Showing papers by "French Institute of Health and Medical Research published in 2002"

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase

Abstract: Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.

Projection of an Immunological Self Shadow Within the Thymus by the Aire Protein

Abstract: Humans expressing a defective form of the transcription factor AIRE (autoimmune regulator) develop multiorgan autoimmune disease. We used aire- deficient mice to test the hypothesis that this transcription factor regulates autoimmunity by promoting the ectopic expression of peripheral tissue- restricted antigens in medullary epithelial cells of the thymus. This hypothesis proved correct. The mutant animals exhibited a defined profile of autoimmune diseases that depended on the absence of aire in stromal cells of the thymus. Aire-deficient thymic medullary epithelial cells showed a specific reduction in ectopic transcription of genes encoding peripheral antigens. These findings highlight the importance of thymically imposed "central" tolerance in controlling autoimmunity.

Excitatory actions of gaba during development: the nature of the nurture.

Abstract: In the immature brain, GABA (gamma-aminobutyric acid) is excitatory, and GABA-releasing synapses are formed before glutamatergic contacts in a wide range of species and structures. GABA becomes inhibitory by the delayed expression of a chloride exporter, leading to a negative shift in the reversal potential for choride ions. I propose that this mechanism provides a solution to the problem of how to excite developing neurons to promote growth and synapse formation while avoiding the potentially toxic effects of a mismatch between GABA-mediated inhibition and glutamatergic excitation. As key elements of this cascade are activity dependent, the formation of inhibition adds an element of nurture to the construction of cortical networks.

Aortic Stiffness Is an Independent Predictor of Primary Coronary Events in Hypertensive Patients: A Longitudinal Study

Abstract: Arterial stiffness may predict coronary heart disease beyond classic risk factors. In a longitudinal study, we assessed the predictive value of arterial stiffness on coronary heart disease in patients with essential hypertension and without known clinical cardiovascular disease. Aortic stiffness was determined from carotid-femoral pulse wave velocity at baseline in 1045 hypertensives. The risk assessment of coronary heart disease was made by calculating the Framingham risk score according to the categories of gender, age, blood pressure, cholesterol, diabetes, and smoking. Mean age at entry was 51 years, and mean follow-up was 5.7 years. Coronary events (fatal and nonfatal myocardial infarction, coronary revascularization, and angina pectoris) and all cardiovascular events served as outcome variables in Cox proportional-hazard regression models. Fifty-three coronary events and 97 total cardiovascular events occurred. In univariate analysis, the relative risk of follow-up coronary event or any cardiovascular event increased with increasing level of pulse wave velocity; for 1 SD, ie, 3.5 m/s, relatives risks were 1.42 (95% confidence interval [CI], 1.10 to 1.82; P P P P P =0.039) or classic risk factors (for 3.5 m/s: relative risk, 1.39; 95% CI, 1.08 to 1.79; P =0.01). Parallel results were observed for all cardiovascular events. This study provides the first direct evidence in a longitudinal study that aortic stiffness is an independent predictor of primary coronary events in patients with essential hypertension.

Wnt/β-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway

Abstract: Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of beta-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by beta-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.

Proneural genes and the specification of neural cell types

Abstract: Certain morphological, physiological and molecular characteristics are shared by all neurons. However, despite these similarities, neurons constitute the most diverse cell population of any organism. Recently, considerable attention has been focused on identifying the molecular mechanisms that underlie this cellular diversity. Parallel studies in Drosophila and vertebrates have revealed that proneural genes are key regulators of neurogenesis, coordinating the acquisition of a generic neuronal fate and of specific subtype identities that are appropriate for the location and time of neuronal generation. These studies reveal that, in spite of differences between invertebrate and vertebrate neural lineages, Drosophila and vertebrate proneural genes have remarkably similar roles.

Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin

Abstract: Renin is an aspartyl protease essential for the control of blood pressure and was long suspected to have cellular receptors We report the expression cloning of the human renin receptor complementary DNA encoding a 350-amino acid protein with a single transmembrane domain and no homology with any known membrane protein Transfected cells stably expressing the receptor showed renin- and prorenin-specific binding The binding of renin induced a fourfold increase of the catalytic efficiency of angiotensinogen conversion to angiotensin I and induced an intracellular signal with phosphorylation of serine and tyrosine residues associated to an activation of MAP kinases ERK1 and ERK2 High levels of the receptor mRNA are detected in the heart, brain, placenta, and lower levels in the kidney and liver By confocal microscopy the receptor is localized in the mesangium of glomeruli and in the subendothelium of coronary and kidney artery, associated to smooth muscle cells and colocalized with renin The renin receptor is the first described for an aspartyl protease This discovery emphasizes the role of the cell surface in angiotensin II generation and opens new perspectives on the tissue renin-angiotensin system and on renin effects independent of angiotensin II

Infection of a human hepatoma cell line by hepatitis B virus

Abstract: Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocyte-like morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.

A Human Genome Diversity Cell Line Panel

Abstract: A resource of 1064 cultured lymphoblastoid cell lines (LCLs) ([1][1]) from individuals in different world populations and corresponding milligram quantities of DNA is deposited at the Foundation Jean Dausset (CEPH) ([2][2]) in Paris. LCLs were collected from various laboratories by the Human Genome

Genetic dissection of immunity to mycobacteria: the human model

Abstract: ▪ Abstract Humans are exposed to a variety of environmental mycobacteria (EM), and most children are inoculated with live Bacille Calmette-Guerin (BCG) vaccine. In addition, most of the world's population is occasionally exposed to human-borne mycobacterial species, which are less abundant but more virulent. Although rarely pathogenic, mildly virulent mycobacteria, including BCG and most EM, may cause a variety of clinical diseases. Mycobacterium tuberculosis, M. leprae, and EM M. ulcerans are more virulent, causing tuberculosis, leprosy, and Buruli ulcer, respectively. Remarkably, only a minority of individuals develop clinical disease, even if infected with virulent mycobacteria. The interindividual variability of clinical outcome is thought to result in part from variability in the human genes that control host defense. In this well-defined microbiological and clinical context, the principles of mouse immunology and the methods of human genetics can be combined to facilitate the genetic dissection of i...

CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease.

Abstract: CARD15/NOD2 encodes a protein involved in bacterial recognition by monocytes. Mutations in CARD15 have recently been found in patients with Crohn disease (CD), a chronic inflammatory condition of the digestive tract. Here, we report the mutational analyses of CARD15 in 453 patients with CD, including 166 sporadic and 287 familial cases, 159 patients with ulcerative colitis (UC), and 103 healthy control subjects. Of 67 sequence variations identified, 9 had an allele frequency >5% in patients with CD. Six of them were considered to be polymorphisms, and three (R702W, G908R, and 1007fs) were confirmed to be independently associated with susceptibility to CD. Also considered as potential disease-causing mutations (DCMs) were 27 rare additional mutations. The three main variants (R702W, G908R, and 1007fs) represented 32%, 18%, and 31%, respectively, of the total CD mutations, whereas the total of the 27 rare mutations represented 19% of DCMs. Altogether, 93% of the mutations were located in the distal third of the gene. No mutations were found to be associated with UC. In contrast, 50% of patients with CD carried at least one DCM, including 17% who had a double mutation. This observation confirmed the gene-dosage effect in CD. The patients with double-dose mutations were characterized by a younger age at onset (16.9 years vs. 19.8 years; P=.01), a more frequent stricturing phenotype (53% vs. 28%; P=.00003; odds ratio 2.92), and a less frequent colonic involvement (43% vs. 62%; P=.003; odds ratio 0.44) than were seen in those patients who had no mutation. The severity of the disease and extraintestinal manifestations were not different for any of the CARD15 genotypes. The proportion of familial and sporadic cases and the proportion of patients with smoking habits were similar in the groups of patients with CD with or without mutation. These findings provide tools for a DNA-based test of susceptibility and for genetic counseling in inflammatory bowel disease.

Functional Neuroimaging of Speech Perception in Infants

Abstract: Human infants begin to acquire their native language in the first months of life. To determine which brain regions support language processing at this young age, we measured with functional magnetic resonance imaging the brain activity evoked by normal and reversed speech in awake and sleeping 3-month-old infants. Left-lateralized brain regions similar to those of adults, including the superior temporal and angular gyri, were already active in infants. Additional activation in right prefrontal cortex was seen only in awake infants processing normal speech. Thus, precursors of adult cortical language areas are already active in infants, well before the onset of speech production.

Functional anatomy of the basal ganglia

Abstract: Four organizational levels of the basal ganglia that could be particularly determinant in terms of functional properties are reviewed: (1) macroscopic anatomy, which is characterized by a dramatic decrease of cerebral tissue volume from the cerebral cortex to the deepest portions of the basal ganglia; (2) connectivity, which consists of both complex loops and a partition into three territories, sensorimotor, associative, and limbic (which process motor, cognitive, and emotional information, respectively); (3) neuronal morphology, characterized by a dramatic numeric and geometric convergence of striatal neurons onto pallidonigral neurons; and (4) dopaminergic innervation of the basal ganglia, which is organized as a dual system that is supposed to have opposite effects on the activity of the system. Current models of the basal ganglia are discussed.

Epidemiology of sepsis and infection in ICU patients from an international multicentre cohort study.

Abstract: Objectives: To examine the incidence of infections and to describe them and their outcome in intensive care unit (ICU) patients. Design and setting: International prospective cohort study in which all patients admitted to the 28 participating units in eight countries between May 1997 and May 1998 were followed until hospital discharge. Patients: A total of 14,364 patients were admitted to the ICUs, 6011 of whom stayed less than 24 h and 8353 more than 24 h. Results: Overall 3034 infectious episodes were recorded at ICU admission (crude incidence: 21.1%). In ICU patients hospitalised longer than 24 h there were 1581 infectious episodes (crude incidence: 18.9%) including 713 (45%) in patients already infected at ICU admission. These rates varied between ICUs. Respiratory, digestive, urinary tracts, and primary bloodstream infections represented about 80% of all sites. Hospital-acquired and ICU-acquired infections were documented more frequently microbiologically than community-acquired infections (71% and 86%, respectively vs. 55%). About 28% of infections were associated with sepsis, 24% with severe sepsis and 30% with septic shock, and 18% were not classified. Crude hospital mortality rates ranged from 16.9% in non-infected patients to 53.6% in patients with hospital-acquired infections at the time of ICU admission and acquiring infection during the ICU stay. Conclusions: The crude incidence of ICU infections remains high, although the rate varies between ICUs and patient subsets, illustrating the added burden of nosocomial infections in the use of ICU resources.

Severe iron deficiency anemia in transgenic mice expressing liver hepcidin

Abstract: We recently reported the hemochromatosis-like phenotype observed in our Usf2 knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the Usf2 knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the Usf2 knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hepcidin action.

Origin of regulatory T cells with known specificity for antigen

Abstract: T cell receptor agonists can induce the differentiation of regulatory T (T(R)) cells. We report here that the immunoglobulin kappa-controlled expression of an agonist in different cell types correlated with the phenotype of the generated T(R) cells. We found that aberrant expression on thymic stroma yielded predominantly CD4(+)CD25(+) T(R) cells, which--under physiological conditions--may be induced by ectopically expressed organ-specific antigens and thus prevent organ-specific autoimmunity. Expression of the agonist antigen by nonactivated hematopoietic cells produced mostly CD4(+)CD25(-) T(R) cells. This subset can be derived from mature monospecific T cells without "tutoring" by other T cells and can be generated in the absence of a functioning thymus. Suppression of CD4(+) T cell proliferative responses by both CD25(+) and CD25(-) subsets was interleukin 10 (IL-10) independent and was overcome by IL-2. These data suggest that distinct pathways can be exploited to interfere with unwanted immune responses.

Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers.

Abstract: The structure of poly(l-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an “in” and “out” diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.

Anatomical Evidence of Multimodal Integration in Primate Striate Cortex

Abstract: The primary visual cortex (area 17 or V1) is not thought to receive input from nonvisual extrastriate cortical areas. However, this has yet to be shown to be the case using sensitive tracers in the part of area 17 subserving the peripheral visual field. Here we show using retrograde tracers that peripheral area 17 subserving the visual field at an eccentricity of 10–20° receives projections from the core and parabelt areas of the auditory cortex as well as from the polysensory area of the temporal lobe (STP). The relative strength of these projections was calculated for each injection by computing the proportions of retrogradely labeled neurons located in the auditory and STP areas with respect to number of labeled neurons constituting the established projection from the superior temporal sulci (STS) motion complex (middle temporal area, medial superior temporal, fundus of the superior temporal area). In peripheral area V1 the projection from auditory cortex corresponds to 9.5% of that of the STS motion complex and STP to 35% of that from the STS motion complex. Compared to peripheral area 17, central and paracentral area 17 showed considerably weaker inputs from auditory cortex (0.2–0.8%) but slightly more from STP cortex (3.5–6.1%). The present results show that the connectivity of area 17 is eccentricity dependent. Direct projections from auditory and STP cortex to peripheral area 17 have important consequences for higher visual functions of area 17, including multimodal integration at early stages of the visual cortical pathway.

New clues about vitamin D functions in the nervous system.

Abstract: Accumulating data have provided evidence that 1 alpha,25 dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] is involved in brain function. Thus, the nuclear receptor for 1,25-(OH)(2)D(3) has been localized in neurons and glial cells. Genes encoding the enzymes involved in the metabolism of this hormone are also expressed in brain cells. The reported biological effects of 1,25-(OH)(2)D(3) in the nervous system include the biosynthesis of neurotrophic factors and at least one enzyme involved in neurotransmitter synthesis. 1,25-(OH)(2)D(3) can also inhibit the synthesis of inducible nitric oxide synthase and increase glutathione levels, suggesting a role for the hormone in brain detoxification pathways. Neuroprotective and immunomodulatory effects of this hormone have been described in several experimental models, indicating the potential value of 1,25-(OH)(2)D(3) pharmacological analogs in neurodegenerative and neuroimmune diseases. In addition, 1,25-(OH)(2)D(3) induces glioma cell death, making the hormone of potential interest in the management of brain tumors. These results reveal previously unsuspected roles for 1,25-(OH)(2)D(3) in brain function and suggest possible areas of future research.

Chronic Infantile Neurological Cutaneous and Articular Syndrome Is Caused by Mutations in CIAS1, a Gene Highly Expressed in Polymorphonuclear Cells and Chondrocytes

Abstract: Chronic infantile neurological cutaneous and articular (CINCA) syndrome is a severe chronic inflammatory disease of early onset, characterized by cutaneous symptoms, central-nervous-system involvement, and arthropathy. In the present study, we report, in seven unrelated patients with CINCA syndrome, distinct missense mutations within the nucleotide-binding site of CIAS1, a gene encoding cryopyrin and previously shown to cause Muckle-Wells syndrome and familial cold urticaria. Because of the severe cartilage overgrowth observed in some patients with CINCA syndrome and the implications of polymorphonuclear cell infiltration in the cutaneous and neurological manifestations of this syndrome, the tissue-specific expression of CIAS1 was evaluated. A high level of expression of CIAS1 was found to be restricted to polymorphonuclear cells and chondrocytes. These findings demonstrate that CIAS1 missense mutations can result in distinct phenotypes with only a few overlapping symptoms and suggest that this gene may function as a potential inducer of apoptosis.

In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation.

Abstract: Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. We took advantage of this differential sensitivity of HDAC6 to TSA and TPX to identify its substrates. Using this approach, α-tubulin was identified as an HDAC6 substrate. HDAC6 deacetylated α-tubulin both in vivo and in vitro. Our investigations suggest that HDAC6 controls the stability of a dynamic pool of microtubules. Indeed, we found that highly acetylated microtubules observed after TSA treatment exhibited delayed drug-induced depolymerization and that HDAC6 overexpression prompted their induced depolymerization. Depolymerized tubulin was rapidly deacetylated in vivo, whereas tubulin acetylation occurred only after polymerization. We therefore suggest that acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules.

Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles

Abstract: The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.

High-resolution full-field optical coherence tomography with a Linnik microscope

Abstract: We describe an original microscope for high-resolution optical coherence tomography applications. Our system is based on a Linnik interference microscope with high-numerical-aperture objectives. Lock-in detection of the interference signal is achieved in parallel on a CCD by use of a photoelastic birefringence modulator and full-field stroboscopic illumination with an infrared LED. Transverse cross-section (en-face, or XY) images can be obtained in real time with better than 1-microm axial (Z) resolution and 0.5-microm transverse (XY) resolution. A sensitivity of approximately 80 dB is reached at a 1-image/s acquisition rate, which allows tomography in scattering media such as biological tissues.