CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

A phosphorylation cycle shapes gradients of the DYRK family kinase Pom1 at the plasma membrane.

Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.

Spontaneous cell polarization: Feedback control of Cdc42 GTPase breaks cellular symmetry.

Spontaneous polarization without spatial cues, or symmetry breaking, is a fundamental problem of spatial organization in biological systems. This question has been extensively studied using yeast models, which revealed the central role of the small GTPase switch Cdc42. Active Cdc42-GTP forms a coherent patch at the cell cortex, thought to result from amplification of a small initial stochastic inhomogeneity through positive feedback mechanisms, which induces cell polarization. Here, I review and discuss the mechanisms of Cdc42 activity self-amplification and dynamic turnover. A robust Cdc42 patch is formed through the combined effects of Cdc42 activity promoting its own activation and active Cdc42-GTP displaying reduced membrane detachment and lateral diffusion compared to inactive Cdc42-GDP. I argue the role of the actin cytoskeleton in symmetry breaking is not primarily to transport Cdc42 to the active site. Finally, negative feedback and competition mechanisms serve to control the number of polarization sites.

Contribution of estrogen and GluN2B subunit to the HtraKO mouse phenotype

Htr1a is one of the most widespread serotonin receptor across the brain, strongly expressed in CAI region of hippocampus. Our laboratory studies the phenotypic alteration in 5HTla- deficient mice (Htr1aK0), characterized an abnormal anxious-like behavior. Our aim is to evaluate the regulation of this cognitive process by understanding the circuitry involved. This phenotype sets up early during development and has durable effect in adulthood. Our laboratory showed that adult Htr1aK0 male mice displaying exuberant dendritic growth of oblique dendrites in a specific layer of a CAI pyramidal neurons, the stratum radiatum. Application of drugs in organotypic cultures and by in vivo injections revealed that GluN2B, a subunit of NMDA receptor highly expressed during development, is responsible for this dendritic exuberance. Immunohistochemistry highlighted in particular a synaptic enrichment of GluN2B in stratum radiatum of Htr1aK0 CAI pyramidal neurons at puberty. Finally, original analysis of Htr1aK0 mouse behavior showed a different response to anxiety between male and female. Htr1a activation down-regulates the CaMKII activity in the CAI pyramidal neurons. CaMKII directly favors the membrane conductance and stability of GluN2B at the synapse. In the context of the Htr1aK0 mouse, GluN2B is the final common pathway of our phenotype. This subunit is well known to regulate the threshold of LTD/LTP and the dendritogenesis during development. In my thesis, I establish a link between the gender differences in the morphology and the physiology in the Htr1aK0 mice during development to understand how these characteristics shape the circuit with prominent cognitive impacts in adulthood. My study highlighted that during development, Htr1aK0 male mice show a constant increase of the dendritic growth of oblique dendrites from early ages until adulthood associated with an increased physiological impact of altered GluN2A/GluN2B ratio. Whereas during puberty, synaptic contribution of GluN2B to NMDA response is higher in Htr1aK0 compared to WT male mice, this ratio comes back to normal values towards adulthood. However, this recovery of the ratio of GluN2A/GluN2B located at the synaptic level is concomitant with the lateral diffusion of excess GluN2B subunits, leading to extrasynaptic enrichment. The main impact was a lowering of the LTP threshold characterized by strong increased potentiation of synaptic strength after 5 Hz low frequency stimulation. Moreover, the extrasynaptic GluN2B overexpression leads to a shift of the maturation phase switch explaining the exuberant morphology. However, Htr1aK0 females characterized during the 3 first weeks of development by an increase of the dendritic growth of oblique dendrites showed starting at puberty that the dendrite arborization returns progressively to WT values. The physiological impact of GluN2B was investigated and directly linked to this morphology, since Htr1aK0 female mice does not show alteration of the synaptic strength during development. These observations show a compensation occurring in Htr1aK0 female, responsible for a rescue of the phenotype morphologically, physiologically and to be tested behaviorally. We highlighted then the biological processes underlying this compensation. During development, sexual hormones such as testosterone and estrogen are responsible to induce sexual differentiation of specific brain regions. I demonstrated that estrogen, but not testosterone, was able to reduce both in vitro and in vivo the dendritic arborization early during development, through activation of GPER-1, a G-coupled protein estrogen receptor, which phenocopy the activation of Htr1a by reducing GluN2B conductance and stability. I then identified a pathway, parallel to Htr1a, able to regulate GluN2B and responsible for the morphological and physiological phenotype in Htr1aK0 female mice. The specific rise of estrogen occurring at puberty in female is responsible for the compensation observed and induces a late rescue of the Htr1aK0 phenotype by activation GPER-1. -- Htr1a est un des récepteurs à la sérotonine les plus répandus dans le cerveau, fortement exprimé dans la région CAI de l'hippocampe. Notre laboratoire étudie les altérations phénotypiques de souris déficientes pour ce récepteur (Htr1aK0), caractérisées par un comportement avec des traits anxieux. Notre objectif est d'évaluer la régulation de ces processus cognitifs en comprenant les connexions nerveuses impliquées. Ce phénotype se met en place tôt au cours du développement et présente un effet durable à l'âge adulte. Notre laboratoire a montré que les souris Htr1aK0 mâles adultes se caractérisent par une croissance exubérante des dendrites obliques dans une couche spécifique des neurones pyramidaux du CAI, le stratum radiatum. L'application de drogues sur cultures organotypiques et par injections in vivo ont révélé que GluN2B, une sous-unité du récepteur NMDA fortement exprimée au cours du développement, est responsable de cette exubérance dendritique. Des expériences d'immunohistochimie ont notamment mis en évidence un enrichissement synaptique de GluN2B durant la puberté dans le stratum radiatum des neurones de la région CAI des souris Htr1aK0. Finalement, l'analyse originale du comportement des souris Htr1aK0 a montré une différence de réponse à l'anxiété entre mâles et femelles. L'activation de Htr1a diminue l'activité de la CaMKII dans les neurones pyramidaux du CAI. La CaMKII favorise directement la conductance et la stabilité de la sous-unité GluN2B à la synapse. Dans le contexte de la souris Htr1aK0, GluN2B est le « médiateur » de notre phénotype. Cette sous-unité est particulièrement connue pour réguler le seuil de LTD-LTP ainsi que la dendritogénèse durant le développement. Dans ma thèse, j'ai établi le lien entre les différences dépendant du genre dans la morphologie et physiologie des souris Htr1aK0 au cours du développement pour comprendre comment ces caractéristiques modulent le circuit accompagnés d'impacts cognitifs visibles à l'âge adulte. Mon étude a mis en évidence que durant le développement, les souris mâles Htr1aK0 montrent une constante augmentation de la croissance des dendrites obliques entre les premières semaines et l'âge adulte associée à une augmentation de l'impact physiologique du ratio GluN2A/GluN2B altéré. Alors que durant la puberté, la contribution synaptique de GluN2B à la réponse NMDA est plus haute chez la souris mâle Htr1aK0 que le WT, ce ratio revient à des valeurs normales à l'âge adulte. Cependant, cette récupération de l'expression du récepteur au niveau synaptique est concomitante avec la diffusion des sous-unités GluN2B excédantes, amenant alors à un enrichissement extrasynaptique. Le principal impact est une diminution du seuil de la LTP caractérisée par une forte potentiation de la plasticité après une stimulation basse fréquence à 5 Hz. De plus, la surexpression des GluN2B extrasynaptiques conduit à un décalage de la bascule à la phase de maturation, expliquant la morphologie dendritique exubérante. Cependant, les femelles Htr1aK0 initialement caractérisées pendant les 3 premières semaines du développement par une augmentation de la croissance des dendrites obliques montrent à partir de la puberté que cette arborisation dendritique retourne à des valeurs WT. L'impact physiologique de GLuN2B a été investigué et mis en lien avec cette morphologie, étant donné que les femelles Htr1aK0 ne montrent pas d'altération de la plasticité durant le développement. Ces observations montrent une compensation se produisant chez la femelle Htr1aK0, responsable d'une récupération du phénotype morphologique, physiologique et peut-être comportemental. Nous avons souligné les processus biologiques sous-jacent à cette compensation. Au cours du développement, les hormones sexuelles telles que la testostérone et l'estrogène sont responsables de la différentiation sexuelle de régions du cerveau spécifiques. J'ai démontré que l'estrogène, mais pas la testostérone, était capable de réduire in vitro et in vivo l'arborisation dendritique tôt dans le développement au travers de l'activation du récepteur GPER-1, un récepteur aux estrogènes couplés à un protéine G, qui phénocopie l'activation de Htr1a en réduisant la conductance et la stabilité de GluN2B à la membrane. J'ai identifié une voie de signalisation parallèle à celle de Htr1a, capable de réguler GluN2B et responsable du phénotype morphologique et physiologique de la souris femelle Htr1aK0. La montée spécifique d'estrogène se déroulant à la puberté chez la femelle est responsable de cette compensation et implique une récupération tardive du phénotype Htr1aK0 par l'activation de GPER-1.

Diffusion Spectrum Imaging after stroke shows structural changes in the contra-lateral motor network correlating with functional recovery

There is growing interest in understanding the role of the non-injured contra-lateral hemisphere in stroke recovery. In the experimental field, histological evidence has been reported that structural changes occur in the contra-lateral connectivity and circuits during stroke recovery. In humans, some recent imaging studies indicated that contra-lateral sub-cortical pathways and functional and structural cortical networks are remodeling, after stroke. Structural changes in the contra-lateral networks, however, have never been correlated to clinical recovery in patients. To determine the importance of the contra-lateral structural changes in post-stroke recovery, we selected a population of patients with motor deficits after stroke affecting the motor cortex and/or sub-cortical motor white matter. We explored i) the presence of Generalized Fractional Anisotropy (GFA) changes indicating structural alterations in the motor network of patientsâeuro? contra-lateral hemisphere as well as their longitudinal evolution ii) the correlation of GFA changes with patientsâeuro? clinical scores, stroke size and demographics data iii) and a predictive model.

Critical evaluation of outcome scales to assess outcome after lateral ankle ligament repair

Introduction: Several scores are commonly used to evaluate patients' postoperative satisfaction after lateral ankle ligament repair, including: AOFAS, FAAM, CAIT and CAIS. Comparing published studies in the literature is difficult, as the same patient can have markedly different results depending on which scoring system is used. The current study aims to address this gap in the literature by developing a system to compare these tests, to allow better analysis and comparison of published studies. Patients and methods: This is a retrospective cohort study of 47 patients following lateral ankle ligament repair using a modified Broström-Gould technique. All patients were operated between 2005 and 2010 by a single surgeon and followed the same post operative rehabilitation protocol. Six patients were excluded from the study because of concomitant surgery. Patients were assessed by an independent observer. We used the Pearson correlation coefficient to analyse the concordance of the scores, as well as scatter plots to assess the linear relationship between them. Results: A linear distribution between the scores was found when the results were analysed using scatter plots. We were thus able to use the Pearson correlation coefficient to evaluate the relationship between each of the different postoperative scores. The correlation was found to be above 0.5 in all cases except for the comparison between the CAIT and the FAAM for the activities of daily living (0.39). We were, therefore, able to compare the results obtained and assess the relative concordance of the scoring systems. The results showed that the more specific the scale is, the worst the score is and inversely. So the CAIT and the CAIS appeared to be more severe than the AOFAS and the FAAM measuring the activities of daily living. The sports subscale of the FAAM demonstrated intermediate results. Conclusion: This study outlines a system to compare different postoperative scores commonly used to evaluate outcome after ankle stabilization surgery. The impact of this study is that it makes comparison of published studies easier, even though they use a variety of different clinical scores, thus facilitating better outcome analysis of operative techniques.

Alveolar echinococcosis of the liver: Diffusion-weighted MRI findings and potential role in lesion characterisation

PURPOSE: To report the diffusion-weighted MRI findings in alveolar echinococcosis (AE) of the liver and evaluate the potential role of apparent diffusion coefficients (ADCs) in the characterisation of lesions. MATERIALS AND METHODS: We retrospectively included 22 patients with 63 AE liver lesions (≥1cm), examined with 3-T liver MRI, including a free-breathing diffusion-weighted single-shot echo-planar imaging sequence (b-values=50, 300 and 600s/mm(2)). Two radiologists jointly assessed the following lesion features: size, location, presence of cystic and/or solid components (according to Kodama's classification system), relative contrast enhancement, and calcifications (on CT). The ADCtotal, ADCmin and ADCmax were measured in each lesion and the surrounding liver parenchyma. RESULTS: Three type 1, 19 type 2, 17 type 3, three type 4 and 21 type 5 lesions were identified. The mean (±SD) ADCtotal, ADCmin and ADCmax for all lesions were 1.73±0.50, 0.76±0.38 and 2.63±0.76×10(-3)mm(2)/s, respectively. The mean ADCtotal for type 1, type 2, type 3, type 4 and type 5 lesions were 1.97±1.01, 1.76±0.53, 1.73±0.41, 1.15±0.42 and 1.76±0.44×10(-3)mm(2)/s, respectively. No significant differences were found between the five lesion types, except for type 4 (p=0.0363). There was a significant correlation between the presence of a solid component and low ADCmin (r=0.39, p=0.0016), whereas an inverse correlation was found between the relative contrast enhancement and ADCtotal (r=-0.34, p=0.0072). CONCLUSION: The ADCs of AE lesions are relatively low compared to other cystic liver lesions, which may help in the differential diagnosis. Although ADCs are of little use to distinguish between the five lesion types, their low value reflects the underlying solid component.

Investigation of field and diffusion time dependence of the diffusion-weighted signal at ultrahigh magnetic fields.

Over the last decade, there has been a significant increase in the number of high-magnetic-field MRI magnets. However, the exact effect of a high magnetic field strength (B0 ) on diffusion-weighted MR signals is not yet fully understood. The goal of this study was to investigate the influence of different high magnetic field strengths (9.4 T and 14.1 T) and diffusion times (9, 11, 13, 15, 17 and 24 ms) on the diffusion-weighted signal in rat brain white matter. At a short diffusion time (9 ms), fractional anisotropy values were found to be lower at 14.1 T than at 9.4 T, but this difference disappeared at longer diffusion times. A simple two-pool model was used to explain these findings. The model describes the white matter as a first hindered compartment (often associated with the extra-axonal space), characterized by a faster orthogonal diffusion and a lower fractional anisotropy, and a second restricted compartment (often associated with the intra-axonal space), characterized by a slower orthogonal diffusion (i.e. orthogonal to the axon direction) and a higher fractional anisotropy. Apparent T2 relaxation time measurements of the hindered and restricted pools were performed. The shortening of the pseudo-T2 value from the restricted compartment with B0 is likely to be more pronounced than the apparent T2 changes in the hindered compartment. This study suggests that the observed differences in diffusion tensor imaging parameters between the two magnetic field strengths at short diffusion time may be related to differences in the apparent T2 values between the pools. Copyright © 2013 John Wiley & Sons, Ltd.

Measuring the diffusion of palliative care in long-term care facilities - a death census

ABSTRACT: BACKGROUND: The dissemination of palliative care for patients presenting complex chronic diseases at various stages has become an important matter of public health. A death census in Swiss long-term care facilities (LTC) was set up with the aim of monitoring the frequency of selected indicators of palliative care. METHODS: The survey covered 150 LTC facilities (105 nursing homes and 45 home health services), each of which was asked to complete a questionnaire for every non-accidental death over a period of six months. The frequency of 4 selected indicators of palliative care (resort to a specialized palliative care service, the administration of opiates, use of any pain measurement scale or other symptom measurement scale) was monitored in respect of the stages of care and analysed based on gender, age, medical condition and place of residence. RESULTS: Overall, 1200 deaths were reported, 29.1% of which were related to cancer. The frequencies of each indicator varied according to the type of LTC, mostly regarding the administration of opiate. It appeared that the access to palliative care remained associated with cancer, terminal care and partly with age, whereas gender and the presence of mental disorders had no effect on the indicators. In addition, the use of drugs was much more frequent than the other indicators. CONCLUSION: The profile of patients with access to palliative care must become more diversified. Among other recommendations, equal access to opiates in nursing homes and in home health services, palliative care at an earlier stage and the systematic use of symptom management scales when resorting to opiates have to become of prime concern.

Acute fractures of medial and lateral great toe sesamoids in an athlete.

We report a case of acute fracture of both sesamoids of the great toe in an athlete. The fractures healed uneventfully after non-surgical treatment.

Diffusion-weighted MR imaging of the spine at 3-T: feasibility, optimization of b-value and utility to differentiate benign from pathological vertebral compression fractures

Purpose: To evaluate the feasibility, determine the optimal b-value, and assess the utility of 3-T diffusion-weighted MR imaging (DWI) of the spine in differentiating benign from pathologic vertebral compression fractures.Methods and Materials: Twenty patients with 38 vertebral compression fractures (24 benign, 14 pathologic) and 20 controls (total: 23 men, 17 women, mean age 56.2years) were included from December 2010 to May 2011 in this IRB-approved prospective study. MR imaging of the spine was performed on a 3-T unit with T1-w, fat-suppressed T2-w, gadolinium-enhanced fat-suppressed T1-w and zoomed-EPI (2D RF excitation pulse combined with reduced field-of-view single-shot echo-planar readout) diffusion-w (b-values: 0, 300, 500 and 700s/mm2) sequences. Two radiologists independently assessed zoomed-EPI image quality in random order using a 4-point scale: 1=excellent to 4=poor. They subsequently measured apparent diffusion coefficients (ADCs) in normal vertebral bodies and compression fractures, in consensus.Results: Lower b-values correlated with better image quality scores, with significant differences between b=300 (mean±SD=2.6±0.8), b=500 (3.0±0.7) and b=700 (3.6±0.6) (all p<0.001). Mean ADCs of normal vertebral bodies (n=162) were 0.23, 0.17 and 0.11×10-3mm2/s with b=300, 500 and 700s/mm2, respectively. In contrast, mean ADCs were 0.89, 0.70 and 0.59×10-3mm2/s for benign vertebral compression fractures and 0.79, 0.66 and 0.51×10-3mm2/s for pathologic fractures with b=300, 500 and 700s/mm2, respectively. No significant difference was found between ADCs of benign and pathologic fractures.Conclusion: 3-T DWI of the spine is feasible and lower b-values (300s/mm2) are recommended. However, our preliminary results show no advantage of DWI in differentiating benign from pathologic vertebral compression fractures.