Autoimmunity-Associated LYP-W620 Does Not Impair Thymic Negative Selection of Autoreactive T Cells.

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.

A simple, robust and rapid approach to detect carbapenemases in Gram-negative isolates by MALDI-TOF mass spectrometry: validation with triple quadripole tandem mass spectrometry, microarray and PCR.

Carbapenemases should be accurately and rapidly detected, given their possible epidemiological spread and their impact on treatment options. Here, we developed a simple, easy and rapid matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based assay to detect carbapenemases and compared this innovative test with four other diagnostic approaches on 47 clinical isolates. Tandem mass spectrometry (MS-MS) was also used to determine accurately the amount of antibiotic present in the supernatant after 1 h of incubation and both MALDI-TOF and MS-MS approaches exhibited a 100% sensitivity and a 100% specificity. By comparison, molecular genetic techniques (Check-MDR Carba PCR and Check-MDR CT103 microarray) showed a 90.5% sensitivity and a 100% specificity, as two strains of Aeromonas were not detected because their chromosomal carbapenemase is not targeted by probes used in both kits. Altogether, this innovative MALDI-TOF-based approach that uses a stable 10-μg disk of ertapenem was highly efficient in detecting carbapenemase, with a sensitivity higher than that of PCR and microarray.

The Beck Cognitive Insight Scale in outpatients with psychotic disorders: further evidence from a French-speaking sample.

OBJECTIVE: The Beck Cognitive Insight Scale (BCIS) evaluates patients' self-report of their ability to detect and correct misinterpretation. Our study aims to confirm the factor structure and the convergent validity of the original scale in a French-speaking environment. METHOD: Outpatients (n = 158) suffering from schizophrenia or schizoaffective disorders fulfilled the BCIS. The 51 patients in Montpellier were equally assessed with the Positive and Negative Syndrome Scale (PANSS) by a psychiatrist who was blind of the BCIS scores. RESULTS: The fit indices of the confirmatory factor analysis validated the 2-factor solution reported by the developers of the scale with inpatients, and in another study with middle-aged and older outpatients. The BCIS composite index was significantly negatively correlated with the clinical insight item of the PANSS. CONCLUSIONS: The French translation of the BCIS appears to have acceptable psychometric properties and gives additional support to the scale, as well as cross-cultural validity for its use with outpatients suffering from schizophrenia or schizoaffective disorders. The correlation between clinical and composite index of cognitive insight underlines the multidimensional nature of clinical insight. Cognitive insight does not recover clinical insight but is a potential target for developing psychological treatments that will improve clinical insight.

The influence of drinking pattern, at individual and aggregate levels, on alcohol-related negative consequences.

AIM: To determine the extent drinking patterns (at the individual and country level) are associated with alcohol-related consequences over and above the total alcohol the person consumes. METHODS: Hierarchical linear models were estimated based on general population surveys conducted in 18 countries participating in the GENACIS project. RESULTS: In general, the positive association between drinking pattern scores and alcohol-related consequences was found at both the individual and country levels, independent of volume of drinking. In addition, a significant interaction effect indicated that the more detrimental the country's drinking pattern, the less steep the association between the volume of drinking and its consequences. CONCLUSION: Drinking patterns have an independent impact on consequences over and above the relationship between volume and consequences.

Determination of picogram levels of midazolam, and 1- and 4-hydroxymidazolam in human plasma by gas chromatography-negative chemical ionization-mass spectrometry.

Midazolam is a widely accepted probe for phenotyping cytochrome P4503A. A gas chromatography-mass spectrometry (GC-MS)-negative chemical ionization method is presented which allows measuring very low levels of midazolam (MID), 1-OH midazolam (1OHMID) and 4-OH midazolam (4OHMID), in plasma, after derivatization with the reagent N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. The standard curves were linear over a working range of 20 pg/ml to 5 ng/ml for the three compounds, with the mean coefficients of correlation of the calibration curves (n = 6) being 0.999 for MID and 1OHMID, and 1.0 for 4OHMID. The mean recoveries measured at 100 pg/ml, 500 pg/ml, and 2 ng/ml, ranged from 76 to 87% for MID, from 76 to 99% for 1OHMID, from 68 to 84% for 4OHMID, and from 82 to 109% for N-ethyloxazepam (internal standard). Intra- (n = 7) and inter-day (n = 8) coefficients of variation determined at three concentrations ranged from 1 to 8% for MID, from 2 to 13% for 1OHMID and from 1 to 14% for 4OHMID. The percent theoretical concentrations (accuracy) were within +/-8% for MID and 1OHMID, within +/-9% for 4OHMID at 500 pg/ml and 2 ng/ml, and within +/-28% for 4OHMID at 100 pg/ml. The limits of quantitation were found to be 10 pg/ml for the three compounds. This method can be used for phenotyping cytochrome P4503A in humans following the administration of a very low oral dose of midazolam (75 microg), without central nervous system side-effects.

Lack of EGFR-activating mutations in European patients with triple-negative breast cancer could emphasise geographic and ethnic variations in breast cancer mutation profiles.

INTRODUCTION: Triple-negative breast cancers (TNBCs) are characterised by lack of expression of hormone receptors and epidermal growth factor receptor 2 (HER-2). As they frequently express epidermal growth factor receptors (EGFRs), anti-EGFR therapies are currently assessed for this breast cancer subtype as an alternative to treatments that target HER-2 or hormone receptors. Recently, EGFR-activating mutations have been reported in TNBC specimens in an East Asian population. Because variations in the frequency of EGFR-activating mutations in East Asians and other patients with lung cancer have been described, we evaluated the EGFR mutational profile in tumour samples from European patients with TNBC. METHODS: We selected from a DNA tumour bank 229 DNA samples isolated from frozen, histologically proven and macrodissected invasive TNBC specimens from European patients. PCR and high-resolution melting (HRM) analyses were used to detect mutations in exons 19 and 21 of EGFR. The results were then confirmed by bidirectional sequencing of all samples. RESULTS: HRM analysis allowed the detection of three EGFR exon 21 mutations, but no exon 19 mutations. There was 100% concordance between the HRM and sequencing results. The three patients with EGFR exon 21 abnormal HRM profiles harboured the rare R836R SNP, but no EGFR-activating mutation was identified. CONCLUSIONS: This study highlights variations in the prevalence of EGFR mutations in TNBC. These variations have crucial implications for the design of clinical trials involving anti-EGFR treatments in TNBC and for identifying the potential target population.

Analytical progresses of the International Olympic Committee and World Anti-Doping Agency Olympic laboratories.

The Summer Olympic Games constitute the biggest concentration of human sports and activities in a particular place and time since 776 BCE, when the written history of the Olympic Games in Olympia began. Summer and Winter Olympic anti-doping laboratories, accredited by the International Olympic Committee in the past and the World Anti-Doping Agency in the present times, acquire worldwide interest to apply all new analytical advancements in the fight against doping in sports, hoping that this major human event will not become dirty by association with this negative phenomenon. This article summarizes the new analytical progresses, technologies and knowledge used by the Olympic laboratories, which for the vast majority of them are, eventually, incorporated into routine anti-doping analysis.

Citizen participation and the Lisbon Treaty : A legal perspective

How to "bring the [European] Union closer to its citizens" is a vexed and vital problem of European integration. Article 11 TEU on participatory democracy, recently introduced by the Lisbon Treaty, is meant to be part of the solution. The EU Economic and Social Committee has gone so far as to define this provision "a milestone on the road to a people's Europe that is real and feasible". This appears to be an overly optimistic assessment - partly because art. 11 relies heavily on the involvement of civil society organisations, which political science literature suggests is conceptually and/or practically irrelevant to citizen involvement; partly because it largely formalizes participatory practices that have been in existence for years without cognizable effects on citizen participation; and partly because even its most innovative element - the European citizens' initiative (ECI) - does not bring significant changes to the Union's constitutional arrangements in terms of redistributing decision-making power. In addition to that, secondary legislation places significant hurdles on the submission of ECIs and might prevent or delay their becoming a standard democratic practice. This is not to say that art. 11 TEU has no potential at all. Its insertion in the Treaty might provide impetus to rethink and develop past participatory practices, such as horizontal civil dialogue. Moreover, the effects of "popular input" in the form of ECIs on EU institutional dynamics is as yet unknown - and perhaps not negligible, to judge from the keen interest that the European Parliament and other bodies have demonstrated in "appropriating" it as a political asset. Finally, art. 11 raises the stakes of the Union's democratic challenge and might pressure EU institutions to make full use of its potential. Or, if eventually proved inadequate, art. 11 might constitute a constitutional experiment on the way to meaningful forms of direct democracy at EU level.

The habenula encodes negative motivational value associated with primary punishment in humans.

Learning what to approach, and what to avoid, involves assigning value to environmental cues that predict positive and negative events. Studies in animals indicate that the lateral habenula encodes the previously learned negative motivational value of stimuli. However, involvement of the habenula in dynamic trial-by-trial aversive learning has not been assessed, and the functional role of this structure in humans remains poorly characterized, in part, due to its small size. Using high-resolution functional neuroimaging and computational modeling of reinforcement learning, we demonstrate positive habenula responses to the dynamically changing values of cues signaling painful electric shocks, which predict behavioral suppression of responses to those cues across individuals. By contrast, negative habenula responses to monetary reward cue values predict behavioral invigoration. Our findings show that the habenula plays a key role in an online aversive learning system and in generating associated motivated behavior in humans.

Bentonite Treatment for Economical Dust Reduction on Limestone Surfaced Secondary Roads, HR-351, 1995

This research project was directed at laboratory and field evaluation of sodium montmorillonite clay (Bentonite) as a dust palliative for limestone surfaced secondary roads. It was postulated that the electrically charged surfaces (negative) of the clay particles could interact with the charged surfaces (positive) of the limestone and act as a bonding agent to agglomerate fine (-#200) particulates, and also to bond the fine particulates to larger (+#200) limestone particles. One mile test roads were constructed in Tama, Appanoose, and Hancock counties in Iowa using Bentonite treatment levels (by weight of aggregate) ranging from 3.0 to 12.0%. Construction was accomplished by adding dry Bentonite to the surfacing material and then dry road mixing. The soda ash/water solution (dispersing agent) was spray applied and the treated surfacing material wet mixed by motor graders to a consistency of 2 to 3 inch slump concrete. Two motor graders working in tandem provided rapid mixing. Following wet mixing the material was surface spread and compacted by local traffic. Quantitative and qualitative periodic evaluations and testing of the test roads was conducted with respect to dust generation, crust development, roughness, and braking characteristics. As the Bentonite treatment level increased dust generation decreased. From a cost/benefit standpoint, an optimum level of treatment is about 8% (by weight of aggregate). For roads with light traffic, one application at this treatment level resulted in a 60-70% average dust reduction in the first season, 40-50% in the second season, and 20-30% in the third season. Crust development was rated at two times better than untreated control sections. No discernible trend was evident with respect to roughness. There was no evident difference in any of the test sections with respect to braking distance and braking handling characteristics, under wet surface conditions compared to the control sections. Chloride treatments are more effective in dust reduction in the short term (3-4 months). Bentonite treatment is capable of dust reduction over the long term (2-3 seasons). Normal maintenance blading operations can be used on Bentonite treated areas. Soda ash dispersed Bentonite treatment is estimated to be more than twice as cost effective per percent dust reduction than conventional chloride treatments, with respect to time. However, the disadvantage is that there is not the initial dramatic reduction in dust generation as with the chloride treatment. Although dust is reduced significantly after treatment there is still dust being generated. Video evidence indicates that the dust cloud in the Bentonite treated sections does not rise as high, or spread as wide as the cloud in the untreated section. It also settles faster than the cloud in the untreated section. This is considered important for driving safety of following traffic, and for nuisance dust invasion of residences and residential areas. The Bentonite appears to be functioning as a bonding agent.

Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4

Rapport de synthèse : L'immunité innée regroupe les mécanismes moléculaires et cellulaires formant la première ligne de défense contre les infections microbiennes. La détection des micro-organismes pathogènes est assurée par des cellules sentinelles (cellules dendritiques et macrophages) qui jouent un rôle fondamental dans l'initiation des mécanismes de défense de l'hôte. Au contact de produits microbiens, ces cellules produisent un large échantillonnage de molécules, dont des cytokines, impliquées dans le développement de la réponse inflammatoire. La régulation de cette réponse relève d'un équilibre délicat, son insuffisance tant que son excès pouvant compromettre le devenir des patients infectés. La sepsis sévère et le choc septique représentent les formes les plus sévères d'infection, et leur mortalité demeure élevée (25 à 30% pour la sepsis sévère et 50 à 60% pour le choc septique). De plus, l'incidence de la sepsis tend à augmenter, atteignant en 2000 plus de 240 cas pour 100'000 personnes en Grande-Bretagne. La sepsis est caractérisée dans sa phase aiguë par une réponse inflammatoire exubérante. La plupart des thérapies visant à la bloquer ont toutefois montré des bénéfices incertains lors de leur application clinique. Il est donc impératif d'identifier de nouvelles cibles thérapeutiques. Les "Toll-like receptors" (TLRs) sont une famille de récepteurs qui jouent un rôle fondamental dans la détection des micro-organismes par les cellules du système immunitaire inné. Parmi eux, TLR4 est indispensable à la reconnaissance du lipopolysaccharide (LPS) des bactéries Gram-négatives. L'interaction entre TLR4 et le LPS représentant un élément précoce de la réponse de l'hôte à l'infection, nous avons émit l'hypothèse que TLR4 pourrait représenter une cible de choix en vue du développement de nouvelles thérapies contre la sepsis. Dans l'objectif de valider ce concept, nous avons, dans un premier temps, démontré que des souris génétiquement déficientes en TLR4 étaient totalement résistantes au choc septique induit par Escherichia coli (E. coli), une bactérie Gram-négative fréquemment responsable de sepsis. Forts de cette observation, nous avons développé une molécule recombinante composée du domaine extracellulaire de TLR4 fusionné à la partie IgGi-Fc. Cette molécule soluble, qui inhibait la réponse des macrophages au LPS in vitro, a été utilisée pour générer des anticorps anti-TLR4 chez le lapin. La spécificité et l'efficacité de ces anticorps ont été prouvées en démontrant que les anti-TLR4 bloquaient les signaux d'activation intracellulaire et la production de TNF et d'IL-6 en réponse au LPS et aux bactéries Gram-négatives in vitro et in vivo. Enfin, l'efficacité des ces anticorps a été testée dans des modèles de sepsis chez la souris. Ainsi, l'injection prophylactique (-lh) ou thérapeutique (+3h) d'anticorps anti-TLR4 réduisait la production de TNF et protégeait les animaux de la mort. De manière spectaculaire, ces anticorps réduisaient également la production de TNF et protégeaient de la sepsis à E. coli lorsqu'ils étaient administrés de manière prophylactique (-4h) et thérapeutique, jusqu'à 13 heures après l'initiation de l'infection. Ces résultats indiquent donc qu'il est possible de bloquer le développement de la réponse inflammatoire et de protéger du choc septique à bactéries Gram-négatives en utilisant des thérapies ciblant TLR4. Par ailleurs, ils suggèrent qu'une fenêtre d'opportunité de plusieurs heures pourrait être mise à profit pour initier un traitement chez les patients septiques. Ces résultats devraient encourager la poursuite des essais cliniques en cours qui visent à tester l'efficacité de thérapies dirigées contre TLR4 comme traitement complémentaire de la sepsis.

Retrofit Methods for Distortion Cracking Problems in Plate Girder Bridges, TR-436, 2003

This report is formatted to independently present four individual investigations related to similar web gap fatigue problems. Multiple steel girder bridges commonly exhibit fatigue cracking due to out-of-plane displacement of the web near the diaphragm connections. This fatigue-prone web gap area is typically located in negative moment regions of the girders where the diaphragm stiffener is not attached to the top flange. In the past, the Iowa Department of Transportation has attempted to stop fatigue crack propagation in these steel girder bridges by drilling holes at the crack tips. Other nondestructive retrofits have been tried; in a particular case on a two-girder bridge with floor beams, angles were bolted between the stiffener and top flange. The bolted angle retrofit has failed in the past and may not be a viable solution for diaphragm bridges. The drilled hole retrofit is often only a temporary solution, so a more permanent and effective retrofit is required. A new field retrofit has been developed that involves loosening the bolts in the connection between the diaphragm and the girders. Research on the retrofit has been initiated; however, no long-term studies of the effects of bolt loosening have been performed. The intent of this research is to study the short-term effects of the bolt loosening retrofit on I-beam and channel diaphragm bridges. The research also addressed the development of a continuous remote monitoring system to investigate the bolt loosening retrofit on an X-type diaphragm bridge over a number of months, ensuring that the measured strain and displacement reductions are not affected by time and continuous traffic loading on the bridge. The testing for the first three investigations is based on instrumentation of web gaps in a negative moment region on Iowa Department of Transportation bridges with I-beam, channel, and X-type diaphragms. One bridge of each type was instrumented with strain gages and deflection transducers. Field tests, using loaded trucks of known weight and configuration, were conducted on the bridges with the bolts in the tight condition and after implementing the bolt loosening retrofit to measure the effects of loosening the diaphragm bolts. Long-term data were also collected on the X-diaphragm bridge by a data acquisition system that collected the data continuously under ambient truck loading. The collected data were retrievable by an off-site modem connection to the remote data acquisition system. The data collection features and ruggedness of this system for remote bridge monitoring make it viable as a pilot system for future monitoring projects in Iowa. Results indicate that loosening the diaphragm bolts reduces strain and out-of-plane displacement in the web gap, and that the reduction is not affected over time by traffic or environmental loading on the bridge. Reducing the strain in the web gap allows the bridge to support more cycles of loading before experiencing fatigue, thus increase the service life of the bridge. Two-girder floor beam bridges may also exhibit fatigue cracking in girder webs.

Bridge Deck Waterproofing Membrane Study, R-262, 1974

One of the main problems of bridge maintenance in Iowa is the spalling and scaling of the decks. This problem stems from the continued use of deicing salts during the winter months. Since bridges will frost or freeze more often than roadways, the use of deicing salts on bridges is more frequent. The salt which is spread onto the bridge dissolves in water and permeates into the concrete deck. When the salt reaches the depth of the reinforcing steel and the concentration at that depth reaches the threshold concentration for corrosion (1.5 lbs./yd. 3 ), the steel will begin to oxidize. The oxidizing steel must then expand within the concrete. This expansion eventually forces undersurface fractures and spalls in the concrete. The spalling increases maintenance problems on bridges and in some cases has forced resurfacing after only a few years of service. There are two possible solutions to this problem. One solution is discontinuing the use of salts as the deicing agent on bridges and the other is preventing the salt from reaching or attacking the reinforcing steel. This report deals with one method which stops the salt from reaching the reinforcing steel. The method utilizes a waterproof membrane on the surface of a bridge deck. The waterproof membrane stops the water-salt solution from entering the concrete so the salt cannot reach the reinforcing steel.