Antibiotics and efflux: combined spectrofluorimetry and mass spectrometry to evaluate the involvement of concentration and efflux activity in antibiotic intracellular accumulation.

Background: In Gram-negative bacteria, passing through the double membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibiotic activity. Spectrofluorimetry has been developed to follow fluoroquinolone accumulation inside bacteria using intrinsic bacterial fluorescence as an internal standard. However, adaptation for non-fluorescent antibiotics is needed; quantitative methods based on MS offer the possibility of expanding the detection range obtained by spectrofluorimetry. Objectives: To validate, with spectrofluorimetry, the use of MS to measure antibiotic accumulation in cells and to determine the relationship between antibiotic concentrations and the amount of intrabacterial accumulation in different efflux backgrounds on the same batch of molecules. Methods: Spectrofluorimetry was performed in parallel with MS on the same samples to measure the ciprofloxacin and fleroxacin accumulation in cells expressing various efflux pump levels. A microplate protocol was set up to determine the antibiotic accumulation as a function of external antibiotic concentrations. Results: A correlation existed between the data obtained with spectrofluorimetry and MS, whatever the efflux pump or tested antibiotic. The results highlighted different dynamics of uptake between ciprofloxacin and fleroxacin as well as the relationship between the level of efflux activity and antibiotic accumulation. Conclusions: We have developed a microplate protocol and cross-validated two complementary methods: spectrofluorimetry, which contains a reliable internal standard; and MS, which allows detection of low antibiotic amounts. These assays allow study of the dose effect and the efflux impact on the intrabacterial accumulation of antibiotics.

Plasma peptide biomarker discovery for amyotrophic lateral sclerosis by MALDI-TOF mass spectrometry profiling.

The diagnostic of Amyotrophic lateral sclerosis (ALS) remains based on clinical and neurophysiological observations. The actual delay between the onset of the symptoms and diagnosis is about 1 year, preventing early inclusion of patients into clinical trials and early care of the disease. Therefore, finding biomarkers with high sensitivity and specificity remains urgent. In our study, we looked for peptide biomarkers in plasma samples using reverse phase magnetic beads (C18 and C8) and MALDI-TOF mass spectrometry analysis. From a set of ALS patients (n=30) and healthy age-matched controls (n=30), C18- or C8-SVM-based models for ALS diagnostic were constructed on the base of the minimum of the most discriminant peaks. These two SVM-based models end up in excellent separations between the 2 groups of patients (recognition capability overall classes > 97%) and classify blinded samples (10 ALS and 10 healthy age-matched controls) with very high sensitivities and specificities (>90%). Some of these discriminant peaks have been identified by Mass Spectrometry (MS) analyses and correspond to (or are fragments of) major plasma proteins, partly linked to the blood coagulation.

Targeting the TLR co-receptor CD14 with TLR2-derived peptides modulates immune responses to pathogens.

Dysregulation of Toll-like receptor (TLR) responses to pathogens can lead to pathological inflammation or to immune hyporesponsiveness and susceptibility to infections, and may affect adaptive immune responses. TLRs are therefore attractive therapeutic targets. We assessed the potential of the TLR co-receptor CD14 as a target for therapeutics by investigating the magnitude of its influence on TLR responses. We studied the interaction of CD14 with TLR2 by conducting peptide screening and site-directed mutagenesis analysis and found TLR2 leucine-rich repeats 5, 9, 15, and 20 involved in interaction with CD14. Peptides representing these regions interacted with CD14 and enhanced TLR2- and TLR4-mediated proinflammatory responses to bacterial pathogens in vitro. Notably, the peptides' immune boosting capacity helped to rescue proinflammatory responses of immunosuppressed sepsis patients ex vivo. In vivo, peptide treatment increased phagocyte recruitment and accelerated bacterial clearance in murine models of Gram-negative and Gram-positive bacterial peritonitis. Up-modulating CD14's co-receptor activity with TLR2-derived peptides also enhanced antigen-induced dendritic cell (DC) maturation and interleukin-2 production and, most notably, differentially affected DC cytokine profile upon antigen stimulation, promoting a T helper 1-skewed adaptive immune response. Biochemical, cell imaging, and molecular docking studies showed that peptide binding to CD14 accelerates microbial ligand transfer from CD14 to TLR2, resulting in increased and sustained ligand occupancy of TLR2 and receptor clustering for signaling. These findings reveal the influence that CD14 exerts on TLR activities and describe a potential therapeutic strategy to amplify responses to different pathogens mediated by different TLRs by targeting the common TLR co-receptor, CD14.

SC5 mAb represents a unique tool for the detection of extracellular vimentin as a specific marker of Sezary cells.

Circulating malignant Sézary lymphocytes result from a clonal proliferation of memory/activated CD4(+)CD45RO(+) T lymphocytes primarily involving the skin. Recently, the CD158k/KIR3DL2 cell surface receptor has been identified to phenotypically characterize these cells. We previously described a mAb termed SC5 that identifies an unknown early activation cell membrane molecule. It is expressed selectively by T lymphocytes isolated from healthy individuals upon activation, and by circulating Sézary syndrome lymphocytes. In addition, we found that SC5 mAb was reactive with all resting T lymphocytes once permeabilized, indicating that SC5 mAb-reactive molecule might present distinct cellular localization according to the T cell activation status. In this study, we show for the first time that SC5 mAb recognizes the intermediate filament protein vimentin when exported to the extracellular side of the plasma membrane of viable Sézary malignant cells. We demonstrate that SC5 mAb is unique as it reacts with both viable malignant lymphocytes and apoptotic T cells. As vimentin is also detected rapidly at the cell membrane surface after normal T lymphocyte activation, it suggests that its extracellular detection on Sézary cells could be a consequence of their constitutive activation status. Finally, as a probable outcome of vimentin cell surface expression, autoantibodies against vimentin were found in the sera of Sézary syndrome patients.