Expression of apoptosis and survival genes in human memory T cell populations

RESUME Introduction: Les cellules T mémoires humaines sont classées en trois sous-populations sur la base de l'expression d'un marqueur de surface cellulaire, CD45RA, et du récepteur aux chimiokines, CCR7. Ces sous-populations, nommées cellules mémoires centrales (TcM), mémoires effectrices (TEM) et mémoires effectrices terminales (ITEM), ont des rôles fonctionnels distincts, ainsi que des capacités de prolifération et de régénération différentes. Cependant, la génération de ces différences reste encore mal comprise et on ignore les mécanismes moléculaires impliqués. Matériaux et Méthodes: Des cellules mononucléaires humaines du sang périphérique ont été séparées par cytométrie de flux selon leur expression de CD4, CD8, CD45RA et CCR7 en sous-populations de cellules CD4+ ou CD8+ naïves, TcM, TEM ou ITEM. Dans chacune de ces sous-populations, 14 gènes impliqués dans l'apoptose, la survie ou la capacité proliférative des cellules T ont été quantifiés par RT-PCR en temps réel, relativement à l'expression d'un gène de référence endogène. L'ARN provenant de 450 cellules T a été utilisé par gène et par sous-population. Les gènes analysés (cibles) comprenaient des gènes de survie (BAFF, APRIL, BAFF-R, BCMA, TACI, IL-15Rα, IL-7Rα), des gènes anti-apoptotiques (Bcl-2, BclxL, FLIP), des gènes pro-apoptotiques (Bad, Bax, Fast) et le gène anti-prolifératif, Tob. A l'aide de la méthode comparative delta-delta-CT, le taux d'expression des gènes cibles de chaque sous-population des cellules T mémoires CD4+ et CD8+, à été comparée à leur taux d'expression dans les cellules T naïves CD4+ et CD8+. Résultats: Dans les cellules CD8+, les gènes pro-apoptotiques Bax et Fast étaient surexprimés dans toutes les sous-populations mémoires, tandis que l'expression des facteurs anti-apoptotiques et de survie comme Bcl-2, APRIL et BAFF-R, étaient diminués. Ces deux tendances étaient particulièrement accentuées dans les sous-groupes des cellules mémoires TEM et TTEM. A noter que malgré le fait que leur expression était également diminuée dans les autres cellules mémoires, le facteur de survie IL-7Ra, était sélectivement surexprimé dans la sous-population de cellules TcM et l'expression d'IL-15Ra était sélectivement augmentée dans les TEM. Dans les cellules CD4+, le taux d'expression des gènes analysés était plus variable entre les sujets étudiés que dans les cellules CD8+, ne permettant pas de définir un profil d'expression spécifique. L'expression du gène de survie BAFF par contre, a été significativement augmentée dans toutes les sous-populations mémoire CD4+. Il en va de même pour l'expression d' APRIL et de BAFF-R, bien que dans moindre degré. A remarquer que l'expression du facteur anti-apoptotique Fast a été observée uniquement dans la souspopulation des TTEM. Discussion et Conclusions: Cette étude montre une nette différence entre les cellules CD8+ et CD4+, en ce qui concerne les profils d'expression des gènes impliqués dans la survie et l'apoptose des cellules T mémoires. Ceci pourrait impliquer une régulation cellulaire homéostatique distincte dans ces deux compartiments de cellules T mémoires. Dans les cellules CD8+ l'expression d'un nombre de gènes impliqués dans la survie et la protection de l'apoptose semblerait être diminuée dans les populations TEM et TTEM en comparaison à celle des sous-populations naïves et TEM, tandis que l'expression des gènes pro-apoptotiques semblerait être augmentée. Comme ceci paraît être plus accentué dans les TTEM, cela pourrait indiquer une plus grande disposition à l'apopotose dans les populations CCR7- (effectrices) et une perte de survie parallèlement à l'acquisition de capacités effectrices. Ceci parlerait en faveur d'un modèle de différentiation linéaire dans les cellules CD8+. De plus, l'augmentation sélective de l'expression d'IL-7Ra observée dans le sous-groupe de cellules mémoires TEM, et d'IL-15Ra dans celui des TEM, pourrait indiquer un moyen de sélection pour des réponses immunitaires mémoires à long terme par une réponse distincte à ces cytokines. Dans les cellules CD4+ par contre, aucun profil d'expression n'a pu être déterminé; les résultats suggèrent même une résistance relative à l'apoptose de la part des cellules mémoires. Ceci pourrait favoriser l'existence d'un modèle de différentiation plus flexible avec des possibilités d'interaction multiples. Ainsi, la surexpression sélective de BAFF, APRIL et BAFF-R dans les sous-populations individuelles des cellules mémoires pourrait être un indice de l'interaction de ces sous-groupes avec des cellules B. ABSTRACT Introduction: Based on their surface expression of the CD45 isoform and of the CCR7 chemokine receptor, memory T cells have been divided into the following three subsets: central memory (TAM), effector memory (TEM) and terminal effector memory (ITEM). Distinct functional roles and different proliferative and regenerative capacities have been attributed to each one of these subpopulations. The molecular mechanisms underlying these differences; however, remain poorly understood. Materials and Methods: According to their expression of CD4, CD8, CD45RA and CCR7, human peripheral blood mononuclear cells were sorted by flow-cytometry into CD4+ or CD8+ naïve, TAM, TEM and ITEM subsets. Using real-time PCR, the expression of 14 genes known to be involved in apoptotis, survival or proliferation of T cells was quantified separately in each individual subset, relative to an endogenous reference gene. The RNA equivalent of 450 T cells was used for each gene and subset. The target gene panel included the survival genes BAFF, APRIL, BAFF-R, BCMA, TACI, IL-15Rα and IL-7Rα, the anti-apoptotic genes Bcl2, Bcl-xL and FLIP, the pro-apoptotic genes Bad, Bax and Fast, as well as the antiproliferative gene Tob. Using the comparative CT-method, the expression of the target genes in the three memory T cell subsets of both CD4+ and CD8+ T cell populations was compared to their expression in the naïve T cells. Results: In CD8+ cells, the pro-apoptotic factors Bax and Fast were found to be upregulated in all memory T cell subsets, whereas the survival and anti-apoptotic factors Bcl-2, APRIL and BAFF-R were downregulated. These tendencies were most accentuated in TEM and TTEM subsets. Even though the survival factor IL-7Rα was also downregulated in these subsets, interestingly, it was selectively upregulated in the CD8+ TAM subset. Similarly, IL-15Rαexpression was shown to be selectively upregulated in the CD8+ TEM subset. In CD4+ cells, the expression levels of the analyzed genes showed a greater inter-individual variability than in CD8+ cells, thus suggesting the absence of any particular expression pattern for CD4+ memory T cells. However, the survival factor BAFF was found to be significantly upregulated in all CD4+ memory T cell subsets, as was also the expression of APRIL and BAFF-R, although to a lesser extent. Furthermore, it was noted that the pro-apoptotic gene Fast was only expressed in the TTEM CD4+ subset. Discussion and Conclusions: Genes involved in apoptosis and survival in human memory T cells have been shown to be expressed differently in CD8+ cells as compared to CD4+ cells, suggesting a distinct regulation of cell homeostasis in these two memory T cell compartments. The present study suggests that, in CD8+ T cells, the expression of various survival and antiapoptotic genes is downregulated in TEM and TTEM subsets, while the expression of proapoptotic genes is upregulated in comparison to the naïve and the TAM populations. These characteristics, potentially translating to a greater susceptibility to apoptosis in the CCR7- (effector) memory populations, are accentuated in the TTEM population, suggesting a loss of survival in parallel to the acquisition of effector capacities. This speaks in favour of a linear differentiation model in CD8+ T memory cells. Moreover, the observed selectively increased expression of IL-7Rα in CD8+ TAM cells - as that of IL-15Rα in CD8+ TEM cells -suggest that differential responsiveness to cytokines could confer a selection bias for distinct long-term memory cell responses. Relative to the results for CD8+ T cells, those for CD4+ T cells seem to indicate a certain resistance of the memory subsets to apoptosis, suggesting the possibility of a more flexible differentiation model with multiple checkpoints and potential interaction of CD4+ memory cells with other cells. Thus, the selective upregulation of BAFF, APRIL and BAFF-R in individual memory subsets could imply an interaction of these subsets with B cells.

Phytochemical investigation of alpine plants : the cottonsedges Eriophorum scheuchzeri Hoppe, E. angustifolium Honck. and E. latifolium Hoppe (CYPERACEAE)

Résumé: Alpine plants living at high altitudes undergo a series of climatic stress factors (chilling, enhanced UV radiation, short growing season, low nutriment supply...) which may influence their secondary compounds composition. Many publications showed in these last years that plants under stress conditions do synthesize a range of specific defence compounds (terpenes, flavonoids, coumarines...). A careful phytochemical investigation of those plants could therefore lead to the discovery of active molecules. Thus, for the biological and chemical screening, about 30 alpine plants have been collected above 2000 metres, in the alpine grass-lands. Eriophorum scheuchzeri Hoppe (Cyperaceae), not yet investigated phytochemically, revealed in its lipophilic and polar extracts the presence of various radical scavengers in a TLC autography with the DPPH (2,2-dipheny1-1- picrylhydrazyl) radical as spray reagent, as well as several antifungal compounds acitve against Cladosporium cucumerinum and Candida albi cans. The first part of this study consisted in the detection, isolation and characterization of the bioactive natural compounds present in the lipophilic extract of Eriophorum scheuchzeri. Among the eight isolated compounds, six were isoflavones. No isoflavones have been reported in the Cyperaceae family yet, nor in related families such as Poaceae or Juncaceae. Besides, isoflavones are generally rare in the plant kingdom and and they occur only in some families, such as Fabaceae, Rosaceae or Myristicaceae. In addition, out of these six isoflavones, three were new isoflavones. The known compounds were parvisoflavone A and B and cajanin which are already known isoflavones in the Fabaceae family. Two of the new isoflavones were particular, as they were C-methylated on the B-ring at the C-3' position. Methylated flavonoids are particularly rare in the plant kingdom. At present, no C-methylated isoflavones with methyl groups on the B-ring have ever been reported. The fourth new compound was a prenylated flavanone. Flavanones are also rare in the Cyperaceae family since they were found only in two genera (Cyperus and Schoenus). Finally, the widespread flavone tricin, characteristic of the Cyperaceae and Poaceae family has also been isolated. The second part of this study consisted in the characterization of the polar components present in the Me0H extract. In order to obtain mass and UV information about the secondary compounds present in the Eriophorum scheuchzeri methanolic extract, a LC-UV/DAD-APCl/MSn analysis has been performed as a first dereplication step. The UV/DAD spectra showed the presence of polyphenol compounds (phenylpropanoids and flavonoids). The LC-APCI/MSn analysis allowed the determination of the molecular weight of these compounds. Moreover, the fragmentation pattern of the [M+H]+ ions indicated presence of mono-, di- and tri-glycosides. LC-UV in combination with UV shift reagents added post-column was used in a second phase for the structural elucidation of the flavonoids. It allowed the positioning of the sugars on the aglycones. Finally, LC-NMR was used for a more detailed structural investigation of the compounds present in the crude MEOH extract. Thus, 10 fiavonoids have been totally or partially characterized by LC-UV-MS and LC-1H-RMN and UV-shift reagents added post column. However, the information obtained on-line was not always sufficient to allow a complete identification of all the compounds. Some of these compounds especially those with more than two sugar units attached to them, have been isolated in order to proceed to their complete characterization. Moreover, the Eriophorum scheuchzeri species was compared to two other species from the same genus. A LC-UV-ESI/MS analysis enabled a survey of the chemical composition of the DCM extracts of two related species E. angustifolium (Honck) and E. latifolium (Hoppe). The chromatograms of the three species showed some similarities in their flavonoid contents, especially by the recurrent presence of three compounds. The MEOH extracts of all three species have been compared by means of LC-UV-APCl/MS analyses. The chromatographic profile of all the three species showed even closer similarities than those found in the DCM extracts. E. angustifolium Honck. and E. latifolium species showed 7 compounds in common. Finally, the pure compounds obtained from the DCM (CH2Cl2) fraction were tested at different concentration, in order to evaluate their chemical and biological activities. All eight compounds showed an anti-scavenger activity against the DPPH radical, and four compounds showed antifungal activities against Cladosporium cucumerinum and Candida albicans. The pure compounds isolated from the MeOH extract were tested only for their biological activities as their antioxidant activity is already well documented in the literature. No compound showed a biological activity against Cladosporium cucumerinum and Candida albicans. Résumé: De nombreux travaux ont démontré ces dernières décennies que les plantes soumises à différents types de stress (basse température, UV, stress hydrique) synthétisent des composés secondaires (fiavonoides, coumarines, terpènes...) de protection et de défense. Les plantes d'altitude par exemple qui sont exposées à des conditions climatiques et environnementales difficiles, ont tendance à synthétiser des substances antioxydantes et antiradicalaires. Une investigation phytochirnique de ces plantes a conduit à la découverte de nouvelles molécules actives. Ainsi plusieurs plantes alpines ont été sélectionnées en fonction de leur habitat en vue de les soumettre aux tests biologiques (antifongiques) et chimiques (antiradicalaires) menés en routine dans notre laboratoire. Dans ce criblage biologique préliminaire, les extraits d'Eriophorum scheuchzeri Hoppe (Cyperaceae) ont réagi positivement aux différents tests. Il a donc été décidé d'entreprendre l'isolement des composés actifs. La première partie de ce travail a consisté à détecter, isoler et caractériser les composés naturels actifs présents dans l'extrait apolaire d' Eriophorum scheuchzeri. Parmi les huit composés isolés, quatre d'entre eux sont nouveaux. Un de ces produits est une flavanone et trois sont de nouvelles isoflavones, particulièrement intéressantes car elles possèdent des groupements C-méthylés au niveau du cycle B. Les flavonoides C-méthylés sont peu répandus dans le règne végétal et les rares exemples connus sont généralement C-méthylés sur le cycle A. Les quatre autres composés isolés n'ont jamais été décrits dans cette famille. Il s'agit d' isoflavones, les parvisoflavones A et B et la cajanine. Enfin, la flavone tricine, flavonoide caractéristique des Cyperaceae et des Poaceae a également été isolée. La deuxième partie de ce travail a consisté à caractériser les constituants polaires présents dans l'extrait methanolique. L'extrait a été analysé par chromatographie analytique couplée à différentes méthodes spectroscopiques (LC-UV-MS et LC-UV-1H RMN). De cette façon, douze flavonoides et un dérivé du phénylpropane, l'acide chlorogénique ont été identifiés. Les flavonoides tri-glycosylés ont dû être isolés afin de déterminer la nature et l'enchaînement des sucres. Finalement, l'espèce Eriophorum scheuchzeri a été comparée à deux autres espèces d' Eriophorum, soit E. angustifolium et E. latifolium. En conclusion, cette étude phytochimique a abouti à l'isolement de plusieurs nouvelles isoflavones aux activités antioxydantes et antifongiques ainsi qu'oestrogéniques.

Human CD8 T-cell responses to Epstein-Barr virus and Cytomegalovirus in healthy donors and melanoma patients

Summary The mechanisms regulating the protective immune T-cell responses generated against the persistent Epstein-Barr virus (EBV) and Cytomegaloviru_s (CNIV) remain poorly understood. We analyzed the dynamics of cellular differentiation and T-cell receptor (TCR) clonotype selection of EBV- and CMV-specific T-cells in healthy adults and melanoma patients. While these responses could be subdivided into four T lymphocyte populations, théir proportions varied between EBV and CMV specific responses. Phenotypic and TCR clonotypic analyses supported a linear model of differentiation from the early-differentiated (EM/CD28pos) subset to the late-differentiatdc (EMRA/CD28neg) subset. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28neg subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, as some clonotypes were selected with differentiation, while others were not. Latedifferentiated CMV-specific clonotypes were mostly characterized by TCRs with lower dependency on CD8 co-receptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of four years. This work was extended to the study of EBV-specific CD8 T-cell responses in melanoma patients undergoing transient lymphodepletion, followed by adoptive cell transfer (ACT) and immune reconstitution for thè treatment of their tumors. Following treatment regimen, we first observed an increase in the proportion of virus-specific T-cells in 3 out of 5 patients, accompanied by a more differentiated phenotype (EMRA/CD28neg), compared to specific cells of healthy individuals. Yet, similarly to healthy donors, clonotype selection and composition of virus-specific T-cells varied along the pathway of cellular differentiation, with some clonotypes being selected with differentiation, while others were not. Intriguingly, no novel clonotypes emerged following transient immuno-suppression and homeostatic proliferation, finding which was subsequently explained by the absence of EBV reactivation. The distribution of each clonotype within early- and late-differentiated T-cell subsets in 4 out 5 patients was highly stable over time, with those clonotypes initially found before the start of treatment that were again present at specific differentiation stages after transient lymphodepletion and ACT. These findings uncover novel features of the highly sophisticated control of steady state protective T-cell immune responses against persistent herpesviruses in healthy adults. Furthermore they reveal the striking stability of these responses in terms of clonotype selection and composition with T-cell differentiation even in situations where the immune system has been. challenged. Résumé : Les mécanismes qui régulent les réponses immunitaires de type protectrices, générées contre les virus chroniquement persistants tels que l'Epstein-Barr (EBV) ou le Cytomegalo (CMV) restent largement inconnus. Nous avons analysé la différenciation des lymphocytes T spécifiques pour ces virus, ainsi que la composition des clonotypes T (par leur récepteur T) chez les donneurs sains. Les réponses immunes peuvent être classifiées en quatre souspopulations majeures de lymphocytes T, cependant, leur proportion varie entre les réponses spécifiques contre EBV ou CMV. Ces analyses soutiennent le modèle linéaire de différenciation, à partir de la population non différenciée (EM/CD28pos) vers la population plus différenciée (ENIIZA/CD28neg). De plus, nos données sur la composition clonale de ces cellules T spécifiques ont révélé des répertoires TCR restreints, pour la réponse anti-CMV, et relativement diversifiés contre EBV. Tous les clonotypes spécifiques de ces virus identifiés dans la sous-population différenciée EMRA/CD28neg, ont également été retrouvés dans la population de cellules "mémoires". Toutefois, de fortes différences ont été observées dans les schémas de domination de ces sous-populations, en effet, certains clonotypes étaient sélectionnés avec la différenciation, alors que d'autres ne l'étaient pas. Nous avons également démontré que ces clonotypes différenciés et spécifiques pour le CMV sont caractérisés par des TCRs à faible dépendance en regard de la coopération du corécepteur CD8. Néanmoins, tous les clonotypes affichent une avidité fonctionnelle similaire, suggérant un rôle compensatoire du CD8, dans le cas des clonotypes avec une faible avidité du TCR En définitive, la composition et la sélection des clonotypes spécifiques pour chaque virus et pour chaque sous-population suit un schéma de différenciation hautement conservé au cours du temps, avec la présence de ces mêmes clonotypes au même stade de différenciation sur une période de quatre ans. Ce travail a été étendu à l'étude des réponses T CD8+ spécifiques pour le virus EBV chez les patients atteints de mélanome et recevant dans le cadre du traitement de leurs tumeurs une lymphodéplétion transitoire, suivie d'un transfert adoptif de cellules et d'une reconstitution immunitaire. Au cours de cette thérapie, nous avons en premier lieu observé pour 3 des 5 patients une augmentation de la proportion de cellules T spécifiques pour le virus, accompagné d'un phénotype plus différencié (EMRA/CD28neg), et ceci comparativement à des cellules spécifiques d'individus sains. Pourtant, comme nous l'avons observé chez les donneurs sains, la sélection et la composition des clonotypes T spécifiques varient tout au long de la différenciation cellulaire, avec certains clonotypes sélectionnés et d'autres qui ne le sont pas. Étonnamment, aucun nouveau clonotype n'a émergé après l'immuno-suppression transitoire et la prolifération homéostatique. Cette observation trouve son explication par une absence de réactivation du virus EBV chez ces patients, et ce malgré leur traitement. De plus, la distribution de chaque clonotype parmi ces sous-populations non-différenciées et différenciées reste stable au cours du traitement. Ainsi, les mêmes clonotypes initialement identifiés avant le début du traitement sont présents aux mêmes stades de différenciation après la lymphodéplétion et la prolifération homéostatique. Ces résultats ont permis d'identifier de nouveaux mécanismes impliqués dans la régulation hautement «sophistiquée » des réponses immunitaires T contre les virus persistants EBV et CMV chez les donneurs sains. En particulier, ils révèlent la grande stabilité de ces réponses en termes de sélection et de composition des clonotypes avec la différenciation cellulaire, et ce dans les situations chroniques, ainsi que dans les situations dans lesquelles le système immunitaire a été profondément perturbé.

Visuospatial working memory deficits and visual pursuit impairments are not directly related in schizophrenia.

OBJECTIVE: Patients with schizophrenia show deficits in visuospatial working memory and visual pursuit processes. It is currently unclear, however, whether both impairments are related to a common neuropathological origin. The purpose of the present study was therefore to examine the possible relations between the encoding and the discrimination of dynamic visuospatial stimuli in schizophrenia. METHOD: Sixteen outpatients with schizophrenia and 16 control subjects were asked to encode complex disc displacements presented on a screen. After a delay, participants had to identify the previously presented disc trajectory from a choice of six static linear paths, among which were five incorrect paths. The precision of visual pursuit eye movements during the initial presentation of the dynamic stimulus was assessed. The fixations and scanning time in definite regions of the six paths presented during the discrimination phase were investigated. RESULTS: In comparison with controls, patients showed poorer task performance, reduced pursuit accuracy during incorrect trials and less time scanning the correct stimulus or the incorrect paths approximating its global structure. Patients also spent less time scanning the leftmost portion of the correct path even when making a correct choice. The accuracy of visual pursuit and head movements, however, was not correlated with task performance. CONCLUSIONS: The present study provides direct support for the hypothesis that active integration of visuospatial information within working memory is deficient in schizophrenia. In contrast, a general impairment of oculomotor mechanisms involved in smooth pursuit did not appear to be directly related to lower visuospatial working memory performance in schizophrenia.

Improvement of allocentric spatial memory resolution in children from 2 to 4 years of age

Allocentric spatial memory, the memory for locations coded in relation to objects comprising our environment, is a fundamental component of episodic memory and is dependent on the integrity of the hippocampal formation in adulthood. Previous research from different laboratories reported that basic allocentric spatial memory abilities are reliably observed in children after 2 years of age. Based on work performed in monkeys and rats, we had proposed that the functional maturation of direct entorhinal cortex projections to the CA1 field of the hippocampus might underlie the emergence of basic allocentric spatial memory. We also proposed that the protracted development of the dentate gyrus and its projections to the CA3 field of the hippocampus might underlie the development of high-resolution allocentric spatial memory capacities, based on the essential contribution of these structures to the process known as pattern separation. Here, we present an experiment designed to assess the development of spatial pattern separation capacities and its impact on allocentric spatial memory performance in children from 18 to 48 months of age. We found that: (1) allocentric spatial memory performance improved with age, (2) as compared to younger children, a greater number of children older than 36 months advanced to the final stage requiring the highest degree of spatial resolution, and (3) children that failed at different stages exhibited difficulties in discriminating locations that required higher spatial resolution abilities. These results are consistent with the hypothesis that improvements in human spatial memory performance might be linked to improvements in pattern separation capacities.

Sentinel nodes and and non sentinel nodes evaluation in T1 melanoma patients

Objective: Because increasing incidence of melanoma and dermatologicsystematic screening, more early superficial melanoma are discovered in Switzerland. Patients with Breslow index more than 1 mm. (T2) represent the classical indication to sentinel node (SN). It has been shown that some ''risky'' T1 patients may have micrometastatic SNs. T1b melanoma are defined by presence of ulceration,Clark IV (ormore) level, signs of melanoma regression (old classification) and high mitotic index (new TNM). The objective of the present study was to review the incidence and risk for metastatic SN in T1 patients and if radical lymph node dissection is justified (evaluation of non sentinel node [NSN]) compared with T2-4 patients.Methods: Retrospective review of a cohort of all patients operated for T1-4 clinically N0 and radiological M0 melanoma patients between 1997 and 2010 in a reference melanoma centre.Results: 599 melanoma patients have been operated with SNdissection. There were 98 T1 patients. Metastatic SN were observed in 2 out of 24 T1a patients and in 5 out of 74 T1b patients. This means overall 7% T1 patients were at least N1a. None of SN+ T1a or T1b patients had metastatic NSN after radical lymph node dissections (RLND). During the follow-up (1998-2010), no patients presented with locoregional disease and only one T1a N1a patient died of metastatic melanoma. These results contrast with the other 591 T2-4 patients: 150 were SN+ (25%) and among them 23 had metastatic NSN after RLND. Overall 23/136 (17%) had metastatic NSN.Conclusion: T1 melanoma patients are at significant risk (7%) for metastatic lymph node in the corresponding drainage basin. T1a and T1b did not differ regarding this risk. However, the benefit for a RLND must be reevaluated regarding surgical morbidity, because none of T1 patients had metastatic NSN.

Older persons' perceptions of general practitioner or specialist primary care physicians : same point of view?

BACKGROUND: Identification of a Primary Care Physician (PCP) by older patients is considered as essential for the coordination of care, but the extent to which identified PCPs are general practitioners or specialists is unknown. This study described older patients' experiences with their PCP and tested the hypothesis of differences between patients who identify a specialist as their PCP (SP PCP) and those who turn to a general practitioner (GP PCP). METHODS: In 2012, a cross-sectional postal survey on care was conducted in the 68+ year old population of the canton of Vaud. Data was provided by 2,276 participants in the ongoing Lausanne cohort 65+ (Lc65+), a study of those born between 1934 and 1943, and by 998 persons from an additional sample drawn to include the population outside of Lausanne or born before 1934. RESULTS: Participants expressed favourable perceptions, at rates exceeding 75% for most items. However, only 38% to 51% responded positively for out-of-hours availability, easy access and at home visits, likelihood of prescribing expensive medication if needed, and doctors' awareness of over-the-counter drugs. 12.0% had an SP PCP, in 95.9% specialised in a discipline implying training in internal medicine. Bivariate and multivariate analyses did not result in significant differences between GP and SP PCPs regarding perceptions of accessibility/availability, doctor-patient relationship, information and continuity of care, prevention, spontaneous use of the emergency department or ambulatory care utilisation. CONCLUSIONS: Experiences of old patients were mostly positive despite some lack in reported hearing, memory testing, and colorectal cancer screening. We found no differences between GP and SP PCP groups.

Genes associated with retinitis pigmentosa and allied diseases are frequently mutated in the general population.

BACKGROUND: Retinitis pigmentosa and other hereditary retinal degenerations (HRD) are rare genetic diseases leading to progressive blindness. Recessive HRD are caused by mutations in more than 100 different genes. Laws of population genetics predict that, on a purely theoretical ground, such a high number of genes should translate into an extremely elevated frequency of unaffected carriers of mutations. In this study we estimate the proportion of these individuals within the general population, via the analyses of data from whole-genome sequencing. METHODOLOGY/PRINCIPAL FINDINGS: We screened complete and high-quality genome sequences from 46 control individuals from various world populations for HRD mutations, using bioinformatic tools developed in-house. All mutations detected in silico were validated by Sanger sequencing. We identified clear-cut, null recessive HRD mutations in 10 out of the 46 unaffected individuals analyzed (∼22%). CONCLUSIONS/SIGNIFICANCE: Based on our data, approximately one in 4-5 individuals from the general population may be a carrier of null mutations that are responsible for HRD. This would be the highest mutation carrier frequency so far measured for a class of Mendelian disorders, especially considering that missenses and other forms of pathogenic changes were not included in our assessment. Among other things, our results indicate that the risk for a consanguineous couple of generating a child with a blinding disease is particularly high, compared to other genetic conditions.

Recruitment, retention and exit from union membership. An analysis of member flows in Swiss union locals

This article enquires into the causes of union growth and decline by analysing flows in and out of membership at the level of 70 Swiss union locals over 2006-2008. Gross flows in union membership are much larger than the resulting net changes: annual membership turnover of 10 per cent is a surprisingly constant feature across unions. Net changes in membership are primarily determined by inflows: successful and languishing union locals differ in their entry rates, whereas exit rates are similar. Variance in union locals' entry rates is not usefully explained by the labour market context, but by differences in union strategy.

High occurrence of hepatitis E virus in samples from wastewater treatment plants in Switzerland and comparison with other enteric viruses.

Hepatitis E virus (HEV) is responsible for many enterically transmitted viral hepatitides around the world. It is currently one of the waterborne diseases of global concern. In industrialized countries, HEV appears to be more common than previously thought, even if it is rarely virulent. In Switzerland, seroprevalence studies revealed that HEV is endemic, but no information was available on its environmental spread. The aim of this study was to investigate -using qPCR- the occurrence and concentration of HEV and three other viruses (norovirus genogroup II, human adenovirus-40 and porcine adenovirus) in influents and effluents of 31 wastewater treatment plants (WWTPs) in Switzerland. Low concentrations of HEV were detected in 40 out of 124 WWTP influent samples, showing that HEV is commonly present in this region. The frequency of HEV occurrence was higher in summer than in winter. No HEV was detected in WWTP effluent samples, which indicates a low risk of environmental contamination. HEV occurrence and concentrations were lower than those of norovirus and adenovirus. The autochthonous HEV genotype 3 was found in all positive samples, but a strain of the non-endemic and highly pathogenic HEV genotype I was isolated in one sample, highlighting the possibility of environmental circulation of this genotype. A porcine fecal marker (porcine adenovirus) was not detected in HEV positive samples, indicating that swine are not the direct source of HEV present in wastewater. Further investigations will be necessary to determine the reservoirs and the routes of dissemination of HEV.

Loss of single HLA class I allospecificities in melanoma cells due to selective genomic abbreviations.

Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8(+) T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA-A2 peptides. To identify the reasons underlying the loss of single HLA-A allospecificities, we searched for genomic alterations at the locus for HLA Class I alpha-chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA-A2 gene sequences as determined by polymerase chain reaction-sequence specific primers (PCR-SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group-specific primer-mixes spanning the 5'-flanking region of the HLA-A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA-A2-restricted cytotoxic T-cell clones were performed in HLA-A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA-A2 negative cells. We could document the occurrence of selective HLA-A2 deficiencies in cultured advanced-stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA-A gene locus.

Comparison of magnetic resonance enterography and video capsule endoscopy in evaluating small bowel disease.

PURPOSE: The goal of this study was to compare magnetic resonance enterography (MRE) and video capsule endoscopy (VCE) in suspected small bowel disease. MATERIALS AND METHODS: Nineteen patients with suspected small bowel disease participated in a prospective clinical comparison of MRE versus VCE. Both methods were evaluated separately and in conjunction with respect to a combined diagnostic endpoint based on clinical, laboratory, surgical, and histopathological findings. The Fisher's exact and j tests were used in comparing MRE and VCE. RESULTS: Small bowel pathologies were found in 15 out of 19 patients: Crohn's disease (n= 5), lymphoma (n= 4), lymphangioma (n= 1), adenocarcinoma (n= 1), postradiation enteropathy (n= 1), NSAID-induced enteropathy (n =1), angiodysplasia (n= 1), and small bowel adhesions (n= 1). VCE and MRE separately and in conjunction showed sensitivities of 92.9, 71.4, and 100% and specificities of 80, 60, and 80% (kappa= 0.73 vs. kappa = 0.29; P= 0.31/kappa = 0.85), respectively. In four patients, VCE depicted mucosal pathologies missed by MRE. MRE revealed 19 extraenteric findings in 11 patients as well as small bowel adhesions not detected on VCE (n= 1). CONCLUSION: VCE can readily depict and characterize subtle mucosal lesions missed at MRE, whereas MRE yields additional mural, perienteric, and extraenteric information. Thus, VCE and MRE appear to be complementary methods which, when used in conjunction, may better characterize suspected small bowel disease.

Phase II study of temozolomide in combination with topotecan (TOTEM) in relapsed or refractory neuroblastoma: A European Innovative Therapies for Children with Cancer-SIOP-European Neuroblastoma study.

PURPOSE: To assess objective response rate (ORR) after two cycles of temozolomide in combination with topotecan (TOTEM) in children with refractory or relapsed neuroblastoma. PATIENTS AND METHODS: This multicenter, non-randomised, phase II study included children with neuroblastoma according to a two-stage Simon design. Eligibility criteria included relapsed or refractory, measurable or metaiodobenzylguanidine (mIBG) evaluable disease, no more than two lines of prior treatment. Temozolomide was administered orally at 150mg/m(2) followed by topotecan at 0.75mg/m(2) intravenously for five consecutive days every 28days. Tumour response was assessed every two cycles according to International Neuroblastoma Response Criteria (INRC), and reviewed independently. RESULTS: Thirty-eight patients were enroled and treated in 15 European centres with a median age of 5.4years. Partial tumour response after two cycles was observed in 7 out of 38 evaluable patients [ORR 18%, 95% confidence interval (CI) 8-34%]. The best ORR whatever the time of evaluation was 24% (95% CI, 11-40%) with a median response duration of 8.5months. Tumour control rate (complete response (CR)+partial response (PR)+mixed response (MR)+stable disease (SD)) was 68% (95% CI, 63-90%). The 12-months Progression-Free and Overall Survival were 42% and 58% respectively. Among 213 treatment cycles (median 4, range 1-12 per patient) the most common treatment-related toxicities were haematologic. Grade 3/4 neutropenia occurred in 62% of courses in 89% of patients, grade 3/4 thrombocytopenia in 47% of courses in 71% of patients; three patients (8%) had febrile neutropenia. CONCLUSION: Temozolomide-Topotecan combination results in very encouraging ORR and tumour control in children with heavily pretreated recurrent and refractory neuroblastoma with favourable toxicity profile.

Predictive ability of an early diagnostic guess in patients presenting with chest pain; a longitudinal descriptive study.

ABSTRACT: BACKGROUND: The intuitive early diagnostic guess could play an important role in reaching a final diagnosis. However, no study to date has attempted to quantify the importance of general practitioners' (GPs) ability to correctly appraise the origin of chest pain within the first minutes of an encounter. METHODS: The validation study was nested in a multicentre cohort study with a one year follow-up and included 626 successive patients who presented with chest pain and were attended by 58 GPs in Western Switzerland. The early diagnostic guess was assessed prior to a patient's history being taken by a GP and was then compared to a diagnosis of chest pain observed over the next year. RESULTS: Using summary measures clustered at the GP's level, the early diagnostic guess was confirmed by further investigation in 51.0% (CI 95%; 49.4% to 52.5%) of patients presenting with chest pain. The early diagnostic guess was more accurate in patients with a life threatening illness (65.4%; CI 95% 64.5% to 66.3%) and in patients who did not feel anxious (62.9%; CI 95% 62.5% to 63.3%). The predictive abilities of an early diagnostic guess were consistent among GPs. CONCLUSIONS: The GPs early diagnostic guess was correct in one out of two patients presenting with chest pain. The probability of a correct guess was higher in patients with a life-threatening illness and in patients not feeling anxious about their pain.

Cost-effectiveness of full-course oral levofloxacin in severe community-acquired pneumonia.

Oral levofloxacin is as efficient as sequential antibiotic treatment in community-acquired pneumonia (CAP). The current authors assessed whether oral levofloxacin treatment of patients with severe CAP, followed-up for 30 days, would save money. Over a 12-month period, 129 hospitalised patients with severe non-intensive care unit CAP were randomly assigned to receive either oral levofloxacin or sequential antibiotic treatment. Direct and indirect costs were compared over a 30-day period from several perspectives. CAP resolved in 71 out of 77 oral levofloxacin (92%) and in 34 out of 37 sequential antibiotic treatment patients (92%). Patients' characteristics, treatment duration, hospital length of stay and mortality were similar in both groups. Drug acquisition costs were 1.7-times smaller in oral levofloxacin patients, who were less often transferred to rehabilitation centres, but they used more physicians' visits during follow-up and their total costs were lower. As only a minority of patients was still active, inability to work and, hence, indirect costs were similar in both groups. In this study, oral levofloxacin for severe non-intensive care unit community-acquired pneumonia was equally effective as sequential antibiotic treatment, but did not lead to major costs savings except for drug acquisition costs. External factors linked with patients' characteristics and/or medical practice are likely to play a role and should be addressed.