A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules.

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.

Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection.

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed β-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.

Chirurgie viscérale. Chirurgie thyroïdienne et parathyroïdienne: place de la chirurgie minimale invasive [Update in thyroid and parathyroid surgery: the role for minimally invasive surgery].

Thyroid and parathyroid surgery takes an important place in general surgery. Preservation of essential cervical structures and cosmetic of outcomes of cervical incisions are major concern. Several minimally invasive strategies have been developed recently but their acceptance in the surgical community is low. Their main advantages are focused tissue dissection and improved cosmetics. In this review, management and surgical indications of thyroid nodules and parathyroid adenomas are discussed. The various minimally invasive techniques are described and their current indications are reviewed.

Role of Notch signaling in epidermis and appendages

Résumé Dans la peau, il a été montré que Notch1 induit l'arrêt de la prolifération et la différentiation des keratinocytes. L'inactivation de Notch1 cause une hyperplasie de l'épiderme et la formation de carcinomes basaux cellulaires. Notre groupe a principalement identifié deux voies de signalisations, la voie Shh et la voie Wnt, qui sont dérégulées en conséquence de l'inactivation de Notch1 dans la peau. Nous avons démontré l'habilité de Notch1 à réprimer la voie Wnt induite par ß-catenin dans les keratinocytes primaires ainsi que dans d'autres types de cellules épithéliales humaines. De plus, nous avons pu déterminer que Notch1 régule cette voie, probablement en favorisant la phosphorylation de ß-catenin par le complexe axin/APC/GSK-3ß. La protéine faisant partie de la voie Wnt, ou la protéine affectant la voie Wnt, qui est régulée par Notch1 est sujette à de plus amples investigations. Un autre but de cette étude a été l'identification de potentiels gènes cibles de Notch1 autres que ceux faisant partie des voies de signalisation Shh et Wnt précédemment évoquées. Ce projet fut abordé par l'analyse de puces à ADN (ISREC et Affymetrix) qui ont été utilisées pour des expériences de gain et de perte de fonction de Notch1 dans des keratinocytes prúmaires. En plus de l'hyperplasie épidermale, les souris Notch1 déficiente ont une perte importante de poils. Nous avons montré que Notch1 est nécessaire pour le développement et l'homéostasie des follicules pileux. En effet, l'inactivation du gène Notch1 mediée par l'activation des kératines 5 ou 14 dans l'épiderme, cause des défauts du cycle ainsi que de la structure des poils. De plus, d'autres appendices de la peau, comme les glandes sudoripares et de Meibomius, ont une structure anormale et sont non fonctionnelles dans les souris Notch1 déficiente. Finalement, nous avons observé que la déficience de Notch1 dans l'épithélium cornéen mène à la formation d'une plaque épidermale opaque sur la cornée. Basé sur l'hypothèse que le défaut des glandes de Meibomius des souris Notch1 déficientes cause des lésions de la surface oculaire, nous avons montré que Notch1 est essentiel pour la cicatrisation de la cornée. Lorsque Notch1 est absent, les cellules souches de l'épithélium cornéen ne sont plus capables de se différentier en cellules cornéennes, mais réparent la blessure en se différentiant en épiderme. Ce résultat indique que Notch1 est essentiel pour la différentiation de cellules souches de la cornée qui sont spécifiquement impliquées dans la réparation de la cornée. De plus, nous avons montré que l'expression de CRBP1 dans l'épithélium cornéen est diminuée en l'absence de Notch1, ceci étant possiblement à l'origine de la formation de la plaque épidermale. Abstract: In the skin, Notch1 has been shown to trigger cell growth arrest and differentiation of keratinocytes. Notch1 inactivation results in epidermal hyperplasia and subsequent formation of basal cell carcinoma-like (BCC-like) tumors. So far our group has identified two main pathways, the Shh and the Wnt pathway, that are deregulated as a consequence of Notch1 inactivation in the skin. We showed the ability of Notch1 to represses ß-catenin-mediated Wnt signaling in primary keratinocytes as well as in other types of human epithelial cells. In addition we were able to determine that Notch1 regulates this pathway possibly by enhancing ß-catenin phosphorylation by the axin/APC/GSK-3ß complex. The exact target protein of the Wnt pathway or target protein that affects the Wnt pathway, and that is regulated by Notch1, is subject of current investigation. Another aim of this study was the identification of possible Notch1 target genes in addition to those of the Shh and Wnt signaling pathways. This was addressed by gene chip analysis using ISREC as well as Affymetrix microarrays for gain and loss of function of Notch1 in mouse primary keratinocytes. In addition to epidermal hyperplasia, Notch1 deficient mice show an important hair loss. We showed that Notch1 is required for postnatal development and homeostasis of hair follicles. Indeed, keratin5 or keratinl4-driven Cre recombinase-mediated inactivation of the Notch1 gene in the epidermis causes perturbations of the hair cycle and structural defects of the hair follicle. Moreover, other skin appendages, like the sweat and Meibomian glands show abnormal morphology and are not functional in the Notch 1 deficient mice. Finally, we observed that Notch1 deficiency in the corneal epithelium leads to the formation of an epidermal corneal plaque. Based on the hypothesis that the Meiboinian gland defect in the Notch1 deficient mice results in lesions of the eye surface, we showed that Notch1 is essential for wound-healing of the cornea. In absence of Notch1 the stem cells of the corneal epithelium are no longer able to differentiate in the corneal fate but instead repair the wound by differentiating into skin-like epidermis. This result indicated that Notch1 is essential for the differentiation of corneal stem cells specifically implicated in corneal wound-healing. Moreover, we showed that CRBP1 expression in the corneal epithelium was lost in the absence of Notch1, possibly being at the origin of plaque formation.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

Molecular and Functional Characterization of Neuroblastoma-Initiating Cells

Neuroblastoma (NB) is the most common extracranial malignant tumor in young children and arises at any site of the sympathetic nervous system. The disease exhibits a remarkable phenotypic diversity ranging from spontaneous regression to fatal disease. Poor outcome results from a rapidly progressive, metastatic and drug-resistant disease. Recent studies have suggested that solid tumors may arise from a minor population of cancer stem cells (CSCs) with stem cell markers and typical properties such as self-renewal ability, asymmetric division and drug resistance. In this model, CSCs possess the exclusive ability to initiate and maintain the tumor, and to produce distant metastases. Tumor cell subpopulations with stem-like phenotypes have indeed been identified in several cancer including leukemia, breast, brain and colon cancers. CSC hypothesis still needs to be validated in the other cancers including NB.NB originates from neural crest-derived malignant sympatho-adrenal cells. We have identified rare cells that express markers in conformity with neural crest stem cells and their derived lineages within primary NB tissue and cell lines, leading us to postulate the existence of CSCs in NB tumors.In the absence of specific markers to isolate CSCs, we adapted to NB tumor cells the sphere functional assay, based on the ability of stem cells to grow as spheres in non-adherent conditions. By serial passages of spheres from bone marrow NB metastases, a subset of cells was gradually selected and its specific gene expression profile identified by micro-array time-course analysis. The differentially expressed genes in spheres are enriched in genes implicated in development including CD133, ABC-transporters, WNT and NOTCH genes, identified in others solid cancers as CSCs markers, and other new markers, all referred by us as the Neurosphere Expression Profile (NEP). We confirmed the presence of a cell subpopulation expressing a combination of the NEP markers within a few primary NB samples.The tumorigenic potential of NB spheres was assayed by in vivo tumor growth analyses using orthotopic (adrenal glands) implantations of tumor cells into immune-compromised mice. Tumors derived from the sphere cells were significantly more frequent and were detected earlier compared to whole tumor cells. However, NB cells expressing the neurosphere-associated genes and isolated from the bulk tumors did not recapitulate the CSC-like phenotype in the orthotopic model. In addition, the NB sphere cells lost their higher tumorigenic potential when implanted in a subcutaneous heterotopic in vivo model.These results highlighted the complex behavior of CSC functions and led us to consider the stem-like NB cells as a dynamic and heterogeneous cell population influenced by microenvironment signals.Our approach identified for the first time candidate genes that may be associated with NB self-renewal and tumorigenicity and therefore would establish specific functional targets for more effective therapies in aggressive NB.

Post-transcriptional down-regulation of expression of transcription factor NF1 by Ha-ras oncogene.

NF1 is a family of polypeptides that binds to discrete DNA motifs and plays varying roles in the regulation of gene expression. These polypeptides are also thought to mediate the expression of differentiation-specific markers such as adipocyte and mammary cell type-specific genes. The expression of a number of cellular differentiation-specific markers is down-regulated during neoplastic transformation. We therefore investigated whether oncogenic transformation interferes with the action of NF1. Stable transfection of activated Ha-ras into a number of murine cells correlated with a down-regulation of the expression of the NF1 genes NF1/CTF and NF1/X. The down-regulation was not at the transcriptional level but at the level of stability of the NF1 mRNAs. The level of the DNA binding activity of the NF1 proteins was also reduced in Ha-v-ras-transformed cells, and the expression of a gene that depends on this family of transcription factors was specifically repressed. These results demonstrate that an activated Ha-ras-induced pathway destabilizes the half-life of mRNAs encoding specific members in the NF1 family of transcription factors, which leads to a decrease in NF1-dependent gene expression.

Molecular and therapeutic characterization of anti-ectodysplasin A receptor (EDAR) agonist monoclonal antibodies.

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC(50) of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.

Jonctions communicantes et sécrétion [Gap junctions and secretion]

The emergence of multicellular organisms has necessitated the development of mechanisms for interactions between adjacent and distant cells. A consistent feature of this network is the expression of gap junction channels between the secretory cells of all glands so far investigated in vertebrates. Here, we reviewed the distribution of the gap junctions proteins, named connexins, in a few mammalian glands, and discussed the recent evidence pointing to the participation of these proteins in the functioning of endocrine and exocrine cells. Specifically, available data indicate the importance of gap junctions for the proper control of glucose-induced insulin secretion. Understanding the functions of beta-cell connexins are crucial for the engineering of surrogate cells, which is necessary for implementation of a replacement cell therapy in diabetic patients.

Leptin inhibits directly glucocorticoid secretion by normal human and rat adrenal gland.

Different interactions have been described between glucocorticoids and the product of the ob gene leptin. Leptin can inhibit the activation of the hypothalamo-pituitary-adrenal axis by stressful stimuli, whereas adrenal glucocorticoids stimulate leptin production by the adipocyte. The present study was designed to investigate the potential direct effects of leptin to modulate glucocorticoid production by the adrenal. Human adrenal glands from kidney transplant donors were dissociated, and isolated primary cells were studied in vitro. These cells were preincubated with recombinant leptin (10(-10)-10(-7) M) for 6 or 24 h, and basal or ACTH-stimulated cortisol secretion was subsequently measured. Basal cortisol secretion was unaffected by leptin, but a significant and dose-dependent inhibition of ACTH-stimulated cortisol secretion was observed [down by 29 +/- 0.1% of controls with the highest leptin dose, P < 0.01 vs. CT (unrelated positive control)]. This effect of leptin was also observed in rat primary adrenocortical cells, where leptin inhibited stimulated corticosterone secretion in a dose-dependent manner (down by 46 +/- 0.1% of controls with the highest leptin dose, P < 0.001 vs. CT). These effects of leptin in adrenal cells are likely mediated by the long isoform of the leptin receptor (OB-R), because its transcript was found to be expressed in the adrenal tissue and leptin had no inhibitory effect in adrenal glands obtained from db/db mice. Therefore, leptin inhibits directly stimulated cortisol secretion from human and rat adrenal glands, and this may represent an important mechanism to modulate glucocorticoid levels in various metabolic states.

Multi-Atlas based Segmentation of Head and Neck CT Images using Active Contour

This paper presents the segmentation of bilateral parotid glands in the Head and Neck (H&N) CT images using an active contour based atlas registration. We compare segmentation results from three atlas selection strategies: (i) selection of "single-most-similar" atlas for each image to be segmented, (ii) fusion of segmentation results from multiple atlases using STAPLE, and (iii) fusion of segmentation results using majority voting. Among these three approaches, fusion using majority voting provided the best results. Finally, we present a detailed evaluation on a dataset of eight images (provided as a part of H&N auto segmentation challenge conducted in conjunction with MICCAI-2010 conference) using majority voting strategy.

Impact of a smoking ban in hospitality venues on second hand smoke exposure: a comparison of exposure assessment methods

BACKGROUND: In May 2010, Switzerland introduced a heterogeneous smoking ban in the hospitality sector. While the law leaves room for exceptions in some cantons, it is comprehensive in others. This longitudinal study uses different measurement methods to examine airborne nicotine levels in hospitality venues and the level of personal exposure of non-smoking hospitality workers before and after implementation of the law. METHODS: Personal exposure to second hand smoke (SHS) was measured by three different methods. We compared a passive sampler called MoNIC (Monitor of NICotine) badge, to salivary cotinine and nicotine concentration as well as questionnaire data. Badges allowed the number of passively smoked cigarettes to be estimated. They were placed at the venues as well as distributed to the participants for personal measurements. To assess personal exposure at work, a time-weighted average of the workplace badge measurements was calculated. RESULTS: Prior to the ban, smoke-exposed hospitality venues yielded a mean badge value of 4.48 (95%-CI: 3.7 to 5.25; n = 214) cigarette equivalents/day. At follow-up, measurements in venues that had implemented a smoking ban significantly declined to an average of 0.31 (0.17 to 0.45; n = 37) (p = 0.001). Personal badge measurements also significantly decreased from an average of 2.18 (1.31-3.05 n = 53) to 0.25 (0.13-0.36; n = 41) (p = 0.001). Spearman rank correlations between badge exposure measures and salivary measures were small to moderate (0.3 at maximum). CONCLUSIONS: Nicotine levels significantly decreased in all types of hospitality venues after implementation of the smoking ban. In-depth analyses demonstrated that a time-weighted average of the workplace badge measurements represented typical personal SHS exposure at work more reliably than personal exposure measures such as salivary cotinine and nicotine.

Hirsutisme: que faire [Hirsutism, what can be done?]

Hirsutism is a relatively frequent condition in an ambulatory setting affecting about 4% of women. A rational clinical and biochemical diagnostic approach assures an optimal treatment directed at etiologic and pathogenetic factors. The diagnosis of hyperandrogenism is evaluated considering the pathophysiologic mechanisms responsible for excessive growth of hair. Ovarian and adrenal tumors are the most serious diseases that have to be excluded by clinical and biochemical tests. The other causes for hirsutism are treatable by a great variety of modalities, available drugs can inhibit pituitary gonadotropins, the hypothalamo-pituitary axis and the conversion of testosterone into biologically active substrate. Finally the binding of androgens to its receptor can be blocked. These possibilities for treatment and their indications for the different etiologies of hirsutism are discussed.

Circadian and wake-dependent effects on the pupil light reflex in response to narrow-bandwidth light pulses.

PURPOSE: Nonvisual light-dependent functions in humans are conveyed mainly by intrinsically photosensitive retinal ganglion cells, which express melanopsin as photopigment. We aimed to identify the effects of circadian phase and sleepiness across 24 hours on various aspects of the pupil response to light stimulation. METHODS: We tested 10 healthy adults hourly in two 12-hour sessions covering a 24-hour period. Pupil responses to narrow bandwidth red (635 ± 18 nm) and blue (463 ± 24 nm) light (duration of 1 and 30 seconds) at equal photon fluxes were recorded, and correlated with salivary melatonin concentrations at the same circadian phases and to subjective sleepiness ratings. The magnitude of pupil constriction was determined from minimal pupil size. The post-stimulus pupil response was assessed from the pupil size at 6 seconds following light offset, the area within the redilation curve, and the exponential rate of redilation. RESULTS: Among the measured parameters, the pupil size 6 seconds after light offset correlated with melatonin concentrations (P < 0.05) and showed a significant modulation over 24 hours with maximal values after the nocturnal peak of melatonin secretion. In contrast, the post-stimulus pupil response following red light stimulation correlated with subjective sleepiness (P < 0.05) without significant changes over 24 hours. CONCLUSIONS: The post-stimulus pupil response to blue light as a marker of intrinsic melanopsin activity demonstrated a circadian modulation. In contrast, the effect of sleepiness was more apparent in the cone contribution to the pupil response. Thus, pupillary responsiveness to light is under influence of the endogenous circadian clock and subjective sleepiness.

Second hand tobacco smoke exposure in Switzerland

A passive sampling device called Monitor of NICotine or "MoNIC", was constructed and evaluated by IST laboratory for determining nicotine in Second Hand Tobacco Smoke (SHTS) or Environmental Tobacco Smoke (ETS). Vapour nicotine was passively collected on a potassium bisulfate treated glass fibre filter as collection medium. Analysis of collected nicotine on the treated filter by gas chromatography equipped with Thermoionic-Specific Detector (GC-TSD) after liquid-liquid extraction of 1mL of 5N NaOH : 1 mL of n-heptane saturated with NH3 using quinoline as internal standard. Based on nicotine amount of 0.2 mg/cigarette as the reference, the inhaled Cigarette Equivalents (CE) by non-smokers can be calculated. Using the detected CE on the badge for non-smokers, and comparing with amount of nicotine and cotinine level in saliva of both smokers and exposed non-smokers, we can confirm the use of the CE concept for estimating exposure to ETS. The regional CIPRET (Center of information and prevention of the addiction to smoking) of different cantons (Valais (VS), Vaud (VD), Neuchâtel (NE) and Fribourg (FR)) are going to organize a big campaign on the subject of the passive addiction to smoking. This campaign took place in 2007-2008 and has for objective to inform clearly the Swiss population of the dangerousness of the passive smoke. More than 3'900 MoNIC badges were gracefully distributed to Swiss population to perform a self-monitoring of population exposure level to ETS, expressed in term of CE. Non-stimulated saliva was also collected to determine ETS biomarkers nicotine/cotinine levels of participating volunteers. Results of different levels of CE in occupational and non-occupational situations in relation with ETS were presented in this study. This study, unique in Switzerland, has established a base map on the population's exposure to SHTS. It underscored the fact that all the Swiss people involved in this campaign (N=1241) is exposed to passive smoke, from <0.2 cig/d (10.8%), 1-2 to more than 10 cig/d (89.2%). In the area of high exposure (15-38 cig/d), are the most workers in public restaurant, cafe, bar, disco. By monitoring ETS tracer nicotine and its biomarkers, salivary nicotine and cotinine, it is demonstrated that the MoNIC badge can serve as indicator of CE passive smoking. The MoNIC badge, accompanied with content of salivary nicotine/cotinine can serve as a tool of evaluation of the ETS passive smoking and contributes to supply useful data for future epidemiological studies. It is also demonstrated that the salivary nicotine (without stimulation) is a better biomarker of ETS exposure than cotinine.