolation & purification , Autoimmune Diseases/immunology , Keratinocytes/immunology , Skin Diseases, Vesiculobullous/immunology , Animals , Autoantigens/biosynthesis , Autoimmune Diseases/metabolism , Basement Membrane/immunology , Cell Differentiation/immunology , Cells, Cultured , Desmosomes/immunology , Humans , Keratinocytes/chemistry , Rats , Retrospective Studies , Skin Diseases, Vesiculobullous/metabolism
7.
Osteoarthritis Cartilage ; 4(2): 139-42, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8806115

ABSTRACT

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.


Subject(s)
Cartilage, Articular/metabolism , Deoxyglucose/antagonists & inhibitors , Deoxyglucose/pharmacokinetics , Hydrocortisone/pharmacology , Interleukin-1/physiology , Cartilage, Articular/cytology , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Recombinant Proteins
10.
J Clin Invest ; 90(4): 1242-7, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1401061

ABSTRACT

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.


Subject(s)
Basophils/physiology , Interleukin-3/physiology , Keratinocytes/physiology , Receptors, IgE/physiology , Antibodies, Monoclonal/immunology , Cells, Cultured , Female , Humans , Interleukin-3/genetics , RNA, Messenger/analysis , Receptors, IgE/genetics
11.
Am J Physiol ; 261(6 Pt 1): C964-72, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1767823

ABSTRACT

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.


Subject(s)
Epidermal Cells , Keratinocytes/cytology , Stem Cells/cytology , Animals , Animals, Newborn , Cell Cycle , Cell Differentiation , Cell Division , Clone Cells , Flow Cytometry , Immunoenzyme Techniques , Keratinocytes/metabolism , Kinetics , Phenotype , Rats , Rats, Inbred Strains , Thymidine/metabolism
13.
J Cell Physiol ; 136(3): 514-8, 1988 Sep.
Article in English | MEDLINE | ID: mdl-3170647

ABSTRACT

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.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Penicillamine/pharmacology , Animals , Cell Cycle/drug effects , Cell Line , DNA/analysis , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Mitotic Index/drug effects , Rabbits
14.
Burns Incl Therm Inj ; 14(4): 326-30, 1988 Aug.
Article in English | MEDLINE | ID: mdl-3066451

ABSTRACT

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.


Subject(s)
Bandages , Biological Dressings , Burns/therapy , Burns/pathology , Culture Techniques , Evaluation Studies as Topic , Fibroblasts , Humans , Skin/pathology , Skin Transplantation
15.
AIDS ; 2(3): 171-7, 1988 Jun.
Article in English | MEDLINE | ID: mdl-2456086

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , HIV/immunology , Antibody Specificity , Brain/immunology , Cross Reactions , Epitopes/analysis , HIV Antibodies , Humans , Immunohistochemistry , Skin/immunology , Thymus Gland/immunology
17.
Ann Rheum Dis ; 46(2): 146-52, 1987 Feb.
Article in English | MEDLINE | ID: mdl-2435251

ABSTRACT

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.


Subject(s)
Cartilage, Articular/drug effects , Dexamethasone/pharmacology , Receptors, Glucocorticoid/drug effects , Animals , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , Cell Division/drug effects , Cell Nucleus/drug effects , Cells, Cultured , DNA/biosynthesis , Flow Cytometry , Macromolecular Substances , RNA/biosynthesis , Rabbits
19.
Ann Rheum Dis ; 43(2): 333-8, 1984 Apr.
Article in English | MEDLINE | ID: mdl-6712306

ABSTRACT

The long-acting antirheumatic drug D-penicillamine was found to inhibit the growth of asynchronous cultures of rabbit articular chondrocytes. This inhibitory effect was dose-related between 5 X 10(-4) M and 5 X 10(-3) M and was time-dependent for a given dose. Flow cytometric analysis showed that drug exposure led to a slowdown in cell cycle progression. This was manifested as a decrease in the number of cells in S phase, due especially to an accumulation of cells in G0 G1 and also to a slight cessation of cell transit through G2 M. Recovery experiments showed that the effect is transitory and reversible. It is suggested that the articular chondrocyte is a target cell for D-penicillamine and that these cells have a D-penicillamine sensitive restriction point in the G0 G1 phase of the cell cycle and to a less extent in the G2 M phase.


Subject(s)
Cartilage, Articular/drug effects , Penicillamine/pharmacology , Animals , Cartilage, Articular/analysis , Cartilage, Articular/cytology , Cell Cycle/drug effects , Cell Division/drug effects , Cells, Cultured , Clone Cells/drug effects , DNA/analysis , Dose-Response Relationship, Drug , Flow Cytometry , Rabbits
20.
Cell Tissue Kinet ; 16(6): 531-7, 1983 Nov.
Article in English | MEDLINE | ID: mdl-6627344

ABSTRACT

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.


Subject(s)
Cartilage, Articular/cytology , Cells, Cultured/cytology , Animals , Cartilage, Articular/metabolism , Cell Division , DNA/analysis , DNA/biosynthesis , Interphase , Kinetics , Mitosis , Rabbits
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