Sylvie Chollet-Martin

Self-assembled squalenoylated penicillin bioconjugates: an original approach for the treatment of intracellular infections.

We describe here new nanoparticles based on the bioconjugation of penicillin G to squalene in order to overcome severe intracellular infections by pathogen bacteria whose mechanism of resistance arises from the poor intracellular diffusion of several antibiotics. Two different squalene-penicillin G conjugates were synthesized (pH-sensitive and pH-insensitive), and their self-assembly as nanoparticles was investigated through morphology and stability studies. These nanoparticles had a size of 140 ± 10 nm (polydispersity index of 0.1) and a negative charge, and they did not display any supramolecular organization. Furthermore, they were found stable in water and in different culture medium. The cellular uptake and localization of these fluorescently labeled nanoparticles were explored on the macrophage cell line J774 by flow cytometry and confocal microscopy analysis. The squalenoylated nanoparticles were found to be cell internalized through clathrin-dependent and -independent endocytic pathways. Moreover, they induced an improved intracellular antibacterial activity on the facultative intracellular pathogen S. aureus, compared with free penicillin G, despite the absence of co-localization between the bacteria and the nanoparticles in the cells. This study suggests that the bioconjugation of an antibiotic to a squalene template could be a valuable approach for overcoming the antibiotic resistance due to intracellular bacterial infections.

An improved strategy to recover large fragments of functional human neutrophil extracellular traps.

Netosis is a recently described neutrophil function that leads to the release of neutrophil extracellular traps (NETs) in response to various stimuli. NETs are filaments of decondensed chromatin associated with granular proteins. In addition to their role against microorganisms, NETs have been implicated in autoimmunity, thrombosis, and tissue injury. Access to a standardized source of isolated NETs is needed to better analyze the roles of NETs. The aim of this study was to develop a procedure yielding soluble, well-characterized NET preparations from fresh human neutrophils. The calcium ionophore A23187 was chosen to induce netosis, and the restriction enzyme AluI was used to prepare large NET fragments. DNA and proteins were detected by electrophoresis and specific labeling. Some NET proteins [histone 3, lactoferrin (LF)] were quantified by western blotting, and double-stranded DNA (dsDNA) was quantified by immunofluorescence. Co-existence of dsDNA and neutrophil proteins confirmed the quality of the NET preparations. Their biological activity was checked by measuring elastase (ELA) activity and bacterial killing against various strains. Interindividual differences in histone 3, LF, ELA, and dsDNA relative contents were observed in isolated NETs. However, the reproducibility of NET preparation and characterization was validated, suggesting that this interindividual variability was rather related to donor variation than to technical bias. This standardized protocol is suitable for producing, isolating, and quantifying functional NETs that could serve as a tool for studying NET effects on immune cells and tissues.

Autoantibodies to citrullinated fibrinogen compared with anti-MCV and anti-CCP2 antibodies in diagnosing rheumatoid arthritis at an early stage: data from the French ESPOIR cohort

Objectives To compare the performance of anticitrullinated peptides/protein antibodies (ACPA) detected by three immunoassays in the French ESPOIR cohort of patients with early rheumatoid arthritis (RA) and undifferentiated arthritis (UA) and to study the relationship between ACPA and disease activity. Methods A diagnosis of RA (1987 American College of Rheumatology (ACR) criteria) was established at baseline in 497 patients and after a 2-year follow-up in 592 patients. At baseline, antibodies to citrullinated fibrinogen (AhFibA), antimutated citrullinated vimentin (anti-MCV) and anticyclic citrullinated peptide (anti-CCP2) were assayed and the individual and combined diagnostic sensitivities and predictive values of the tests were determined. Relationships between ACPA positivity and the 28-joint disease activity score and Health Assessment Questionnaire scores were analysed. Results At a diagnostic specificity of at least 98%, the three tests exhibited similar diagnostic sensitivities (47–48.5%). When considering as positive patients with at least one positive test, the sensitivity increased to 53.5% with a probable loss of specificity. Among the patients classified as having UA at baseline, 30% were positive for one ACPA, the positive predictive values for RA of the three tests ranging from 73% to 80% but increasing when two tests were associated. Whatever the test used, the addition of ACPA positivity to the 1987 criteria enhanced their sensitivity by 6%, close to that of the 2010 ACR/European League Against Rheumatism (EULAR) criteria. Conclusions In early arthritis, AhFibA, anti-MCV and anti-CCP2 showed similar diagnostic sensitivity with a high diagnostic specificity and a similar high positive predictive value for RA. Adding ACPA to the 1987 ACR criteria significantly increased the number of patients classified as having RA, confirming the validity of the recent inclusion of the serological criterion in the ACR/EULAR criteria.

Molecular Epidemiology of Chronic Granulomatous Disease in a Series of 80 Kindreds: Identification of 31 Novel Mutations

Chronic granulomatous disease (CGD) results from constitutional inactivating mutations in the CYBB, NCF1, CYBA or NCF2 genes that encode subunits of phagocyte NADPH oxidase. We report the findings of molecular analysis of 80 kindred. In 75 unrelated male and 5 female probands, CGD was suspected on the basis of clinical symptoms, and biological samples were referred to our laboratory between 2000 and 2007. Seventy seven patients were found to have mutations in CYBB, NCF1, CYBA or NCF2 (52 different mutations including 31 mutations not previously reported). CYBB was the most frequently mutated gene (58 males and 3 females, 76%). In autosomal recessive forms of the disease, mutations were found in NCF1 (11 patients), NCF2 (3 patients) and CYBA (2 patients). We observed that significantly fewer females were affected by autosomal recessive CGD than expected (2 females/14 males; p=0.002), suggesting that female patients with CGD may be under diagnosed.

Neutrophil Extracellular Traps Downregulate Lipopolysaccharide-Induced Activation of Monocyte-Derived Dendritic Cells

Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell–cell contacts. Neutrophil extracellular traps (NETs) released by PMN could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-α, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4+ T lymphocyte polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-γ and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation.

Apical ballooning syndrome following perioperative anaphylaxis is likely related to high doses of epinephrine

Apical ballooning syndrome, a reversible left ventricle dysfunction, has been reported following anaphylaxis and, during this clinical circumstance, is seemingly linked to the use of either low or high doses of epinephrine. We report a severe succinylcholine-induced IgE-mediated anaphylaxis in a 65-year-old woman, in whom the diagnosis of apical ballooning syndrome following anaphylaxis was established. As a thorough description of the clinical features and resuscitative measures could be obtained, we discuss the reasons for apical ballooning syndrome occurrence and highlight the fact that optimal care management of anaphylaxis should include a progressive titration of epinephrine.

Anaphylaxis to amidotrizoate proved by skin testing and flow cytometry-based basophil activation test

hypersensitivity to bosentan. This higher sensitivity of the LTT has been observed in other studies concerning non immediate reactions to b-lactams (7) and other drugs, such as non b-lactam antibiotics and anticonvulsants (8). On the other hand, the activation of drug-specific T cells plays a central role in many non immediate drug hypersensitivity reactions, such as MPE, toxic epidermal necrolysis (TEN), DRESS, and acute generalized exanthematous pustolosis (AGEP) (8, 9). Therefore, a more extensive diagnostic use of the LTT – which is still generally considered merely a research tool – could improve the sensitivity of the diagnostic workup for non immediate reactions to drugs, reducing the number of drug provocation tests, which are not only harmful, but are also contraindicated in severe reactions such as TEN, DRESS, and AGEP. However, a recent study by Kano et al. (9) proved that the LTT sensitivity varies depending on the type of drug reaction and the time interval between such reactions and testing. In fact, positive results were obtained when the test was performed at the acute stage, but not at the recovery stage, in MPE and TEN, while positive responses were observed at the recovery stage, but not at the acute one, in DRESS, as in our case. All authors have no relationships to declare.

Effect of rapid desensitization on platelet inhibition and basophil activation in patients with aspirin hypersensitivity and coronary disease.

AimsnTo determine antiplatelet efficacy after desensitization in patients with a history of aspirin hypersensitivity.nnnMethods and resultsnWe conducted a case-control study to evaluate the efficacy of aspirin 1u2009day (D1) and 6-8 weeks (W6-8) after desensitization. We also assessed ex vivo basophil reactivity to aspirin after desensitization. Cases were patients with coronary artery disease (CAD) and documented history of aspirin hypersensitivity who underwent rapid successful oral desensitization to aspirin. Controls were patients with stable CAD without hypersensitivity and receiving aspirin. Among 56 cases, 27 received aspirin for acute coronary syndromes and 29 were treated for stable CAD. Aspirin was effective (defined as light transmission aggregometry induced by arachidonic acidu2009≤20%) at D1 in 86% of cases (Pu2009=u20090.045 vs. controls) and in 95% at W6-8, vs. 100% of controls (Pu2009=u20090.39). Urinary excretion of thromboxane B2 diminished substantially in cases (Pu2009<u20090.0001, D0 vs. W6-8) but remained higher than in controls (Pu2009=u20090.03). Platelet reactivity (defined by platelet P-selectin expression, activated glycoprotein IIb/IIIa inhibitors, and platelet-monocyte aggregates) was similar in cases between D0 and D1 but decreased at W6-8. Basophil activation (quantified by upregulation of CD203c in response to aspirin) was higher in cases at W6-8 than in controls (Pu2009=u20090.0002).nnnConclusionnThus, following rapid desensitization, aspirin achieves rapid biological efficacy, which is slightly lower at D1, but becomes indistinguishable from chronically treated patients at W6-8. Persistent basophil activation several weeks after desensitization suggests infraclinical hypersensitivity and the need to continue aspirin to maintain desensitization.

Miraculous catch by neutrophils NETs

Neutrophil extracellular traps (NET) account for a new mechanism of anti-infectious innate immunity that may lead to important tissue damages. Indeed, a growing number of studies demonstrate the involvement of NET in the pathogenesis of several human diseases as diverse as autoimmune diseases, thrombotic disorders or some inflammatory diseases. After a short description of molecular mechanisms of NETosis and its host-defense function, we will review their detrimental effects. We will then examine the potential therapeutic approaches for modulating excessive or inappropriate NETosis and thereby minimize tissue injury.

Does Percutaneous Transluminal Coronary Angioplasty Activate Neutrophils ? Preliminary Clinical Result.

AbstractIt has been suggested that activation of neutrophils and the complement system plays a role in post-ischemic myocardial dysfunction and restenosis after coronary angioplasty. Furthermore, recent experimental evidence suggests activation of the neutrophils in angina pectoris and acute myocardial infarction. Percutaneous transluminal coronary angioplasty (PICA) represents both a controlled injury to the coronary arterial wall, and a model of transient ischemia. Both of these processes are liable to activate neutrophils.The aim of this study was to investigate ex vivo the morphological and functional characteristics of coronary sinus neutrophils, before and immediately after PTCA. Neutrophil activation was assessed by 1.measurement of plasma lactoferrin (LF) and plasma myeloperoxidase, to study release of specific and azurophilic granules respectively, 2.whole blood chemiluminescence stimulated in vitro by phorbol-myristate acetate and opsonized zymosan, 3.plasma fluorescent thiobarbituric acid-reactive substances (TBA-RS). These measurements were performed in blood drawn from the coronary sinus, before and immediately after PICA (7 Pts). To ensure that changes observed were related to PTCA but not to the catheterization procedure, controls undergoing coronary angiography without PICA were also studied. (4 Pts).After PTCA, LF increased 2 fold (p