Profiling of signal transduction in human memory T cells

Résumé pour un large public: La vaccination a eu un impact énorme sur la santé mondiale. Mais, quel est le principe d'un vaccin? Il est basé sur la 'mémoire immunologique', qui est une particularité exclusive des systèmes immunitaires des organismes évolués. Suite à une infection par un pathogène, des cellules spécialisées de notre système immunitaire (les lymphocytes) le reconnaissent et initient une réaction immunitaire qui a pour but son élimination. Pendant cette réaction se développent aussi des cellules, appelées cellules lymphocytaires mémoire, qui persistent pour longue durée et qui ont la capacité de stimuler une réaction immunitaire très efficace immédiatement après une seconde exposition à ce même pathogène. Ce sont ces cellules mémoires (lymphocytes B et T) qui sont à la base de la 'mémoire immunologique' et qui sont stimulées lors de la vaccination. Chez l'homme, deux populations distinctes des lymphocytes T mémoires ont été identifiées: les cellules centrales (CM) et effectrices (EM) mémoires. Ces populations sont fonctionnellement hétérogènes et exercent des rôles distincts et essentiels dans l'immunité protectrice. Typiquement, les cellules effectrices mémoires sont capables de tuer immédiatement le pathogène tandis que les cellules centrales mémoires sont responsables d'initier une réponse immunitaire complète. Pourtant, les mécanismes biochimiques qui contrôlent les fonctions de ces cellules ont été jusqu'à présent peu étudiés à cause de la faible fréquence de ces cellules et de la quantité limitée de tissus humains disponibles pour les analyses. La compréhension de ces mécanismes est cruciale pour la réalisation de vaccins efficaces et pour le développement de nouveaux médicaments capables de moduler la réponse immunitaire lymphocytaire. Dans cette thèse, nous avons d'abord développé et amélioré une technologie appelée 'protéine array en phase inverse' qui possède un niveau de sensibilité beaucoup plus élevé par rapport aux technologies classiquement utilisées dans l'étude des protéines. Grâce à cette technique, nous avons pu comparer la composition protéique du système de transmission des signaux d'activation des cellules CM et EM humaines. L'analyse de 8 à 13 sujets sains a montré que ces populations des cellules mémoires possèdent un système de signalisation protéique différent. En effet, les cellules EM possèdent, par rapport aux cellules CM, des niveaux réduits d'une protéine régulatrice (appelée c-Cbl) que nous avons démontré comme étant responsable des fonctions spécifiques de ces cellules. En effet, en augmentant artificiellement l'expression de cette protéine régulatrice dans les cellules EM jusqu'au niveau de celui des cellules CM, nous avons induit dans les cellules EM des capacités fonctionnelles caractéristiques des cellules CM. En conclusion, notre étude a identifié, pour la première fois chez l'homme, un mécanisme biochimique qui contrôle les fonctions des populations des cellules mémoires. Résumé en Français: Les cellules mémoires persistent inertes dans l'organisme et produisent des réactions immunitaires rapides et robustes contre les pathogènes précédemment rencontrés. Deux populations distinctes des cellules mémoires ont été identifiées chez l'homme: les cellules centrales (CM) et effectrices (EM) mémoires. Ces populations sont fonctionnellement hétérogènes et exercent des rôles distincts et critiques dans l'immunité protectrice. Les mécanismes biochimiques qui contrôlent leurs fonctions ont été jusqu'à présent peu étudiés, bien que leur compréhension soit cruciale pour le développement des vaccins et des nouveaux traitements/médicaments. Les limites majeures à ces études sont la faible fréquence de ces populations et la quantité limitée de tissus humains disponibles. Dans cette thèse nous avons d'abord développé et amélioré la technologie de 'protéine array en phase inverse' afin d'analyser les molécules de signalisation des cellules mémoires CD4 et CD8 humaines isolées ex vivo. L'excellente sensibilité, la reproductibilité et la linéarité de la détection, ont permis de quantifier des variations d'expression protéiques supérieures à 20% dans un lysat équivalent à 20 cellules. Ensuite, grâce à l'analyse de 8 à 13 sujets sains, nous avons prouvé que les cellules mémoires CD8 ont une composition homogène de leur système de signalisation tandis que les cellules CD4 EM expriment significativement de plus grandes quantités de SLP-76 et des niveaux réduits de c-Cbl, Syk, Fyn et LAT par rapport aux cellules CM. En outre, l'expression réduite du régulateur négatif c-Cbl est corrélée avec l'expression des SLP-76, PI3K et LAT uniquement dans les cellules EM. L'évaluation des propriétés fonctionnelles des cellules mémoires a permis de démontrer que l'expression réduite du c-Cbl dans les cellules EM est associé à une diminution de leur seuil d'activation. En effet, grâce a la technique de transduction cytosolique, nous avons augmenté la quantité de c-Cbl des cellules EM à un niveau comparable à celui des cellules CM et constaté une réduction de la capacité des cellules EM à proliférer et sécréter des cytokines. Ce mécanisme de régulation dépend principalement de l'activité d'ubiquitine ligase de c-Cbl comme démontré par l'impact réduit du mutant enzymatiquement déficient de c-Cbl sur les fonctions de cellules EM. En conclusion, cette thèse identifie c-Cbl comme un régulateur critique des réponses fonctionnelles des populations de cellules T mémoires et fournit, pour la première fois chez l'homme, un mécanisme contrôlant l'hétérogénéité fonctionnelle des ces cellules. De plus, elle valide l'utilisation combinée des 'RPP arrays' et de la transduction cytosolique comme outil puissant d'analyse quantitative et fonctionnel des protéines de signalisation. Summary : Memory cells persist in a quiescent state in the body and mediate rapid and vigorous immune responses toward pathogens previously encountered. Two subsets of memory cells, namely central (CM) and effector (EM) memory cells, have been identified in humans. These subsets display high functional heterogeneity and assert critical and distinct roles in the control of protective immunity. The biochemical mechanisms controlling their functional properties remain so far poorly investigated, although their clarification is crucial for design of effective T-cell vaccine and drug development. Major limitations to these studies lie in the low frequency of memory T cell subsets and the limited amount of human specimen available. In this thesis we first implemented the innovative reverse phase protein array approach to profile 15 signalling components in human CD8 and CD4 memory T cells isolated ex vivo. The high degree of sensitivity, reproducibility and linearity achieved, allowed an excellent quantification of variations in protein expression higher than 20% in as few as 20-cell equivalent per spot. Based on the analysis of 8 to 13 healthy subjects, we showed that CD8 memory cells have a homogeneous composition of their signaling machinery while CD4 EM cells express statistically significant increased amounts of SLP-76 and reduced levels of c- Cbl, Syk, Fyn and LAT as compared to CM cells. Moreover, in EM but not CM cells, reduced expression of negative regulator c-Cbl correlated with the expression of SLP-76, PI3K and LAT. Subsequently, we demonstrated that the higher functional properties and the lower functional threshold of EM cells is associated with reduced expression of c-Cbl. Indeed, by increasing c-Cbl content of EM cells to the same level of CM cells using cytosolic transduction, we impaired their proliferation and cytokine production. This regulatory mechanism was primarily dependent on c-Cbl E3 ubiquitin ligase activity as evidenced by the weaker impact of enzymatically deficient c-Cbl C381A mutant on EM cell functions. Together, these results identify c-Cbl as a critical regulator of the functional responses of memory T cell subsets and provides, for the first time in humans, a mechanism controlling the functional heterogeneity of memory CD4 cells. Moreover it validates the combined use of RPP arrays and cytosolic transduction approaches as a powerful tool to quantitatively analyze signalling proteins and functionally assess their roles

IL-25 participates in keratinocyte-driven dermal matrix turnover and is reduced in systemic sclerosis epidermis

OBJECTIVES: Evidence shows that dysfunctional SSc keratinocytes contribute to fibrosis by altering dermal homeostasis. Whether IL-25, an IL-17 family member regulating many epidermal functions, takes part in skin fibrosis is unknown. Here we address the role of IL-25 in skin fibrosis. METHODS: The expression of IL-25 was evaluated by immunofluorescence and in situ hybridization in 10 SSc and seven healthy donor (HD) skin biopsies. Epidermal equivalents (EE) reconstituted by primary HD keratinocytes were used as a model to study transcriptomic changes induced by IL-25 in the epidermis. RNA expression profile in EEs was characterized by RNAseq. The conditioned medium (CM) from primary SSc and HD keratinocytes primed with IL-25 was used to stimulate fibroblasts. IL-6, IL-8, MMP-1, type-I collagen (Col-I), and fibronectin production by fibroblasts was assessed by ELISA. RESULTS: SSc epidermis expressed lower levels of IL-25 compared with HDs. In EEs, IL-25 regulated several molecular pathways related to wound healing and extracellular matrix remodelling. Compared with control CM, the CM from IL-25-primed keratinocytes enhanced the fibroblast production of MMP-1, IL-6 and IL-8, but not of Col-I nor fibronectin. However, IL-25 significantly reduced the production of Col-I when applied directly to fibroblasts. The activation of keratinocytes by IL-25 was receptor-dependent and evident after a very short incubation time (10 min), largely mediated by IL-1, suggesting enhanced and specific release of preformed mediators. CONCLUSIONS: These results show that IL-25 participates in skin homeostasis, and its decreased expression in SSc may contribute to skin fibrosis by favouring extracellular matrix deposition over degradation

Algoritmos de Detecçao de Taquicardias Incorporado a Desfibriladores Automáticos Implantáveis. 1) Desfibriladores Monocamerais

Diversos algoritmos foram incorporados aos cardioversores-desfibriladores automáticos implantáveis (CDIs) para identificar os distúrbios do ritmo ventricular e, sobretudo, para os diferenciar de taquicardias supraventriculares que nao necessitam terapia. Esses benefícios também sao encontrados nos CDIs bicamerais que têm como benefício a detecçao atrial acoplada à detecçao do ventrículo. O objetivo dos algoritmos é de identificar todas as arritmias ventriculares (sensibilidade de 100%), para que sejam tratadas corretamente. Devem ainda evitar erros de identificaçao de arritmias supraventriculares (especificidade máxima). Infelizmente, nao é possível alcançar 100% de sensibilidade e especificidade. Além disso, todo aumento da especificidade será acompanhado por uma diminuiçao da sensibilidade. Essa diminuiçao de especificidade pode conduzir a falha na detecçao dos distúrbios do ritmo ventricular, e como conseqüência, isto é pior que o tratamento inadequado de uma taquicardia sinusal ou supraventricular

Algoritmos de Detecçao de Taquicardias Incorporado a Desfibriladores Automáticos Implantáveis. 1) Desfibriladores Monocamerais

Diversos algoritmos foram incorporados aos cardioversores-desfibriladores automáticos implantáveis (CDIs) para identificar os distúrbios do ritmo ventricular e, sobretudo, para os diferenciar de taquicardias supraventriculares que nao necessitam terapia. Esses benefícios também sao encontrados nos CDIs bicamerais que têm como benefício a detecçao atrial acoplada à detecçao do ventrículo. O objetivo dos algoritmos é de identificar todas as arritmias ventriculares (sensibilidade de 100%), para que sejam tratadas corretamente. Devem ainda evitar erros de identificaçao de arritmias supraventriculares (especificidade máxima). Infelizmente, nao é possível alcançar 100% de sensibilidade e especificidade. Além disso, todo aumento da especificidade será acompanhado por uma diminuiçao da sensibilidade. Essa diminuiçao de especificidade pode conduzir a falha na detecçao dos distúrbios do ritmo ventricular, e como conseqüência, isto é pior que o tratamento inadequado de uma taquicardia sinusal ou supraventricular

Assessing daylight performance in use: A comparison between long-term daylight measurements and simulations

Climate-Based Daylight Modelling (CBDM) methods have been validated against long-term measurements in laboratory settings and found to exhibit errors small enough to make such assessments useful for daylight performance prediction. However, real occupied spaces are affected by a higher number of uncertainties than laboratory or controlled conditions. This study aims at validating CBDM methods against measurements collected in an occupied classroom space, where a monitoring system based on High Dynamic Range Imaging was installed. Four vertical regions were identified on two of the room's walls, and mean illuminance was calculated for these regions at every time step, both from HDR images and from simulated results. Two simulation methods were evaluated: the 2-phase and the 4-component methods. Sun and sky conditions for the simulations were derived from simultaneous monitored irradiation measurements. Both simulation methods led to moderate over-prediction of HDR-derived results, when considering instantaneous illuminance means and when looking at long-term metrics (cumulative irradiation and Useful Daylight Illuminance). Wall regions exposed to more direct sky- and sunlight were characterised by smaller systematic errors (rMBE = 4%) but similar variance (r2 = 0.83) than regions situated at the back of the room (rMBE = 17–34% and rMAE = 27–37%). Further studies are needed to identify and separate the sources of such errors.Building Physic

Interleukin-17E, inducible nitric oxide synthase and arginase1 as new biomarkers in the identification of neutrophilic dermatoses.

BACKGROUND Neutrophilic dermatoses (ND) are a heterogeneous group of diseases, but can often have a relatively similar histological appearance. AIM To identify a combination of biomarkers allowing a better differentiation of ND types. METHODS Biopsies were obtained from normal human skin (NS; n = 4), chronic plaque-type psoriasis (PsO; n = 7), paradoxical psoriasis (PP; n = 8), generalized pustular psoriasis (GPP; n = 9), subcorneal pustular dermatosis of Sneddon-Wilkinson (SPD; n = 3), acute generalized exanthematous pustulosis (AGEP; n = 3), hidradenitis suppurativa (HS; n = 7), Sweet syndrome (SS; n = 8) and pyoderma gangrenosum (PG; n = 8). Samples were analysed by immunofluorescence using three biomarkers, interleukin (IL)-17E, inducible nitric oxide synthase (iNOS) and arginase1, each one in combination with two cell markers, myeloperoxidase (MPO) and CD68, which allow the identification of neutrophils and macrophages, respectively. RESULTS We found that SS is characterized by high expression of IL-17E and iNOS in the epidermis, while PG exhibits low expression. The density of the neutrophil infiltrate helps to differentiate PP (high-density infiltrate) from PsO (low-density infiltrate). High expression of arginase1 in the granular layer of the epidermis is a hallmark of SPD. Finally, mature neutrophils and proinflammatory macrophages are readily detectable in PP, SPD and PG, whereas immature neutrophils and anti-inflammatory macrophages are more frequent in GPP, AGEP, HS and SS. CONCLUSIONS The analysis of ND by immunofluorescence using IL-17E, iNOS and arginase1 in combination with MPO and CD68 allows for characterization of differential expression patterns in the epidermis as well as the determination of the polarization status of the dermal neutrophils and macrophages. The appropriate markers may help in the differentiation of ND in clinical practice

Wolff-Parkinson-White syndrome in the elderly: clinical and electrophysiological findings

SummaryBackgroundScreening for Wolff-Parkinson-White (WPW) syndrome is recommended in children and young adults. The aim of this study was to evaluate the clinical and electrophysiological characteristics of patent WPW syndrome in subjects ≥60 years of age.MethodsFour-hundred and fifty-nine consecutive patients with WPW syndrome, aged 8–80 years, were recruited; 32 (7%) of these patients were ≥60 years of age. The clinical, electrophysiological and therapeutic data for these patients were evaluated.ResultsSixteen men and 16 women, aged 60–81 years (67±4.5), were admitted for resuscitated sudden death (1), rapid atrial fibrillation (4), syncope (4), or junctional tachycardia (13); 10 patients were asymptomatic (10). Left lateral bundles of Kent were detected more frequently in patients over 60 years (56%) than in those < 60 years of age (40.5%). Reciprocal tachycardia was induced in 58% of subjects <60 years of age and 53% of those ≥60 years old (difference not significant); atrial fibrillation was more frequent in subjects ≥ 60 years of age (37.5%vs. 19%) (p<0.05). The incidence of malignant forms of WPW syndrome was identical in older and younger subjects. Ablation of the accessory pathway was indicated 18 times; effective ablation of a left bundle of Kent required a second intervention more often in patients ≥60 years of age (22%vs. 5%) (p<0.05).ConclusionWPW syndrome is not uncommon in subjects over 60 years of age (7%). Left lateral accessory pathways, that have similar conduction properties to those in much younger subjects, are common. Ablation of the bundle of Kent is often difficult but is indicated in symptomatic subjects or those with more serious forms of WPW syndrome

A Stepwise Approach to the Management of Postinfarct Ventricular Tachycardia Using Catheter Ablation as the First-Line Treatment: A Single-Center Experience

International audienceBACKGROUND:The occurrence of ventricular tachycardia (VT) after myocardial infarction is associated with poorer prognosis. In such patients, implantable cardioverter-defibrillators are recommended. Catheter ablation of VT is currently recommended only as an adjunctive therapy. Whether a successful VT ablation alone might be a viable strategy in some of these patients, however, remains unknown. The aim of the present study was to evaluate this strategy.METHODS AND RESULTS:Between January 2002 and December 2011, 189 patients with cardiomyopathy underwent 259 VT ablations in our center. Forty-five patients (mean age, 65.2±9.6 years; 91% men) with a history of myocardial infarction and mean left ventricular ejection fraction of 39.7±9.7% matched the study criteria and were included in this analysis. Acute success was obtained in 40 of 45 patients (88.9%). During a follow-up, on the basis of our stepwise algorithm (using acute success, repeat electrophysiological study, and recurrence of VT), 19 of 45 patients (42.2%) underwent implantable cardioverter-defibrillators implantation. During a median follow-up of 4.5 (interquartile range, 2.1-7.0) years, all-cause mortality occurred in 14 of 45 patients (31.1%). Using multivariate Cox regression analysis, age (hazard ratio, 1.13; 95% confidence interval, 1.03-1.22; P=0.007) was the only independent predictor of mortality, whereas implantable cardioverter-defibrillators implantation was not (hazard ratio, 0.54; 95% confidence interval, 0.18-1.64; P=0.28)CONCLUSIONS:Our results suggest that a stepwise approach to the management of VT with ablation as a first-line treatment in postinfarct patients presenting with VT might be a reasonable option. Further studies are required to confirm these results