Expression and secretion of the atrial natriuretic peptide in human adipose tissue and preadipocytes.

OBJECTIVE: Atrial natriuretic peptide (ANP) is a secretory hormone displaying diuretic, natriuretic, and vasorelaxant activities. Recently, its lipolytic activity has been reported. Since the expression of ANP in adipose tissue has not been documented, we used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to investigate the expression of ANP in human adipose tissue and preadipocytes. RESEARCH METHODS AND PROCEDURES: RNA was extracted from the human adipose tissue of severely obese premenopausal women as well as from human preadipocytes. For human preadipocytes, two cell systems were investigated: the human preadipose immortalized (Chub-S7) cells, a well-characterized human preadipose cell line, and primary preadipocytes derived from the stromal vascular fraction of the human adipose tissue. We measured the mRNA of ANP, of corin (a transmembrane serine protease involved in the conversion of pro-ANP to ANP) and of uncoupling protein 2 (UCP2; a control gene known to be ubiquitously expressed). The expression of ANP was also investigated using immunofluorescence and radioimmunoassay in Chub-S7 cells and human primary preadipocytes in culture. RESULTS: Our results indicate that ANP and corin are expressed at the mRNA level in human adipose tissue and preadipocytes. Immunofluorescence experiments demonstrated that pro-ANP was expressed in Chub-S7 cells. In addition, ANP secretion could be measured in Chub-S7 cells and human primary preadipocytes in culture. Rosiglitazone, a selective peroxisome proliferator-activated receptor type gamma (PPAR-gamma) agonist promoting adipocyte differentiation, was found to modulate both ANP expression and secretion in preadipocytes. DISCUSSION: Our findings suggest the existence of an autocrine/paracrine system for ANP in the human adipose tissue whose implications in lipolysis and cardiovascular function need to be further explored.

Functions of the human papillomavirus type 16 E4 protein

1. Abstract Cervical cancer is thought to be the consequence of infection by human papillomaviruses (HPV). In the majority of cases, DNA from HPV type 16 (HPV16) is found in malignant cervical lesions. The initial steps leading to transformation of an infected cell are not clearly understood but in most cases, disruption and integration of the episomal viral DNA must take place. As a consequence, the E2 and E4 genes are usually not expressed whereas the E6 and E7 oncogenes are highly expressed. However, in a normal infection in which the viral DNA is maintained as an episome, all viral genes are expressed. The pattern according to which the viral proteins are made, and therefore the life cycle of the virus, is tightly linked to the differentiation process of the host keratinocyte. The study of the viral oncogenes E6 and E7 has revealed crucial functions in the process of malignant transformation such as degradation of the p53 tumor suppressor protein, deregulation of the Retinoblastoma protein pathway and activation of the telomerase ribonucleoprotein. All these steps are necessary for cancerous lesions to develop. However, the loss of the E2 gene product seems to be necessary for sufficient expression of E6 and E7 in order to achieve such effects. In normal infections, the E4 protein is made abundantly in the later stages of the viral life cycle. Though extensive amounts of work have been carried out to define the function of E4, it still remains unclear. In this study, several approaches have been used to try and determine the functions of E4. First, a cell-penetrating fusion protein was designed and produced in order to circumvent the chronic difficulties of expressing E4 in mammalian cells. Unfortunately, this approach was not successful due to precipitation of the purified fusion protein. Second, the observation that E4 accumulates in cells having modified their adhesion properties led to the hypothesis that E4 might be involved in the differentiation process of keratinocytes. Preliminary results suggest that E4 triggers differentiation. Last, as E4 has been reported to collapse the cytokeratin network of keratinocytes, a direct approach using atomic force microscopy has allowed us to test the potential modification of mechanical properties of cells harboring reorganized cytokeratin networks. If so, a potential role for E4 in viral particle release could be hypothesized. 2. Résumé Il a été établi que le cancer du col de l'utérus se développe essentiellement à la suite d'une infection par le virus du papillome humain (HPV). Dans la majorité des cas analysés, de l'ADN du HPV de type 16 (HPV16) est détecté. Les étapes initiales de la transformation d'une cellule infectée sont mal connues mais il semble qu'une rupture du génome viral, normalement épisomal, suivi d'une intégration dans le génome de la cellule hôte soient des étapes nécessaires dans la plupart des cas. Or il semble qu'il y ait une sélection pour les cas où l'expression des oncogènes viraux E6 et E7 soit favorisée alors que l'expression des gènes E2 et E4 est en général impossible. Par contre, dans une infection dite normale où le génome viral n'est pas rompu, il n'y pas développement de cancer et tous les gènes viraux sont exprimés. L'ordre dans lequel les protéines virales sont produites, et donc le cycle de réplication du virus, est intimement lié au processus de différentiation de la cellule hôte. L'étude des protéines oncogènes E6 et E7 a révélé des fonctions clés dans le processus de transformation des cellules infectées telles que la dégradation du suppresseur de tumeur p53, la dérégulation de la voie de signalisation Rb ainsi que l'activation de la télomérase. Toutes ces activités sont nécessaires au développement de lésions cancéreuses. Toutefois, il semble que l'expression du gène E2 doit être empêchée afin que suffisamment des protéines E6 et E7 soient produites. Lorsque le gène E2 est exprimé, et donc lorsque le génome viral n'est pas rompu, les protéines E6 et E7 n'entraînent pas de telles conséquences. Le gène E4, qui se trouve dans la séquence codante de E2, a aussi besoin d'un génome viral intact pour être exprimé. Dans une infection normale, le gène E4 est exprimé abondamment dans les dernières étapes de la réplication du virus. Bien que de nombreuses études aient été menées afin de déterminer la fonction virale à E4, aucun résultat n'apparaît évident. Dans ce travail, plusieurs approches ont été utilisées afin d'adresser cette question. Premièrement, une protéine de fusion TAT-E4 a été produite et purifiée. Cette protéine, pouvant entrer dans les cellules vivantes par diffusion au travers de la membrane plasmique, aurait permis d'éviter ainsi les problèmes chroniques rencontrés lors de l'expression de E4 dans les cellules mammifères. Malheureusement, cette stratégie n'a pas pu être utilisée à cause de la précipitation de la protéine purifiée. Ensuite, l'observation que E4 s'accumule dans les cellules ayant modifié leurs propriétés d'adhésion a suggéré que E4 pourrait être impliqué dans le procédé de différentiation des kératinocytes. Des résultats préliminaires supportent cette possibilité. Enfin, il a été montré que E4 pouvait induire une réorganisation du réseau des cytokératines. Une approche directe utilisant le microscope à force atomique nous a ainsi permis de tester une potentielle modification des propriétés mécaniques de cellules ayant modifié leur réseau de cytokératines en présence de E4. Si tel est le cas, un rôle dans la libération de particules virales peut être proposé pour E4.

Assessment of inflammatory Bowel Disease patient's needs and problems from a nursing perspective

BACKGROUND: In this study, we aimed at assessing Inflammatory Bowel Disease patients' needs and current nursing practice to investigate to what extent consensus statements (European Crohn's and Colitis Organization) on the nursing roles in caring for patients with IBD concur with local practice. METHODS: We used a mixed-method convergent design to combine quantitative data prospectively collected in the Swiss IBD cohort study and qualitative data from structured interviews with IBD healthcare experts. Symptoms, quality of life, and anxiety and depression scores were retrieved from physician charts and patient self-reported questionnaires. Descriptive analyses were performed based on quantitative and qualitative data. RESULTS: 230 patients of a single center were included, 60% of patients were males, and median age was 40 (range 18-85). The prevalence of abdominal pain was 42%. Self-reported data were obtained from 75 out of 230 patients. General health was perceived significantly lower compared with the general population (p < 0.001). Prevalence of tiredness was 73%; sleep problems, 78%; issues related to work, 20%; sexual constraints, 35%; diarrhea, 67%; being afraid of not finding a bathroom, 42%; depression, 11%; and anxiety symptoms, 23%. According to experts' interviews, the consensus statements are found mostly relevant with many recommendations that are not yet realized in clinical practice. CONCLUSION: Identified prevalence may help clinicians in detecting patients at risk and improve patient management. © 2015 S. Karger AG, Basel.

Transcranial Magnetic Stimulation Combined with EEG Reveals Covert States of Elevated Excitability in the Human Epileptic Brain.

BACKGROUND: Transcranial magnetic stimulation combined with electroencephalogram (TMS-EEG) can be used to explore the dynamical state of neuronal networks. In patients with epilepsy, TMS can induce epileptiform discharges (EDs) with a stochastic occurrence despite constant stimulation parameters. This observation raises the possibility that the pre-stimulation period contains multiple covert states of brain excitability some of which are associated with the generation of EDs. OBJECTIVE: To investigate whether the interictal period contains "high excitability" states that upon brain stimulation produce EDs and can be differentiated from "low excitability" states producing normal appearing TMS-EEG responses. METHODS: In a cohort of 25 patients with Genetic Generalized Epilepsies (GGE) we identified two subjects characterized by the intermittent development of TMS-induced EDs. The high-excitability in the pre-stimulation period was assessed using multiple measures of univariate time series analysis. Measures providing optimal discrimination were identified by feature selection techniques. The "high excitability" states emerged in multiple loci (indicating diffuse cortical hyperexcitability) and were clearly differentiated on the basis of 14 measures from "low excitability" states (accuracy = 0.7). CONCLUSION: In GGE, the interictal period contains multiple, quasi-stable covert states of excitability a class of which is associated with the generation of TMS-induced EDs. The relevance of these findings to theoretical models of ictogenesis is discussed.

Cooperative and Competitive Spreading Dynamics on the Human Connectome.

Increasingly detailed data on the network topology of neural circuits create a need for theoretical principles that explain how these networks shape neural communication. Here we use a model of cascade spreading to reveal architectural features of human brain networks that facilitate spreading. Using an anatomical brain network derived from high-resolution diffusion spectrum imaging (DSI), we investigate scenarios where perturbations initiated at seed nodes result in global cascades that interact either cooperatively or competitively. We find that hub regions and a backbone of pathways facilitate early spreading, while the shortest path structure of the connectome enables cooperative effects, accelerating the spread of cascades. Finally, competing cascades become integrated by converging on polysensory associative areas. These findings show that the organizational principles of brain networks shape global communication and facilitate integrative function.

Brain metastases in gastro-oesophageal adenocarcinoma: insights into the role of the human epidermal growth factor receptor 2 (HER2).

BACKGROUND: Gastro-oesophageal adenocarcinomas rarely metastasize to the central nervous system (CNS). The role of the human epidermal growth factor receptor 2 (HER2) in patients with these cancers and CNS involvement is presently unknown. PATIENTS AND METHODS: A multicentre registry was established to collect data from patients with gastro-oesophageal adenocarcinomas and CNS involvement both retrospectively and prospectively. Inclusion in the study required a predefined clinical data set, a central neuro-radiological or histopathological confirmation of metastatic CNS involvement and central assessment of HER2 by immunohistochemistry (IHC) and in situ hybridisation (ISH). In addition, expression of E-cadherin and DNA mismatch repair (MMR) proteins were assessed by IHC. RESULTS: One hundred patients fulfilled the inclusion criteria. The population's median age was 59 years (interquartile range: 54-68), of which 85 (85%) were male. Twenty-five patients were of Asian and 75 of Caucasian origin. HER2 status was positive in 36% (95% CI: 26.6-46.2) of cases. Median time from initial diagnosis to the development of brain metastases (BMets) or leptomeningeal carcinomatosis (LC) was 9.9 months (95% CI: 8.5-15.0). Median overall survival from diagnosis was 16.9 months (95% CI: 14.0-20.7) and was not related to the HER2 status. E-cadherin loss was observed in 9% of cases and loss of expression in at least one DNA MMR proteins in 6%. CONCLUSIONS: The proportion of a positive HER2 status in patients with gastro-oesophageal adenocarcinoma and CNS involvement was higher than expected. The impact of anti-HER2 therapies should be studied prospectively.

Intrahemispheric cortico-cortical connections of the human auditory cortex.

The human auditory cortex comprises the supratemporal plane and large parts of the temporal and parietal convexities. We have investigated the relevant intrahemispheric cortico-cortical connections using in vivo DSI tractography combined with landmark-based registration, automatic cortical parcellation and whole-brain structural connection matrices in 20 right-handed male subjects. On the supratemporal plane, the pattern of connectivity was related to the architectonically defined early-stage auditory areas. It revealed a three-tier architecture characterized by a cascade of connections from the primary auditory cortex to six adjacent non-primary areas and from there to the superior temporal gyrus. Graph theory-driven analysis confirmed the cascade-like connectivity pattern and demonstrated a strong degree of segregation and hierarchy within early-stage auditory areas. Putative higher-order areas on the temporal and parietal convexities had more widely spread local connectivity and long-range connections with the prefrontal cortex; analysis of optimal community structure revealed five distinct modules in each hemisphere. The pattern of temporo-parieto-frontal connectivity was partially asymmetrical. In conclusion, the human early-stage auditory cortical connectivity, as revealed by in vivo DSI tractography, has strong similarities with that of non-human primates. The modular architecture and hemispheric asymmetry in higher-order regions is compatible with segregated processing streams and lateralization of cognitive functions.

Immune Memory and Exhaustion: Clinically Relevant Lessons from the LCMV Model.

The development of dysfunctional or exhausted T cells is characteristic of immune responses to chronic viral infections and cancer. Exhausted T cells are defined by reduced effector function, sustained upregulation of multiple inhibitory receptors, an altered transcriptional program and perturbations of normal memory development and homeostasis. This review focuses on (a) illustrating milestone discoveries that led to our present understanding of T cell exhaustion, (b) summarizing recent developments in the field, and (c) identifying new challenges for translational research. Exhausted T cells are now recognized as key therapeutic targets in human infections and cancer. Much of our knowledge of the clinically relevant process of exhaustion derives from studies in the mouse model of Lymphocytic choriomeningitis virus (LCMV) infection. Studies using this model have formed the foundation for our understanding of human T cell memory and exhaustion. We will use this example to discuss recent advances in our understanding of T cell exhaustion and illustrate the value of integrated mouse and human studies and will emphasize the benefits of bi-directional mouse-to-human and human-to-mouse research approaches.

How to get rid of a pathobiontic bacterium from the stomach mucosa : role of inflammatory monocytes and IL-22 in the vaccine-induced reduction of helicobacter infection

Helicobacter pylori is a bacterium colonizing the human stomach. To prevent or cure this potentially detrimental infection, vaccination might be a suitable alternative to antibiotic therapies. Recently, a study has demonstrated that a vaccine efficiently prevented H pylori infection in human. However, the mechanisms leading to protection remain elusive. In mice, the vaccine-induced protective response relies on CD4+ T cells and especially on Thl7 response. Nevertheless, the factors mediating the reduction of H pylori infection are not fully characterized. Hence, the aim of my thesis was to characterize the factors associated with the Thl7 response. In the context of the vaccine-induced reduction of Helicobacter infection, I first focused on the role of inflammatory monocytes. I showed that CDllb+Ly6CLOW inflammatory monocytes accumulated in the stomach of vaccinated mice in association with the reduction of Helicobacter infection. Remarkably, the depletion of inflammatory monocytes delayed the vaccine-induced protective response. Concerning the role of these cells, I demonstrated that inflammatory monocytes extracted from the stomach of vaccinated mice produced iNOS and killed H pylori in vitro. In a next step, I evaluated the role of IL-22 during the vaccine-induced response. IL-22, which is linked to the Thl7 response, increases innate defense mechanisms of epithelial cells. I demonstrated that IL-22 produced by antigen- specific Thl7 was increased in the stomach of vaccinated mice during the protective response. Interestingly, neutralization of IL-22 was associated with an impaired vaccine-induced protective response. Then, I demonstrated that IL-22 induced antimicrobial peptides (AMPs) secretion by epithelial cells. These AMPs killed H pylori in vitro. In conclusion, I showed that both inflammatory monocytes and IL-22 participated to the vaccine induced reduction of Helicobacter infection. In addition, I demonstrated that the epithelium along with inflammation induced by Thl7 response is a critical factor mediating reduction of Helicobacter infection.

Characterisation of anatomical properties of the human basal ganglia using magnetic resonance imaging

The detailed in-vivo characterization of subcortical brain structures is essential not only to understand the basic organizational principles of the healthy brain but also for the study of the involvement of the basal ganglia in brain disorders. The particular tissue properties of basal ganglia - most importantly their high iron content, strongly affect the contrast of magnetic resonance imaging (MRI) images, hampering the accurate automated assessment of these regions. This technical challenge explains the substantial controversy in the literature about the magnitude, directionality and neurobiological interpretation of basal ganglia structural changes estimated from MRI and computational anatomy techniques. My scientific project addresses the pertinent need for accurate automated delineation of basal ganglia using two complementary strategies: ? Empirical testing of the utility of novel imaging protocols to provide superior contrast in the basal ganglia and to quantify brain tissue properties; ? Improvement of the algorithms for the reliable automated detection of basal ganglia and thalamus Previous research demonstrated that MRI protocols based on magnetization transfer (MT) saturation maps provide optimal grey-white matter contrast in subcortical structures compared with the widely used Tl-weighted (Tlw) images (Helms et al., 2009). Under the assumption of a direct impact of brain tissue properties on MR contrast my first study addressed the question of the mechanisms underlying the regional specificities effect of the basal ganglia. I used established whole-brain voxel-based methods to test for grey matter volume differences between MT and Tlw imaging protocols with an emphasis on subcortical structures. I applied a regression model to explain the observed grey matter differences from the regionally specific impact of brain tissue properties on the MR contrast. The results of my first project prompted further methodological developments to create adequate priors for the basal ganglia and thalamus allowing optimal automated delineation of these structures in a probabilistic tissue classification framework. I established a standardized workflow for manual labelling of the basal ganglia, thalamus and cerebellar dentate to create new tissue probability maps from quantitative MR maps featuring optimal grey-white matter contrast in subcortical areas. The validation step of the new tissue priors included a comparison of the classification performance with the existing probability maps. In my third project I continued investigating the factors impacting automated brain tissue classification that result in interpretational shortcomings when using Tlw MRI data in the framework of computational anatomy. While the intensity in Tlw images is predominantly