Protective role of peroxisome proliferator-activated receptor-β/δ in septic shock.

Rationale: Peroxisome proliferator activated receptor (PPAR)-beta/delta is a transcription factor that belongs to the PPAR nuclear hormone receptor family, but the role of PPAR-beta/delta in sepsis is unknown. Objectives: We investigated the role of PPAR-beta/delta in murine models of LPS-induced organ injury and dysfunction and cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Methods: Wild-type (WT) and PPAR-beta/delta knockout (1(0) mice and C57BL/6 mice were subjected to LPS for 16 hours. C57BL/6 mice received the PPAR-beta/delta agonist GW0742 (0.03 mg/kg intravenously, 1 h after LPS) or GW0742 plus the PPAR-beta/delta antagonist GSK0660 (0.1 mg/kg intravenously, 30 min before LPS). CD-1 mice subjected to CLP received GW0742 or GW0742 plus GSK0660. Measurements and Main Results: In PPAR-beta/delta KO mice, endotoxemia exacerbated organ injury and dysfunction (cardiac, renal, and hepatic) and inflammation (lung) compared with WT mice. In C57BL/6 mice subjected to endotoxemia, GW0742 significantly (1) attenuated organ (cardiac and renal) dysfunction and inflammation (lung); (2) increased the phosphorylation of Akt and glycogen synthase kinase (GSK)-3 beta; (3) attenuated the increase in extracellular signal-regulated kinase (ERK)1/2 and signal transducer and activator of transcription (STAT)-3 phosphorylation; and (4) attenuated the activation of nuclear factor (NF)-kappa B and the expression of inducible nitric oxide synthase (iNOS). In CD-1 mice subjected to CLP, GW0742 improved 10-day survival. All the observed beneficial effects of GW0742 were attenuated by the PPAR-beta/delta antagonist GSK0660. Conclusions: PPAR-beta/delta protects against multiple organ injury and dysfunction, and inflammation caused by endotoxic shock and improves survival in polymicrobial sepsis by a mechanism that may involve activation of Akt and inhibition of GSK-3 beta and NF-kappa B.

The sirtuin inhibitor cambinol impairs MAPK signaling, inhibits inflammatory and innate immune responses and protects from septic shock.

Sirtuins (SIRT1-7) are NAD(+)-dependent histone deacetylases (HDACs) that play an important role in the control of metabolism and proliferation and the development of age-associated diseases like oncologic, cardiovascular and neurodegenerative diseases. Cambinol was originally described as a compound inhibiting the activity of SIRT1 and SIRT2, with efficient anti-tumor activity in vivo. Here, we studied the effects of cambinol on microbial sensing by mouse and human immune cells and on host innate immune responses in vivo. Cambinol inhibited the expression of cytokines (TNF, IL-1β, IL-6, IL-12p40, and IFN-γ), NO and CD40 by macrophages, dendritic cells, splenocytes and whole blood stimulated with a broad range of microbial and inflammasome stimuli. Sirtinol, an inhibitor of SIRT1 and SIRT2 structurally related to cambinol, also decreased macrophage response to TLR stimulation. On the contrary, selective inhibitors of SIRT1 (EX-527 and CHIC-35) and SIRT2 (AGK2 and AK-7) used alone or in combination had no inhibitory effect, suggesting that cambinol and sirtinol act by targeting more than just SIRT1 and SIRT2. Cambinol and sirtinol at anti-inflammatory concentrations also did not inhibit SIRT6 activity in in vitro assay. At the molecular level, cambinol impaired stimulus-induced phosphorylation of MAPKs and upstream MEKs. Going well along with its powerful anti-inflammatory activity, cambinol reduced TNF blood levels and bacteremia and improved survival in preclinical models of endotoxic shock and septic shock. Altogether, our data suggest that pharmacological inhibitors of sirtuins structurally related to cambinol may be of clinical interest to treat inflammatory diseases.

On the mechanism of protective action of cold acclimatization against carbon tetrachloride - and ethionine-induced fatty liver

In this study the hepatic lipoprotein lipase (LPL), activity was evaluated in adult female mice acclimatized at 5-C and submitted to carbon tetrachloride (CCI) or ethionine, in order to determine the possible role of this enzuyme in the fatty liver. The results were compared with those obtained in mice kept at room temperature (27-C) that the same hepatoesteatosis inducing agent. In contrast to animals kept at room temperature, in cold aclimatized mice neither the enhancement of the LPL-liver activity by the action of CCI or ethionine occurred nor the development of fatty infiltration in the liver was observed. We conclude that the low temperature induced a protective effect against CCI or ethionine-induced fatty liver that was correlated with the no-increase of the hepatic LPL activity.

Thermal niches are more conserved at cold than warm limits in arctic-alpine plant species

Aim Understanding the stability of realised niches is crucial for predicting the responses of species to climate change. One approach is to evaluate the niche differences of populations of the same species that occupy regions that are geographically disconnected. Here, we assess niche conservatism along thermal gradients for 26 plant species with a disjunct distribution between the Alps and the Arctic. Location European Alps and Norwegian Finnmark. Methods We collected a comprehensive dataset of 26 arctic-alpine plant occurrences in two regions. We assessed niche conservatism through a multi-species comparison and analysed species rankings at cold and warm thermal limits along two distinct gradients corresponding to (1) air temperatures at 2 meters above ground level and (2) elevation distances to the treeline (TLD) for the two regions. We assessed whether observed relationships were close to those predicted under thermal limit conservatism. Results We found a weak similarity in species ranking at the warm thermal limits. The range of warm thermal limits for the 26 species was much larger in the Alps than in Finnmark. We found a stronger similarity in species ranking and correspondence at the cold thermal limit along the gradients of 2-m temperature and TLD. Yet, along the 2-m temperature gradient, the cold thermal limits of species in the Alps were lower on average than those in Finnmark. Main conclusion We found low conservatism of the warm thermal limits but a stronger conservatism of the cold thermal limits. We suggest that biotic interactions at the warm thermal limit likely modulate species responses more strongly than at the cold limit. The differing biotic context between the two regions is likely responsible for the observed differences in realised niches.

Association of mutations in the NALP3/CIAS1/PYPAF1 gene with a broad phenotype including recurrent fever, cold sensitivity, sensorineural deafness, and AA amyloidosis.

OBJECTIVE: Familial cold urticaria (FCU) and Muckle-Wells syndrome (MWS) are dominantly inherited autoinflammatory disorders that cause rashes, fever, arthralgia, and in some subjects, AA amyloidosis, and have been mapped to chromosome 1q44. Sensorineural deafness in MWS, and provocation of symptoms by cold in FCU, are distinctive features. This study was undertaken to characterize the genetic basis of FCU, MWS, and an overlapping disorder in French Canadian, British, and Indian families, respectively. METHODS: Mutations in the candidate gene NALP3, which has also been named CIAS1 and PYPAF1, were sought in the study families, in a British/Spanish patient with apparent sporadic MWS, and in matched population controls. Identified variants were sought in 50 European subjects with uncharacterized, apparently sporadic periodic fever syndromes, 48 subjects with rheumatoid arthritis (RA), and 19 subjects with juvenile idiopathic arthritis (JIA). RESULTS: Point mutations, encoding putative protein variants R262W and L307P, were present in all affected members of the Indian and French Canadian families, respectively, but not in controls. The R262W variant was also present in the subject with sporadic MWS. The V200M variant was present in all affected members of the British family with MWS, in 2 of the 50 subjects with uncharacterized periodic fevers, and in 1 of 130 Caucasian and 2 of 48 Indian healthy controls. No mutations were identified among the subjects with RA or JIA. CONCLUSION: These findings confirm that mutations in the NALP3/CIAS1/PYPAF1 gene are associated with FCU and MWS, and that disease severity and clinical features may differ substantially within and between families. Analysis of this gene will improve classification of patients with inherited or apparently sporadic periodic fever syndromes.

The Growth of Brown Adipose Tissue in Cold-acclimatized Rats after Depletion of Mast Cell Histamine by Compound 48/80

Cold acclimatization (4-5°C) is accompanied by 2-3 fold increase of brown adipose tissue (BAT). This rapid growth of interscapular BAT was studied after histamine depletion. In control rats maintained at room temperature (28 ± 2°C) the BAT histamine content was 23.4 ± 5.9 (mean ± SD) µg/g of tissue and cold acclimatization (5±1°C) produced a significant increase of BAT weight, but reduced the histamine content to 8.4 ± 1.9 µg/g. The total weight of BAT after 20 days of acclimatization was unaffected by depletion of histamine due to compound 48/80. The low level of histamine in BAT of cold acclimatized rats could be due to a fast rate of amine utilization; alternatively an altered synthesis or storage process may occur during acclimatization.

Activation of the heat shock response in plants by chlorophenols: transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants.

The ability to detect early molecular responses to various chemicals is central to the understanding of biological impact of pollutants in a context of varying environmental cues. To monitor stress responses in a model plant, we used transgenic moss Physcomitrella patens expressing the beta-glucuronidase reporter (GUS) under the control of the stress-inducible promoter hsp17.3B. Following exposure to pollutants from the dye and paper industry, GUS activity was measured by monitoring a fluorescent product. Chlorophenols, heavy metals and sulphonated anthraquinones were found to specifically activate the hsp17.3B promoter (within hours) in correlation with long-term toxicity effects (within days). At mildly elevated physiological temperatures, the chemical activation of this promoter was strongly amplified, which considerably increased the sensitivity of the bioassay. Together with the activation of hsp17.3B promoter, chlorophenols induced endogenous chaperones that transiently protected a recombinant thermolabile luciferase (LUC) from severe heat denaturation. This sensitive bioassay provides an early warning molecular sensor to industrial pollutants under varying environments, in anticipation to long-term toxic effects in plants. Because of the strong cross-talk between abiotic and chemical stresses that we find, this P. patens line is more likely to serve as a direct toxicity bioassay for pollutants combined with environmental cues, than as an indicator of absolute toxicity thresholds for various pollutants. It is also a powerful tool to study the role of heat shock proteins (HSPs) in plants exposed to combined chemical and environmental stresses.

Lactate and glucose metabolism in severe sepsis and cardiogenic shock.

OBJECTIVE: To evaluate the relative importance of increased lactate production as opposed to decreased utilization in hyperlactatemic patients, as well as their relation to glucose metabolism. DESIGN: Prospective observational study. SETTING: Surgical intensive care unit of a university hospital. PATIENTS: Seven patients with severe sepsis or septic shock, seven patients with cardiogenic shock, and seven healthy volunteers. INTERVENTIONS: C-labeled sodium lactate was infused at 10 micromol/kg/min and then at 20 micromol/kg/min over 120 mins each. H-labeled glucose was infused throughout. MEASUREMENTS AND MAIN RESULTS: Baseline arterial lactate was higher in septic (3.2 +/- 2.6) and cardiogenic shock patients (2.8 +/- 0.4) than in healthy volunteers (0.9 +/- 0.20 mmol/L, p < .05). Lactate clearance, computed using pharmacokinetic calculations, was similar in septic, cardiogenic shock, and controls, respectively: 10.8 +/- 5.4, 9.6 +/- 2.1, and 12.0 +/- 2.6 mL/kg/min. Endogenous lactate production was determined as the initial lactate concentration multiplied by lactate clearance. It was markedly enhanced in the patients (septic 26.2 +/- 10.5; cardiogenic shock 26.6 +/- 5.1) compared with controls (11.2 +/- 2.7 micromol/kg/min, p < .01). C-lactate oxidation (septic 54 +/- 25; cardiogenic shock 43 +/- 16; controls 65 +/- 15% of a lactate load of 10 micromol/kg/min) and transformation of C-lactate into C-glucose were not different (respectively, 15 +/- 15, 9 +/- 18, and 10 +/- 7%). Endogenous glucose production was markedly increased in the patients (septic 14.8 +/- 1.8; cardiogenic shock 15.0 +/- 1.5) compared with controls (7.2 +/- 1.1 micromol/kg/min, p < .01) and was not influenced by lactate infusion. CONCLUSIONS: In patients suffering from septic or cardiogenic shock, hyperlactatemia was mainly related to increased production, whereas lactate clearance was similar to healthy subjects. Increased lactate production was concomitant to hyperglycemia and increased glucose turnover, suggesting that the latter substantially influences lactate metabolism during critical illness.

Recombinant " Physcomitrella patens " as a sensitive tool to study heat sensing and heat-shock signal transduction in plants

SUMMARY When exposed to heat stress, plants display a particular set of cellular and molecular responses, such as chaperones expression, which are highly conserved in all organisms. In chapter 1, I studied the ability of heat shock genes to become transiently and abundantly induced under various temperature regimes. To this aim, I designed a highly sensitive heat-shock dependent conditional gene expression system in the moss Physcomitrella patens, using the soybean heatinducible promoter (hsp17.3B). Heat-induced expression of various reporter genes was over three orders of magnitude, in tight correlation with the intensity and duration of the heat treatments. By performing repeated heating/cooling cycles, a massive accumulation of recombinant proteins was obtained. Interestingly, the hsp17.3B promoter was also activated by specific organic chemicals. Thus, in chapter 2, I took advantage of the extreme sensitivity of this promoter to small temperature variations to further address the role of various natural and organic chemicals and develop a plant based-bioassay that can serve as an early warning indicator of toxicity by pollutants and heavy metals. A screen of several organic pollutants from textile and paper industry showed that chlorophenols as well as sulfonated anthraquinones elicited a heat shock like response at noninducing temperatures. Their effects were synergistically amplified by mild elevated temperatures. In contrast to standard methods of pollutant detection, this plant-based biosensor allowed to monitor early stress-responses, in correlation with long-term toxic effect, and to attribute effective toxicity thresholds for pollutants, in a context of varying environmental cues. In chapter 3, I deepened the study of the primary mechanism by which plants sense mild temperature variations and trigger a cellular signal leading to the heat shock response. In addition to the above described heat-inducible reporter line, I generated a P. patens transgenic line to measure, in vivo, variations of cytosolic calcium during heat treatment, and another line to monitor the role of protein unfolding in heat-shock sensing and signalling. The heat shock signalling pathway was found to be triggered by the plasma membrane, where temperature up shift specifically induced the transient opening of a putative high afimity calcium channel. The calcium influx triggered a signalling cascade leading to the activation of the heat shock genes, independently on the presence of misfolded proteins in the cytoplasm. These results strongly suggest that changes in the fluidity of the plasma membrane are the primary trigger of the heatshocksignalling pathway in plants. The present thesis contributes to the understanding of the basic mechanism by which plants perceive and respond to heat and chemical stresses. This may contribute to developing appropriate better strategies to enhance plant productivity under the increasingly stressful environment of global warming. RÉSUME Les plantes exposées à des températures élevées déclenchent rapidement des réponses cellulaires qui conduisent à l'induction de gènes codant pour les heat shock proteins (HSPs). En fonction de la durée d'exposition et de la vitesse à laquelle la température augmente, les HSPs sont fortement et transitoirement induites. Dans le premier chapitre, cette caractéristique aété utilisée pour développer un système inductible d'expression de gènes dans la mousse Physcomitrella patens. En utilisant plusieurs gènes rapporteurs, j'ai montré que le promoteur du gène hsp17.3B du Soja est activé d'une manière. homogène dans tous les tissus de la mousse proportionnellement à l'intensité du heat shock physiologique appliqué. Un très fort taux de protéines recombinantes peut ainsi être produit en réalisant plusieurs cycles induction/recovery. De plus, ce promoteur peut également être activé par des composés organiques, tels que les composés anti-inflammatoires, ce qui constitue une bonne alternative à l'induction par la chaleur. Les HSPs sont induites pour remédier aux dommages cellulaires qui surviennent. Étant donné que le promoteur hsp17.3B est très sensible à des petites augmentations de température ainsi qu'à des composés chimiques, j'ai utilisé les lignées développées dans le chapitre 1 pour identifier des polluants qui déclenchent une réaction de défense impliquant les HSPs. Après un criblage de plusieurs composés, les chlorophénols et les antraquinones sulfonés ont été identifiés comme étant activateurs du promoteur de stress. La détection de leurs effets a été réalisée seulement après quelques heures d'exposition et corrèle parfaitement avec les effets toxiques détectés après de longues périodes d'exposition. Les produits identifiés montrent aussi un effet synergique avec la température, ce qui fait du biosensor développé dans ce chapitre un bon outil pour révéler les effets réels des polluants dans un environnement où les stress chimiques sont combinés aux stress abiotiques. Le troisième chapitre est consacré à l'étude des mécanismes précoces qui permettent aux plantes de percevoir la chaleur et ainsi de déclencher une cascade de signalisation spécifique qui aboutit à l'induction des gènes HSPs. J'ai généré deux nouvelles lignées afin de mesurer en temps réel les changements de concentrations du calcium cytosolique ainsi que l'état de dénaturation des protéines au cours du heat shock. Quand la fluidité de la membrane augmente après élévation de la température, elle semble induire l'ouverture d'un canal qui permet de faire entrer le calcium dans les cellules. Ce dernier initie une cascade de signalisation qui finit par activer la transcription des gènes HSPs indépendamment de la dénaturation de protéines cytoplasmiques. Les résultats présentés dans ce chapitre montrent que la perception de la chaleur se fait essentiellement au niveau de la membrane plasmique qui joue un rôle majeur dans la régulation des gènes HSPs. L'élucidation des mécanismes par lesquels les plantes perçoivent les signaux environnementaux est d'une grande utilité pour le développement de nouvelles stratégies afin d'améliorer la productivité des plantes soumises à des conditions extrêmes. La présente thèse contribue à décortiquer la voie de signalisation impliquée dans la réponse à la chaleur.

Fluidoterapia guiada con PICCO en pacientes con shock séptico

El tratamiento de la hipovolemia es uno de los principios en la resucitación inicial de la sepsis severa y su tratamiento inicial es mediante la infusión de líquidos endovenosos. A menudo, esta infusión de líquidos resulta inefectiva, y si seguimos insistiendo, puede provocar edema a diferentes órganos e incrementar el agua extravascular pulmonar (EVLW) favoreciendo la aparición del síndrome de distres respiratorio del adulto. En este trabajo se avalúa la utilidad de los parámetros del PICCO (Volumétricos y EVLW) como guía para la administración de fluidos en las primeras 48horas del shock séptico en pacientes postoperados con shock séptico.

Nuclear phenotype changes after heat shock in Panstrongylus megistus (Burmeister)

The nuclear phenotypes of Malpighian tubule epithelial cells of male nymphs of the blood-sucking insect, Panstrongylus megistus, subjected to short- and long-duration heat shocks at 40ºC were analyzed immediately after the shock and 10 and 30 days later. Normal nuclei with a usual heterochromatic body as well as phenotypes indicative of survival (unravelled heterochromatin, giants) and death (apoptosis, necrosis) responses were observed in control and treated specimens. However, all nuclear phenotypes, except the normal ones, were more frequent in shocked specimens. Similarly altered phenotypes have also been reported in Triatoma infestans following heat shock, although at different frequencies. The frequency of the various nuclear phenotypes observed in this study suggests that the forms of cell survival observed were not sufficient or efficient enough to protect all of the Malpighian tubule cells from the deleterious effects of stress. In agreement with studies on P. megistus survival following heat shock, only long-duration shock produced strongly deleterious effects.

Macrophage migration inhibitory factor: a counter-regulator of glucocorticoid action and critical mediator of septic shock.

Recent studies have led to the discovery of a mediator that acts as an endogenous counter-regulator of glucocorticoid action within the immune system. Isolated as a product of anterior pituitary cells, this protein was found to have the sequence of macrophage migration inhibitory factor (MIF), one of the first cytokine activities to be described. Macrophages and T cells release MIF in response both to various inflammatory stimuli and upon incubation with low concentrations of glucocorticoids. The glucocorticoid-induced secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once secreted, MIF "overrides" the anti-inflammatory and immunosuppressive effects of steroids on macrophage and T-cell cytokine production. The physiological role of MIF thus appears to be to counter-balance steroid inhibition of the inflammatory response. Anti-MIF antibodies fully protect animals from experimentally induced gram-negative or gram-positive septic shock, an effect that may be the result of the increased anti-inflammatory effects of glucocorticoids after neutralization of endogenous MIF. Anti-MIF therapeutic strategies are presently under development and may prove to be a means to modulate cytokine production in septic shock as well as in other inflammatory disease states.

Schistosoma mansoni heat shock protein 70 elicits an early humoral immune response in S. mansoni infected baboons

A study was undertaken to search for DNA recombinant Schistosoma mansoni proteins responsible for eliciting an antibody response from the host at a very early phase after infection. A S. mansoni adult worm cDNA expression library was screened using pooled sera from baboons with four weeks of infection. Based on their specific reactivity with the S. mansoni infected sera and no reactivity when tested against the pre-infection sera from the same baboons, four clones were selected for further studies. Sequence analysis revealed that they were homologous to the S. mansoni heat shock protein 70 (hsp70). The insert sizes of the four selected clones varied from 1150 to 2006 bp. The preliminary characterization for antibody reactivity against a panel of baboon sera showed that the longest clone was the most reactive, eight out of eight acute and three out of four chronic sera reacting positively to this clone. The shortest clone was the least reactive. Our results suggest that the S. mansoni hsp70 elicits an early and strong antibody response in baboons and that antibodies to this protein can be detected in chronically infected animals. Therefore S. mansoni hsp70 may be a valid target for immunodiagnosis. However further studies are needed to identify the portion of the hsp70 that best fits the requirements for a valuable diagnostic antigen.