One Health Surveillance: perceived benefits and workforce motivations.

One Health Surveillance (OHS) implements the One Health approach to improving health by collecting data and producing information to support integrated action across the animal health, human health and environment sectors. The purpose of this study was to survey the biosurveillance community to assess its OHS practices and capabilities, its attitudes towards OHS (perceived value), and the factors that motivate its members to implement OHS practices. The authors used a convenience sample of 185 professionals from multiple domains and 44 nations. They examined the extent to which these professionals implemented OHS, gathered their opinions on the value of OHS, assessed their perceptions of the capacity to perform specific OHS tasks and identified their priorities for change. Over 85% of all respondents said that they considered OHS to be beneficial, with no significant differences between work domains or country income groups; over 50% indicated that they already applied OHS. Obtaining access to data collected by other domains was both the most frequent challenge and the most difficult to improve. The highest priority for improvement was having the ability to send and receive electronic data. Respondents from low-income or middle-income countries were more motivated to make improvements than stakeholders from high-income countries. These findings provide a snapshot of current opinions and practices and, together with suggestions for improvements from professionals in the field, can help to target priority needs for OHS information, training and resources.

La surveillance Une seule santé opérationnalise la méthode Une seule santé pour une meilleure santé à travers la collecte de données et la production d'informations visant à soutenir la mobilisation transversale des secteurs de la santé animale, de la santé publique et de la santé environnementale en vue d'une action intégrée. Les auteurs présentent les résultats d'une enquête menée auprès des professionnels en charge de la biosurveillance afin d'évaluer leurs pratiques et capacités en matière de surveillance Une seule santé, leurs attitudes à l'égard de cette surveillance (c'est-à-dire leur perception de l'intérêt de la démarche) et les facteurs susceptibles de les motiver à la mettre en oeuvre. Les auteurs ont procédé à un échantillonnage de commodité de 185 intervenants issus de plusieurs secteurs dans 44 pays. Ils ont ensuite analysé le niveau de mise en oeuvre de la surveillance Une seule santé chez ces intervenants, recueilli leurs opinions concernant l'intérêt de la démarche, évalué la perception qu'ils avaient de leur capacité à mener à bien certaine tâches spécifiques dans ce domaine et identifié leurs priorités en vue du changement. Plus de 85 % des répondants ont déclaré considérer la surveillance Une seule santé comme étant bénéfique, résultat ne présentant pas de corrélation significative avec le secteur professionnel des personnes interrogées ni avec le niveau de revenu de leur pays ; plus de 50 % des répondants ont par ailleurs indiqué qu'ils appliquaient déjà les principes d'une surveillance Une seule santé. La difficulté la plus fréquente et qui paraissait la plus difficile à résoudre était celle de pouvoir accéder aux données enregistrées par d'autres secteurs. La première des priorités identifiées en vue d'une amélioration concernait la capacité d'envoyer et de recevoir des données électroniques. La motivation à introduire des améliorations était plus forte chez les répondants des pays à revenu faible ou intermédiaire que chez les parties prenantes des pays à revenus élevés. Ces résultats, qui offrent un instantané des opinions et des pratiques actuelles assorti de propositions concrètes d'amélioration formulées par les professionnels de terrain devraient pouvoir contribuer à cibler les besoins prioritaires en matière d'information, de formation et de ressources dédiées à la surveillance Une seule santé.

Practicar la vigilancia en clave de Una sola salud significa traducir esta idea en la práctica con el fin de mejorar la salud reuniendo datos y generando información a partir de la cual actuar de forma integrada en los sectores de la sanidad animal, la salud humana y el medio ambiente. Los autores describen un estudio de los círculos dedicados a la vigilancia biológica que tenía por objetivo evaluar sus procedimientos y capacidades de vigilancia en clave de Una sola salud, sus actitudes al respecto (valor atribuido) y los factores que los motivan a instaurar procedimientos concebidos desde la lógica de Una sola salud. Para ello los autores utilizaron una muestra de conveniencia de 185 profesionales de múltiples disciplinas y 44 países. Tras determinar en qué medida esos profesionales practicaban la vigilancia en clave de Una sola salud, les pidieron su opinión sobre la utilidad de este tipo de vigilancia, evaluaron la capacidad que subjetivamente se atribuían de efectuar labores específicas de vigilancia en clave de Una sola salud y determinaron aquellos cambios que esas personas juzgaban prioritarios. Más de un 85% de los encuestados dijo considerar beneficiosa la vigilancia en clave de Una sola salud, sin que se observaran diferencias significativas por ámbito de trabajo o por países según el grupo de ingresos. Más de un 50% afirmó que ya aplicaba este tipo de vigilancia. El problema señalado con más frecuencia y juzgado a la vez más difícil de resolver era el del acceso a datos obtenidos desde otros ámbitos de trabajo. El aspecto que más urgía mejorar era el de la capacidad de enviar y recibir datos electrónicos. Los encuestados de países de nivel bajo o medio de ingresos mostraban mayor motivación a la hora de introducir mejoras que sus homólogos de países de ingresos altos. Estas conclusiones, que ofrecen una «instantánea¼ de las opiniones y prácticas imperantes, pueden ayudar, junto con las propuestas de mejora procedentes de esos profesionales que trabajan sobre el terreno, a seleccionar las necesidades prioritarias de información, formación y recursos para la práctica de la vigilancia en clave de Una sola salud.

Diel Rhythm Does Not Shape the Vertical Distribution of Bacterial and Archaeal 16S rRNA Transcript Diversity in Intertidal Sediments: a Mesocosm Study.

In intertidal sediments, circadian oscillations (i.e., tidal and diel rhythms) and/or depth may affect prokaryotic activity. However, it is difficult to distinguish the effect of each single force on active community changes in these natural and complex intertidal ecosystems. Therefore, we developed a tidal mesocosm to control the tidal rhythm and test whether diel fluctuation or sediment depth influence active prokaryotes in the top 10 cm of sediment. Day- and nighttime emersions were compared as they are expected to display contrasting conditions through microphytobenthic activity in five different sediment layers. A multiple factor analysis revealed that bacterial and archaeal 16S ribosomal RNA (rRNA) transcript diversity assessed by pyrosequencing was similar between day and night emersions. Potentially active benthic Bacteria were highly diverse and influenced by chlorophyll a and phosphate concentrations. While in oxic and suboxic sediments, Thaumarchaeota Marine Group I (MGI) was the most active archaeal phylum, suggesting the importance of the nitrogen cycle in muddy sediments, in anoxic sediments, the mysterious archaeal C3 group dominated the community. This work highlighted that active prokaryotes organize themselves vertically within sediments independently of diel fluctuations suggesting adaptation to physicochemical-specific conditions associated with sediment depth.

IL-1beta mediates MMP secretion and IL-1beta neosynthesis via upregulation of p22(phox) and NOX4 activity in human articular chondrocytes.

OBJECTIVES: Osteoarthritis (OA) is characterized by a progressive alteration of the biochemical properties of the articular cartilage. Inflammation plays a major role in OA, particularly through the cytokine Interleukine-1ß, promoting reactive oxygen species (ROS) generation and matrix metalloproteinases (MMP) synthesis by the chondrocytes, orchestrating matrix proteolysis. NADPH oxidases (NOX) are membrane enzymes dedicated to the production of ROS. Role of oxidative stress is well established in OA; however, contribution of NOX in this process is still poorly documented. In this study, we addressed the role of NOX in primary human articular chondrocytes (HAC) upon inflammatory conditions--namely IL-1ß and OA. DESIGN: HAC were collected from patients undergoing hip surgery. Chondrocytes were treated with IL-1ß and NOX inhibitors Diphenylene Iodonium, GKT136901, Tiron and Heme oxygenase-1 before MMP expression and NOX activity assessment. Finally, NOX4 expression was compared between OA and non OA parts of hip cartilage (n = 14). RESULTS: This study establishes for the first time in human that NOX4 is the main NOX isoform expressed in chondrocytes. We found a significant upregulation of NOX4 mRNA in OA chondrocytes. Expression of NOX4/p22(phox) as well as ROS production is enhanced by IL-1ß. On the other hand, the use of NOX4 inhibitors decreased IL-1ß-induced collagenase synthesis by chondrocytes. Moreover, our study support the existence of a redox dependant loop sustaining pro-catabolic pathways induced by IL-1ß. CONCLUSIONS: This study points out NOX4 as a new putative target in OA and suggests that NOX-targeted therapies could be of interest for the causal treatment of the pathology.

Pilot simulation study using meat inspection data for syndromic surveillance: use of whole carcass condemnation of adult cattle to assess the performance of several algorithms for outbreak detection.

The objective of this study was to assess the performance of several algorithms for outbreak detection based on weekly proportions of whole carcass condemnations. Data from one French slaughterhouse over the 2005-2009 period were used (177 098 slaughtered cattle, 0.97% of whole carcass condemnations). The method involved three steps: (i) preparation of an outbreak-free historical baseline over 5 years, (ii) simulation of over 100 years of baseline time series with injection of artificial outbreak signals with several shapes, durations and magnitudes, and (iii) assessment of the performance (sensitivity, specificity, outbreak detection precocity) of several algorithms to detect these artificial outbreak signals. The algorithms tested included the Shewart p chart, confidence interval of the negative binomial model, the exponentially weighted moving average (EWMA); and cumulative sum (CUSUM). The highest sensitivity was obtained using a negative binomial algorithm and the highest specificity with CUSUM or EWMA. EWMA sensitivity was too low to select this algorithm for efficient outbreak detection. CUSUM's performance was complementary to the negative binomial algorithm. The use of both algorithms on real data for a prospective investigation of the whole carcass condemnation rate as a syndromic surveillance indicator could be relevant. Shewart could also be a good option considering its high sensitivity and simplicity of implementation.

Intravitreal dexamethasone implant versus anti-VEGF injection for treatment-naïve patients with retinal vein occlusion and macular edema: a 12-month follow-up study.

PURPOSE: The objective of his study was to compare the visual and anatomical outcomes in treatment-naïve patients with macular edema secondary to retinal vein occlusion after intravitreal injections of dexamethasone implants (DEX) and anti-VEGF. METHODS: One hundred two patients (64 in the anti-VEGF group, 38 in the DEX group) without previous treatment were included in this multi-center retrospective study and evaluated at baseline and 1, 3, 6, and 12 months after the onset of treatment. Patients were defined as "good responders" if central macular thickness (CMT) was less than or equal to 250 µm in TD-OCT or 300 µm in SD-OCT after the injections. RESULTS: At month 3 (n = 102), BCVA had increased significantly, by 0.1 ± 0.3 logMAR in the anti-VEGF group (p = 0.04) and 0.4 ± 0.4 logMAR in the DEX group (p < 0.001); the difference between the two groups was statistically significant (p = 0.007). CMT decreased significantly, by 138 ± 201 µm (-19 %, p < 0.001) in the anti-VEGF group and 163 ± 243 µm (-21 %, p < 0.001) in the DEX group. After 3 months, five patients (13 %) in the DEX group and 20 (31 %) in the anti-VEGF group (p < 0.001) changed treatment. Among the 77 patients who did not switch from their initial treatment, no significant functional or anatomical difference between the two groups was observed at months 6 and 12. Elevation of intraocular pressure > 21 mmHg was more frequent in the DEX group (21 %) than in the anti-VEGF group (3 %, p = 0.008). CONCLUSIONS: Visual acuity recovery was better in the DEX group than in the anti-VEGF group at month 3, but with no difference in CMT. In patients who did not change treatment, the long-term anatomical and visual outcome was similar between the DEX and anti-VEGF groups.

Microbial mats as model to decipher climate change effect on microbial communities through a mesocosm study.

Marine environments are expected to be one of the most affected ecosystems by climate change, notably with increasing ocean temperature and ocean acidification. In marine environments, microbial communities provide important ecosystem services ensuring biogeochemical cycles. They are threatened by the modification of environmental parameters induced by climate change that, in turn, affect their activities. Microbial mats, ensuring important ecosystem services in coastal areas, are well-organized communities of diverse microorganisms representing accurate microbial models. It is hypothesized that their microbial diversity and metabolic versatility will reveal various adaptation strategies in response to climate change. Thus, understanding how climate change affects microbial mats will provide valuable information on microbial behaviour and functioning in changed environment. Experimental ecology, based on mesocosm approaches, provides the opportunity to control physical-chemical parameters, as close as possible to those observed in the environment. The exposure of microbial mats to physical-chemical conditions mimicking the climate change predictions will help to decipher the modification of the microbial community structure and function in response to it. Here, we present how to expose microbial mats, following a mesocosm approach, to study the impact of climate change on microbial community.

Climate change influences chlorophylls and bacteriochlorophylls metabolism in hypersaline microbial mat.

This study aimed to determine the effect of the climatic change on the phototrophic communities of hypersaline microbial mats. Ocean acidification and warming were simulated alone and together on microbial mats placed into mesocosms. As expected, the temperature in the warming treatments increased by 4 °C from the initial temperature. Surprisingly, no significance difference was observed between the water pH of the different treatments despite of a decrease of 0.4 unit pH in the water reserves of acidification treatments. The salinity increased on the warming treatments and the dissolved oxygen concentration increased and was higher on the acidification treatments. A total of 37 pigments were identified belonging to chlorophylls, carotenes and xanthophylls families. The higher abundance of unknown chlorophyll molecules called chlorophyll derivatives was observed in the acidification alone treatment with a decrease in chlorophyll a abundance. This change in pigmentary composition was accompanied by a higher production of bound extracellular carbohydrates but didn't affect the photosynthetic efficiency of the microbial mats. A careful analysis of the absorption properties of these molecules indicated that these chlorophyll derivatives were likely bacteriochlorophyll c contained in the chlorosomes of green anoxygenic phototroph bacteria. Two hypotheses can be drawn from these results: 1/ the phototrophic communities of the microbial mats were modified under acidification treatment leading to a higher relative abundance of green anoxygenic bacteria, or 2/ the highest availability of CO2 in the environment has led to a shift in the metabolism of green anoxygenic bacteria being more competitive than other phototrophs.

Transfer of a chromosomal Maverick to endogenous bracovirus in a parasitoid wasp.

Bracoviruses are used by parasitoid wasps to allow development of their progeny within the body of lepidopteran hosts. In parasitoid wasps, the bracovirus exists as a provirus, integrated in a wasp chromosome. Viral replication occurs in wasp ovaries and leads to formation of particles containing dsDNA circles (segments) that are injected into the host body during wasp oviposition. We identified a large DNA transposon Maverick in a parasitoid wasp bracovirus. Closely related elements are present in parasitoid wasp genomes indicating that the element in CcBV corresponds to the insertion of an endogenous wasp Maverick in CcBV provirus. The presence of the Maverick in a bracovirus genome suggests the possibility of transposon transfers from parasitoids to lepidoptera via bracoviruses.

[Interpersonal and social rhythm therapy (IPSRT)]. / Thérapie interpersonnelle et aménagement des rythmes sociaux (TIPARS) : du concept anglo-saxon à l'expérience française.

Bipolar disorder is common, recurrent, often severe and debiliting disorder. All types of bipolar disorder have a common determinant: depressive episode. It is justify to propose a psychotherapy which shown efficacy in depression. Howewer, perturbations in circadian rhythms have been implicated in the genesis of each episode of the illness. Biological circadian dysregulation can be encouraged by alteration of time-givers (Zeitgebers) or occurrence of time-disturbers (Zeitstörers). Addition of social rhythm therapy to interpersonal psychotherapy leads to create a new psychotherapy adaptated to bipolar disorders: InterPersonal and Social Rhythm Therapy (IPSRT). IPSRT, in combinaison with medication, has demonstrated efficacy as a treatment for bipolar disorders. IPSRT combines psychoeducation, behavioral strategy to regularize daily routines and interpersonal psychotherapy which help patients cope better with the multiple psychosocial and relationship problems associated with this chronic disorder. The main issues of this psychotherapy are: to take the history of the patient's illness and review of medication, to help patient for "grief for the lost healthy self" translated in the french version in "acceptance of a long-term medical condition", to give the sick role, to examinate the current relationships and changes proximal to the emergence of mood symptoms in the four problem areas (unresolved grief, interpersonal disputes, role transitions, role déficits), to examinate and increase daily routines and social rhythms. French version of IPSRT called TIPARS (with few differences), a time-limited psychotherapy, in 24 sessions during approximatively 6 months, is conducted in three phases. In the initial phase, the therapist takes a thorough history of previous episodes and their interpersonal context and a review of previous medication, provides psychoeducation, evaluates social rhythms, introduces the Social Rhythm Metric, identifies the patient's main interpersonal problem area, and contractualizes the therapy. In the second phase, the therapist focuses work with patient toward regulating the patient's daily routines as well as resolving the interpersonal problem areas relevant to episodes (mainly interpersonal disputes and role transitions). In the third or terminaison phase, the therapist evaluates efficacy of the therapy, enhances the patient's independent functioning and develops strategies for relapse prevention. The further maintenance phase suggests differents strategies as maintenance therapy or focused intensive interventions on specific topics.

Comparative evaluation of field samples using 2 in-clinic assays for heartworm antigen detection in dogs.

Dirofilaria immitis (heartworm) antigen testing is routinely performed in veterinary practices to detect canine heartworm infections. The purpose of this study was to evaluate the performance of two in-clinic assays to detect heartworm antigen on field samples from practices in heartworm endemic regions. Veterinary staff in 3 practices located in the Southern United States performed a side by side comparison of the SNAP® 4Dx® Plus Test (IDEXX) and the VETSCAN FLEX4® Test (Zoetis) on samples from canine patients presented for vector-borne disease screening. Assays were performed according to the manufacturer's instructions. The remaining plasma sample was submitted for confirmatory testing using the PetChek® Heartworm Test (IDEXX) including immune complex dissociation (ICD) by heat treatment. A total of 232 samples were evaluated by the two in-clinic assays and PetChek Test. SNAP 4Dx Plus was significantly more sensitive for the detection of heartworm antigen in this study; sensitivity was 97.4 % for the SNAP 4Dx Plus test and 76.9 % for VETSCAN FLEX4 test (p < 0.01). The specificity of both tests was 99.5 %. This study reveals significant difference in detecting canine heartworm antigen in field samples.

A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.

Listeria monocytogenes is responsible for severe food-borne infections, but the mechanisms by which bacteria cross the intestinal barrier are unknown. Listeria monocytogenes expresses a surface protein, internalin, that interacts with a host receptor, E-cadherin, to promote entry into human epithelial cells. Murine E-cadherin, in contrast to guinea pig E-cadherin, does not interact with internalin, excluding the mouse as a model for addressing internalin function in vivo. In guinea pigs and transgenic mice expressing human E-cadherin, internalin was found to mediate invasion of enterocytes and crossing of the intestinal barrier. These results illustrate how relevant animal models for human infections can be generated.

Modelling the functioning of a coupled microphytobenthic-EPS-bacterial system in intertidal mudflats.

A mechanistic and biogeochemical model was developed to analyze the interactions between microphytobenthos (MPB), bacteria and nutrients in a tidal system. Behavioral vertical migration was hypothesized as being controlled by exogenous factors (tide and light) but also by endogenous factors (carbon and nitrogen requirements). The secretion of Extracellular Polymeric Substances (EPS) during photosynthesis (overflow metabolism) and migration of diatoms was also formulated. Similarities in MPB dynamics between observations and simulations support the assumption that carbon and nitrogen ratios are additional key processes behind the vertical migration of diatoms in the sediment. The model satisfactorily reproduced the three growth phases of the MPB development observed in a mesocosm (the lag phase, the logarithmic growth, and the plateau). Besides, nutrient availability, which could be induced by faunal bioturbation, significantly determined the extent of MPB biomass and development. The plateau phase observed in the last days of simulations appeared to be attributed to a nutrient depletion in the system, emphasizing the importance of nutrient availability. The model, although improvable especially on the formulation of the EPS excretion and bacteria development, already updated understanding of several aspects of benthic-system functioning during experimental conditions.

Reactive oxygen species and NADPH oxidase 4 involvement in osteoarthritis.

Osteoarthritis (OA) is a degenerative chronic disease affecting >300,000 million people around the world as of 2016. Symptomatic measures exist, but there are hardly any curative treatments available. Disruption of the cartilage homeostasis in favor of catabolism leads to cartilage destruction. ROS-macromolecular-induced damage is significantly greater in OA cartilage and OA is described as low-grade chronic systemic inflammation. This review aimed to assess the critical role of cartilage ageing and oxidative stress in the OA process, focusing in particular on NADPH oxidase and especially Nox4 involvement. With age, hypertrophic senescent cells with an altered redox cell profile accumulated. Chondrocytes are more sensitive to oxidant-mediators and the serum level of pro-inflammatory mediators increases. Age-related advanced glycation end products impact on extra cellular matrix (ECM) properties leading to the apoptosis of chondrocytes. A focus on NADPH oxidase-mediated-ROS signaling highlighted the very specific Nox4 isoform, which plays a role on the final common pathway targeting chondrocyte cells. IL-1ß-mediated Nox4 stimulation induced an increase in the levels released by the chondrocyte of MMP-1 and MMP-13 proteins, which are involved in ECM degradation. In comparison with the other Nox isoforms, Nox4 remains unusual, since it is constitutively active, does not depend on cytosolic activator proteins and seems to generate H2O2 thanks to the specific conformation of the Nox4 E-loop. Nox4-induced ROS production appears an essential actor in the OA process and it could be relevant to focus on this target in the aim of discovering and developing new therapeutic strategies.

[Understanding the evolution of polydnaviruses]. / Vers la compréhension de l'histoire évolutive des polydnavirus.

Described in thousands of parasitoid wasp species polydnaviruses (PDVs) are unique viruses having both a segmented DNA genome in viral particles and an integrated form that persists as a provirus in the wasp genome. Parasitoid wasps inject their eggs in another insect host and along with them, the virus particles that are essential to ensure parasitism success. Two phylogenetically unrelated genera of polydnaviruses exist, the bracoviruses (BVs) and the ichnoviruses (IVs) associated with braconid and ichneumonid wasps respectively. New data on the genomes of two bracoviruses (Microplitis demolitor BV and Cotesia congregata BV) and an ichnovirus associated with Campoletis sonorensis (CsIV) offers us new elements to discuss the central questions concerning the origin and the evolution of these viral entities. The results indicate that the tens of millions of years of mutualistic associations between PDVs and wasps has had a strong impact on PDV genomes that now ressemble an eukaryotic region.

Telematic expert system Diabeto. New tool for diet self-monitoring for diabetic patients.

OBJECTIVE: To evaluate Diabeto, a computer-assisted diet education system. RESEARCH DESIGN AND METHODS: One hundred five patients with insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM) were divided into two randomized groups to participate in the evaluation of Diabeto. With free access through Minitel, the French public videotex network, Diabeto helps diabetic patients self-monitor their diets and balance their meals with personalized counseling. RESULTS: During the first 6-mo study, group A (54 patients) used Diabeto, whereas group B (51 patients) were control subjects. For the second 6-mo study, group B used the system. Evaluation was based on patients' dietetic knowledge, dietary habits, and metabolic balance. CONCLUSIONS: Diabeto led to a significant improvement of dietetic, knowledge in group A (P less than 0.0005) and also to improved dietary habits; decreased caloric intake in patients initially overeating (P less than 0.05), increase of dietary carbohydrate from 39.7 +/- 0.7 to 42.9 +/- 0.9% in patients with an initial intake less than 45% carbohydrate, and decrease of fat intake from 41.9 +/- 0.9 to 37.4 +/- 1.1% in patients with an initial intake of greater than 35% fat (P less than 0.0005). In the second study, in addition to similar improvements to those observed in the first study, HbA1 decreased from 11.0 +/- 0.4 to 9.9 +/- 0.4% (P less than 0.005) and fructosamine from 5.00 +/- 0.17 to 4.57 +/- 0.17% (P less than 0.001). Diabeto appears to be an effective therapeutic tool in the control of metabolic diseases.

The Ca2+- and reduced nicotinamide adenine dinucleotide phosphate-dependent hydrogen peroxide generating system is induced by thyrotropin in porcine thyroid cells.

Hydrogen peroxide (H2O2) is an essential electron acceptor for thyroid peroxidase-catalyzed iodination and coupling reactions. In the presence of iodide, its production is a limiting step in thyroid hormone biosynthesis. Several studies have demonstrated that the thyroid particulate fraction contains a Ca2+- and NADPH- dependent H@O@ generator (NADPH-O2:oxidoreductase), the so- called thyroid NADPH-oxidase. It has recently been demonstrated that cellular H2O2 release is under the tonic control of TSH in primary cultures of dog thyrocytes. The present study evaluates the effect of TSH on the thyroid NADPH-oxidase and cytochrome c reductase activities, two enzymes believed to be involved on H2O2 generation in the thyroid gland. There was almost no detectable NADPH-dependent H2O2 generator in the membranes of cells grown for 18 h without TSH. But cells grown in the presence of TSH (0.1 mU/ml) had a CA2+- and NADPH-dependent H2O2-generating activity that increased up to the third day in culture, as did the cell iodide organification capacity. This increase was also partially blocked by 12-O-tetradecanoylphorbol 13-acetate and cycloheximide. Forskolin and 8-bromo-cAMP both reproduced the action of TSH on the Ca2+- and NADPH-dependent H2O2 generator. In contrast, the thyroid NADPH-cytochrome c reductase activity in particles from control cells was similar to that of TSH-treated cells and was unaffected by forskolin or 12-O-tetradecanoylphorbol 13-acetate. These results suggest that NADPH-cytochrome c reductase activity is not regulated by TSH and, thus, reinforce the idea that this enzyme is not involved in thyroid H2O2 generation. On the other hand, the Ca2+- and NADPH-dependent H2O2 generator, so-called thyroid NADPH- oxidase, is induced by TSH through the cAMP cascade. Thus, it seems to be another marker of thyroid differentiation, in addition to thyroperoxidase and thyroglobulin, and could play a key role in thyroid hormone production.

Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase (ThoX, LNOX, Duox) genes and proteins in human thyroid tissues.

The large homolog of NADPH oxidase flavoprotein LNOX2, and probably LNOX1, are flavoproteins involved in the thyroid H(2)O(2) generator. Western blot analysis of membrane proteins from normal human thyroid, using antipeptide antibodies, indicated that LNOX1,2 are 164-kDa glycoproteins and that N-glycosylated motifs account for at least 10-20 kDa of their total apparent molecular mass. Northern blot analysis of 23 different human tissues demonstrated that LNOX2 messenger RNA (mRNA) is strongly expressed only in the thyroid gland, although blast analysis of expressed sequence tags databases indicated that LNOX genes are also expressed in some nonthyroid cells. We investigated LNOX1,2 gene and protein expressions in normal and pathological human thyroid tissues using real-time kinetic quantitative PCR and antipeptide antibodies, respectively. In normal tissue, LNOX1,2 are localized at the apical pole of thyrocytes. Immunostaining for LNOX1,2 was heterogeneous, inside a given follicle, with 40-60% of positive follicular cells. Among normal and pathological tissues, variations of LNOX1 and LNOX2 mRNA levels were parallel, suggesting a similar regulation of both gene expressions. Whereas LNOX mRNAs seemed slightly affected in benign disease, the expression of protein was highly variable. In multinodular goiters, 40-60% of cells were stained. In hypofunctioning adenomas, LNOX immunostaining was highly variable among follicles, whereas sodium/iodide (Na+/I-) symporter immunostaining was decreased. In hyperfunctioning thyroid tissues, only few cells (0-10%) were weakly stained, whereas sodium/iodide symporter staining was found in the majority of follicular cells. In conclusion, LNOX proteins are new apical glycoproteins with a regulation of expression that differs from other thyroid markers.

Ca2+ regulation of thyroid NADPH-dependent H2O2 generation.

A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s).

The relationship between HDL-, LDL-, liposomes-free cholesterol, biliary cholesterol and bile salts in the rat.

In order to study the relationship between bile cholesterol and free cholesterol carried by high and low density lipoproteins (HDL and LDL), 10 male Wistar rats, 11 weeks old and fed with a standard diet were divided into 3 groups which received an intravenous infusion (jugular vein) of either LDL, HDL or liposomes. Liposomes were used for comparison because they are assimilated by hepatocytes, but are not recognized by specific receptors. HDL isolated from rat sera were labeled with [14C]cholesterol by molecular exchange and LDL were labeled by exchange with [14C]cholesterol incorporated into phosphatidyl choline/cholesterol liposomes. The peaks of radioactivity appeared in bile 30 min after the HDL or liposome injection and after 210 min for the LDL injection. The kinetic behavior of the cholesterol carried by the liposomes was quite similar to that of cholesterol carried by HDL. Cholesterol carried by HDL was metabolized in bile salts faster than that carried by LDL: cholesterol-HDL or cholesterol-liposomes contributed to the same extent to the secretion of bile cholesterol (15 and 11%, respectively, of the injected dose), LDL (20% of the injected dose). However, the main part of [14C]cholesterol from HDL, LDL or liposomes was metabolized in bile salts. Thus, cholesterol from an exogenous source seemed to be used mainly as a substrate for bile salts. Our study revealed a difference between the hepatic metabolism of HDL, liposomes and LDL in the rat: the kinetic difference between the secretions of the radioactive compounds in bile may be explained by differences in assimilation, intracellular pathways or bile secretion.

Biochemical characterization of a Ca2+/NAD(P)H-dependent H2O2 generator in human thyroid tissue.

An NAD(P)H-dependent H2O2 forming activity has been evidenced in thyroid tissue from patients with Grave's disease. Its biochemical properties were compared to those of the NADPH oxidase previously described in pig thyroid gland. Both were Ca2+-dependent and activated by inorganic phosphate anions in the same range of concentrations. Both are flavoproteins using FAD as cofactor, but the human enzyme was also able to utilize FMN. The apparent Km for NADPH of the human enzyme (100 microM) was 5-10 times higher than that of porcine enzyme. Vm was 3 to 10 times higher in pig (150 nmol x h(-1) x mg(-1)) than in man (14 to 45). Total content in human tissue was 7 to 9% of that in porcine tissue. An unidentified inhibitor has been detected in the 3000 g particulate fraction from most patients, which could account for this apparently low enzyme content. An NADH-dependent H2O2 production has also been observed in porcine and human thyroid tissues. This activity was only partly Ca2+-dependent (man, 50-70%; pig, 80-90%) and presented similar apparent Km values for NADH (man, 100 microM; pig, 200 microM). In pig thyrocytes, the expression of the Ca2+-dependent part of the NADH-oxidase activity was induced by TSH and down-regulated by TGFbeta, as was the NADPH oxidase activity. Furthermore, NADPH and NADH-dependent activities were not additive. We conclude that a single, inducible, NAD(P)H-oxidase can use NADPH or NADH as substrate to catalyse H2O2 formation, and that human and porcine NAD(P)H-oxidases are highly similar. Differences observed could be attributed to minor differences in enzyme structure and/or in membrane microenvironment. The NADH-dependent Ca2+-independent activity observed in human and porcine thyroid fractions could be attributed to a distinct and constitutive enzyme.