Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Mechanisms of the protective effect of 3-Deazaadenosine in a rat model of lipopolysaccharide-induced myocardial dysfunction

Sepsis und septischer Schock gehen noch immer mit einer hohen Mortalität einher. Neben einer generellen Organdysfunktion kommt es zu einer Verschlechterung der kardialen Funktion, die als septische Kardiomyopathie bezeichnet wird und zu der hohen Mortalität beiträgt. Ursächlich steht eine überschießende Entzündungsreaktion im Vordergrund. Die Extravasation und Akkumulation von aktivierten Leukozyten im Gewebe spielt dabei eine wichtige Rolle, da es dadurch zu einer exzessiven lokalen Freisetzung von Entzündungsmediatoren im Herzgewebe kommt. Für 3-Deazaadenosin (c3Ado), ein potenter Inhibitor der S-Adenosylhomocysteinhydrolase, sind immunmodulatorische und antiinflammatorische Eigenschaften bekannt. Ziel dieser Arbeit war es, diese Eigenschaften von c3Ado bei der septischen Kardiomyopathie zu untersuchen und Mechanismen der protektiven Wirkung von c3Ado auf die Myokarddepression aufzuzeigen. Die Untersuchungen wurden am Rattenmodell mit Lipopolysaccharid (LPS) induzierter Sepsis durchgeführt.Durch Behandlung mit c3Ado konnte die LPS-induzierte Endothelzellaktivierung/vermehrte Expression der Adhäsionsmoleküle ICAM-1 und VCAM-1 verhindert werden. Die Expression von P-Selektin, das bereits vorgeformt in den Weibel-Palade-Körperchen im Endothel vorliegt, wurde nicht beeinflusst. Durch immunhistochemische Färbung gegen CD11b, ED1 und gp91phox konnte eine Hemmung der Migration aktivierter Leukozyten aus den postkapillären Venolen in das Myokard nachgewiesen werden. Dies führte zu einer verminderten Akkumulation iNOS positiver polymorphkerniger Neutrophiler und konsekutiver NO-Produktion sowie einer verminderten lokalen Sauerstoffradikalfreisetzung. Die Aktivität der endothelialen Stickstoffsynthetase (eNOS) wurde durch c3Ado nicht beeinflusst. 3-Deazaadenosin beeinflusst vor allem die endotheliale Seite der Leukozyten-Endothelzell-Interaktion: Mittels FACS-Analyse war eine Hemmung der CD11b-Expression auf den zirkulierenden aktivierten Neutrophilen nicht nachweisbar. Wir konnten im Myokard einen inhibitorischen Effekt von c3Ado auf die Bindungsaktivität des Transkriptionsfaktors NF-κB als einen zentralen Entzündungsmediator zeigen. 3-Deazaadenosin greift somit auf verschiedenen Ebenen in die Entzündungskaskade bei der septischen Kardiomyopathie ein. Die kardioprotektive Wirkung macht c3Ado zu einer interessanten potentiellen Substanz zur Behandlung der kardialen Dysfunktion bei Sepsis.Mortality from severe sepsis and septic shock remains high. In addition to general organ dysfunction, myocardial depression is a characteristic feature of cardiocirculatory changes during severe sepsis. The so called septic cardiomyopathy contributes to the high mortality of the syndrome. The leading process is an excessive inflammatory response to an infection. The migration to extravascular sites and accumulation of activated leukocytes in the myocardium contributes to an excessive local release of NO and reactive oxygen species (ROS). 3-Deazaadenosine (c3Ado) is a strong inhibitor of the S-adenosylhomocysteine hydrolase. Immune-modulating and anti-inflammatory effects of c3Ado have been described. This study aimed to elucidate the underlying mechanisms of the positive effect of c3Ado in a rat model of LPS-induced cardiomyopathy. The treatment with c3Ado prevented the LPS-induced upregulation of the adhesion molecules ICAM-1 and VCAM-1. 3-Deazaadenosine was not able to prevent LPS-induced expression of P-selectin, which is already preformed in the Weibel-Palade bodies of the endothelium. Immunohistochemical staining for CD11b, ED1 and gp91phox demonstrated that c3Ado prevented local recruitment of monocytes and polymorph nuclear neutrophils to the myocardium. Accordingly, significantly fewer leukocytes producing nitric oxide or reactive oxygen species accumulated within the myocardium. The activity of the endothelial nitric oxide synthase (eNOS) was not affected by c3Ado. 3-Deazaadenosine has its main impact on the endothelial site of the leukocytes-endothelium-interaction: FACS analysis demonstrated, that c3Ado was not able to prevent the CD11b expression on circulating activated neutrophils. 3-Deazaadenosine was able to prevent the activation of NF-κB within the myocardium of LPS-treated rats. NF-κB is one of the main mediators of inflammatory response. 3-Deazaadenosine interferes with different levels of the inflammatory cascade leading to septic cardiomyopathie. The cardioprotective effect makes c3Ado an interesting potential drug for the treatment of myocardial dysfunction in sepsis

\u3cem\u3eIn Vitro\u3c/em\u3e evaluation of leukemia inhibitory factor receptor antagonists as candidate therapeutics for inflammatory arthritis

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) are found in appreciable concentrations in synovial fluid from patients with rheumatoid arthritis (RA) but not osteoarthritis. Accordingly, both are potential therapeutic targets in inflammatory diseases of the joints. Several LIF antagonists have been developed. They have the capacity to inhibit the biologic activities of not only LIF but also other interleukin-6 (IL-6) subfamily cytokines, including OSM. Both LIF and OSM share the same receptor, which is part of a cytokine receptor super family in which the glycoprotein 130 (gp130) subunit is a common constituent. The aim of this study was to evaluate the antagonistic potentials of two LIF mutants, LIF05 and MH35-BD. Both are mutant forms of human LIF with reduced affinity for gp130 and greater LIF receptor (LIFR) binding affinity. The results, using Ba/F3 cell proliferation assay, acute-phase protein (haptoglobin) induction analysis in HepG2 human hepatoma cells, a porcine cartilage glycosaminoglycan release assessment for proteoglycan degradation, and a collagen release assay, show that these antagonists inhibit relevant LIF, OSM, and other IL-6 subfamily cytokines in vitro albeit with differential potencies and have, therefore, therapeutic potential for treatment of RA and perhaps other diseases

Auto-phosphorylation represses protein kinase R activity

The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity

Generation of mutant leukaemia inhibitory factor (LIF)-IgG heavy chain fusion proteins as bivalent antagonists of LIF

Two leukaemia inhibitory factor (LIF) mutants, designated MH35-BD and LIF05, have been shown to have a capacity to inhibit the biological activities of not only human LIF (hLIF) but also other interleukin-6 (IL-6) subfamily cytokines such as human oncostatin M (hOSM). These cytokines share the same receptor complex in which the glycoprotein 130 (gp130) subunit is a common constituent. However, at low concentrations and in their monomeric forms, such molecules have a relatively short plasma half-life due to rapid clearance from the kidneys. Here, to prolong their serum half-lives, we have used a multi-step polymerase chain reaction (PCR) to fuse each of the LIF05 and MH35-BD cDNA fragments to a sequence encoding the Fc portion, and the hinge region, of the human immunoglobulin G (hIgG) heavy chain. The linking was achieved through an oligomer encoding a thrombin-sensitive peptide linker thus generating MH35-BD:Fc and LIF05:Fc, respectively. Both Fc fusion constructs were expressed in insect cell Sf21 and the proteins were purified by two successive affinity chromatography steps using nickel&ndash;nitrilotriacetic acid (Ni&ndash;NTA) agarose and protein A beads. The Ba/F3 cell-based proliferation assay was used to confirm that the proteins were biologically active. In addition, preliminary pharmacokinetics indicates that the Fc fusion constructs have a longer serum half-life compared to their non-fusion counterparts.<br /

Purification and biological characterization of soluble, recombinant mouse IFNβ expressed in insect cells

Interferon β (IFNβ) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNβ utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNβ. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNβ purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNβ than any other reported eukaryotic-based expression system. Recombinant murine IFNβ produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNβ activity in vivo. Recombinant IFNβ also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNβ

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN ? receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2