Unconjugated TAT carrier peptide protects against excitotoxicity.

We report in this article for the first time the neuroprotective effects of unconjugated TAT carrier peptide against a mild excitotoxic stimulus both in vitro and in vivo. In view of the widespread use of TAT peptides to deliver neuroprotectants into cells, it is important to know the effects of the carrier itself. Unconjugated TAT carrier protects dissociated cortical neurons against NMDA but not against kainate, suggesting that TAT peptides may interfere with NMDA signaling. Furthermore, a retro-inverso form of the carrier peptide caused a reduction in lesion volume (by about 50%) in a rat neonatal cerebral ischemia model. Thus, even though TAT is designed merely as a carrier, its own pharmacological activity will need to be considered in the analysis of TAT-linked neuroprotectant peptides.

Systemic exposure to tobramycin after local antibiotic treatment with calcium sulphate as carrier material.

INTRODUCTION: Osteoset(®) T is a calcium sulphate void filler containing 4% tobramycin sulphate, used to treat bone and soft tissue infections. Despite systemic exposure to the antibiotic, there are no pharmacokinetic studies in humans published so far. Based on the observations made in our patients, a model predicting tobramycin serum levels and evaluating their toxicity potential is presented. METHODS: Following implantation of Osteoset(®) T, tobramycin serum concentrations were monitored systematically. A pharmacokinetic analysis was performed using a non-linear mixed effects model based on a one compartment model with first-degree absorption. RESULTS: Data from 12 patients treated between October 2006 and March 2008 were analysed. Concentration profiles were consistent with the first-order slow release and single-compartment kinetics, whilst showing important variability. Predicted tobramycin serum concentrations depended clearly on both implanted drug amount and renal function. DISCUSSION AND CONCLUSION: Despite the popularity of aminoglycosides for local antibiotic therapy, pharmacokinetic data for this indication are scarce, and not available for calcium sulphate as carrier material. Systemic exposure to tobramycin after implantation of Osteoset(®) T appears reassuring regarding toxicity potential, except in case of markedly impaired renal function. We recommend in adapting the dosage to the estimated creatinine clearance rather than solely to the patient's weight.

The impact of penicillinase on cefamandole treatment and prophylaxis of experimental endocarditis due to methicillin-resistant Staphylococcus aureus.

Beta-lactams active against methicillin-resistant Staphylococcus aureus (MRSA) must resist penicillinase hydrolysis and bind penicillin-binding protein 2A (PBP 2A). Cefamandole might share these properties. When tested against 2 isogenic pairs of MRSA that produced or did not produce penicillinase, MICs of cefamandole (8-32 mg/L) were not affected by penicillinase, and cefamandole had a > or =40 times greater PBP 2A affinity than did methicillin. In rats, constant serum levels of 100 mg/L cefamandole successfully treated experimental endocarditis due to penicillinase-negative isolates but failed against penicillinase-producing organisms. This suggested that penicillinase produced in infected vegetations might hydrolyze the drug. Indeed, cefamandole was slowly degraded by penicillinase in vitro. Moreover, its efficacy was restored by combination with sulbactam in vivo. Cefamandole also uniformly prevented MRSA endocarditis in prophylaxis experiments, a setting in which bacteria were not yet clustered in the vegetations. Thus, while cefamandole treatment was limited by penicillinase, the drug was still successful for prophylaxis of experimental MRSA endocarditis.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.

Which subgroup of patients with rheumatoid arthritis benefits from switching to rituximab versus alternative anti-tumour necrosis factor (TNF) agents after previous failure of an anti-TNF agent?

BACKGROUND: Patients with rheumatoid arthritis (RA) with an inadequate response to TNF antagonists (aTNFs) may switch to an alternative aTNF or start treatment from a different class of drugs, such as rituximab (RTX). It remains unclear in which clinical settings these therapeutic strategies offer most benefit. OBJECTIVE: To analyse the effectiveness of RTX versus alternative aTNFs on RA disease activity in different subgroups of patients. METHODS: A prospective cohort study of patients with RA who discontinued at least one aTNF and subsequently received either RTX or an alternative aTNF, nested within the Swiss RA registry (SCQM-RA) was carried out. The primary outcome, longitudinal improvement in 28-joint count Disease Activity Score (DAS28), was analysed using multivariate regression models for longitudinal data and adjusted for potential confounders. RESULTS: Of the 318 patients with RA included; 155 received RTX and 163 received an alternative aTNF. The relative benefit of RTX varied with the type of prior aTNF failure: when the motive for switching was ineffectiveness to previous aTNFs, the longitudinal improvement in DAS28 was significantly better with RTX than with an alternative aTNF (p = 0.03; at 6 months, -1.34 (95% CI -1.54 to -1.15) vs -0.93 (95% CI -1.28 to -0.59), respectively). When the motive for switching was other causes, the longitudinal improvement in DAS28 was similar for RTX and alternative aTNFs (p = 0.40). These results were not significantly modified by the number of previous aTNF failures, the type of aTNF switches, or the presence of co-treatment with a disease-modifying antirheumatic drug. CONCLUSION: This observational study suggests that in patients with RA who have stopped a previous aTNF treatment because of ineffectiveness changing to RTX is more effective than switching to an alternative aTNF.

Fast screening and confirmation of doping agents by UHPLC-QTOF-MS/MS

Introduction: The general strategy to perform anti-doping analysis starts with a screening followed by a confirmatory step when a sample is suspected to be positive. The screening step should be fast, generic and able to highlight any sample that may contain a prohibited substance by avoiding false negative and reducing false positive results. The confirmatory step is a dedicated procedure comprising a selective sample preparation and detection mode. Aim: The purpose of the study is to develop rapid screening and selective confirmatory strategies to detect and identify 103 doping agents in urine. Methods: For the screening, urine samples were simply diluted by a factor 2 with ultra-pure water and directly injected ("dilute and shoot") in the ultrahigh- pressure liquid chromatography (UHPLC). The UHPLC separation was performed in two gradients (ESI positive and negative) from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. The gradient analysis time is 9 min including 3 min reequilibration. Analytes detection was performed in full scan mode on a quadrupole time-of-flight (QTOF) mass spectrometer by acquiring the exact mass of the protonated (ESI positive) or deprotonated (ESI negative) molecular ion. For the confirmatory analysis, urine samples were extracted on SPE 96-well plate with mixed-mode cation (MCX) for basic and neutral compounds or anion exchange (MAX) sorbents for acidic molecules. The analytes were eluted in 3 min (including 1.5 min reequilibration) with a S1-25 Ann Toxicol Anal. 2009; 21(S1) Abstracts gradient from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. Analytes confirmation was performed in MS and MS/MS mode on a QTOF mass spectrometer. Results: In the screening and confirmatory analysis, basic and neutral analytes were analysed in the positive ESI mode, whereas acidic compounds were analysed in the negative mode. The analyte identification was based on retention time (tR) and exact mass measurement. "Dilute and shoot" was used as a generic sample treatment in the screening procedure, but matrix effect (e.g., ion suppression) cannot be avoided. However, the sensitivity was sufficient for all analytes to reach the minimal required performance limit (MRPL) required by the World Anti Doping Agency (WADA). To avoid time-consuming confirmatory analysis of false positive samples, a pre-confirmatory step was added. It consists of the sample re-injection, the acquisition of MS/MS spectra and the comparison to reference material. For the confirmatory analysis, urine samples were extracted by SPE allowing a pre-concentration of the analyte. A fast chromatographic separation was developed as a single analyte has to be confirmed. A dedicated QTOF-MS and MS/MS acquisition was performed to acquire within the same run a parallel scanning of two functions. Low collision energy was applied in the first channel to obtain the protonated molecular ion (QTOF-MS), while dedicated collision energy was set in the second channel to obtain fragmented ions (QTOF-MS/MS). Enough identification points were obtained to compare the spectra with reference material and negative urine sample. Finally, the entire process was validated and matrix effects quantified. Conclusion: Thanks to the coupling of UHPLC with the QTOF mass spectrometer, high tR repeatability, sensitivity, mass accuracy and mass resolution over a broad mass range were obtained. The method was sensitive, robust and reliable enough to detect and identify doping agents in urine. Keywords: screening, confirmatory analysis, UHPLC, QTOF, doping agents

Gold nanoparticles functionalized with gadolinium chelates as high-relaxivity MRI contrast agents

Water-dispersible gold nanoparticles functionalized with paramagnetic gadolinium have been fully characterized, and the NMRD profiles show very high relaxivities up to 1.5 T. Characterization using TEM images and dynamic light scattering indicate a particle size distribution from 2 to 15 nm. The gold cores of the nanoparticles do not contribute significantly to the overall magnetic moment.

Less frequent dosing of erythropoiesis stimulating agents in patients undergoing dialysis: a European multicentre cost study.

OBJECTIVE: To calculate the variable costs involved with the process of delivering erythropoiesis stimulating agents (ESA) in European dialysis practices. METHODS: A conceptual model was developed to classify the processes and sub-processes followed in the pharmacy (ordering from supplier, receiving/storing/delivering ESA to the dialysis unit), dialysis unit (dose determination, ordering, receipt, registration, storage, administration, registration) and waste disposal unit. Time and material costs were recorded. Labour costs were derived from actual local wages while material costs came from the facilities' accounting records. Activities associated with ESA administration were listed and each activity evaluated to determine if dosing frequency affected the amount of resources required. RESULTS: A total of 21 centres in 8 European countries supplied data for 142 patients (mean) per hospital (range 42-648). Patients received various ESA regimens (thrice-weekly, twice-weekly, once-weekly, once every 2 weeks and once-monthly). Administering ESA every 2 weeks, the mean costs per patient per year for each process and the estimates of the percentage reduction in costs obtainable, respectively, were: pharmacy labour (10.1 euro, 39%); dialysis unit labour (66.0 euro, 65%); dialysis unit materials (4.11 euro, 61%) and waste unit materials (0.43 euro, 49%). LIMITATION: Impact on financial costs was not measured. CONCLUSION: ESA administration has quantifiable labour and material costs which are affected by dosing frequency.

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines.

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

When nightclub security agents assault clients.

In 2006, a medico-legal consultation service devoted to adult victims of interpersonal violence was set up at the Lausanne University Hospital Centre, Switzerland: the Violence Medical Unit. Most patients are referred to the consultation by the Emergency Department. They are received by forensic nurses for support, forensic examination (in order to establish medical report) and community orientation. Between 2007 and 2009, among community violence, aggressions by security agents of nightclubs on clients have increased from 6% to 10%. Most of the victims are young men who had drunk alcohol before the assault. 25.7% presented one or several fractures, all of them in the head area. These findings raise questions about the ability of security agents of nightclubs to deal adequately with obviously risky situations and ensure client security.

The NLRP3 inflammasome in health and disease: the good, the bad and the ugly.

While interleukin (IL)-1β plays an important role in combating the invading pathogen as part of the innate immune response, its dysregulation is responsible for a number of autoinflammatory disorders. Large IL-1β activating platforms, known as inflammasomes, can assemble in response to the detection of endogenous host and pathogen-associated danger molecules. Formation of these protein complexes results in the autocatalysis and activation of caspase-1, which processes precursor IL-1β into its secreted biologically active form. Inflammasome and IL-1β activity is required to efficiently control viral, bacterial and fungal pathogen infections. Conversely, excess IL-1β activity contributes to human disease, and its inhibition has proved therapeutically beneficial in the treatment of a spectrum of serious, yet relatively rare, heritable inflammasomopathies. Recently, inflammasome function has been implicated in more common human conditions, such as gout, type II diabetes and cancer. This raises the possibility that anti-IL-1 therapeutics may have broader applications than anticipated previously, and may be utilized across diverse disease states that are linked insidiously through unwanted or heightened inflammasome activity.