P619Role of Toll-like receptor 5 in the development of post-myocardial infarction inflammation

Background: Inflammatory processes play a key role in the pathophysiology of myocardial infarction (MI). Genetic deletion of toll-like recpetors (TLRs), especially TLR2 and TLR4 have shown protective role in murine models of MI. The role of other TLRs remains unknown. We have previously shown that cardiomyocytes express TLR5 and that the ligand of TLR5, flagellin, activates the NF-kappaB and MAPK pathways in cardiomyocytes. We also have shown that injection of flagellin induces acute systolic dysfunction in vivo in mice. Aim: Determine the role of TLR5 in the development of post-MI inflammation. Methods: A murine model of myocardial infarction was done by a 30 minutes ligation of the left anterior descending coronary artery followed by 2 hours of reperfusion. Infarct size was measured by standard Evans blue/TTC staining. Plasma creatine kinase (CK) was quantified as a read out of myocardial necrosis. Tissue and plasma cytokines (MIP-2, MCP-1, IL-6) were quantified by ELISA. To determine the extent of tissue lipid peroxidation we used malondialdehyde and 4-hydroxynonenal-HIS adduct assays. Tissue protein oxidation was tested by protein carbonyl ELISA kit. Phosphorylation of MAPK was analyzed by western blot. Results: Genetic suppression of TLR5 induced a significant increase of myocardial infarct size and plasma CK, of biochemical markers of myocardial oxidative stress, and cytokine levels in the heart and the plasma after MI. These effects were associated with a marked enhancement of p38 phosphorylation in the heart from TLR5 KO mice. Conclusion: TLR5 protects from acute myocardial injury and reduces local and systemic inflammation during myocardial infarction. The mechanisms may involve reduced p38 signaling, decreased oxidative stress and attenuated cytokine expression. Research supported by the Swiss National Science Foundation, Grant n° 310030_135394/

P90Necrotic cardiomyocytes release soluble pro-inflammatory molecule(s) inducing il1r/myd88-dependent inflammatory responses in cardiac fibroblasts

Background: Inflammation comes out to be a critical biological process in the pathophysiology of myocardial infarction (MI). We hypothesize that this inflammation is triggered by necrotic cardiomyocytes (Cmc) that release a set of endogenous molecules (DAMPs: danger-associated molecular patterns) activating inflammatory responses in cardiac fibroblasts. Aim: Analyze in vitro the immune activation of cardiac fibroblasts exposed to necrotic Cmc conditioned media. Methods: Primary neonatal murine cardiac fibroblasts and Cmc were obtained by digestion of neonatal hearts and differential plating technique allowing a selection for cardiomyocytes and cardiac fibroblasts. Cmc were killed by necrotic stimuli including oxidants (hydrogen peroxide) and mechanic stresses (freeze-thaw). Necrosis was assessed using Hoechst/PI stainings. Fibroblasts were exposed to necrotic Cmc conditioned media and mRNA expression of inflammatory genes was measured by real-time PCR and ELISA. Activation of signaling pathways was analyzed by western blot. We used cardiac cells from Myd88-/-, Trif-/- and Nlrp3-/- animals to evaluate the contribution of TLRs/IL1-R and NLRP3 inflammasome in the sensing of necrotic DAMPs. Results: mRNA expression of chemokines such as MCP-1, MIP-2 and IP-10 were induced in fibroblasts exposed to necrotic Cmc conditioned media. Alternatively, fibroblasts exposed to necrotic fibroblasts conditioned media showed a lower increase in mRNA expression of these chemokines. In addition, in fibroblasts from Myd88-/- mice, response to Cmc conditioned media was fully abrogated whereas no difference was observed in Trif-/- and Nlrp3-/- fibroblasts. Conclusion: Cardiac fibroblasts are able to produce a rapid and specific inflammatory response to necrotic Cmc conditioned media involving the expression of neutrophil and monocyte chemoattractants. The dependence on MyD88 adaptor protein strongly suggests that this response relies on TLR/IL-1R signaling. These results engage cardiac fibroblasts as key players in post-MI inflammatory responses as they are able to sense DAMPs from necrotic Cmc and possibly recruit inflammatory cells. Research supported by the Swiss National Science Foundation, Grant n° 310030_135394/

Homocysteine induces cell death in H9C2 cardiomyocytes through the generation of peroxynitrite

Homocysteine (HCY) is toxic on blood vessels, but a potential direct toxicity of HCY on the heart is unknown. We addressed this issue by exposing H9C2 cardiomyocytes to HCY (0.1-5 mM) for up to 6h. At these concentrations, HCY reduced cell viability, induced necrosis and apoptosis and triggered the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). This was associated with the intracellular generation of the potent oxidant peroxynitrite. Removing peroxynitrite by the decomposition catalyst FeTPPS considerably reduced LDH release, DNA fragmentation, cleavage of caspase-3 and PARP, and restored normal cell morphology. In additional experiments performed in primary rat ventricular cardiomyocytes, HCY (1 mM, 6h) activated the phosphorylation of the MAP kinases ERK and JNK, two essential stress signaling kinases regulating myocardial apoptosis, hypertrophy and remodeling. These results provide the first demonstration that HCY kills cardiomyocytes through the generation of peroxynitrite and can activate key signaling cascades in the myocardium

Peroxynitrite is a potent inhibitor of NF-{kappa}B activation triggered by inflammatory stimuli in cardiac and endothelial cell lines.

Peroxynitrite is a potent oxidant and nitrating species proposed as a direct effector of myocardial damage in numerous cardiac pathologies. Whether peroxynitrite also acts indirectly, by modulating cell signal transduction in the myocardium, has not been investigated. Therefore, we examined a possible role for peroxynitrite on the activation of NF-kappaB, a crucial pro-inflammatory transcription factor, in cultured H9C2 cardiomyocytes. H9C2 cells were stimulated with tumor necrosis factor-alpha or lipopolysaccharide following a brief (20-min) exposure to peroxynitrite. NF-kappaB activation (phosphorylation and degradation of its inhibitor IkappaBalpha, nuclear translocation of NF-kappaB p65, and NF-kappaB DNA binding) triggered by lipopolysaccharide or tumor necrosis factor-alpha was abrogated by peroxynitrite. Peroxynitrite also inhibited NF-kappaB in two human endothelial cell lines activated with tumor necrosis factor-alpha or interleukin-1beta. These effects were related to oxidative but not nitrative chemistry and were still being observed while nitration was suppressed by epicatechin. The mechanism of NF-kappaB inhibition by peroxynitrite was a complete blockade of phosphorylation and activation of the upstream kinase IkappaB kinase (IKK) beta, required for canonical, pro-inflammatory NF-kappaB activation. At the same time, peroxynitrite activated phosphorylation of NF-kappaB-inducing kinase and IKKalpha, considered as part of an alternative, noncanonical NF-kappaB activation pathway. Suppression of IKKbeta-dependent NF-kappaB activation translated into a marked inhibition of the transcription of NF-kappaB-dependent genes by peroxynitrite. Thus, peroxynitrite has a dual effect on NF-kappaB, inhibiting canonical IKKbeta-dependent NF-kappaB activation while activating NF-kappaB-inducing kinase and IKKalpha phosphorylation, which suggests its involvement in an alternative pathway of NF-kappaB activation. These findings offer new perspectives for the understanding of the relationships between redox stress and inflammation

Seelische Belastung bei Menschen mit umweltbezogenen Störungen: Ein Vergleich zwischen Selbstbild und Fremdeinschätzung

Zusammenfassung: Umweltbezogene Gesundheitsstörungen stellen wissenschaftlich und klinisch ein diagnostisches und therapeutisches Konfliktfeld dar. Ein hoher subjektiver Leidensdruck, ein somatisches Krankheitsmodell sowie das Festhalten an einer Umweltursache gefährden die therapeutische Beziehung, wenn die Krankheitsmodelle von Arzt und Patient nicht übereinstimmen. Unsere Untersuchung greift diese empirisch noch kaum erforschte Diskrepanz auf, indem sie die Selbsteinschätzung von Patienten mit umweltbezogenen Krankheitsstörungen (n=61) systematisch der Fremdeinschätzung durch ein multidisziplinäres Expertenteam gegenüberstellt. Die Resultate weisen darauf hin, dass in Selbst- und Fremdeinschätzung psychisch unauffällige Probanden wenig psychiatrische Störungen zeigen, über stabile psychische Strukturen verfügen und v.a. unter umweltbedingten oder medizinischen Ursachen leiden. Wenn Selbst- und Fremdeinschätzung beide eine psychische Auffälligkeit beschreiben, sind entsprechend psychiatrische Störungen gehäuft, psychische Strukturen labil, und die Beschwerden werden psychiatrisch verursacht. Divergieren Selbst- und Fremdeinschätzung, erlaubt die Fremdeinschätzung akkuratere Aussagen bezüglich der psychiatrischen Diagnosen, der innerpsychischen Struktur und der Ursachenzuschreibun

A proposal for ethical research conduct in Madagascar

This journal provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. The attached file is the published version of the article

Cutting edge: IL-1α is a crucial danger signal triggering acute myocardial inflammation during myocardial infarction.

Myocardial infarction (MI) induces a sterile inflammatory response that contributes to adverse cardiac remodeling. The initiating mechanisms of this response remain incompletely defined. We found that necrotic cardiomyocytes released a heat-labile proinflammatory signal activating MAPKs and NF-κB in cardiac fibroblasts, with secondary production of cytokines. This response was abolished in Myd88(-/-) fibroblasts but was unaffected in nlrp3-deficient fibroblasts. Despite MyD88 dependency, the response was TLR independent, as explored in TLR reporter cells, pointing to a contribution of the IL-1 pathway. Indeed, necrotic cardiomyocytes released IL-1α, but not IL-1β, and the immune activation of cardiac fibroblasts was abrogated by an IL-1R antagonist and an IL-1α-blocking Ab. Moreover, immune responses triggered by necrotic Il1a(-/-) cardiomyocytes were markedly reduced. In vivo, mice exposed to MI released IL-1α in the plasma, and postischemic inflammation was attenuated in Il1a(-/-) mice. Thus, our findings identify IL-1α as a crucial early danger signal triggering post-MI inflammation

Beta amyloid and hyperphosphorylated tau deposits in the pancreas in type 2 diabetes.

Strong epidemiologic evidence suggests an association between Alzheimer disease (AD) and type 2 diabetes. To determine if amyloid beta (Abeta) and hyperphosphorylated tau occurs in type 2 diabetes, pancreas tissues from 21 autopsy cases (10 type 2 diabetes and 11 controls) were analyzed. APP and tau mRNAs were identified in human pancreas and in cultured insulinoma beta cells (INS-1) by RT-PCR. Prominent APP and tau bands were detected by Western blotting in pancreatic extracts. Aggregated Abeta, hyperphosphorylated tau, ubiquitin, apolipoprotein E, apolipoprotein(a), IB1/JIP-1 and JNK1 were detected in Langerhans islets in type 2 diabetic patients. Abeta was co-localized with amylin in islet amyloid deposits. In situ beta sheet formation of islet amyloid deposits was shown by infrared microspectroscopy (SIRMS). LPS increased APP in non-neuronal cells as well. We conclude that Abeta deposits and hyperphosphorylated tau are also associated with type 2 diabetes, highlighting common pathogenetic features in neurodegenerative disorders, including AD and type 2 diabetes and suggesting that Abeta deposits and hyperphosphorylated tau may also occur in other organs than the brain