25 results on '"Christine Miossec"'
Search Results
2. Characterisation of Early Positive mcr-1 Resistance Gene and Plasmidome in Escherichia coli Pathogenic Strains Associated with Variable Phylogroups under Colistin Selection
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Guerrino Macori, Scott V. Nguyen, Ankita Naithani, Daniel Hurley, Li Bai, Farid El Garch, Frédérique Woehrlé, Christine Miossec, Benjamin Roques, Peadar O’Gaora, James L. Bono, and Séamus Fanning
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antimicrobial resistance ,Escherichia coli ,colistin resistance ,whole genome sequencing ,mcr-1 gene ,plasmid ,Therapeutics. Pharmacology ,RM1-950 - Abstract
An antibiotic susceptibility monitoring programme was conducted from 2004 to 2010, resulting in a collection of 143 Escherichia coli cultured from bovine faecal samples (diarrhoea) and milk-aliquots (mastitis). The isolates were subjected to whole-genome sequencing and were distributed in phylogroups A, B1, B2, C, D, E, and G with no correlation for particular genotypes with pathotypes. In fact, the population structure showed that the strains belonging to the different phylogroups matched broadly to ST complexes; however, the isolates are randomly associated with the diseases, highlighting the necessity to investigate the virulence factors more accurately in order to identify the mechanisms by which they cause disease. The antimicrobial resistance was assessed phenotypically, confirming the genomic prediction on three isolates that were resistant to colistin, although one isolate was positive for the presence of the gene mcr-1 but susceptible to colistin. To further characterise the genomic context, the four strains were sequenced by using a single-molecule long read approach. Genetic analyses indicated that these four isolates harboured complex and diverse plasmids encoding not only antibiotic resistant genes (including mcr-1 and bla) but also virulence genes (siderophore, ColV, T4SS). A detailed description of the plasmids of these four E. coli strains, which are linked to bovine mastitis and diarrhoea, is presented for the first time along with the characterisation of the predicted antibiotic resistance genes. The study highlighted the diversity of incompatibility types encoding complex antibiotic resistance elements such as Tn6330, ISEcp1, Tn6029, and IS5075. The mcr-1 resistance determinant was identified in IncHI2 plasmids pCFS3273-1 and pCFS3292-1, thus providing some of the earliest examples of mcr-1 reported in Europe, and these sequences may be a representative of the early mcr-1 plasmidome characterisation in the EU/EEA.
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- 2021
- Full Text
- View/download PDF
3. Multidrug-Resistant Escherichia coli in Bovine Animals, Europe
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Evan Brennan, Marta Martins, Matthew P. McCusker, Juan Wang, Bruno Martins Alves, Daniel Hurley, Farid El Garch, Frédérique Woehrlé, Christine Miossec, Leisha McGrath, Shabarinath Srikumar, Patrick Wall, and Séamus Fanning
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multidrug-resistant ,MDR ,Escherichia coli ,colistin resistance ,food-producing animals ,bovine ,Medicine ,Infectious and parasitic diseases ,RC109-216 - Abstract
Of 150 Escherichia coli strains we cultured from specimens taken from cattle in Europe, 3 had elevated MICs against colistin. We assessed all 3 strains for the presence of the plasmid-mediated mcr-1 gene and identified 1 isolate as mcr-1–positive and co-resistant to β-lactam, florfenicol, and fluoroquinolone antimicrobial compounds.
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- 2016
- Full Text
- View/download PDF
4. Characterisation of Early Positive mcr-1 Resistance Gene and Plasmidome in Escherichia coli Pathogenic Strains Associated with Variable Phylogroups under Colistin Selection
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James L. Bono, Frédérique Woehrlé, Farid El Garch, Scott V. Nguyen, Benjamin Roques, Daniel Hurley, Li Bai, Guerrino Macori, Séamus Fanning, Ankita Naithani, Peadar O'Gaora, and Christine Miossec
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Microbiology (medical) ,Virulence ,Context (language use) ,RM1-950 ,Biology ,medicine.disease_cause ,Biochemistry ,Microbiology ,Article ,Plasmid ,Antibiotic resistance ,fluids and secretions ,SDG 3 - Good Health and Well-being ,plasmid ,medicine ,Escherichia coli ,Pharmacology (medical) ,antimicrobial resistance ,General Pharmacology, Toxicology and Pharmaceutics ,Genetics ,whole genome sequencing ,mcr-1 gene ,Infectious Diseases ,colistin resistance ,Colistin ,MCR-1 ,Plasmidome ,Therapeutics. Pharmacology ,medicine.drug - Abstract
An antibiotic susceptibility monitoring programme was conducted from 2004 to 2010, resulting in a collection of 143 Escherichia coli cultured from bovine faecal samples (diarrhoea) and milk-aliquots (mastitis). The isolates were subjected to whole-genome sequencing and were distributed in phylogroups A, B1, B2, C, D, E, and G with no correlation for particular genotypes with pathotypes. In fact, the population structure showed that the strains belonging to the different phylogroups matched broadly to ST complexes, however, the isolates are randomly associated with the diseases, highlighting the necessity to investigate the virulence factors more accurately in order to identify the mechanisms by which they cause disease. The antimicrobial resistance was assessed phenotypically, confirming the genomic prediction on three isolates that were resistant to colistin, although one isolate was positive for the presence of the gene mcr-1 but susceptible to colistin. To further characterise the genomic context, the four strains were sequenced by using a single-molecule long read approach. Genetic analyses indicated that these four isolates harboured complex and diverse plasmids encoding not only antibiotic resistant genes (including mcr-1 and bla) but also virulence genes (siderophore, ColV, T4SS). A detailed description of the plasmids of these four E. coli strains, which are linked to bovine mastitis and diarrhoea, is presented for the first time along with the characterisation of the predicted antibiotic resistance genes. The study highlighted the diversity of incompatibility types encoding complex antibiotic resistance elements such as Tn6330, ISEcp1, Tn6029, and IS5075. The mcr-1 resistance determinant was identified in IncHI2 plasmids pCFS3273-1 and pCFS3292-1, thus providing some of the earliest examples of mcr-1 reported in Europe, and these sequences may be a representative of the early mcr-1 plasmidome characterisation in the EU/EEA.
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- 2021
5. A study of the correlation between phenotypic antimicrobial susceptibility testing methods and the associated genotypes determined by whole genome sequencing for a collection of Escherichia coli of bovine origin
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Emmanuel Cuinet, Séamus Fanning, Frédérique Woehrlé, Scott V. Nguyen, Farid El Garach, Christine Miossec, Dagmara A. Niedziela, and Thomas J Maunsell
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Whole genome sequencing ,Genetics ,Minimum inhibitory concentration ,Antibiotic resistance ,Nalidixic acid ,medicine.drug_class ,Tetracycline ,Antibiotics ,medicine ,Biology ,Antimicrobial ,Kappa ,medicine.drug - Abstract
Antimicrobial resistance (AMR) has increased at an alarming pace in the recent years. Molecular-based methods such as whole genome sequencing (WGS) offer a potential alternative to the conventional labour-intensive methods traditionally used to characterise AMR phenotypes. The aim of this study was to investigate whether WGS could be used as a predictor of AMR in Escherichia coli isolates of bovine origin.Genomes of 143 E. coli cultured from cattle presenting with diarrhoea or mastitis were sequenced on an Illumina MiSeq platform. AMR genes were identified using the ResFinder and AMRFinder databases. Antimicrobial susceptibility testing by disk diffusion was performed on a panel of 10 antibiotics, covering 7 antimicrobial classes. Minimum inhibitory concentration (MIC) measurements were made using the Sensititre plate with 6 antibiotics, covering 5 antimicrobial classes. Correlation between genotype and phenotype was assessed statistically by means of a two-by-two table analysis and Cohen’s kappa (κ) test.The overall κ correlation between WGS and disk diffusion was 0.81, indicating a near perfect agreement, and the average positive predicted value was 77.4 %. Correlation for individual antimicrobial compounds varied, with five yielding near perfect agreement (κ = 0.81–1.00; amoxicillin, florfenicol, gentamicin, tetracycline and trimethoprim-sulfamethoxazole), one showing substantial agreement (κ = 0.65; nalidixic acid), and four showing moderate agreement (κ = 0.41– 0.60). The overall κ correlation between WGS and MIC was 0.55 indicating moderate agreement, and the average positive predicted value was 68.6 %. Three antibiotics yielded near perfect agreement (gentamicin, tetracycline and trimethoprim-sulfamethoxazole) and a further three showed fair agreement (κ = 0.21–0.40).WGS is a useful tool that can be used for the prediction of AMR phenotypes, and correlates well with disk diffusion results. MIC measurements may be necessary for antimicrobial compounds with a high proportion of intermediately resistant isolates recorded, such as cephalothin.HighlightsCulture based antimicrobial susceptibility testing is used to identify therapeutics in the treatment of clinical veterinary isolatesWhole genome sequencing is increasingly adopted for surveillance, epidemiological traceback investigations, and detection of antimicrobial resistance genesLittle is known in correlations between antimicrobial resistance genotypes and disk diffusion antimicrobial susceptibility testingThis study finds that whole genome sequencing is a useful predictor for antimicrobial susceptibility however, minimum inhibitory concentration measurements may still be needed for intermediately resistant isolates
- Published
- 2021
6. Investigation of the effect of the adsorbent DAV131A on the propensity of moxifloxacin to induce simulated Clostridioides (Clostridium) difficile infection (CDI) in an in vitro human gut model
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Antoine Andremont, G S Crowther, C H Chilton, Mark H. Wilcox, J de Gunzburg, and Christine Miossec
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Microbiology (medical) ,Moxifloxacin ,Gut flora ,medicine.disease_cause ,Microbiology ,Clostridium ,Clostridioides ,medicine ,Animals ,Humans ,Pharmacology (medical) ,Feces ,Pharmacology ,biology ,Toxin ,Clostridioides difficile ,Clostridium difficile ,biology.organism_classification ,Antimicrobial ,Anti-Bacterial Agents ,Gastrointestinal Tract ,Infectious Diseases ,Clostridium Infections ,Bacteroides fragilis ,medicine.drug - Abstract
Background Clostridioides difficile infection (CDI) remains a high burden worldwide. DAV131A, a novel adsorbent, reduces residual gut antimicrobial levels, reducing CDI risk in animal models. Objectives We used a validated human gut model to investigate the efficacy of DAV131A in preventing moxifloxacin-induced CDI. Methods C. difficile (CD) spores were inoculated into two models populated with pooled human faeces. Moxifloxacin was instilled (43 mg/L, once daily, 7 days) alongside DAV131A (5 g in 18 mL PBS, three times daily, 14 days, Model A), or PBS (18 mL, three times daily, 14 days, Model B). Selected gut microbiota populations, CD total counts, spore counts, cytotoxin titre and antimicrobial concentrations (HPLC) were monitored daily. We monitored for reduced susceptibility of CD to moxifloxacin. Growth of CD in faecal filtrate and medium in the presence/absence of DAV131A, or in medium pre-treated with DAV131A, was also investigated. Results DAV131A instillation reduced active moxifloxacin levels to below the limit of detection (50 ng/mL), and prevented microbiota disruption, excepting Bacteroides fragilis group populations, which declined by ∼3 log10 cfu/mL. DAV131A delayed onset of simulated CDI by ∼2 weeks, but did not prevent CD germination and toxin production. DAV131A prevented emergence of reduced susceptibility of CD to moxifloxacin. In batch culture, DAV131A had minor effects on CD vegetative growth, but significantly reduced toxin/spores (P Conclusions DAV131A reduced moxifloxacin-induced microbiota disruption and emergence of antibiotic-resistant CD. Delayed onset of CD germination and toxin production indicates further investigations are warranted to understand the clinical benefits of DAV131A in CDI prevention.
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- 2019
7. High genetic diversity among methicillin-susceptible Staphylococcus pseudintermedius in dogs in Europe
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Benoît Valot, Christine Miossec, Jean-Yves Madec, Marisa Haenni, Didier Hocquet, Farid El Garch, Unité Antibiorésistance et Virulence Bactériennes, Laboratoire de Lyon [ANSES], Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Vétoquinol, Laboratoire Chrono-environnement - CNRS - UBFC (UMR 6249) (LCE), Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), and Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon)
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Microbiology (medical) ,Genetics ,0303 health sciences ,Genetic diversity ,Staphylococcus pseudintermedius ,030306 microbiology ,Staphylococcus ,Immunology ,Genetic Variation ,Biology ,biology.organism_classification ,Microbiology ,QR1-502 ,3. Good health ,Europe ,03 medical and health sciences ,Methicillin ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,Dogs ,Immunology and Allergy ,Animals ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology - Abstract
International audience
- Published
- 2019
8. In Vitro Antibacterial Activity of the Ceftazidime-Avibactam Combination against Enterobacteriaceae, Including Strains with Well-Characterized β-Lactamases
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Christine Miossec, Ken Coleman, Anne-Marie Girard, John L. Pace, and Premavathy Levasseur
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Imipenem ,Cefotaxime ,Avibactam ,Cefepime ,Ceftazidime ,Microbial Sensitivity Tests ,Pharmacology ,Biology ,beta-Lactamases ,Microbiology ,chemistry.chemical_compound ,Enterobacteriaceae ,polycyclic compounds ,medicine ,Pharmacology (medical) ,Enzyme Inhibitors ,Beta-Lactamase Inhibitors ,Enterobacteriaceae Infections ,Drug Synergism ,Ceftazidime/avibactam ,Anti-Bacterial Agents ,Drug Combinations ,Infectious Diseases ,chemistry ,Susceptibility ,Ceftriaxone ,beta-Lactamase Inhibitors ,Azabicyclo Compounds ,medicine.drug - Abstract
The novel β-lactamase inhibitor avibactam is a potent inhibitor of class A, class C, and some class D enzymes. The in vitro antibacterial activity of the ceftazidime-avibactam combination was determined for a collection of Enterobacteriaceae clinical isolates; this collection was enriched for resistant strains, including strains with characterized serine β-lactamases. The inhibitor was added either at fixed weight ratios to ceftazidime or at fixed concentrations, with the latter type of combination consistently resulting in greater potentiation of antibacterial activity. In the presence of 4 μg/ml of avibactam, the ceftazidime MIC 50 and MIC 90 (0.25 and 2 μg/ml, respectively) were both below the CLSI breakpoint for ceftazidime. Further comparisons with reference antimicrobial agents were performed using this fixed inhibitor concentration. Against most ceftazidime-susceptible and -nonsusceptible isolates, the addition of avibactam resulted in a significant increase in ceftazidime activity, with MICs generally reduced 256-fold for extended-spectrum β-lactamase (ESBL) producers, 8- to 32-fold for CTX-M producers, and >128-fold for KPC producers. Overall, MICs of a ceftazidime-avibactam combination were significantly lower than those of the comparators piperacillin-tazobactam, cefotaxime, ceftriaxone, and cefepime and similar or superior to those of imipenem.
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- 2015
9. Efficacy of a Ceftazidime-Avibactam Combination in a Murine Model of Septicemia Caused by Enterobacteriaceae Species Producing AmpC or Extended-Spectrum β-Lactamases
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Kenneth Coleman, Premavathy Levasseur, Ludovic Lavallade, Christine Miossec, Anne-Marie Girard, and John L. Pace
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Male ,Klebsiella pneumoniae ,Avibactam ,Penicillanic Acid ,Ceftazidime ,Microbial Sensitivity Tests ,medicine.disease_cause ,beta-Lactamases ,Microbiology ,Mice ,chemistry.chemical_compound ,Bacterial Proteins ,Sepsis ,Escherichia coli ,medicine ,Animals ,Experimental Therapeutics ,Pharmacology (medical) ,Beta-Lactamase Inhibitors ,Escherichia coli Infections ,Piperacillin ,Pharmacology ,Mice, Inbred ICR ,biology ,biology.organism_classification ,Ceftazidime/avibactam ,Enterobacteriaceae ,Anti-Bacterial Agents ,Klebsiella Infections ,Drug Combinations ,Piperacillin, Tazobactam Drug Combination ,Infectious Diseases ,chemistry ,beta-Lactamase Inhibitors ,Azabicyclo Compounds ,medicine.drug - Abstract
Avibactam is a novel non-β-lactam β-lactamase inhibitor that has been shown in vitro to inhibit class A, class C, and some class D β-lactamases. It is currently in phase 3 of clinical development in combination with ceftazidime. In this study, the efficacy of ceftazidime-avibactam was evaluated in a murine septicemia model against five ceftazidime-susceptible (MICs of 0.06 to 0.25 μg/ml) and 15 ceftazidime-resistant (MICs of 64 to >128 μg/ml) species of Enterobacteriaceae , bearing either TEM, SHV, CTX-M extended-spectrum, or AmpC β-lactamases. In the first part of the study, ceftazidime-avibactam was administered at ratios of 4:1 and 8:1 (wt/wt) to evaluate the optimal ratio for efficacy. Against ceftazidime-susceptible isolates of Klebsiella pneumoniae and Escherichia coli , ceftazidime and ceftazidime-avibactam demonstrated similar efficacies (50% effective doses [ED 50 ] of 50 of >90 mg/kg), the addition of avibactam restored efficacy to ceftazidime (ED 50 dropped to 50 values ranging from 2 to 27 mg/kg compared to >90 mg/kg and 14 to >90 mg/kg for piperacillin-tazobactam and cefotaxime-avibactam, respectively. This study demonstrates that the potent in vitro activity observed with the ceftazidime-avibactam combination against ceftazidime-resistant Enterobacteriaceae species bearing class A and class C β-lactamases translated into good efficacy in the mouse septicemia model.
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- 2014
10. Protection of hamsters from mortality by reducing fecal moxifloxacin concentration with DAV131A in a model of moxifloxacin-induced Clostridium difficile colitis
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Charles Burdet, Sakina Sayah-Jeanne, Thu Thuy Nguyen, Christine Miossec, Nathalie Saint-Lu, Mark Pulse, William Weiss, Antoine Andremont, France Mentré, Jean de Gunzburg, Département d’Epidémiologie, de Biostatistique et de Recherche Clinique [AP-HP Hôpital Bichat - Claude Bernard] (DEBRC), AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Infection, Anti-microbiens, Modélisation, Evolution (IAME (UMR_S_1137 / U1137)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris 13 (UP13)-Université Paris Diderot - Paris 7 (UPD7)-Université Sorbonne Paris Cité (USPC), Da Volterra, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7), UNT Health Science Center [Fort Worth, USA], University of North Texas (UNT), Laboratoire de bactériologie, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7), and Comets, Emmanuelle
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0301 basic medicine ,Antibiotics ,Quantitative Biology - Quantitative Methods ,Gastroenterology ,prevention ,Moxifloxacin ,Cricetinae ,Pharmacology (medical) ,Quantitative Methods (q-bio.QM) ,Enterocolitis, Pseudomembranous ,C. difficile infection ,[SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] ,[SDV.MHEP] Life Sciences [q-bio]/Human health and pathology ,Mortality rate ,[SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences ,[SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] ,3. Good health ,[SDV.SP] Life Sciences [q-bio]/Pharmaceutical sciences ,Infectious Diseases ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,Charcoal ,moxifloxacin ,Fluoroquinolones ,medicine.drug ,medicine.medical_specialty ,medicine.drug_class ,030106 microbiology ,Hamster ,Clostridium Difficile Colitis ,03 medical and health sciences ,Internal medicine ,medicine ,Animals ,Experimental Therapeutics ,[SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology ,Feces ,Pharmacology ,Dose-Response Relationship, Drug ,Clostridioides difficile ,business.industry ,mortality ,Gastrointestinal Microbiome ,Gastrointestinal Tract ,Disease Models, Animal ,Regimen ,hamster animal model ,Activated charcoal ,FOS: Biological sciences ,Clostridium Infections ,Dysbiosis ,business ,[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology - Abstract
Lowering the gut exposure to antibiotics during treatments can prevent microbiota disruption. We evaluated the effects of an activated charcoal-based adsorbent, DAV131A, on the fecal free moxifloxacin concentration and mortality in a hamster model of moxifloxacin-induced Clostridium difficile infection. A total of 215 hamsters receiving moxifloxacin subcutaneously (day 1 [D 1 ] to D 5 ) were orally infected at D 3 with C. difficile spores. They received various doses (0 to 1,800 mg/kg of body weight/day) and schedules (twice a day [BID] or three times a day [TID]) of DAV131A (D 1 to D 8 ). Moxifloxacin concentrations and C. difficile counts were determined at D 3 , and mortality was determined at D 12 . We compared mortality rates, moxifloxacin concentrations, and C. difficile counts according to DAV131A regimen and modeled the links between DAV131A regimen, moxifloxacin concentration, and mortality. All hamsters that received no DAV131A died, but none of those that received 1,800 mg/kg/day died. Significant dose-dependent relationships between DAV131A dose and (i) mortality, (ii) moxifloxacin concentration, and (iii) C. difficile count were evidenced. Mathematical modeling suggested that (i) lowering the moxifloxacin concentration at D 3 , which was 58 μg/g (95% confidence interval [CI] = 50 to 66 μg/g) without DAV131A, to 17 μg/g (14 to 21 μg/g) would reduce mortality by 90%; and (ii) this would be achieved with a daily DAV131A dose of 703 mg/kg (596 to 809 mg/kg). In this model of C. difficile infection, DAV131A reduced mortality in a dose-dependent manner by decreasing the fecal free moxifloxacin concentration.
- Published
- 2017
11. In vitro activity of the -lactamase inhibitor NXL104 against KPC-2 carbapenemase and Enterobacteriaceae expressing KPC carbapenemases
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Marie-Claude Péchereau, Anne-Marie Girard, Christine Miossec, Michael T. Black, Thérèse Stachyra, Monique Claudon, and Premavathy Levasseur
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Microbiology (medical) ,Imipenem ,carbapenem resistance ,Ceftazidime ,Microbial Sensitivity Tests ,Biology ,beta-Lactams ,beta-Lactamases ,Microbiology ,Inhibitory Concentration 50 ,Enterobacteriaceae ,KPC inhibition ,polycyclic compounds ,medicine ,Humans ,Nitrocefin ,Pharmacology (medical) ,Enzyme Inhibitors ,Beta-Lactamase Inhibitors ,Original Research ,Antibacterial agent ,Pharmacology ,Molecular Structure ,Broth microdilution ,biochemical phenomena, metabolism, and nutrition ,β-lactamase ,bacterial infections and mycoses ,Antimicrobial ,Anti-Bacterial Agents ,Cephalosporins ,Infectious Diseases ,beta-Lactamase Inhibitors ,Azabicyclo Compounds ,Piperacillin ,medicine.drug - Abstract
Background: NXL104 is a novel-structure b-lactamase inhibitor with potent activity against both class A and class C enzymes. Among the class A carbapenemases, KPC-type enzymes are now spreading rapidly and KPC-related carbapenemase resistance is an emerging phenomenon of great clinical importance. The activity of NXL104 against KPC b-lactamases was examined. Methods: Enzymatic activity of purified recombinant KPC-2 was measured with nitrocefin as reporter substrate and inhibition by NXL104 was measured by determination of IC50 values. Antimicrobial susceptibility testing of various b-lactams combined with a fixed concentration of NXL104 at 4 mg/L against strains producing KPC enzymes was performed by the broth microdilution method. Results: NXL104 was a potent inhibitor of KPC-2 with an IC50 of 38 nM. NXL104 restored the antimicrobial activity of ceftazidime, ceftriaxone, imipenem and piperacillin against Enterobacteriaceae strains producing KPC-2 or KPC-3. MIC values of ceftazidime against KPC producers were reduced by up to 1000-fold by combination with NXL104. Conclusions: NXL104 inhibitory activity is unique in terms of spectrum, encompassing class A extended-spectrum b-lactamases, class C enzymes and class A carbapenemases. Given the limited therapeutic options available for infections caused by multiresistant Enterobacteriaceae isolates, NXL104 b-lactamase inhibitor is a promising agent to be used in combination with a b-lactam to protect its antibacterial activity.
- Published
- 2009
12. NXL104 combinations versus Enterobacteriaceae with CTX-M extended-spectrum -lactamases and carbapenemases
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Neil Woodford, Christine Miossec, Marina Warner, Shazad Mushtaq, and David M. Livermore
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Microbiology (medical) ,Cefotaxime ,medicine.drug_class ,Cephalosporin ,Ceftazidime ,Microbial Sensitivity Tests ,Biology ,beta-Lactams ,Enterobacter aerogenes ,medicine.disease_cause ,beta-Lactamases ,Microbiology ,chemistry.chemical_compound ,Enterobacteriaceae ,polycyclic compounds ,medicine ,Pharmacology (medical) ,Enzyme Inhibitors ,Escherichia coli ,Pharmacology ,Molecular Structure ,β lactamases ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,biology.organism_classification ,Anti-Bacterial Agents ,Infectious Diseases ,chemistry ,bacteria ,Ertapenem ,medicine.drug - Abstract
The beta-lactamase landscape is changing radically, with CTX-M types now the most prevalent extended-spectrum beta-lactamases (ESBLs) worldwide, except maybe in the USA. In addition, there are growing numbers of Enterobacteriaceae with KPC and metallo-carbapenemases. We examined whether combinations of oxyimino-cephalosporins with NXL104, a novel non-beta-lactam beta-lactamase inhibitor, overcame these resistances.NXL104 was tested at 4 mg/L in combination with cefotaxime and ceftazidime versus: (i) Escherichia coli transconjugants and wild-type Enterobacteriaceae with CTX-M ESBLs; (ii) Enterobacteriaceae with ertapenem resistance contingent on combinations of impermeability and ESBLs or AmpC; and (iii) Enterobacteriaceae with KPC, SME, metallo- or OXA-48 carbapenemases.MICs of cefotaxime + NXL104 wereor = 1 mg/L for most Enterobacteriaceae with CTX-M, KPC or OXA-48 enzymes and wereor = 2 mg/L for those that also had ertapenem resistance contingent on combinations of beta-lactamase and impermeability. MICs of the ceftazidime + NXL104 combination wereor = 4 mg/L, except for a single Enterobacter aerogenes with KPC and AmpC enzymes together with porin loss, which required an MIC of 32 mg/L. The major gap was that NXL104 could not potentiate cephalosporins against Enterobacteriaceae with IMP or VIM metallo-enzymes.Oxyimino-cephalosporin + NXL104 combinations have potential against strains with the prevalent ESBLs and non-metallo-carbapenemases.
- Published
- 2008
13. Activities of Ceftazidime and Avibactam against β-Lactamase-Producing Enterobacteriaceae in a Hollow-Fiber Pharmacodynamic Model
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Ken Coleman, Premavathy Levasseur, David M. Shlaes, Wright W. Nichols, Anne-Marie Girard, Henri Merdjan, Christine Miossec, Monica Borgonovi, and George L. Drusano
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Avibactam ,Ceftazidime ,Microbial Sensitivity Tests ,Pharmacology ,Bacterial growth ,beta-Lactamases ,chemistry.chemical_compound ,Enterobacteriaceae ,Enterobacter cloacae ,Medicine ,Humans ,Pharmacology (medical) ,Test organism ,Experimental Therapeutics ,Intravenous dose ,biology ,business.industry ,Enterobacteriaceae Infections ,biology.organism_classification ,Anti-Bacterial Agents ,Citrobacter freundii ,Klebsiella pneumoniae ,Infectious Diseases ,Single bolus ,chemistry ,Pharmacodynamics ,Drug Therapy, Combination ,business ,beta-Lactamase Inhibitors ,Azabicyclo Compounds ,medicine.drug - Abstract
Avibactam is a novel non-β-lactam β-lactamase inhibitor that is currently undergoing phase 3 clinical trials in combination with ceftazidime. Ceftazidime is hydrolyzed by a broad range of β-lactamases, but avibactam is able to inhibit the majority of these enzymes. The studies described here attempt to provide insight into the amount of avibactam required to suppress bacterial growth in an environment where the concentrations of both agents are varying as they would when administered to humans. Following the simulation of a single intravenous dose of the drug, ceftazidime alone had no effect on any test organism, but a ceftazidime-avibactam combination resulted in rapid killing of all of the strains, with growth suppressed for the 8 h of the study. For seven of eight strains, this was achieved with a 1-g–250-mg profile, but a 2-g–500-mg profile was necessary to completely suppress a high-level-AmpC-producing isolate. When ceftazidime was infused continuously for 24 h with a single bolus dose of avibactam, rapid killing of all of the strains was again observed, with growth suppressed for 10 to >24 h. Regrowth appeared to commence once the avibactam concentration dropped below a critical concentration of approximately 0.3 μg/ml. In a third series of studies, ceftazidime was administered every 8 h for 24 h with avibactam administered at fixed concentrations for short periods during each ceftazidime dose profile. Simulating a 1-g dose of ceftazidime, an avibactam pulse of >0.25 and
- Published
- 2014
14. The New Ketolide HMR3647 Accumulates in the Azurophil Granules of Human Polymorphonuclear Cells
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Elsa-Noah N’Diaye, Pascale Peyron, Lydie Pilatre, Christine Miossec-Bartoli, Isabelle Maridonneau-Parini, Véronique Collart-Dutilleul, and Anita Diu-Hercend
- Subjects
Ketolides ,Neutrophils ,Centrifugation ,Granulocyte ,Biology ,Cytoplasmic Granules ,Peripheral blood mononuclear cell ,Cell Line ,Azurophilic granule ,medicine ,Humans ,Pharmacology (medical) ,Ketolide ,Antibacterial agent ,Pharmacology ,hemic and immune systems ,Anti-Bacterial Agents ,Erythromycin ,Cell biology ,Infectious Diseases ,medicine.anatomical_structure ,Biochemistry ,Cell culture ,Leukocytes, Mononuclear ,Macrolides ,Cell fractionation ,Intracellular ,Subcellular Fractions ,medicine.drug - Abstract
HMR3647 is a semisynthetic representative of a new group of drugs, the ketolides, derived from erythromycin A. Since macrolides have been shown to accumulate in human polymorphonuclear cells (PMNs), we have investigated the ability of the molecule HMR3647 to enter human PMNs as well as other cell types, such as peripheral blood mononuclear cells and cell lines of hematopoietic and nonhematopoietic origin. In these experiments, HMR3647 was compared to erythromycin A, azithromycin, clarithromycin, and roxithromycin. Our results show that HMR3647 is specifically trapped in PMNs, where it is concentrated up to 300 times. In addition, it is poorly released by these cells, 80% of the compound remaining cell associated after 2 h in fresh medium. By contrast, it is poorly internalized and quickly released by the other cell types studied. This differs from the results obtained with the macrolide molecules, which behaved similarly in the different cells studied. In addition, subcellular fractionation of PMNs allowed us to identify the intracellular compartment where HMR3647 was trapped. In PMNs, more than 75% of the molecule was recovered in the azurophil granule fraction. Similarly, in NB4 cells differentiated into PMN-like cells, almost 60% of the molecules accumulated in the azurophil granule fraction. In addition, when HMR3647 was added to disrupted PMNs, 63% accumulated in the azurophil granules. Therefore, this study shows that the ketolide HMR3647 specifically accumulates in PMN azurophil granules, thus favoring its delivery to bacteria phagocytosed in these cells.
- Published
- 1999
15. A novel human protease similar to the interleukin-1 beta converting enzyme induces apoptosis in transfected cells
- Author
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A.W. Chan, Anne-Marie Blanchet, J.A. Lippke, R.A. Aldape, V. Collard-Dutilleul, F. Hervé, Christine Miossec, Chi Faucheu, Y. Gu, and A. Diu
- Subjects
Models, Molecular ,Proteases ,DNA, Complementary ,medicine.medical_treatment ,Molecular Sequence Data ,Apoptosis ,Transfection ,Polymerase Chain Reaction ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Complementary DNA ,Endopeptidases ,medicine ,Animals ,Humans ,Computer Simulation ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Molecular Biology ,Peptide sequence ,Caspase ,COS cells ,Protease ,Base Sequence ,Sequence Homology, Amino Acid ,General Immunology and Microbiology ,biology ,General Neuroscience ,Caspase 1 ,Cysteine protease ,Molecular biology ,Caspases, Initiator ,Cysteine Endopeptidases ,Caspases ,Multigene Family ,biology.protein ,Research Article - Abstract
We have identified a novel cDNA encoding a protein (named TX) with > 50% overall sequence identity with the interleukin-1 beta converting enzyme (ICE) and approximately 30% sequence identity with the ICE homologs NEDD-2/ICH-1L and CED-3. A computer homology model of TX was constructed based on the X-ray coordinates of the ICE crystal recently published. This model suggests that TX is a cysteine protease, with the P1 aspartic acid substrate specificity retained. Transfection experiments demonstrate that TX is a protease which is able to cleave itself and the p30 ICE precursor, but not to generate mature IL-1 beta from pro-IL-1 beta. In addition, this protein induces apoptosis in transfected COS cells. TX therefore delineates a new member of the growing Ice/ced-3 gene family coding for proteases with cytokine processing activity or involved in programmed cell death.
- Published
- 1995
16. In Vitro Antibacterial Activity of the Ceftazidime-Avibactam (NXL104) Combination against Pseudomonas aeruginosa Clinical Isolates
- Author
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Kenneth Coleman, Premavathy Levasseur, Monique Claudon, Christine Miossec, Herman Goossens, Michael T. Black, and Anne-Marie Girard
- Subjects
Imipenem ,Avibactam ,Ceftazidime ,Microbial Sensitivity Tests ,Pharmacology ,medicine.disease_cause ,beta-Lactamases ,Microbiology ,chemistry.chemical_compound ,Bacterial Proteins ,medicine ,Humans ,Pharmacology (medical) ,Pseudomonas Infections ,Beta-Lactamase Inhibitors ,Biology ,chemistry.chemical_classification ,Pseudomonas aeruginosa ,Pharmacology. Therapy ,Ceftazidime/avibactam ,Anti-Bacterial Agents ,Drug Combinations ,Infectious Diseases ,Enzyme ,chemistry ,Susceptibility ,Antibacterial activity ,beta-Lactamase Inhibitors ,Azabicyclo Compounds ,medicine.drug - Abstract
The β-lactamase inhibitor avibactam (NXL104) displays potent inhibition of both class A and C enzymes. The in vitro antibacterial activity of the combination ceftazidime-avibactam was evaluated against a clinical panel of Pseudomonas aeruginosa isolates. Avibactam offered efficient protection from hydrolysis since 94% of isolates were susceptible to ceftazidime when combined with 4 μg/ml avibactam, compared with 65% susceptible to ceftazidime alone. Ceftazidime-avibactam also demonstrated better antipseudomonal activity than imipenem (82% susceptibility), a common reference treatment.
- Published
- 2012
17. Mechanistic studies of the inactivation of TEM-1 and P99 by NXL104, a novel non-beta-lactam beta-lactamase inhibitor
- Author
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Kenneth Coleman, Jean-Marie Frère, Jean-Michel Bruneau, Michael T. Black, Marie-Claude Péchereau, Thérèse Stachyra, Monique Claudon, and Christine Miossec
- Subjects
Pharmacology ,Spectrometry, Mass, Electrospray Ionization ,Molecular Structure ,Stereochemistry ,medicine.medical_treatment ,Sulbactam ,Tazobactam ,beta-Lactamases ,Dissociation constant ,chemistry.chemical_compound ,Inhibitory Concentration 50 ,Infectious Diseases ,Reaction rate constant ,chemistry ,Beta-lactamase ,medicine ,Lactam ,Pharmacology (medical) ,beta-Lactamase Inhibitors ,Beta-Lactamase Inhibitors ,Azabicyclo Compounds ,Mechanisms of Action: Physiological Effects ,Antibacterial agent ,medicine.drug - Abstract
NXL104 is a potent inhibitor of class A and C serine β-lactamases, including KPC carbapenemases. Native and NXL104-inhibited TEM-1 and P99 β-lactamases analyzed by liquid chromatography-electrospray ionization-time of flight mass spectrometry revealed that the inactivated enzymes formed a covalent adduct with NXL104. The principal inhibitory characteristics of NXL104 against TEM-1 and P99 β-lactamases were determined, including partition ratios, dissociation constants ( K ), rate constants for deactivation ( k 2 ), and reactivation rates. NXL104 is a potent inhibitor of TEM-1 and P99, characterized by high carbamylation efficiencies ( k 2 / K of 3.7 × 10 5 M −1 s −1 for TEM-1 and 1 × 10 4 M −1 s −1 for P99) and slow decarbamylation. Complete loss of β-lactamase activity was obtained at a 1/1 enzyme/NXL104 ratio, with a k 3 value (rate constant for formation of product and free enzyme) close to zero for TEM-1 and P99. Fifty percent inhibitory concentrations (IC 50 s) were evaluated on selected β-lactamases, and NXL104 was shown to be a very potent inhibitor of class A and C β-lactamases. IC 50 s obtained with NXL104 (from 3 nM to 170 nM) were globally comparable on the β-lactamases CTX-M-15 and SHV-4 with those obtained with the comparators (clavulanate, tazobactam, and sulbactam) but were far lower on TEM-1, KPC-2, P99, and AmpC than those of the comparators. In-depth studies on TEM-1 and P99 demonstrated that NXL104 had a comparable or better affinity and inactivation rate than clavulanate and tazobactam and in all cases an improved stability of the covalent enzyme/inhibitor complex.
- Published
- 2010
18. Mechanism of action of the antibiotic NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II topoisomerases
- Author
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Jean-Michel Bruneau, Thérèse Stachyra, Christine Miossec, Anne-Marie Girard, Monique Claudon, Denis Platel, and Michael T. Black
- Subjects
Models, Molecular ,Staphylococcus aureus ,Topoisomerase IV ,medicine.drug_class ,medicine.disease_cause ,DNA gyrase ,Microbiology ,Inhibitory Concentration 50 ,Moxifloxacin ,Drug Resistance, Bacterial ,medicine ,Escherichia coli ,Point Mutation ,Topoisomerase II Inhibitors ,Pharmacology (medical) ,Enzyme Inhibitors ,Mechanisms of Action: Physiological Effects ,Pharmacology ,biology ,Topoisomerase ,Quinolone ,Anti-Bacterial Agents ,Ciprofloxacin ,Infectious Diseases ,DNA Topoisomerases, Type II ,DNA Gyrase ,biology.protein ,Quinolines ,Topoisomerase-II Inhibitor ,medicine.drug ,Fluoroquinolones - Abstract
NXL101 is one of a new class of quinoline antibacterial DNA gyrase and topoisomerase IV inhibitors showing potent activity against gram-positive bacteria, including methicillin- and fluoroquinolone-resistant strains. NXL101 inhibited topoisomerase IV more effectively than gyrase from Escherichia coli , whereas the converse is true of enzymes from Staphylococcus aureus . This apparent target preference is opposite to that which is associated with most fluoroquinolone antibiotics. In vitro isolation of S. aureus mutants resistant to NXL101 followed by cloning and sequencing of the genes encoding gyrase and topoisomerase IV led to the identification of several different point mutations within, or close to, the quinolone resistance-determining region (QRDR) of GyrA. However, the mutations were not those that are most frequently associated with decreased sensitivity to quinolones. A fluoroquinolone-resistant mutant variant of gyrase generated in vitro was highly resistant to inhibition by the fluoroquinolones ciprofloxacin and moxifloxacin but remained fully susceptible to inhibition by NXL101. Two mutant gyrases constructed in vitro, with mutations in gyrA engineered according to those most frequently found in S. aureus strains resistant to NXL101, were insensitive to inhibition by NXL101 and had a diminished sensitivity to ciprofloxacin and moxifloxacin. Certain combinations of mutations giving rise to NXL101 resistance and those giving rise to fluoroquinolone resistance may be mutually exclusive.
- Published
- 2008
19. The β-lactamase inhibitor avibactam (NXL104) does not induce ampC β -lactamase in Enterobacter cloacae
- Author
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Premavathy Levasseur, Michael T. Black, Christine Miossec, and Monique Claudon
- Subjects
Imipenem ,medicine.drug_class ,Avibactam ,Antibiotics ,Microbiology ,NXL104 ,chemistry.chemical_compound ,Clavulanic acid ,polycyclic compounds ,medicine ,Pharmacology (medical) ,Inducer ,Cefoxitin ,avibactam ,induction ,Original Research ,Pharmacology ,chemistry.chemical_classification ,biology ,business.industry ,biochemical phenomena, metabolism, and nutrition ,β-lactamase ,bacterial infections and mycoses ,biology.organism_classification ,Infectious Diseases ,Enzyme ,chemistry ,Infection and Drug Resistance ,ampC ,bacteria ,business ,Enterobacter cloacae ,medicine.drug - Abstract
Christine Miossec, Monique Claudon, Premavathy Levasseur, Michael T Black Novexel, Romainville, France Abstract: Induction of ampC β-lactamase expression can often compromise antibiotic treatment and is triggered by several β-lactams (such as cefoxitin and imipenem) and by the β-lactamase inhibitor clavulanic acid. The novel β-lactamase inhibitor avibactam (NXL104) is a potent inhibitor of both class A and class C enzymes. The potential of avibactam for induction of ampC expression in Enterobacter cloacae was investigated by ampC messenger ribonucleic acid quantitation. Cefoxitin and clavulanic acid were confirmed as ampC inducers, whereas avibactam was found to exert no effect on ampC expression. Thus, avibactam is unlikely to diminish the activity of any partner β-lactam antibiotic against AmpC-producing organisms. Keywords: β-lactamase, ampC, induction, NXL104, avibactam
- Published
- 2013
20. Enzymatic activity of two caspases related to interleukin-1beta-converting enzyme
- Author
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Bentou Komara, Anita Diu-Hercend, Anne-Marie Blanchet, Odile Krebs, Chi Faucheu, Corinne Gillard, Christopher Yea, Florence Fassy, Hélène Rey, Christine Miossec, and Cécile Capdevila
- Subjects
Proteases ,Insecta ,In Vitro Techniques ,Biochemistry ,Cell Line ,Substrate Specificity ,Aspartic acid ,Animals ,Humans ,Amino Acid Sequence ,Protein Precursors ,Protein precursor ,Caspase ,chemistry.chemical_classification ,biology ,Caspase 1 ,Molecular biology ,Cysteine protease ,Recombinant Proteins ,Amino acid ,Cysteine Endopeptidases ,Kinetics ,Enzyme ,chemistry ,biology.protein ,Baculoviridae ,Oligopeptides ,Cysteine ,Interleukin-1 - Abstract
Interleukin-1beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occurred within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1beta. TX cleaved the same synthetic substrates as ICE (Km of 90 microM and k(cat) of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 microM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1beta cleavage.
- Published
- 1998
21. Evidence for CPP32 activation in the absence of apoptosis during T lymphocyte stimulation
- Author
-
Anita Diu-Hercend, Florence Fassy, Véronique Dutilleul, and Christine Miossec
- Subjects
Programmed cell death ,Proteases ,T-Lymphocytes ,Cell ,Apoptosis ,Biology ,Lymphocyte Activation ,Biochemistry ,TCIRG1 ,Jurkat Cells ,medicine ,Cytotoxic T cell ,Humans ,Molecular Biology ,Enzyme Precursors ,Caspase 3 ,Cell Biology ,T lymphocyte ,Mixed lymphocyte reaction ,Cell biology ,Enzyme Activation ,Cysteine Endopeptidases ,Kinetics ,medicine.anatomical_structure ,Caspases ,Carcinogens ,Tetradecanoylphorbol Acetate ,Cell Division - Abstract
Cysteine proteases of the interleukin-1beta-converting enzyme family have been implicated in the effector process of apoptosis in several systems. Among these, CPP32 has been shown to be processed to active enzyme at the onset of apoptosis. Here, we show that CPP32 precursor is cleaved into its active form during phytohaemaglutinin A activation of T lymphocytes. Maximal processing is observed between day 3 and day 4 following addition of mitogen and is a transient process. Precursor cleavage is associated with the appearance of a CPP32-like enzymatic activity in cell lysates. At this time in the culture, almost no apoptotic cell and no dead cell can be detected, and T lymphocytes are actively proliferating. CPP32 processing also occurs when lymphocytes are stimulated through an allogeneic primary mixed lymphocyte reaction. Our results suggest that proteolytic activation of CPP32 could be a physiological step during T lymphocyte activation. In addition, these data indicate that CPP32 activation can occur independently of programmed cell death in T lymphocytes.
- Published
- 1997
22. Evidence for an interleukin-1beta converting enzyme (ICE)-like activity distinct from ICE and responsible for ICE and CPP32 cleavage during apoptosis
- Author
-
Christine Miossec, V. Dutilleul, Anita Diu-Hercend, Chi Faucheu, J. Golec, Laurence Durand, L. Pilatre, and Florence Fassy
- Subjects
Pharmacology ,chemistry.chemical_classification ,Cancer Research ,Proteases ,U937 cell ,medicine.diagnostic_test ,Biochemistry (medical) ,Clinical Biochemistry ,Pharmaceutical Science ,Cell Biology ,Biology ,Cleavage (embryo) ,Jurkat cells ,Molecular biology ,Enzyme ,Western blot ,chemistry ,Apoptosis ,medicine ,Receptor ,human activities - Abstract
IL-1beta converting enzyme (ICE) and ICE-related proteases (IRPs) have been suggested to play a central role in apoptosis. We report the use of peptidic ICE inhibitors to reassess the role of this enzyme in the apoptosis induced by Fas or TNFalpha receptor ligation in Jurkat cells, U937 cells or monocytes. Our results show that inhibition of IL-1beta processing can be dissociated from inhibition of apoptosis. Indeed, two out of three com-pounds active on ICE are not inhibitory for apoptosis. This shows that ICE is not required for progression in the apoptotic pathway, but that one or several IRPs are necessary. In addition, Western blot analysis of cell lysates shows that both ICE and CPP32 precursors disappear rapidly after apoptosis induction, while ICH-1L precursor remains intact. Concomitant appearance of cleavage products can be visualized for CPP32, but not for ICE, suggesting that the former is proteolytically activated. In addition, this precursor cleavage can be blocked by an ICE inhibitor active on apoptosis. Altogether, our data support the hypothesis that one or several IRPs are necessary for apoptosis and are responsible for ICE and CPP32 cleavage during this process.
- Published
- 1997
23. Use of monoclonal antibodies to study interleukin-1 beta-converting enzyme expression: only precursor forms are detected in interleukin-1 beta-secreting cells
- Author
-
Marie‐Claude Decoen, Anita Diu-Hercend, Laurence Durand, Florence Fassy, and Christine Miossec
- Subjects
Lipopolysaccharides ,medicine.drug_class ,Protein subunit ,Immunology ,Molecular Sequence Data ,Mice, Inbred Strains ,Recombinant Interleukin ,Biology ,Monoclonal antibody ,Transfection ,Cell Line ,Mice ,medicine ,Tumor Cells, Cultured ,Immunology and Allergy ,Animals ,Humans ,Amino Acid Sequence ,COS cells ,Caspase 1 ,Antibodies, Monoclonal ,Molecular biology ,Blot ,Enzyme Activation ,Cysteine Endopeptidases ,Cell culture ,Cytoplasm ,Leukocytes, Mononuclear ,Interleukin-1 - Abstract
We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1 beta-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67,000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1 beta was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1 beta cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.
- Published
- 1996
24. Cloned CD3.
- Author
-
Jitsukawa, Setsuko, Triebel, Frédéric, Faure, Florence, And, Christine Miossec, and Hercend, Thierry
- Published
- 1988
- Full Text
- View/download PDF
25. Cloned CD3+ TcRα/β− TiγA− peripheral blood lymphocytes compared to the Ti γA+ counterparts: structural differences of the γ/δ receptor and functional heterogeneity
- Author
-
Jitsukawa, Setsuko, primary, Triebel, Frédéric, additional, Faure, Florence, additional, And, Christine Miossec, additional, and Hercend, Thierry, additional
- Published
- 1988
- Full Text
- View/download PDF
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