Amoebae as a tool to isolate new bacterial species, to discover new virulence factors and to study the host-pathogen interactions.

Amoebae are unicellular protozoan present worldwide in several environments mainly feeding on bacteria. Some of them, the amoebae-resistant bacteria (ARBs), have evolved mechanisms to survive and replicate inside amoebal species. These mainly include legionella, mycobacteria and Chlamydia-related bacteria. Amoebae can provide a replicative niche, can act as reservoir for bacteria whereas the cystic form can protect the internalized bacteria. Moreover, the amoebae represent a Trojan horse for ARBs to infect animals. The long interaction between amoebae and bacteria has likely selected for bacterial virulence traits leading to the adaptation towards an intracellular lifestyle, and some ARBs have acquired the ability to infect mammals. This review intends to highlight the important uses of amoebae in several fields in microbiology by describing the main tools developed using amoebal cells. First, amoebae such as Acanthamoeba are used to isolate and discover new intracellular bacterial species by two main techniques: the amoebal co-culture and the amoebal enrichment. In the second part, taking Waddlia chondrophila as example, we summarize some important recent applications of amoebae to discover new bacterial virulence factors, in particular thanks to the amoebal plaque assay. Finally, the genetically tractable Dictyostelium discoideum is used as a model organism to study host-pathogen interactions, in particular with the development of several approaches to manipulate its genome that allowed the creation of a wide range of mutated strains largely shared within the Dictyostelium community.

Peut-on traiter la maladie d'Alzheimer [Is there a treatment for Alzheimer's disease?].

Alzheimer's disease is a frequent neurodegenerative disease, which affects more than one third of elderly persons over 80 years. No curative treatment is currently available for this disease, but symptomatic treatments have produced significant improvements in patients' condition. Cholinesterase inhibitors should be prescribed for early and moderate stages and memantine for more severe stages of the disease. These drugs have an impact on cognitive performances, may delay functional decline and improve behaviour disturbances. From a preventive perspective, evidence of benefit from early management of vascular risk factors is accumulating. In the near future, the improved comprehension of the underlying mechanisms of Alzheimer's disease will hopefully bring new treatments, thats will delay or modify its course.

Efficacy of daptomycin in the treatment of experimental endocarditis due to susceptible and multidrug-resistant enterococci.

OBJECTIVES: Daptomycin was tested in vitro and in rats with experimental endocarditis against the ampicillin-susceptible and vancomycin-susceptible Enterococcus faecalis JH2-2, the vancomycin-resistant (VanA type) mutant of strain JH2-2 (strain JH2-2/pIP819), and the ampicillin-resistant and vancomycin-resistant (VanB type) Enterococcus faecium D366. METHODS: Rats with catheter-induced aortic vegetations were treated with doses simulating intravenously kinetics in humans of daptomycin (6 mg/kg every 24 h), amoxicillin (2 g every 6 h), vancomycin (1 g every 12 h) or teicoplanin (12 mg/kg every 12 h). Treatment was started 16 h post-inoculation and continued for 2 days. RESULTS: MICs of daptomycin were 1, 1 and 2 mg/L, respectively, for strains JH2-2, JH2-2/pIP819 and D366. In time-kill studies, daptomycin showed rapid (within 2 h) bactericidal activity against all strains. Daptomycin was highly bound to rat serum proteins (89%). In the presence of 50% rat serum, simulating free concentrations, daptomycin killing was maintained but delayed (6-24 h). In vivo, daptomycin treatment resulted in 10 of 12 (83%), 9 of 11 (82%) and 11 of 12 (91%) culture-negative vegetations in rats infected with strains JH2-2, JH2-2/pIP819 and D366, respectively (P < 0.001 compared to controls). Daptomycin efficacy was comparable to that of amoxicillin and vancomycin for susceptible isolates. Daptomycin, however, was significantly (P < 0.05) more effective than teicoplanin against the glycopeptide-susceptible strain JH2-2 and superior to all comparators against resistant isolates. CONCLUSIONS: These results support the use of the newly proposed daptomycin dose of 6 mg/kg every 24 h for treatment of enterococcal infections in humans.

La quête du consentement éclairé en médecine comme construction sociale [The application of informed consent in medicine as social construction].

The aim of this article is to propose an anthropological point of view about informed consent in medicine. This quest for legitimacy should be read as a relational and social construction. In the heart of clinical complexity we find on one side various techniques employed by the medical community to validate research and to obtain the consent of patients. On the other side patients offer plural and subjective answers due to the doctor patient hierarchical and long relationship. Between constraints and freedoms, informed consent brings to light social relation.

Indications à la coronarographie en urgence. Partie II: syndromes coronariens aigus sans élévation du segment ST [Indications for urgent coronary angiography. Part II: Acute coronary syndromes without ST-segment elevation]

The optimal treatment strategy for patients presenting with an acute coronary syndrome without ST elevation is controversial and different therapeutic approaches are recognized. Currently, given the literature available, it is not possible to recommend a universal systematic invasive approach. It is essential to individually risk stratify patients in order to identify those high risk patients that have been shown to benefit from an invasive strategy. Compared to conservative medical treatment, patients at low risk have not been shown to benefit from an invasive strategy. Urgent coronary angiography remains recommended for those patients with persistent or recurrent ischemic symptoms under optimal medical treatment.

Reversible and irreversible pollutant-induced bacterial cellular stress effects measured by ethidium bromide uptake and efflux.

Chemical pollution is known to affect microbial community composition but it is poorly understood how toxic compounds influence physiology of single cells that may lay at the basis of loss of reproductive fitness. Here we analyze physiological disturbances of a variety of chemical pollutants at single cell level using the bacterium Pseudomonas fluorescens in an oligotrophic growth assay. As a proxy for physiological disturbance we measured changes in geometric mean ethidium bromide (EB) fluorescence intensities in subpopulations of live and dividing cells exposed or not exposed to different dosages of tetradecane, 4-chlorophenol, 2-chlorobiphenyl, naphthalene, benzene, mercury chloride, or water-dissolved oil fractions. Because ethidium bromide efflux is an energy-dependent process any disturbance in cellular energy generation is visible as an increased cytoplasmic fluorescence. Interestingly, all pollutants even at the lowest dosage of 1 nmol/mL culture produced significantly increased ethidium bromide fluorescence compared to nonexposed controls. Ethidium bromide fluorescence intensities increased upon pollutant exposure dosage up to a saturation level, and were weakly (r(2) = 0.3905) inversely correlated to the proportion of live cells at that time point in culture. Temporal increase in EB fluorescence of growing cells is indicative for toxic but reversible effects. Cells displaying high continued EB fluorescence levels experience constant and permanent damage, and no longer contribute to population growth. The procedure developed here using bacterial ethidium bromide efflux pump activity may be a useful complement to screen sublethal toxicity effects of chemicals.

RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-kappa B and is processed during apoptosis.

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-kappa B activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-kappa B and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-kappa B induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-kappa B activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-kappa B and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-kappa B-dependent pro-survival or pro-inflammatory signals.

Cloning, functional expression, and chromosomal localization of the human pancreatic islet glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is a hormone secreted by the endocrine K-cells from the duodenum that stimulates glucose-induced insulin secretion. Here, we present the molecular characterization of the human pancreatic islet GIP receptor. cDNA clones for the GIP receptor were isolated from a human pancreatic islet cDNA library. They encoded two different forms of the receptor, which differed by a 27-amino acid insertion in the COOH-terminal cytoplasmic tail. The receptor protein sequence was 81% identical to that of the rat GIP receptor. When expressed in Chinese hamster lung fibroblasts, both forms of the receptor displayed high-affinity binding for GIP (180 and 600 pmol/l). GIP binding was displaced by &lt; 20% by 1 mumol/l glucagon, glucagon-like peptide (GLP-I)(7-36) amide, vasoactive intestinal peptide, and secretin. However exendin-4 and exendin-(9-39) at 1 mumol/l displaced binding by approximately 70 and approximately 100% at 10 mumol/l. GIP binding to both forms of the receptor induced a dose-dependent increase in intracellular cAMP levels (EC50 values of 0.6-0.8 nmol/l) but no elevation of cytoplasmic calcium concentrations. Interestingly, both exendin-4 and exendin-(9-39) were antagonists of the receptor, inhibiting GIP-induced cAMP formation by up to 60% when present at a concentration of 10 mumol/l. Finally, the physical and genetic chromosomal localization of the receptor gene was determined to be on 19q13.3, close to the ApoC2 gene. These data will help study the physiology and pathophysiology of the human GIP receptor.

Improved diagnosis of periprosthetic joint infection by multiplex PCR of sonication fluid from removed implants.

The microbiological diagnosis of periprosthetic joint infection (PJI) is crucial for successful antimicrobial treatment. Cultures have limited sensitivity, especially in patients receiving antibiotics. We evaluated the value of multiplex PCR for detection of microbial DNA in sonication fluid from removed orthopedic prostheses. Cases of PJI in which the prosthesis (or part of it) was removed were prospectively included. The removed implant was sonicated, and the resulting sonication fluid was cultured and subjected to multiplex PCR. Of 37 PJI cases (17 hip prostheses, 14 knee prostheses, 4 shoulder prostheses, 1 elbow prosthesis, and 1 ankle prosthesis), pathogens were identified in periprosthetic tissue in 24 (65%) cases, in sonication fluid in 23 (62%) cases, and by multiplex PCR in 29 (78%) cases. The pathogen was detected in 5 cases in sonication fluid only (Propionibacterium acnes in all cases; none of these patients had previously received antibiotics) and in 11 cases by multiplex PCR only (all of these patients had previously received antibiotics). After exclusion of 8 cases caused by P. acnes or Corynebacterium species, which cannot be detected due to the absence of specific primers in the PCR kit, sonication cultures were positive in 17 cases and multiplex PCR sonication cultures were positive in 29 cases (59% versus 100%, respectively; P < 0.01). Among 19 cases (51%) receiving antibiotics, multiplex PCR was positive in all 19 (100%), whereas sonication cultures grew the organism in 8 (42%) (P < 0.01). Multiplex PCR of sonication fluid is a promising test for diagnosis of PJI, particularly in patients who previously received antibiotics. With modified primer sets, multiplex PCR has the potential for further improvement of the diagnosis of PJI.

The expression of estrogen receptors as well as GREB1, c-MYC, and cyclin D1, estrogen-regulated genes implicated in proliferation, is increased in peritoneal endometriosis.

OBJECTIVE: To analyze the expression of estrogen receptors α and β as well as their target genes implicated in proliferation, c-myc, cyclin D1, and GREB1, in the endometrium of women with or without endometriosis. DESIGN: Expression analysis in human tissue. SETTING: University hospitals and a clinic. PATIENT(S): Ninety-one premenopausal women (59 patients with endometriosis and 32 controls) undergoing laparoscopic surgery. INTERVENTION(S): Biopsies were obtained at time of surgery, performed during the proliferative phase of the cycle. MAIN OUTCOME MEASURE(S): Estrogen receptors α and β as well as c-myc, cyclin D1, and GREB1 mRNA expression levels were determined by quantitative reverse transcriptase-polymerase chain reaction. Tissue localization of these estrogen-regulated genes was analyzed by immunohistochemistry. RESULT(S): Estrogen receptors α and β as well as c-myc, cyclin D1, and GREB1 mRNA expression levels were increased in ectopic tissue in comparison with both normal and eutopic endometrium. Estrogen receptor mRNA levels also were upregulated in the eutopic peritoneal tissue of patients with endometriosis. Cyclin D1 and GREB1 expression was augmented in eutopic endometrium. c-myc, cyclin D1, and GREB1 proteins exhibited a nuclear localization in ectopic endometrial tissue. CONCLUSION(S): This constitutes the first report of increased expression of GREB1, as well as cyclin D1 and c-myc, in peritoneal endometriotic lesions, implicating these proteins in estrogen-dependent growth in this context.

Sputum containing zinc enhances carbapenem resistance, biofilm formation and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa chronic lung infections are the leading cause of mortality in cystic fibrosis patients, a serious problem which is notably due to the numerous P. aeruginosa virulence factors, to its ability to form biofilms and to resist the effects of most antibiotics. Production of virulence factors and biofilm formation by P. aeruginosa is highly coordinated through complex regulatory systems. We recently found that CzcRS, the zinc and cadmium-specific two-component system is not only involved in metal resistance, but also in virulence and carbapenem antibiotic resistance in P. aeruginosa. Interestingly, zinc has been shown to be enriched in the lung secretions of cystic fibrosis patients. In this study, we investigated whether zinc might favor P. aeruginosa pathogenicity using an artificial sputum medium to mimic the cystic fibrosis lung environment. Our results show that zinc supplementation triggers a dual P. aeruginosa response: (i) it exacerbates pathogenicity by a CzcRS two-component system-dependent mechanism and (ii) it stimulates biofilm formation by a CzcRS-independent mechanism. Furthermore, P. aeruginosa cells embedded in these biofilms exhibited increased resistance to carbapenems. We identified a novel Zn-sensitive regulatory circuit controlling the expression of the OprD porin and modifying the carbapenem resistance profile. Altogether our data demonstrated that zinc levels in the sputum of cystic fibrosis patients might aggravate P. aeruginosa infection. Targeting zinc levels in sputum would be a valuable strategy to curb the increasing burden of P. aeruginosa infections in cystic fibrosis patients.

Sublingual sugar for infant hypoglycaemia

Refers To Deborah L Harris, Philip J Weston, Matthew Signal, J Geoffrey Chase, Jane E Harding Dextrose gel for neonatal hypoglycaemia (the Sugar Babies Study): a randomised, double-blind, placebo-controlled trial The Lancet, Volume 382, Issue 9910, 21 December 2013-3 January 2014, Pages 2077-2083 Referred to by Jane E Harding, Deborah L Harris, Philip J Weston, Matthew Signal, Geoffrey Chase Sublingual sugar for infant hypoglycaemia - Authors' reply The Lancet, Volume 383, Issue 9924, 5-11 April 2014, Pages 1208-1209

Decreasing educational differences in mortality over 40 years: evidence from the Turin Longitudinal Study (Italy)

BACKGROUND: Recent studies suggest that inequalities in premature mortality have continued to rise over the last decade in most European countries, but not in southern European countries. METHODS: In this study, we assess long-term trends (1971-2011) in absolute and relative educational inequalities in all-cause and cause-specific mortality in the Turin Longitudinal Study (Turin, Italy), a record-linkage study including all individuals resident in Turin in the 1971, 1981, 1991 and 2001 censuses, and aged 30-99 years (more than 2 million people). We examined mortality for all causes, cardiovascular disease (CVD), all cancers and specific cancers (lung, breast), as well as smoking and alcohol-related mortality. RESULTS: Overall mortality substantially decreased in all educational groups over the study period, although cancer rates only slightly declined. Absolute inequalities decreased for both genders (SII=962/694 in men/women in 1972-1976 and SII=531/259 in 2007-2011, p<0.01). Among men, absolute inequalities for CVD and alcohol-related causes declined (p<0.05), while remaining stable for other causes of death. Among women, declines in absolute inequalities were observed for CVD, smoking and alcohol-related causes and lung cancer (p<0.05). Relative inequalities in all-cause mortality remained stable for men and decreased for women (RII=1.92/2.03 in men/women in 1972-1976 and RII=2.15/1.32 in 2007-2011). Among men, relative inequalities increased for smoking-related causes, while among women they decreased for all cancers, CVD, smoking-related causes and lung cancer (p<0.05). CONCLUSIONS: Absolute inequalities in mortality strongly declined over the study period in both genders. Relative educational inequalities in mortality were generally stable among men; while they tended to narrow among women. In general, this study supports the hypothesis that educational inequalities in mortality have decreased in southern European countries.