Cutaneous and renal vasodilatory response to local pressure application: A comparative study in mice.

BACKGROUND AND AIM: We have reported a novel relationship involving mechanical stimulation and vasodilation in rodent and human skin, referred to as pressure-induced vasodilation (PIV). It is unknown whether this mechanism exists in kidney and reflects the microcirculation in deep organs. Therefore, we compared the skin and kidney PIV to determine whether their changes were similar. METHODS: In anesthetized mice fed a normal salt-diet, laser Doppler flux (LDF) signals were measured when an increase in local pressure was applied to the surface of the head skin with the rate of 2.2Pa/s (1mmHg/min) and to the left kidney with a rate of 4.4Pa/s (2mmHg/min). The mechanism underlying renal PIV was also investigated. The skin and kidney PIV were also compared during salt load (4% NaCl diet). RESULTS: The kidney had higher baseline LDF and vascular conductance compared to those of the skin. Pressure application increased the LDF in the kidney as well as in the skin with a comparable maximal magnitude (about 25% from baseline value), despite different kinetics of PIV evolution. As we previously reported in the skin, the kidney PIV response was mediated by the activation of transient receptor potential vanilloid type 1 channels, the release of calcitonin gene-related peptide, and the participation of prostaglandins and nitric oxide. In the absence of hypertension, high salt intake abolished the cutaneous PIV response and markedly impaired the renal one. CONCLUSION: PIV response in the mouse kidney results from a neuro-vascular interaction. Despite some differences between the skin and the kidney PIV, the similarities in their response and signaling mechanisms suggest that the cutaneous microcirculation could reflect, in part, the microcirculation of the renal cortex.

A comparison of the abilities of natural rubber (NR) and synthetic polyisoprene cis-1,4 rubber (IR) to crystallize under strain at high strain rates.

Strain induced crystallization (SIC) of a natural rubber (NR) and a synthetic rubber (IR) with a high amount of cis-1,4 units (98.6%) is studied, thanks to in situ wide angle X-ray (WAXS) experiments at room temperature performed in a large range of strain rates. During stretching at a low strain rate (4.2 × 10(-3) s(-1)), SIC in IR occurs at a larger stretching ratio than in NR. As a result, the crystallinity index at a given stretching ratio is lower in IR than in NR, in spite of the similar crosslink densities of the chains involved in the crystallization in both materials. This lower ability for crystallization in IR is attributed to the presence of branching along its backbone and its lower stereoregularity. Conversely, dynamic experiments performed at high strain rates (10(1)/10(2) s(-1)) show for both materials a similar ability to crystallize. This unexpected result is confirmed by monotonic tensile tests performed in a large range of strain rates. The reason is thermodynamic: the chain extension plays a predominant role compared to the role of the microstructure defects when the strain rate is high, i.e. when the kinetics of the crystallite nucleation forces the crystallization to occur at a large stretching ratio. A thermodynamic model enables qualitative reproduction of the experimental results.

Strain induced crystallization and melting of natural rubber during dynamic cycles.

Strain-induced crystallization (SIC) of natural rubber (NR) is studied during dynamic cycles at high frequencies (with equivalent strain rates ranging from 7.2 s(-1) to 290 s(-1)). The testing parameters are varied: the frequency, the temperature and the stretching ratio domain. It is found that an increase of the frequency leads to an unexpected form of the CI-λ curve, with a decrease of the crystallinity during both loading and unloading steps of the cycle. Nevertheless, the interpretation of the curves needs to take into account several phenomena such as (i) instability of the crystallites generated during the loading step, which increases with the frequency, (ii) the memory of the previous alignment of the chains, which depends on the minimum stretching ratio of the cycle λmin and the frequency, and (iii) self-heating which makes the crystallite nucleation more difficult and their melting easier. Thus, when the stretching ratio domain is above the expected stretching ratio at complete melting λmelt, the combination of these phenomena, at high frequencies, leads to unexpected results such as complete melting at λmin, and hysteresis in the CI-λ curves.

ER egress of invariant chain isoform p35 requires direct binding to MHCII molecules and is inhibited by the NleA virulence factor of enterohaemorrhagic Escherichia coli.

Four invariant chain (Ii) isoforms assist the folding and trafficking of human MHC class II (MHCIIs). The main isoforms, Iip33 and Iip35, assemble in the ER into homo- and/or hetero-trimers. The sequential binding of up to three MHCII αß heterodimers to Ii trimers results in the formation of pentamers, heptamers and nonamers. MHCIIs are required to overcome the p35-encoded di-arginine (RxR) ER retention motif and to allow anterograde trafficking of the complex. Here, we show that inactivation of the RxR motif requires a direct cis interaction between p35 and the MHCII, precluding ER egress of some unsaturated Ii trimers. Interestingly, as opposed to MHCII/p33 complexes, those including p35 remained in the ER when co-expressed with the NleA protein of enterohaemorrhagic Escherichia coli. Taken together, our results demonstrate that p35 influences distinctively MHCII/Ii assembly and trafficking.

The D-6 mouse monoclonal antibody recognizes the CD74 cytoplasmic tail.

The invariant chain (Ii; CD74) is a multifunctional protein of the immune system and a major player in the presentation of exogenous antigens to T cells. In the endoplasmic reticulum (ER), Ii assists the folding and trafficking of MHC class II molecules. In the present study, we characterized the recently commercialized D-6 monoclonal antibody (MAb) made against a polypeptide spanning the entire sequence of the p33 isoform of human Ii. Using transgenic mice expressing the human p35 isoform, we showed by flow cytometry that D-6 only slightly cross-reacts with mouse Ii in permeabilized splenocytes. Analysis of the human B lymphoblastoid cell line LG2 revealed that D-6 recognizes Ii only upon membrane permeabilization. Variants of Ii bearing specific mutations or deletions were transfected in human cells to map the D-6 epitope. Our results showed that this MAb binds to the N-terminal cytoplasmic domain of Ii and that the epitope was destroyed upon mutagenesis of the two leucine-based endosomal targeting motifs. Thus, D-6 cannot be used for rapid flow cytometric assessment of CD74 cell surface expression and would be ineffective as a drug conjugate for the treatment of hematological malignancies.

The invariant chain p35 isoform promotes formation of nonameric complexes with MHC II molecules.

Four different isoforms of the human invariant chain (Ii) have been described (p33, p35, p41 and p43). These heterotrimerize in the endoplasmic reticulum (ER) before associating with MHC class II molecules (MHCIIs). However, the final stoichiometry of the Ii/MHCII complex remains debated. This is particularly interesting as both p35 and p43 include a di-arginine motif that requires masking by MHCII to allow ER egress. Here, to functionally address the requirement for stoichiometric interactions, we used a recombinant DR heterodimer bearing its own cytoplasmic di-lysine ER-retention motif (DRKKAA). When coexpressed with p33 and a control myc-tagged DR (DRmyc), DRKKAA was retained in the ER but had little impact on surface expression of DRmyc. However, when coexpressed with p35, DRKKAA restricted the surface expression of DRmyc, indicating that Ii trimers can be loaded with more than one MHCII. Similar results were obtained using HLA-DQ instead of DRmyc, showing that a single trimeric Ii scaffold can include distinct MHCII isotypes. Altogether, these results demonstrate that the subunit stoichiometry of oligomeric Ii/MHCII complexes is influenced by p35.

MMTV superantigens coerce an unconventional topology between the TCR and MHC class II.

Mouse mammary tumor virus superantigens (vSAGs) are notorious for defying structural characterization, and a consensus has yet to be reached regarding their ability to bridge the TCR to MHC class II (MHCII). In this study, we determined the topology of the T cell signaling complex by examining the respective relation of vSAG7 with the MHCII molecule, MHCII-associated peptide, and TCR. We used covalently linked peptide/MHCII complexes to demonstrate that vSAG presentation is tolerant to variation in the protruding side chains of the peptide, but can be sensitive to the nature of the protruding N-terminal extension. An original approach in which vSAG was covalently linked to either MHCII chain confirmed that vSAG binds outside the peptide binding groove. Also, whereas the C-terminal vSAG segment binds to the MHCII α-chain in a conformation-sensitive manner, the membrane-proximal N-terminal domain binds the ß-chain. Because both moieties of the mature vSAG remain noncovalently associated after processing, our results suggest that vSAG crosslinks MHCII molecules. Comparing different T cell hybridomas, we identified key residues on the MHCII α-chain that are differentially recognized by the CDR3ß when engaged by vSAG. Finally, we show that the highly conserved tyrosine residue found in the vSAg TGXY motif is required for T cell activation. Our results reveal a novel SAG/MHCII/TCR architecture in which vSAGs coerce a near-canonical docking between MHCII and TCR that allows eschewing of traditional CDR3 binding with the associated peptide in favor of MHCII α-chain binding. Our findings highlight the plasticity of the TCR CDRs.

Use of precision-cut renal cortical slices in nephrotoxicity studies.

1.Unlike cell lines and primary cells in culture, precision-cut tissue slices remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. 2.In this article, we illustrate the use of such an experimental model to study the nephrotoxic effects of (i) chloroacetaldehyde, a metabolite of the anticancer drug ifosfamide, (ii) of cobalt chloride, a potential leakage product of the cobalt-containing nanoparticles, and (iii) of valproate, a widely used antiepileptic drug. 3.Since all the latter test compounds, like many toxic compounds, negatively interact with cellular metabolic pathways, we also illustrate our biochemical toxicology approach in which we used not only enzymatic but also carbon 13 NMR measurements and mathematical modelling of metabolic pathways. 4.This original approach, which can be applied to any tissue, allows to predict the nephrotoxic effects of milligram amounts of test compounds very early during the research and development processes of drugs and chemicals. This approach, combined with the use of cells that retain their in vivo metabolic properties and, therefore, are predictive, reduces the risk, the time and cost of such processes.

Le symptôme dans la psychothérapie d'un enfant dysphasique. analyse de sa fonction dynamique dans une perspective psychanalytique / O sintoma na psicoterapia de uma criança disfásica. análise da sua função dinâmica a partir de uma perspectiva psicanalítica / The symptom in the psychotherapy of a dysphasic child. analysis of his dynamic function from a psychoanalytical perspective / El síntoma en la psicoterapia de un niño disfásico. análisis de su función dinámica desde una perspectiva psicoanalítica

Dans cet article, à partir d'un cas clinique, nous analysons la spécificité de la prise en charge psychothérapeutique de l'enfant dysphasique. Notre objectif principal est de montrer comment la succession de moments logiques donne une place à chaque fois nouvelle au symptôme. L'étude du déroulement de la séquence clinique, permet d'interroger la valeur du symptôme et son lien indissociable à la parole, dans la relation transférentielle, pour rendre compte du processus de subjectivation à l'œuvre chez cet enfant. En outre, ce travail amène à préciser la particularité de la démarche psychothérapeutique par rapport à celles des autres professionnels intervenant auprès des enfants dysphasiques.

Neste artigo analisamos a especificidade da cobertura psicoterapêutica de crianças com disfasia a partir de um caso clínico. Trata-se aqui de mostrar a sucessão de momentos lógicos que dão um lugar cada vez novo aos sintomas. A seqüência clínica vai permitir-nos a questionar o valor do sintoma e sua ligação inseparável com a palavra na relação de transferência para refletir o processo de historicização da subjetividade da criança. Além disso, este percurso vai permitir-nos situar a especificidade da abordagem psicoterapêutico comparados aos de outros profissionais que trabalham com crianças com disfagia.

In this article we analyze the specificity of the psychotherapy of a dysphasic child, based on a clinical case. Our aim is to show the succession of logical moments that give ever newer places to the symptom. The study of a particular clinical sequence will allow us to question the value of the symptom and its inseparable association with speech in the transference relationship, in order to discuss the "subjectivation" process at work in this child. This discussion will also allow us to situate the main specific characteristics of the psychotherapeutic approach as compared to approaches of other professionals who treat dysphasic children.

En este artículo, a partir de un caso clínico, analizaremos la especificidad del tratamiento psicoterapéutico de un niño disfásico. Nuestro objetivo principal es mostrar como la sucesión de momentos lógicos confiere en cada momento una nueva posición al síntoma. El estudio del desarrollo de la secuencia clínica, permite interrogar el valor del síntoma y su relación indisociable con la palabra, en la relación transferencial, lo que permite reflexionar sobre el proceso de historización de la subjetividad del niño en el caso considerado. Además, este recorrido nos permitirá precisar la particularidad del trabajo psicoterapéutico respecto al de otros profesionales que intervienen en el tratamiento de niños con disfasia.

Le symptôme dans la psychothérapie d'un enfant dysphasique. Analyse de sa fonction dynamique dans une perspective psychanalytique / O sintoma na psicoterapia de uma criança disfásica. Análise da sua função dinâmica a partir de uma perspectiva psicanalítica / The symptom in the psychotherapy of a dysphasic child. Analysis of his dynamic function from a psychoanalytical perspective / El síntoma en la psicoterapia de un niño disfásico. Análisis de su función dinámica desde una perspectiva psicoanalítica

Dans cet article, à partir d'un cas clinique, nous analysons la spécificité de la prise en charge psychothérapeutique de l'enfant dysphasique. Notre objectif principal est de montrer comment la succession de moments logiques donne une place à chaque fois nouvelle au symptôme. L'étude du déroulement de la séquence clinique, permet d'interroger la valeur du symptôme et son lien indissociable à la parole, dans la relation transférentielle, pour rendre compte du processus de subjectivation à l'œuvre chez cet enfant. En outre, ce travail amène à préciser la particularité de la démarche psychothérapeutique par rapport à celles des autres professionnels intervenant auprès des enfants dysphasiques.(AU)

Neste artigo analisamos a especificidade da cobertura psicoterapêutica de crianças com disfasia a partir de um caso clínico. Trata-se aqui de mostrar a sucessão de momentos lógicos que dão um lugar cada vez novo aos sintomas. A seqüência clínica vai permitir-nos a questionar o valor do sintoma e sua ligação inseparável com a palavra na relação de transferência para refletir o processo de historicização da subjetividade da criança. Além disso, este percurso vai permitir-nos situar a especificidade da abordagem psicoterapêutico comparados aos de outros profissionais que trabalham com crianças com disfagia.(AU)

In this article we analyze the specificity of the psychotherapy of a dysphasic child, based on a clinical case. Our aim is to show the succession of logical moments that give ever newer places to the symptom. The study of a particular clinical sequence will allow us to question the value of the symptom and its inseparable association with speech in the transference relationship, in order to discuss the "subjectivation" process at work in this child. This discussion will also allow us to situate the main specific characteristics of the psychotherapeutic approach as compared to approaches of other professionals who treat dysphasic children.(AU)

En este artículo, a partir de un caso clínico, analizaremos la especificidad del tratamiento psicoterapéutico de un niño disfásico. Nuestro objetivo principal es mostrar como la sucesión de momentos lógicos confiere en cada momento una nueva posición al síntoma. El estudio del desarrollo de la secuencia clínica, permite interrogar el valor del síntoma y su relación indisociable con la palabra, en la relación transferencial, lo que permite reflexionar sobre el proceso de historización de la subjetividad del niño en el caso considerado. Además, este recorrido nos permitirá precisar la particularidad del trabajo psicoterapéutico respecto al de otros profesionales que intervienen en el tratamiento de niños con disfasia.(AU)

Protocols and applications of cellular metabolomics in safety studies using precision-cut tissue slices and carbon 13 NMR.

Numerous xenobiotics are toxic to human and animal cells by interacting with their metabolism, but the precise metabolic step affected and the biochemical mechanism behind such a toxicity often remain unknown. In an attempt to reduce the ignorance in this field, we have developed a new approach called cellular metabolomics. This approach, developed in vitro, provides a panoramic view not only of the pathways involved in the metabolism of physiologic substrates of any normal or pathologic human or animal cell but also of the beneficial and adverse effects of xenobiotics on these metabolic pathways. Unlike many cell lines, precision-cut tissue slices, for which there is a renewed interest, remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. Cellular metabolomics (or cellular metabonomics), which combines enzymatic and carbon 13 NMR measurements with mathematical modeling of metabolic pathways, is illustrated in this brief chapter for studying the effect of insulin on glucose metabolism in rat liver precision-cut slices, and of valproate on glutamine metabolism in human renal cortical precision-cut slices. The use of very small amounts of test compounds allows to predict their toxic effect and eventually their beneficial effects very early in the research and development processes. Cellular metabolomics is complementary to other omics approaches, but, unlike them, provides functional and dynamic pieces of information by measuring enzymatic fluxes.

Mechanical properties and cytocompatibility of poly(ε-caprolactone)-infiltrated biphasic calcium phosphate scaffolds with bimodal pore distribution.

Biphasic calcium phosphate scaffolds have attracted interest because they have good osteoconductivity and a resorption rate close to that of new bone ingrowth, but their brittleness limits their potential applications. In this study, we show how the infiltration of biphasic calcium phosphate scaffolds with poly(ε-caprolactone) improves their mechanical properties. It was found that the polymer effectively contributes to energy to failure enhancement in bending, compressive and tensile tests. The main toughening mechanism in these composites is crack bridging by polymer fibrils. The presence of fibrils at two different size scales--as found in scaffolds with a bimodal pore distribution--results in a more effective toughening effect as compared to scaffolds with a monomodal pore size distribution, especially in the early stage of mechanical deformation. An optimized infiltration process allowed the preservation of micropore interconnection after infiltration, which is beneficial for cells adhesion. In addition, it is shown that biphasic calcium phosphates infiltrated with poly(ε-caprolactone) are cytocompatible with human bone marrow stromal cells, which makes them good candidates for bone substitution.

Metastable and unstable cellular solidification of colloidal suspensions.

Colloidal particles are often seen as big atoms that can be directly observed in real space. They are therefore becoming increasingly important as model systems to study processes of interest in condensed-matter physics such as melting, freezing and glass transitions. The solidification of colloidal suspensions has long been a puzzling phenomenon with many unexplained features. Here, we demonstrate and rationalize the existence of instability and metastability domains in cellular solidification of colloidal suspensions, by direct in situ high-resolution X-ray radiography and tomography observations. We explain such interface instabilities by a partial Brownian diffusion of the particles leading to constitutional supercooling situations. Processing under unstable conditions leads to localized and global kinetic instabilities of the solid/liquid interface, affecting the crystal morphology and particle redistribution behaviour.

Latex imaging by environmental STEM: application to the study of the surfactant outcome in hybrid alkyd/acrylate systems.

Among other uses, latexes are a successful alternative to solvent-borne binders for coatings. Efforts are made to produce hybrid nanostructured latexes containing an acrylic phase and an alkyd phase. However, after the film-forming process, the surfactant used to stabilize these latexes remains in the film, and its location can have a drastic effect on the application properties. Among the processing parameters, the alkyd hydrophobicity can strongly influence this location. This article aims at the imaging of these surfactant molecules in two hybrid latexes with different hydrophobicity level of the alkyd resin. A first part of this paper is dedicated to the understanding of the contrast provided by the surfactant in environmental STEM imaging of latexes. Then, the influence of surfactant-polymer affinity on the surfactant location after film-forming of those hybrid alkyd/acrylate latexes is studied by this technique. It is shown that in the hybrid latex with an alkyd shell (obtained with the most hydrophilic resin), the surfactant molecules tend to remain buried in the alkyd phase. Conversely, in the hybrid latex with an acrylate shell (in the case of the most hydrophobic resin), the surfactant molecules tend to gather into islands like in pure acrylate latex films.

Grafting of polystyrene on nitrogen-doped multi-walled carbon nanotubes.

Polymer grafting of polystyrene (PS) on nitrogen-doped multiwall carbon nanotubes (CNx) was successfully obtained by a "grafting from" technique. The production method involves the immobilization of initiators, using wet chemistry, onto the nanotube surface, followed by an in situ surface-initiated polymerization. The polymer-grafting carbon nanotubes synthesis includes the free radical functionalization of CNx and the "controlled/living" Nitroxide Mediated Radical Polymerization (NMRP). The obtained products were studied using several microscopic techniques as scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), and electron energy loss spectroscopy (EELS). The characterization also includes thermogravimetric analysis (TGA), Raman spectroscopy, infrared spectroscopy, and electron spin resonance (ESR), among others. The analyzed samples were also compared with solutions fabricated by physical blending of the polymer and CNx nanotubes. These results indicate that the nanotube radical functionalization, the chemical grafting, and the polymerization reaction were obtained over CNx when NMRP method was successfully used, giving rise to a uniform PS layer of several nanometers grafted on the outer surface of the CNx nanotubes. Several properties of the PS-grafted CNx nanotubes were also studied. It is shown that the production method leads to a narrower distribution of the external diameters. Moreover, their solubilization in organic solvents is greatly improved. Finally, the dispersion of PS-grafted CNx into a PS matrix is studied to determine the differences in filler dispersion and interfacial adhesion strength, in comparison with nanocomposites elaborated with as-produced CNx.

Characteristics of glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study.

The metabolism of glutamine, a physiological substrate of the human kidney, plays a major role in systemic acid-base homoeostasis. Not only because of the limited availability of human renal tissue but also in part due to the lack of adequate cellular models, the mechanisms regulating the renal metabolism of this amino acid in humans have been poorly characterized. Therefore given the renewed interest in their use, human precision-cut renal cortical slices were incubated in Krebs-Henseleit medium (118 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4*7H2O, 24.9 mM NaHCO3 and 2.5 mM CaCl2*2H2O) with 2 mM unlabelled or 13C-labelled glutamine residues. After incubation, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. Glutamate accumulation tended to plateau but glutamine removal and ammonia, alanine and lactate production as well as flux through GLDH (glutamate dehydrogenase) increased to various extents with time for up to 4 h of incubation indicating the metabolic viability of the slices. Valproate, a stimulator of renal glutamine metabolism, markedly and in a dose-dependent fashion increased ammonia production. With [3-13C]glutamine as a substrate, and in the absence and presence of valproate, [13C]glutamate, [13C]alanine and [13C]lactate accounted for 81 and 96%, 34 and 63%, 30 and 46% of the glutamate, alanine and lactate accumulations measured enzymatically respectively. The slices also metabolized glutamine and retained their reactivity to valproate during incubations lasting for up to 48 h. These results demonstrate that, although endogenous metabolism substantially operates in the presence of glutamine, human precision-cut renal cortical slices are metabolically viable and strongly respond to the ammoniagenic effect of valproate. Thus, this experimental model is suitable for metabolic and pharmaco-toxicological studies.

Metabolic viability and pharmaco-toxicological reactivity of cryopreserved human precision-cut renal cortical slices.

We have tested the suitability of cryopreserved human precision-cut renal cortical slices for metabolic and pharmaco-toxicological studies. The viability of these slices and their pharmaco-toxicological reactivity were assessed using intracellular ATP and protein contents, lactate dehydrogenase (LDH) leakage, lactate and glutamine metabolism and the ammoniagenic effect of valproate. Despite a decrease in ATP and protein contents when compared with those of fresh slices, cryopreserved slices did not show any LDH leakage and retained the capacity to metabolize glutamine and lactate. Glutamine removal and ammonia, lactate and alanine production were similar in fresh and cryopreserved slices; by contrast, cryopreserved slices accumulated more glutamate as a result of decreased flux through glutamate dehydrogenase which catalyses an oxygen-dependent reaction. Valproate markedly and similarly stimulated glutamine metabolism in fresh and cryopreserved slices. Cryopreservation did not alter lactate removal but inhibited lactate gluconeogenesis. In conclusion, these results demonstrate that, although their mitochondrial oxidative metabolism seems to be diminished, cryopreserved human precision-cut renal cortical slices remain metabolically viable and retain the capacity to respond to the ammoniagenic effect of valproate. Thus, this experimental model may be helpful to optimize the use of human renal tissue for metabolic and pharmaco-toxicological studies.