Consommation d'aliments riches en calcium dans la population adulte de Suisse romande et du Tessin. [Consumption of calcium-rich food in the adult population of French-speaking Switzerland and of Tessin].

Food intake high in calcium content is important in the development of skeleton and the prevention of osteoporosis. From a public health perspective, it is therefore important to know the dietary calcium intake of a population. Two population surveys in the French and Italian parts of Switzerland (MONICA, 1988-1989, and Geneva, 1991), were combined to study nutritional habits related to calcium intake. A random population sample, aged 35 et 65 years, answered to a 24 hour recall questionnaire, either self-administered (MONICA, cantons of Vaud, Fribourg and Tessin, N = 2734) or by phone (canton of Geneva, N = 475). In the 4 cantons, for the previous day, 60% of participants ate dairy products, particularly whole milk and cheese. They drunk 3-4 dl/day of milk. A significant proportion (10%) of french and italian speaking Swiss did not consume any food high in calcium content. In Geneva, the mean daily calcium intake was 656 mg in men and 489 mg in women. In conclusion 1) eating habits related to calcium intake are similar across cantons; 2) women consume dairy products more frequently than men, but in smaller quantities; 3) about 60% of men and 80% of women do not get the daily amount of calcium recommended for the prevention of osteoporosis.

Dairy calcium supplementation in overweight or obese persons: its effect on markers of fat metabolism

BACKGROUND: Dairy calcium supplementation has been proposed to increase fat oxidation and to inhibit lipogenesis. OBJECTIVE: We aimed to investigate the effects of calcium supplementation on markers of fat metabolism. DESIGN: In a placebo-controlled, crossover experiment, 10 overweight or obese subjects who were low calcium consumers received 800 mg dairy Ca/d for 5 wk. After 4 wk, adipose tissue was taken for biopsy for analysis of gene expression. Respiratory exchange, glycerol turnover, and subcutaneous adipose tissue microdialysis were performed for 7 h after consumption of 400 mg Ca or placebo, and the ingestion of either randomized slow-release caffeine (SRC; 300 mg) or lactose (500 mg). One week later, the test was repeated with the SRC or lactose crossover. RESULTS: Calcium supplementation increased urinary calcium excretion by 16% (P = 0.017) but did not alter plasma parathyroid hormone or osteocalcin concentrations. Resting energy expenditure (59.9 +/- 3.0 or 59.6 +/- 3.3 kcal/h), fat oxidation (58.4 +/- 2.5 or 53.8 +/- 2.2 mg/min), plasma free fatty acid concentrations (0.63 +/- 0.02 or 0.62 +/- 0.03 mmol/L), and glycerol turnover (3.63 +/- 0.41 or 3.70 +/- 0.38 micromol . kg(-1) . min(-1)) were similar with or without calcium, respectively. SRC significantly increased free fatty acid concentrations, resting fat oxidation, and resting energy expenditure. During microdialysis, epinephrine increased dialysate glycerol concentrations by 250% without and 254% with calcium. Expression of 7 key metabolic genes in subcutaneous adipose tissue was not affected by calcium supplementation. CONCLUSION: Dairy calcium supplementation in overweight subjects with habitually low calcium intakes failed to alter fat metabolism and energy expenditure under resting conditions and during acute stimulation by caffeine or epinephrine

Cloning, functional expression, and chromosomal localization of the human pancreatic islet glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is a hormone secreted by the endocrine K-cells from the duodenum that stimulates glucose-induced insulin secretion. Here, we present the molecular characterization of the human pancreatic islet GIP receptor. cDNA clones for the GIP receptor were isolated from a human pancreatic islet cDNA library. They encoded two different forms of the receptor, which differed by a 27-amino acid insertion in the COOH-terminal cytoplasmic tail. The receptor protein sequence was 81% identical to that of the rat GIP receptor. When expressed in Chinese hamster lung fibroblasts, both forms of the receptor displayed high-affinity binding for GIP (180 and 600 pmol/l). GIP binding was displaced by < 20% by 1 mumol/l glucagon, glucagon-like peptide (GLP-I)(7-36) amide, vasoactive intestinal peptide, and secretin. However exendin-4 and exendin-(9-39) at 1 mumol/l displaced binding by approximately 70 and approximately 100% at 10 mumol/l. GIP binding to both forms of the receptor induced a dose-dependent increase in intracellular cAMP levels (EC50 values of 0.6-0.8 nmol/l) but no elevation of cytoplasmic calcium concentrations. Interestingly, both exendin-4 and exendin-(9-39) were antagonists of the receptor, inhibiting GIP-induced cAMP formation by up to 60% when present at a concentration of 10 mumol/l. Finally, the physical and genetic chromosomal localization of the receptor gene was determined to be on 19q13.3, close to the ApoC2 gene. These data will help study the physiology and pathophysiology of the human GIP receptor.

Roles of astrocytic sodium dynamics in the neurometabolic coupling : a study by cellular imaging

RESUME LARGE PUBLIC Le système nerveux central est principalement composé de deux types de cellules :les neurones et les cellules gliales. Ces dernières, bien que l'emportant en nombre sur les neurones, ont longtemps été considérées comme des cellules sans intérêts par les neuroscientifiques. Hors, les connaissances modernes à leurs sujets indiquent qu'elles participent à la plupart des tâches physiologiques du cerveau. Plus particulièrement, elles prennent part aux processus énergétiques cérébraux. Ceux-ci, en plus d'être vitaux, sont particulièrement intrigants puisque le cerveau représente seulement 2 % de la masse corporelle mais consomme environ 25 % du glucose (substrat énergétique) corporel. Les astrocytes, un type de cellules gliales, jouent un rôle primordial dans cette formidable utilisation de glucose par le cerveau. En effet, l'activité neuronale (transmission de l'influx nerveux) est accompagnée d'une augmentation de la capture de glucose, issu de la circulation sanguine, par les astrocytes. Ce phénomène est appelé le «couplage neurométabolique » entre neurones et astrocytes. L'ion sodium fait partie des mécanismes cellulaires entrant en fonction lors de ces processus. Ainsi, dans le cadre de cette thèse, les aspects dynamiques de la régulation du sodium astrocytaire et leurs implications dans le couplage neurométabolique ont été étudiés par des techniques d'imagerie cellulaires. Ces études ont démontré que les mitochondries, machineries cellulaires convertissant l'énergie contenue dans le glucose, participent à la régulation du sodium astrocytaire. De plus, ce travail de thèse a permis de découvrir que les astrocytes sont capables de se transmettre, sous forme de vagues de sodium se propageant de cellules en cellules, un message donnant l'ordre d'accroître leur consommation d'énergie. Cette voie de signalisation leur permettrait de fournir de l'énergie aux neurones suite à leur activation. RESUME Le glutamate libéré dans la fente synaptique pendant l'activité neuronale, est éliminé par les astrocytes environnants. Le glutamate est co-transporté avec des ions sodiques, induisant une augmentation intracellulaire de sodium (Na+i) dans les astrocytes. Cette élévation de Na+i déclenche une cascade de mécanismes moléculaires qui aboutissent à la production de substrats énergétiques pouvant être utilisés par les neurones. Durant cette thèse, la mesure simultanée du sodium mitochondrial (Na+mit) et cytosolique par des techniques d'imagerie utilisant des sondes fluorescentes spécifiques, a indiqué que les variations de Na+i induites par le transport du glutamate sont transmises aux mitochondries. De plus, les voies d'entrée et de sortie du sodium mitochondrial ont été identifiées. L'échangeur de Na+ et de Ca2+ mitochondrial semble jouer un rôle primordial dans l'influx de Na+mit, alors que l'efflux de Na+mit est pris en charge par l'échangeur de Na+ et de H+ mitochondrial. L'étude du Na+mit a nécessité l'utilisation d'un système de photoactivation. Les sources de lumière ultraviolette (UV) classiques utilisées à cet effet (lasers, lampes à flash) ayant plusieurs désavantages, une alternative efficace et peu coûteuse a été développée. Il s'agit d'un système compact utilisant une diode électroluminescente (LED) à haute puissance et de longueur d'onde de 365nm. En plus de leurs rôles dans le couplage neurométabolique, les astrocytes participent à la signalisation multicellulaire en transmettant des vagues intercellulaires de calcium. Ce travail de thèse démontre également que des vagues intercellulaires de sodium peuvent être évoquées en parallèle à ces vagues calciques. Le glutamate, suite à sa libération par un mécanisme dépendent du calcium, est réabsorbé par les transporteurs au glutamate. Ce mécanisme a pour conséquence la génération de vagues sodiques se propageant de cellules en cellules. De plus, ces vagues sodiques sont corrélées spatialement avec une consommation accrue de glucose par les astrocytes. En conclusion, ce travail de thèse a permis de montrer que le signal sodique astrocytaire, déclenché en réponse au glutamate, se propage à la fois de façon intracellulaire aux mitochondries et de façon intercellulaire. Ces résultats suggèrent que les astrocytes fonctionnent comme un réseau de cellules nécessaire au couplage énergétique concerté entre neurones et astrocytes et que le sodium est un élément clé dans les mécanismes de signalisations cellulaires sous-jacents. SUMMARY Glutamate, released in the synaptic cleft during neuronal activity, is removed by surrounding astrocytes. Glutamate is taken-up with Na+ ions by specific transporters, inducing an intracellular Na+ (Na+i) elevation in astrocytes which triggers a cascade of molecular mechanisms that provides metabolic substrates to neurons. Thus, astrocytic Na+i homeostasis represents a key component of the so-called neurometabolic coupling. In this context, the first part of this thesis work was aimed at investigating whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Simultaneous monitoring of both mitochondrial Na+ (Na+mit) and cytosolic Na+ changes with fluorescent dyes revealed that glutamate-evoked cytosolic Na+ elevations are indeed transmitted to mitochondria. The mitochondrial Na+/Ca2+ exchangers have a prominent role in the regulation of Na+mit influx pathway, and Na+mit extrusion appears to be mediated by Na+/H+ exchangers. To demonstrate the implication of Na+/Ca2+ exchangers, this study has required the technical development of an UV-flash photolysis system. Because light sources for flash photolysis have to be powerful and in the near UV range, the use of UV lasers or flash lamps is usually required. As an alternative to these UV sources that have several drawbaks, we developped a compact, efficient and lowcost flash photolysis system which employs a high power 365nm light emitting diode. In addition to their role in neurometabolic coupling, astrocytes participate in multicellular signaling by transmitting intercellular Ca2+ waves. The third part of this thesis show that intercellular Na+ waves can be evoked in parallel to Ca2+ waves. Glutamate released by a Ca2+ wave-dependent mechanism is taken up by glutamate transporters, resulting in a regenerative propagation of cytosolic Na+ increases. Na+ waves in turn lead to a spatially correlated increase in glucose uptake. In conclusion, the present thesis demonstrates that glutamate-induced Na+ changes occurring in the cytosol of astrocytes propagate to both the mitochondrial matrix and the astrocytic network. These results furthermore support the view that astrocytic Na+ is a signal coupled to the brain energy metabolism.

Pathogenic role of basic calcium phosphate crystals in destructive arthropathies.

BACKGROUND: basic calcium phosphate (BCP) crystals are commonly found in osteoarthritis (OA) and are associated with cartilage destruction. BCP crystals induce in vitro catabolic responses with the production of metalloproteases and inflammatory cytokines such as interleukin-1 (IL-1). In vivo, IL-1 production induced by BCP crystals is both dependant and independent of NLRP3 inflammasome. We aimed to clarify 1/ the role of BCP crystals in cartilage destruction and 2/ the role of IL-1 and NLRP3 inflammasome in cartilage degradation related to BCP crystals. METHODOLOGY PRINCIPAL FINDINGS: synovial membranes isolated from OA knees were analysed by alizarin Red and FTIR. Pyrogen free BCP crystals were injected into right knees of WT, NLRP3 -/-, ASC -/-, IL-1α -/- and IL-1β-/- mice and PBS was injected into left knees. To assess the role of IL-1, WT mice were treated by intra-peritoneal injections of anakinra, the IL-1Ra recombinant protein, or PBS. Articular destruction was studied at d4, d17 and d30 assessing synovial inflammation, proteoglycan loss and chondrocyte apoptosis. BCP crystals were frequently found in OA synovial membranes including low grade OA. BCP crystals injected into murine knee joints provoked synovial inflammation characterized by synovial macrophage infiltration that persisted at day 30, cartilage degradation as evidenced by loss of proteoglycan staining by Safranin-O and concomitant expression of VDIPEN epitopes, and increased chondrocyte apoptosis. BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β. Similarly, treatment with anakinra did not prevent BCP crystal effects. In vitro, BCP crystals elicited enhanced transcription of matrix degrading and pro-inflammatory genes in macrophages. CONCLUSIONS SIGNIFICANCE: intra-articular BCP crystals can elicit synovial inflammation and cartilage degradation suggesting that BCP crystals have a direct pathogenic role in OA. The effects are independent of IL-1 and NLRP3 inflammasome.

Basic calcium phosphate crystals induce IL-1B secretion in vitro through activation of the Nalp3 inflammasome

Objectives: We tested the effects of the three forms of basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbonate-substituted apatite (CA) and hydroxyapatite (HA)) on monocytes and macrophages on IL-1β secretion. The requirement for the NALP3 inflammasome and TLR2 and TLR4 receptors in this acute response was analyzed.

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

Structure of intracellular mature vaccinia virus observed by cryoelectron microscopy.

Intracellular mature vaccinia virus, also called intracellular naked virus, and its core envelope have been observed in their native, unfixed, unstained, hydrated states by cryoelectron microscopy of vitrified samples. The virion appears as a smooth rounded rectangle of ca. 350 by 270 nm. The core seems homogeneous and is surrounded by a 30-nm-thick surface domain delimited by membranes. We show that surface tubules and most likely also the characteristic dumbbell-shaped core with the lateral bodies which are generally observed in negatively stained or conventionally embedded samples are preparation artifacts.

Systemic exposure to tobramycin after local antibiotic treatment with calcium sulphate as carrier material.

INTRODUCTION: Osteoset(®) T is a calcium sulphate void filler containing 4% tobramycin sulphate, used to treat bone and soft tissue infections. Despite systemic exposure to the antibiotic, there are no pharmacokinetic studies in humans published so far. Based on the observations made in our patients, a model predicting tobramycin serum levels and evaluating their toxicity potential is presented. METHODS: Following implantation of Osteoset(®) T, tobramycin serum concentrations were monitored systematically. A pharmacokinetic analysis was performed using a non-linear mixed effects model based on a one compartment model with first-degree absorption. RESULTS: Data from 12 patients treated between October 2006 and March 2008 were analysed. Concentration profiles were consistent with the first-order slow release and single-compartment kinetics, whilst showing important variability. Predicted tobramycin serum concentrations depended clearly on both implanted drug amount and renal function. DISCUSSION AND CONCLUSION: Despite the popularity of aminoglycosides for local antibiotic therapy, pharmacokinetic data for this indication are scarce, and not available for calcium sulphate as carrier material. Systemic exposure to tobramycin after implantation of Osteoset(®) T appears reassuring regarding toxicity potential, except in case of markedly impaired renal function. We recommend in adapting the dosage to the estimated creatinine clearance rather than solely to the patient's weight.

Functional genomics of intracellular bacteria.

During the genomic era, a large amount of whole-genome sequences accumulated, which identified many hypothetical proteins of unknown function. Rapidly, functional genomics, which is the research domain that assign a function to a given gene product, has thus been developed. Functional genomics of intracellular pathogenic bacteria exhibit specific peculiarities due to the fastidious growth of most of these intracellular micro-organisms, due to the close interaction with the host cell, due to the risk of contamination of experiments with host cell proteins and, for some strict intracellular bacteria such as Chlamydia, due to the absence of simple genetic system to manipulate the bacterial genome. To identify virulence factors of intracellular pathogenic bacteria, functional genomics often rely on bioinformatic analyses compared with model organisms such as Escherichia coli and Bacillus subtilis. The use of heterologous expression is another common approach. Given the intracellular lifestyle and the many effectors that are used by the intracellular bacteria to corrupt host cell functions, functional genomics is also often targeting the identification of new effectors such as those of the T4SS of Brucella and Legionella.

Economics of Using Calcium Chloride vs. Sodium Chloride for Deicing/Anti-Icing, TR-488, 2008

The use of chemicals is a critical part of a pro-active winter maintenance program. However, ensuring that the correct chemicals are used is a challenge. On the one hand, budgets are limited, and thus price of chemicals is a major concern. On the other, performance of chemicals, especially at lower pavement temperatures, is not always assured. Two chemicals that are used extensively by the Iowa Department of Transportation (Iowa DOT) are sodium chloride (or salt) and calcium chloride. While calcium chloride can be effective at much lower temperatures than salt, it is also considerably more expensive. Costs for a gallon of salt brine are typically in the range of $0.05 to $0.10, whereas calcium chloride brine may cost in the range of $1.00 or more per gallon. These costs are of course subject to market forces and will thus change from year to year. The idea of mixing different winter maintenance chemicals is by no means new, and in general discussions it appears that many winter maintenance personnel have from time to time mixed up a jar of chemicals and done some work around the yard to see whether or not their new mix “works.” There are many stories about the mixture turning to “mayonnaise” (or, more colorfully, to “snot”) suggesting that mixing chemicals may give rise to some problems most likely due to precipitation. Further, the question of what constitutes a mixture “working” in this context is a topic of considerable discussion. In this study, mixtures of salt brine and calcium chloride brine were examined to determine their ice melting capability and their freezing point. Using the results from these tests, a linear interpolation model of the ice melting capability of mixtures of the two brines has been developed. Using a criterion based upon the ability of the mixture to melt a certain thickness of ice or snow (expressed as a thickness of melt-water equivalent), the model was extended to develop a material cost per lane mile for the full range of possible mixtures as a function of temperature. This allowed for a comparison of the performance of the various mixtures. From the point of view of melting capacity, mixing calcium chloride brine with salt brine appears to be effective only at very low temperatures (around 0° F and below). However, the approach described herein only considers the material costs, and does not consider application costs or other aspects of the mixture performance than melting capacity. While a unit quantity of calcium chloride is considerably more expensive than a unit quantity of sodium chloride, it also melts considerably more ice. In other words, to achieve the same result, much less calcium chloride brine is required than sodium chloride brine. This is important in considering application costs, because it means that a single application vehicle (for example, a brine dispensing trailer towed behind a snowplow) can cover many more lane miles with calcium chloride brine than with salt brine before needing to refill. Calculating exactly how much could be saved in application costs requires an optimization of routes used in the application of liquids in anti-icing, which is beyond the scope of the current study. However, this may be an area that agencies wish to pursue for future investigation. In discussion with winter maintenance personnel who use mixtures of sodium chloride and calcium chloride, it is evident that one reason for this is because the mixture is much more persistent (i.e. it stays longer on the road surface) than straight salt brine. Operationally this persistence is very valuable, but at present there are not any established methods to measure the persistence of a chemical on a pavement. In conclusion, the study presents a method that allows an agency to determine the material costs of using various mixtures of salt brine and calcium chloride brine. The method is based upon the requirement of melting a certain quantity of snow or ice at the ice-pavement interface, and on how much of a chemical or of a mixture of chemicals is required to do that.

In situ fluorescence imaging of glutamate-evoked mitochondrial Na+ responses in astrocytes.

Astrocytes can experience large intracellular Na+ changes following the activation of the Na+-coupled glutamate transport. The present study investigated whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Mitochondrial Na+ (Na+(mit)) changes were monitored using the Na+-sensitive fluorescent probe CoroNa Red (CR) in intact primary cortical astrocytes, as opposed to the classical isolated mitochondria preparation. The mitochondrial localization and Na+ sensitivity of the dye were first verified and indicated that it can be safely used as a selective Na+(mit) indicator. We found by simultaneously monitoring cytosolic and mitochondrial Na+ using sodium-binding benzofuran isophthalate and CR, respectively, that glutamate-evoked cytosolic Na+ elevations are transmitted to mitochondria. The resting Na+(mit) concentration was estimated at 19.0 +/- 0.8 mM, reaching 30.1 +/- 1.2 mM during 200 microM glutamate application. Blockers of conductances potentially mediating Na+ entry (calcium uniporter, monovalent cation conductances, K+(ATP) channels) were not able to prevent the Na+(mit) response to glutamate. However, Ca2+ and its exchange with Na+ appear to play an important role in mediating mitochondrial Na+ entry as chelating intracellular Ca2+ with BAPTA or inhibiting Na+/Ca2+ exchanger with CGP-37157 diminished the Na+(mit) response. Moreover, intracellular Ca2+ increase achieved by photoactivation of caged Ca2+ also induced a Na+(mit) elevation. Inhibition of mitochondrial Na/H antiporter using ethylisopropyl-amiloride caused a steady increase in Na+(mit) without increasing cytosolic Na+, indicating that Na+ extrusion from mitochondria is mediated by these exchangers. Thus, mitochondria in intact astrocytes are equipped to efficiently sense cellular Na+ signals and to dynamically regulate their Na+ content.

The fate and persistence of Leishmania major in mice of different genetic backgrounds: an example of exploitation of the immune system by intracellular parasites.

Leishmania spp. are intracellular protozoan parasites that are delivered within the dermis of their vertebrate hosts. Within this peripheral tissue and the draining lymph node, they find and/or rapidly create dynamic microenvironments that determine their ultimate fate, namely their more or less successful expansion, and favour their transmission to another vertebrate host though a blood-feeding vector. Depending on their genetic characteristics as well as the genetic make-up of their hosts, once within the dermis Leishmania spp. very rapidly drive and maintain sustained T cell-dependent immune responses that arbitrate their ultimate fate within their hosts. The analysis of the parasitism exerted by Leishmania major in mice of different genetic backgrounds has allowed us to recognize some of the early and late mechanisms driven by this parasite that lead to either uncontrolled or restricted parasitism. Uncontrolled parasitism by Leishmania major characterizing mice from a few inbred strains (e.g. BALB/c) is associated with the expansion of parasite reactive Th2 CD4 lymphocytes and results from their rapid and sustained activity. In contrast, restricted parasitism characteristic of mice from the majority of inbred strains results from the development of a polarized parasite-specific Th1 CD4 response. This murine model of infection has already been and will continue to be particularly instrumental in dissecting the rules controlling the pathway of differentiation of T cells in vivo. In the long run, the understanding of these rules should contribute to the rational development of novel immunotherapeutic interventions against severe infectious diseases.