How to strengthen the presence of patients in health technology assessments conducted by the health authorities.

The constant development of health technologies, combined with the increase in the cost of treatment, means that States must continually make choices about the introduction of new technologies into their healthcare system and how they are to be funded. In France, the systematic participation of patients in these processes is one of the targets to be met in terms of healthcare democracy. Although, on an international level, patient involvement in these assessments is constantly growing, it is difficult to define due to the presence of unstabilised elements in terms of both terminology and assessment methods. As a result, patient and public involvement in health technology assessments varies considerably from one country to the next, from one field to the next and even from one type of technology to the next. Several types of involvement exist, ranging from studies conducted to collect patient "insight" (experience, perception, needs, preferences, attitudes to treatment and health, etc.) to processes aimed at including patients in assessments (as individuals, as representatives of associations, etc.). Given the scope and complexity of the subject, and the difficulty involved in understanding all the different aspects of health technologies and innovations, the members of the Round Table chose to concentrate on health technology assessments (medicinal products and medical devices) to develop national recommendations on all possible types of patient involvement in the health technology assessment processes conducted by the health authorities in France.

What should be done to combat misinformation about health products?

The increasing role of digital technology, social media, the wide range of channels and the volume of information, the role of medicine as a societal subject, public information that is insufficient and poorly suited to situations of uncertainty are all observations which led to the theme of this round table. After discussing the definition of disinformation, which is not limited to fake news, and talking about contributors who misinform, whether intentionally or not, the participants of this round table made nine recommendations (R) to combat disinformation about health products: create a collaborative platform, information/training on health products, a platform with five major characteristics, namely accessibility, flexibility, objectivity, transparency and independence, as well as media suited to the different targets (R1); promote basic knowledge on health products: education/training to restore the particularly poor image of medication, and teach the public how to use basic concepts appropriately (R2); improve communication to the public based on the observation that information is the main weapon against misinformation and entails, in particular, coordinating communication from the different institutions to make public information more audible, making institutional messages clearer, ensuring they are more factual and prioritising them (R3); know how to communicate using the correct codes and tools (R4), because, to be understood, the substance and the form are inseparable; develop research on communication in the field of health products (R5); acquire tools to identify and regulate as soon as possible (R6); keep check of content by developing critical thinking (R7); define quality criteria for information sources (R8); identify, assess and reference initiatives for the public that could be placed on the platform (R9).

Apelin stimulation of the vascular skeletal muscle stem cell niche enhances endogenous repair in dystrophic mice.

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here, we showed that defects in the endothelial cell (EC) compartment of the vascular stem cell niche in mouse models of Duchenne MD, laminin α2-related MD, and collagen VI-related myopathy were associated with inefficient mobilization of MuSCs after tissue damage. Using chemoinformatic analysis, we identified the 13-amino acid form of the peptide hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. Systemic administration of AP-13 using osmotic pumps generated a pro-proliferative EC-rich niche that supported MuSC function through angiocrine factors and markedly improved tissue regeneration and muscle strength in all three dystrophic mouse models. Moreover, EC-specific knockout of the apelin receptor led to regenerative defects that phenocopied key pathological features of MD, including vascular defects, fibrosis, muscle fiber necrosis, impaired MuSC function, and reduced force generation. Together, these studies provide in vivo proof of concept that enhancing endogenous skeletal muscle repair by targeting the vascular niche is a viable therapeutic avenue for MD and characterized AP-13 as a candidate for further study for the systemic treatment of MuSC dysfunction.

Anti-mycobacterial immunity induced by a single injection of M. leprae Hsp65-encoding plasmid DNA in biodegradable microparticles.

A single sub-cutaneous injection of a plasmid DNA encoding a mycobacterial heat shock protein 65 (Hsp65) entrapped in biodegradable poly(lactic-co-glycolic acid) microspheres produced high titers of antibodies, measured 5 months after the injection in BALB/c mice. Splenocytes secreted IFN-gamma and exerted an anti-bacterial effect on macrophages infected in vitro with Mycobacterium tuberculosis. The results are encouraging with regard to obtaining good compliance and vaccination coverage with candidate plasmid DNA vaccines, especially in developing countries.

lmo1273, a novel gene involved in Listeria monocytogenes virulence.

Listeria monocytogenes is a foodborne pathogen able to infect humans and many other mammalian species, leading to serious, often fatal disease. We have previously identified a five-gene locus in the genome of L. monocytogenes EGD-e which comprised three contiguous genes encoding paralogous type I signal peptidases. In the present study, we focused on the two distal genes of the locus (lmo1272 and lmo1273), encoding proteins sharing significant similarities with the YlqF and RnhB proteins, respectively, of Bacillus subtilis. lmo1273 could complement an Escherichia coli rnhA-rnhB thermosensitive growth phenotype, suggesting that it encodes a functional RNase H. Strikingly, inactivation of lmo1273 provoked a strong attenuation of virulence in the mouse model, and kinetic studies in infected mice revealed that multiplication of the lmo1273 mutant in target organs was significantly impaired. However, the mutation did not impair L. monocytogenes intracellular multiplication or cell-to-cell spread in cell culture models. Transcriptional profiles obtained with an lmo1273-overexpressing strain were compared to those of the wild-type strain, using microarray analyses. The data obtained suggest a pleiotropic regulatory role of Lmo1273 and possible links with amino acid uptake.

Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity.

Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS.

Exploring the role of the CTL epitope region of listeriolysin O in the pathogenesis of Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen responsible for severe opportunistic infections in humans and animals. The secreted cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates phagosomal escape and allows bacterial growth in the cytosol of infected cells. In order to identify new LLO determinants participating in bacterial pathogenesis, this study focused on a major target of LLO proteolytic cleavage in vitro, the CTL epitope region (residues 91-99). Mutations were generated by site-directed mutagenesis in the epitope or in the two clusters of positive charges flanking the epitope. Two LLO mutants (a single mutation K103A and a double mutation R89G, K90G) were normally and stably secreted by L. monocytogenes. In contrast, a mutant carrying four amino acid substitutions in the epitope itself (Y92K, D94A, E97K, Y98F) was highly susceptible to proteolytic degradation. While these three LLO mutant proteins showed a reduced haemolytic activity, they all promoted efficient phagosomal escape and intracellular multiplication in different cell types, and were non-cytotoxic. The deletion of the epitope (Delta91-99), as well as the substitution of two, three or four of the four lysine residues (K103 to K106) by alanine residues did not lead to the production of a detectable protein. These results confirm the lack of correlation between haemolytic activity and phagosomal membrane disruption. They reveal the importance of the 91-99 region in the production of a stable and functional LLO. LD(50) determinations in the mouse model suggest a possible link between LLO stability and virulence.

Sevoflurane-remifentanil versus propofol-remifentanil anesthesia at a similar bispectral level for off-pump coronary artery surgery: no evidence of reduced myocardial ischemia.

OBJECTIVE: Sevoflurane could decrease myocardial ischemic injury in patients undergoing off-pump coronary artery bypass surgery. This study was designed to compare postoperative troponin I (cTnI) concentrations after sevoflurane-remifentanil versus propofol-remifentanil anesthesia. DESIGN: Prospective, randomized single-blind clinical study. SETTING: University hospital. PARTICIPANTS: Eighteen patients. INTERVENTIONS: General anesthesia was conducted with sevoflurane-remifentanil (n = 9) or propofol-remifentanil (n = 9). Administration of sevoflurane and propofol was adjusted to maintain the bispectral index (BIS) between 40 and 60. MEASUREMENTS AND MAIN RESULTS: Groups were comparable regarding the patients' characteristics. The objective of BIS was maintained in both groups except during the period of coronary artery grafts (p < 0.001) when the BIS number in the propofol group fell below 40 and was significantly lower than in the sevoflurane group. Intraoperative hemodynamic variables were similar between groups. No patient required cardiopulmonary bypass. Need for inotropic and vasoactive support during the first graft was not necessary in the propofol group and occurred in 4 patients in the sevoflurane group (not significant). During the second graft, 2 patients in the propofol group and 3 in the sevoflurane group needed hemodynamic support. Postoperative hemodynamic variables were comparable between groups. Areas under the curve of postoperative increases in cTnI were 27.0 +/- 38.6 and 17.4 +/- 14.6 ng/mL/hour in the sevoflurane and propofol groups, respectively (not significant). CONCLUSION: This study does not support cardioprotective effects of sevoflurane. The particularly short total cumulative duration of ischemia and the relatively low administered end-tidal sevoflurane concentrations may explain this result.

Regulation of expression of type I signal peptidases in Listeria monocytogenes.

The role of type I signal peptidases (SPases I) is to remove the signal peptides of preproteins exported by the general secretory pathway. The genome of Listeria monocytogenes contains a locus encoding three contiguous SPases I (denoted SipX, SipY and SipZ). The authors recently showed that SipX and SipZ perform distinct functions in protein secretion and bacterial pathogenicity. Here, the regulation of sip gene expression in broth and in infected eukaryotic cells was studied. The results show that expression of the three sip genes is (i) controlled by two distinct promoter regions that respond differently to growth phase and temperature variations, and (ii) influenced by PrfA (the transcriptional activator regulating most of the virulence genes of L. monocytogenes) and the stress proteins ClpC and ClpP. It was found that sip gene expression was strongly upregulated upon infection of eukaryotic cells when bacteria were still entrapped in the phagosomal compartment. This upregulation is compatible with the need of L. monocytogenes to optimize its production of virulence factors in the early stage of the intracellular cycle.

Role of FliF and FliI of Listeria monocytogenes in flagellar assembly and pathogenicity.

Flagellar structures have been shown to participate in virulence in a variety of intestinal pathogens. Here, we have identified two potential flagellar genes of Listeria monocytogenes: lmo0713, encoding a protein similar to the flagellar basal body component FliF, and lmo0716, encoding a protein similar to FliI, the cognate ATPase energizing the flagellar export apparatus. Expression of fliF and fliI appears to be downregulated at 37 degrees C, like that of flaA, encoding flagellin. By constructing two chromosomal deletion mutants, we show that inactivation of either fliF or fliI (i) abolishes bacterial motility and flagella production, (ii) impairs adhesion and entry into nonphagocytic epithelial cells, and (iii) also reduces uptake by bone marrow-derived macrophages. However, the DeltafliF and DeltafliI mutations have only a minor impact on bacterial virulence in the mouse model, indicating that the flagellar secretion apparatus itself is not essential for survival in this animal model. Finally, among 100 human clinical isolates of L. monocytogenes tested, we found 20 strains still motile at 37 degrees C. Notably, all these strains adhered less efficiently than strain EGD-e to Caco-2 cells at 37 degrees C but showed no defect of intracellular multiplication. These data suggest that expression of the flagella at 37 degrees C might hinder optimal adhesion to epithelial cells but has no impact on intracytosolic survival of L. monocytogenes.

The svpA-srtB locus of Listeria monocytogenes: fur-mediated iron regulation and effect on virulence.

In Listeria monocytogenes the promoter region of the svpA-srtB locus contains a well-conserved Fur box. We characterized the iron-regulation of this locus: real-time polymerase chain reaction analyses and anti-SvpA immunoblots showed that, in response to iron deprivation svpA transcription and SvpA production markedly increased (80-fold and 10-fold respectively), when initiated by either the addition of the iron chelator 2,2'-bipyridyl to BHI media, or by growth in iron-restricted minimal media. Green fluorescent protein (GFP) reporter constructs also showed increased activity of the svpA-srtB promoter in Escherichia coli (37-fold) and in L. monocytogenes (two- to threefold) when the bacteria were grown in iron-deficient conditions. A Deltafur mutant of L. monocytogenes constitutively synthesized SvpA, as well as GFP fused to the svpA-srtB promoter. Cellular fractionation data revealed that in iron-rich media wild-type SvpA was exclusively secreted to the culture supernatant. However, both the Deltafur derivative and wild-type L. monocytogenes grown in iron-deficient media anchored a fraction of the SvpA proteins (approximately 5%) to peptidoglycan, and produced a lower-molecular weight, wholly secreted form of SvpA. Together these data establish that iron availability controls transcription of the svpA-srtB locus (through Fur-mediated regulation), and attachment of SvpA to the cell wall (through SrtB-mediated covalent linkage). SvpA bears homology to IsdC, a haemin-binding protein of Staphylococcus aureus, and haemin bound to SvpA in solution. However, site-directed deletions of four structural genes and the promoter of the svpA-srtB locus did not impair haemin, haemoglobin or ferrichrome utilization in nutrition tests. We did not find strong evidence to support the notion that the svpA-srtB locus participates in haemin acquisition, as was reported for the homologous isd operon of S. aureus. Furthermore, the svpA-srtB mutant strains showed no significant attenuation of virulence in an intravenous mouse model system, but we found that the mutations reduced the persistence of L. monocytogenes in murine liver, spleen and intestines after oral administration.

Identification of the agr locus of Listeria monocytogenes: role in bacterial virulence.

Listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM) to identify genes involved in the virulence of L. monocytogenes. A new round of STM allowed us to identify a new locus encoding a protein homologous to AgrA, the well-studied response regulator of Staphylococcus aureus and part of a two-component system involved in bacterial virulence. The production of several secreted proteins was modified in the agrA mutant of L. monocytogenes grown in broth, indicating that the agr locus influenced protein secretion. Inactivation of agrA did not affect the ability of the pathogen to invade and multiply in cells in vitro. However, the virulence of the agrA mutant was attenuated in the mouse (a 10-fold increase in the 50% lethal dose by the intravenous route), demonstrating for the first time a role for the agr locus in the virulence of L. monocytogenes.

Mechanism of isoniazid uptake in Mycobacterium tuberculosis.

Initial transport kinetics of isoniazid (INH) and its uptake at the plateau were studied in Mycobacterium tuberculosis H37Rv under various experimental conditions. The initial uptake velocity increased linearly with INH concentration from 2 x 10(-6) M to 10(-2) M. It was modified neither by addition of a protonophore that abolished proline transport, nor following ATP depletion by arsenate, which inhibited glycerol uptake, two transport processes taken as controls for secondary active transport and facilitated diffusion, respectively. Microaerobiosis or low temperature (4 degrees C) were without effect on initial uptake. It is thus likely that INH transport in M. tuberculosis proceeds by a passive diffusion mechanism, and that catalase-peroxidase (KatG) is not involved in the actual transport. However, conditions inhibiting KatG activity (high INH concentration, microaerobiosis, low temperature) decrease cell radioactivity at the uptake plateau. It is proposed that INH transport occurs by passive diffusion. KatG is involved only in the intracellular accumulation of oxidized derivatives of INH, especially of isonicotinic acid, which is trapped inside cells in its ionized form. This model explains observed and previously known characteristics of the accumulation of radioactivity in the presence of [14C]INH for various species and strains of mycobacteria.

Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis.

To evaluate the potential contribution of extracellular enzymes to the pathogenicity of mycobacteria, the presence of selected enzyme activities was investigated in the culture filtrates of the obligate human pathogen Mycobacterium tuberculosis, M. bovis BCG, the opportunistic pathogens M. kansasii and M. fortuitum, and the non-pathogenic species M. phlei and M. smegmatis. For M. tuberculosis and M. bovis, 22 enzyme activities were detected in the culture filtrates and/or cell surfaces, of which eight were absent from the culture fluids of non-pathogens: alanine dehydrogenase, glutamine synthetase, nicotinamidase, isonicotinamidase, superoxide dismutase, catalase, peroxidase and alcohol dehydrogenase. These activities, which correspond to secreted enzymes, formed a significant part (up to 92%) of the total enzyme activities of the bacteria and were absent from the culture fluids and the cell surfaces of the non-pathogenic species M. smegmatis and M. phlei. The extracellular location of superoxide dismutase and glutamine synthetase seemed to be restricted to the obligate pathogens examined. The difference in the enzyme profiles was not attributable to the growth rates of the two groups of bacteria. The presence of the eight enzyme activities in the outermost compartments of obligate pathogens and their absence in those of non-pathogens provides further evidence that these enzymes may be involved in the pathogenicity of mycobacteria. In addition, the eight enzyme activities were demonstrated in the cell extract of M. smegmatis. Stepwise erosion of the cell surface of M. smegmatis to expose internal capsular constituents showed that the various enzyme activities, with the possible exception of superoxide dismutase, were located more deeply in the cell envelope of this bacterium. This suggests that the molecular architecture of the mycobacterial envelopes may play an important role in the pathogenicity of these organisms.

Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity.

Mycobacteria are known to acquire resistance to the antituberculous drug pyrazinamide (PZA) through mutations in the gene encoding pyrazinamidase (PZase), an enzyme that converts PZA into pyrazinoic acid, the presumed active form of PZA against bacteria. Additional mechanisms of resistance to the drug are known to exist but have not been fully investigated. Among these is the non-uptake of the pro-drug, a possibility investigated in the present study. The uptake mechanism of PZA, a requisite step for the activation of the pro-drug, was studied in Mycobacterium tuberculosis. The incorporation of [14C]PZA by the bacilli was followed in both neutral and acidic environments since PZA activity is known to be optimal at acidic pH. By using a protonophore (carbonyl cyanide m-chlorophenylhydrazone; CCCP), valinomycin, arsenate and low temperature, it was shown that an ATP-dependent transport system is involved in the uptake of PZA. Whilst the structurally analogous compound nicotinamide inhibited the transport system of PZA, other structurally related compounds such as pyrazinoic acid, isoniazid and cytosine did not. Acidic conditions were also without effect. Based on diffusion experiments in liposomes, it was found that PZA diffuses rapidly through membrane bilayers, faster than glycerol, whilst the presence of OmpATb, the porin-like protein of M. tuberculosis, in proteoliposomes slightly increased the diffusion of the drug. This finding may explain why the cell wall mycolate hydrophobic layer does not represent the limiting step in the diffusion of PZA, as judged from comparative experiments using a M. tuberculosis strain and its isogenic mutant elaborating 40% less covalently linked mycolates. PZase activity, and PZA uptake and susceptibility in different mycobacterial species were compared. M. tuberculosis, a naturally PZA-susceptible species, was the only species that exhibited both PZase activity and PZA uptake; no such correlation was observed with the four naturally resistant species examined. Mycobacterium smegmatis possessed a functional PZase but did not take up PZA; the reverse was true for the PZase-negative strain of Mycobacterium avium used, with PZA uptake comparable to that of M. tuberculosis. Mycobacterium bovis BCG and Mycobacterium kansasii exhibited neither a PZase activity nor PZA uptake. These data clearly demonstrate that one of the mechanisms of resistance to PZA resides in the failure of strains to take up the drug, indicating that susceptibility to PZA in mycobacteria requires both the presence of a functional PZase and a PZA transport system. No correlation was observed between the occurrence and cellular location of PZase and of nicotinamidase in the strains examined, suggesting that one or both amides can be hydrolysed by other mycobacterial amidases.

Differential roles of multiple signal peptidases in the virulence of Listeria monocytogenes.

Most bacteria contain one type I signal peptidase (Spase I) for cleavage of signal peptides from exported and secreted proteins. Here, we identified a locus encoding three contiguous Spase I genes in the genome of Listeria monocytogenes. The deduced Sip proteins (denoted SipX, SipY and SipZ) are significantly similar to SipS and SipT, the major SPase I proteins of Bacillus subtilis (38% to 44% peptidic identity). We studied the role of these multiple signal peptidases in bacterial pathogenicity by constructing a series of single- and double-chromosomal knock-out mutants. Inactivation of sipX did not affect intracellular multiplication of L. monocytogenes but significantly reduced bacterial virulence (approximately 100-fold). Inactivation of sipZ impaired the secretion of phospholipase C (PC-PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD(50) of 10(8.3)), inactivation of sipY had no detectable effects. Most importantly, a mutant expressing only SipX was impaired in intracellular survival and strongly attenuated in the mouse (LD(50) of 10(7.2)), whereas, a mutant expressing only SipZ behaved like wild-type EGD in all the assays performed. The data establish that SipX and SipZ perform distinct functions in bacterial pathogenicity and that SipZ is the major Spase I of L. monocytogenes. This work constitutes the first report on the differential role of multiple Spases I in a pathogenic bacterium and suggests a possible post-translational control mechanism of virulence factors expression.

Phospholipases C are involved in the virulence of Mycobacterium tuberculosis.

Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild-type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the Delta plcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT-PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.

Tracking the putative biosynthetic precursors of oxygenated mycolates of Mycobacterium tuberculosis. Structural analysis of fatty acids of a mutant strain deviod of methoxy- and ketomycolates.

Disruption of the mma4 gene (renamed hma) of Mycobacterium tuberculosis has yielded a mutant strain defective in the synthesis of both keto- and methoxymycolates, with an altered cell-wall permeability to small molecules and a decreased virulence in the mouse model of infection (Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle, M. A., Yu, K., Quémard, A., Smith, I., and Daffé, M. (2000) Mol. Microbiol. 36, 630-637). Assuming that the mutant would accumulate the putative precursors of the oxygenated mycolates of M. tuberculosis, a detailed structural analysis of mycolates from the hma-inactivated strain was performed using a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, proton NMR spectroscopy, and chemical degradation techniques. These consisted most exclusively of alpha-mycolates, composed of equal amounts of C(76)-C(82) dicyclopropanated (alpha(1)) and of C(77)-C(79) monoethylenic monocyclopropanated (alpha(2)) mycolates, the double bond being located at the "distal" position. In addition, small amounts of cis-epoxymycolates, structurally related to alpha(2)-mycolates, was produced by the mutant strain. Complementation of the hma-inactivated mutant with the wild-type gene resulted in the disappearance of the newly identified mycolates and the production of keto- and methoxymycolates of M. tuberculosis. Introduction of the hma gene in Mycobacterium smegmatis led to the lowering of diethylenic alpha mycolates of the recipient strain and the production of keto- and hydroxymycolates. These data indicate that long-chain ethylenic compounds may be the precursors of the oxygenated mycolates of M. tuberculosis. Because the lack of production of several methyltransferases involved in the biosynthesis of mycolates is known to decrease the virulence of the tubercle bacillus, the identification of the substrates of these enzymes should help in the design of inhibitors of the growth of M. tuberculosis.