No description available.

Background: Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration dependently. Results: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24–48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8–12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12–24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. Conclusions: Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.

Adipositas und ihre Folgeerkrankungen stellen eine wachsende medizinische Herausforde- rung globalen Ausmaßes dar. Im Rahmen der Adipositasforschung wurde das Fettgewebe als endokrines Organ identifiziert. Von ihm sezernierte Proteine, die sogenannten Adipo- kine, beeinflussen maßgeblich Insulinresistenz und Gefäßverletzbarkeit. Adipositas geht im Fettgewebe mit einer subklinischen chronischen Entzündung einher, die zu einer erhöhten Sekretion von proinflammatorischen Adipokinen führt. Die verstärkte Anwesenheit dieser Proteine ist mit den Komplikationen der Adipositas assoziiert. Die vorliegende Arbeit be- fasst sich mit dem Einfluss von Interleukin (IL)-1β, einem wichtigen Entzündungsmediator des Organismus, auf die Sekretion der proinflammatorischen Adipokine tissue inhibitor of metalloproteinase (TIMP)-1, serum amyloid A (SAA)-3, Lipocalin-2 und Chemerin. Die zugrundeliegenden Untersuchungen wurden mit 3T3-L1- und braunen Adipozyten durch- geführt. Es erfolgte der Nachweis auf mRNA- sowie auf Proteinebene. Der Einsatz von spe- zifischen Inhibitoren erlaubte den Rückschluss auf grundlegende Signalwege. Für alle vier untersuchten Adipokine konnte eine signifikante dosis- und zeitabhängige Steige- rung der mRNA- und Proteinexpression durch IL-1β nachgewiesen werden. Die Transduktion des IL-1β-Signals erfolgte im Falle von Lipocalin-2 und SAA-3 über nuclear factor (NF)-κB und janus kinase (Jak)-2, bei TIMP-1 lediglich über Jak-2 und in Bezug auf Chemerin über NFκB, Jak-2, p44/42 mitogen-activated protein kinase und Phosphatidylinositol-3-Kinase. Die in dieser Arbeit nachgewiesenen Expressions- und Sekretionssteigerungen von TIMP-1, SAA-3, Lipocalin-2 und Chemerin in braunen und weißen Adipozyten festigen IL-1β als einen entscheidenden Mediator proinflammatorischer Prozesse im Fettgewebe. Eine umfassende Bewertung der Funktion von IL-1β im Fettgewebe, insbesondere im Zustand der Adipositas, muss jedoch in weitergehenden Studien erfolgen.

The innate immune system is the first line of defense against invading pathogens. Recognition of microbial ligands by the innate immune system relies on germ-line encoded, evolutionarily conserved receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are one such family of PRRs and are involved in innate defenses to a variety of microbes. At the core of TLR signaling pathways are Toll interleukin-1 receptor (TIR) domain containing adapter proteins. Much of the specificity of TLR pathways arise from the differential use of these adapter proteins. The TLR signaling cascade that ensues upon ligand recognition is marked by finely orchestrated molecular interactions between the receptor and the TIR domain containing adapter proteins, as well as various downstream kinases and effector molecules. Conserving the structural integrity of the TLR components is thus essential for maintaining a robust host defense system. Sometimes, changes in a protein can be brought about by single nucleotide polymorphisms (SNPs). Studies carried out in this thesis focus on polymorphisms in MyD88 adapter-like (Mal) and myeloid differentiation protein 88 (MyD88), two TIR domain-containing adapter proteins, which incidentally are also highly polymorphic. Mal is a 235 amino acid protein that is involved in TLR2 and TLR4 signaling. The known polymorphisms in the coding region of Mal were screened with an aim to identify SNPs with altered signaling potential. A TIR domain polymorphism, D96N, was found to be completely defective in TLR2 and TLR4 signaling. Immortalized macrophage-like cell lines expressing D96N have impaired cytokine production as well as NF-κB activation. The reason for this loss-of-function phenotype is the inability of Mal D96N to bind the downstream adapter MyD88, an event necessary for signaling to occur. Genotyping studies reveal a very low frequency of this polymorphism in the population. Similar SNP analysis was carried out in myeloid differentiation protein 88 (MyD88). MyD88 is a key signaling adapter in TLR signaling; critical for all TLR pathways except TLR3. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knockout mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. S34Y mutant has a dramatically different localization pattern as compared to wild type MyD88. Unlike wild type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. IRAK4, a downstream kinase, colocalizes with MyD88 in these aggregates or “Myddosomes”. S34Y MyD88, however, is unable to assemble into Myddosomes, thus demonstrating that proper cellular localization of MyD88 is a feature required for MyD88 function. This thesis thus describes two loss‐of‐function polymorphisms in TLR adapter proteins Mal and MyD88. It sheds light not only on the structural aspects of signaling by these two proteins, but also has implications for the development of novel pharmaceutical agents.

Transplantation of multipotent mesenchymal progenitor cells is a valuable option for treating tendon disease. Tenogenic differentiation leading to cell replacement and subsequent matrix modulation may contribute to the regenerative effects of these cells, but it is unclear whether this occurs in the inflammatory environment of acute tendon disease. Equine adipose-derived stromal cells (ASC) were cultured as monolayers or on decellularized tendon scaffolds in static or dynamic conditions, the latter represented by cyclic stretching. The impact of different inflammatory conditions, as represented by supplementation with interleukin-1β and/or tumor necrosis factor-α or by co-culture with allogeneic peripheral blood leukocytes, on ASC functional properties was investigated. High cytokine concentrations increased ASC proliferation and osteogenic differentiation, but decreased chondrogenic differentiation and ASC viability in scaffold culture, as well as tendon scaffold repopulation, and strongly influenced musculoskeletal gene expression. Effects regarding the latter differed between the monolayer and scaffold cultures. Leukocytes rather decreased ASC proliferation, but had similar effects on viability and musculoskeletal gene expression. This included decreased expression of the tenogenic transcription factor scleraxis by an inflammatory environment throughout culture conditions. The data demonstrate that ASC tenogenic properties are compromised in an inflammatory environment, with relevance to their possible mechanisms of action in acute tendon disease.

No description available.

La naissance prématurée (NPM) est la cause primaire de morbidité et de mortalité néonatales. Les études ont révélé que l’interleukine-1 (IL-1) possède un rôle primordial dans la pathophysiologie de la NPM, puisqu’elle induit la production de médiateurs pro-inflammatoires et des protéines activatrices de l’utérus menant au travail pré-terme. L’inflammation utéroplacentaire, néfaste pour le fœtus en voie de développement, entraine des séquelles à vie chez ces nouveau-nés. Présentement, le traitement de l’accouchement prématuré repose principalement sur l’administration d’agents tocolytiques, dont la nifédipine, qui ne permettent de prolonger la gestation que de quelques heures. De plus, les études cliniques ont démontré à maintes reprises que les agents tocolytiques ne préviennent pas la NPM, ni les morbidités néonatales. Notre groupe a développé un peptide allostérique (rytvela) qui antagonise le récepteur d'IL-1. Rytvela s'est révélé efficace à prévenir la NPM en inhibant l’inflammation via l’inhibition des voies signalétiques activant le facteur de transcription AP-1 tout en préservant l’activation de la voie NF-B, qui est impliquée dans l’immunovigilence innée et la cytoprotection. Des études antérieures ont démontré l’efficacité de rytvela en mode prophylactique à inhiber la réponse pro-inflammatoire ainsi que l’activation utérine tout en préservant le développement fœtal. Cette étude a pour but de faire progresser le développement pré-clinique de rytvela et de caractériser ses propriétés pharmacodynamiques. Nous avons évalué la dose maximale efficace et la durée de traitement minimale de rytvela pouvant inhiber l’inflammation ainsi que prévenir la NPM et les lésions tissulaires néonatales lorsqu’administré comme agent prophylactique. Nous avons ensuite déterminé le délai maximal d’administration de rytvela à la suite de l’induction du travail pré-terme. Les efficacités de rytvela et de la nifédipine permettant de prévenir les issues gestationnelles et néonatales indésirables ont été comparées. Nous démontrons que lorsqu’administré en mode prophylactique, rytvela présente une efficacité maximale à une dose de 2 mg/kg/jour à une durée minimale de traitement de 36 heures dans des modèles murins de travail pré-terme induit par une infection systémique (LPS) ou de l’inflammation stérile utéroplacentaire (IL-1). Dans le but de représenter une intervention clinique pertinente, nous avons déterminé que rytvela démontrait une efficacité maximale lorsqu’administré jusqu’à 2 heures après l’induction du travail pré-terme. De plus, rytvela possède une efficacité supérieure à celle de l’agent tocolytique, la nifédipine, puisque ce peptide a prévenu les issues gestationnelles et néonatales indésirables associées à la NPM. En somme, à ses propriétés anti-inflammatoires de rytvela, rytvela diminue l’activation utérine afin de prolonger efficacement la gestation ainsi que de prévenir la NPM et les atteintes tissulaires néonatales. Cette étude représente un développement pré-clinique majeur permettant de prévenir les complications causées par la NPM et de s’attaquer à ce besoin non-comblé, puisque les agents tocolytiques demeurent inefficaces. En conclusion, rytvela présente des propriétés désirables pour prévenir et traiter le travail pré-terme afin de promouvoir le développement néonatal. / Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality and affects over 15 million births every year. Studies have shown that interleukin-1 (IL-1) plays a key role in the pathophysiology of PTB as it induces the production of pro-inflammatory mediators and uterine activating proteins leading to labor. More importantly, uteroplacental inflammation, associated with the parturition pathways of PTB, is detrimental to fragile fetal tissues and leads to long term sequalae. Clinical studies have repeatedly shown that tocolytic agents, such as nifedipine, do not prevent PTB or neonatal morbidities. Our group has developed an allosteric peptide (rytvela) that antagonizes the IL-1 receptor. Rytvela is potent and safe in preventing PTB by suppressing inflammation via the inhibition of pathways activating AP-1 while preserving the activation of the NF-B pathway, important in immune-vigilance and cytoprotection. Past work has shown that the prophylactic administration of rytvela inhibits inflammatory upregulation as well as uterine activation and preserves fetal development. This present study aimed to further pre-clinical development of rytvela and to characterize its pharmacodynamic properties. We evaluated the maximal effective dose and minimal duration of treatment of rytvela to inhibit the inflammatory cascade, prevent PTB and neonatal tissue injury when administered as a prophylactic agent. We then determined the maximal delay to administer rytvela after inducing preterm labor (PTL), to inhibit inflammatory upregulation, prolong gestation, and promote neonatal outcome. The efficacies of rytvela and nifedipine in preventing PTB as well as adverse gestational and neonatal outcomes were compared. We demonstrate that when administered prophylactically, rytvela exerts a maximal efficacy at a dose of 2 mg/kg/day at a minimal duration of treatment of 36 hours in murine models of PTL induced by systemic infection (LPS) or by intrauterine sterile inflammation (IL-1). In a clinically relevant treatment approach, rytvela exhibited a maximal efficacy when administered up to 2 hours after the induction of PTL. Moreover, rytvela displayed a superior efficacy in preventing adverse gestational and neonatal outcomes when compared to the tocolytic agent nifedipine. Altogether, through the anti-inflammatory properties of rytvela, rytvela decreases uterine activation to effectively prolong gestation and prevent PTB and neonatal tissue injury. This work represents a relevant treatment approach to prevent complications caused by PTB and would set in place a major preclinical development to address this unmet medical need as tocolytic agents are ineffective. Therefore, rytvela exhibits desirable properties for the prevention and treatment of PTL to prevent adverse gestational and neonatal outcomes.

Dans ce mémoire, je présente mes études sur la synthèse, la caractérisation et l’évaluation biologique de différentes séries d’analogues du D-heptapeptide appelé 101.10, un modulateur négatif allostérique du récepteur de l’interleukine-1β (IL-1β). Sachant que les peptides ont généralement de faibles propriétés pharmacologiques, le but de ce projet portait sur l’examen des structures nécessaires à la bioactivité, la conformation tridimensionnelle de ces derniers afin d’améliorer la droguabilité du peptide parent. Les stratégies d’optimisation du 101.10 utilisées furent : la coupure N- et C-terminale; la substitution par la proline, α-amino-γ-lactame (Agl), β-amino-γ-lactame (Bgl) et α-amino-β-hydroxy-γ-lactame (Hgl); et la rigidification du squelette à l’aide d’un bicycle, l’indolozidin-2-one (I2aa). Afin de clarifier certaines relations de structure-activité, quelques modifications furent apportées au peptide, incluant l’échange de la thréonine pour la valine, la permutation de la stéréochimie de certains résidus clés ainsi que le remplacement de certaines chaînes latérales par un méthyle. Pour pallier aux difficultés de reproductibilité des résultats avec des échantillons provenant de différentes sources, des études sur l’identité du contre-anion et la pureté du peptide furent conduites. Afin d’évaluer l’effet des modifications sur la conformation aqueuse et l’activité biologique du peptide, des analyses de dichroïsme circulaire et des tests in vitro mesurant l’inhibition de certains effets de l’IL-1β furent effectués. Ces essais cellulaires comportaient l’inhibition de la prolifération de cellules immunes et de l’activation des voies de signalisation inflammatoires du facteur nucléaire κB (NF-κB) et de la protéine kinase activée par mitogène (MAPK), toutes deux stimulées par l’IL-1β. La compilation de ces données a permis de déceler certaines tendances entre la structure, la conformation et l’activité anti-IL-1β des peptidomimétiques. / In this thesis, I present my studies toward the synthesis, characterisation and biological evaluation of different series of analogues of the D-heptapeptide called 101.10, a negative allosteric modulator of the interleukin-1β (IL-1β) receptor. Considering that peptides generally exhibit poor pharmacological properties, the objective of this project consisted in: the examination of the peptidic structures essential to elicit bioactivity; the investigation of the three-dimensional arrangement of these moieties; and the improvement of the “drug-like” properties of the parent peptide. The optimisation strategies that were used include: N- and C-terminal truncation; positional scanning using monocycles such as proline, α-amino-γ-lactam (Agl), β-amino-γ-lactam (Bgl) and α-amino-β-hydroxy-γ-lactam (Hgl); and backbone rigidification with indolizidin-2-one (I2aa). Moreover, in order to validate certain structure-activity relationships, further modifications were performed on the peptide: substitution of threonine for valine, exchange of stereochemistry, and substitution of certain side-chain for a methyl group. Lastly, due to divergent behaviour between peptide samples obtained from different sources, studies on the identity of the counter-anion and on the sample purity were conducted. In order to evaluate the influence of these modifications on the aqueous conformation and on the biological activity of the peptide, circular dichroism analyses and in vitro tests measuring the inhibition of certain IL-1β-mediated effects were performed. These cellular assays comprised the inhibition of IL-1β-stimulated proliferation of immune cells, as well as activation of the inflammatory pathways of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Compiling these data revealed certain trends existing between the structure, conformation and anti-IL-1β activity of the peptidomimetics.

Dans ce mémoire, je présente mes études sur la synthèse, la caractérisation et l’évaluation biologique de différentes séries d’analogues du D-heptapeptide appelé 101.10, un modulateur négatif allostérique du récepteur de l’interleukine-1β (IL-1β). Sachant que les peptides ont généralement de faibles propriétés pharmacologiques, le but de ce projet portait sur l’examen des structures nécessaires à la bioactivité, la conformation tridimensionnelle de ces derniers afin d’améliorer la droguabilité du peptide parent. Les stratégies d’optimisation du 101.10 utilisées furent : la coupure N- et C-terminale; la substitution par la proline, α-amino-γ-lactame (Agl), β-amino-γ-lactame (Bgl) et α-amino-β-hydroxy-γ-lactame (Hgl); et la rigidification du squelette à l’aide d’un bicycle, l’indolozidin-2-one (I2aa). Afin de clarifier certaines relations de structure-activité, quelques modifications furent apportées au peptide, incluant l’échange de la thréonine pour la valine, la permutation de la stéréochimie de certains résidus clés ainsi que le remplacement de certaines chaînes latérales par un méthyle. Pour pallier aux difficultés de reproductibilité des résultats avec des échantillons provenant de différentes sources, des études sur l’identité du contre-anion et la pureté du peptide furent conduites. Afin d’évaluer l’effet des modifications sur la conformation aqueuse et l’activité biologique du peptide, des analyses de dichroïsme circulaire et des tests in vitro mesurant l’inhibition de certains effets de l’IL-1β furent effectués. Ces essais cellulaires comportaient l’inhibition de la prolifération de cellules immunes et de l’activation des voies de signalisation inflammatoires du facteur nucléaire κB (NF-κB) et de la protéine kinase activée par mitogène (MAPK), toutes deux stimulées par l’IL-1β. La compilation de ces données a permis de déceler certaines tendances entre la structure, la conformation et l’activité anti-IL-1β des peptidomimétiques. / In this thesis, I present my studies toward the synthesis, characterisation and biological evaluation of different series of analogues of the D-heptapeptide called 101.10, a negative allosteric modulator of the interleukin-1β (IL-1β) receptor. Considering that peptides generally exhibit poor pharmacological properties, the objective of this project consisted in: the examination of the peptidic structures essential to elicit bioactivity; the investigation of the three-dimensional arrangement of these moieties; and the improvement of the “drug-like” properties of the parent peptide. The optimisation strategies that were used include: N- and C-terminal truncation; positional scanning using monocycles such as proline, α-amino-γ-lactam (Agl), β-amino-γ-lactam (Bgl) and α-amino-β-hydroxy-γ-lactam (Hgl); and backbone rigidification with indolizidin-2-one (I2aa). Moreover, in order to validate certain structure-activity relationships, further modifications were performed on the peptide: substitution of threonine for valine, exchange of stereochemistry, and substitution of certain side-chain for a methyl group. Lastly, due to divergent behaviour between peptide samples obtained from different sources, studies on the identity of the counter-anion and on the sample purity were conducted. In order to evaluate the influence of these modifications on the aqueous conformation and on the biological activity of the peptide, circular dichroism analyses and in vitro tests measuring the inhibition of certain IL-1β-mediated effects were performed. These cellular assays comprised the inhibition of IL-1β-stimulated proliferation of immune cells, as well as activation of the inflammatory pathways of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Compiling these data revealed certain trends existing between the structure, conformation and anti-IL-1β activity of the peptidomimetics.

Acute spinal cord injury is often caused by collisions with motor vehicles, falls or violence. This injury could potentially lead to paraplegia or tetraplegia, causing great economic and personal loss. The patophysiology is biphasic, with primary and secondary mechanisms. Regarding secondary spinal cord injury, glutamate and interleukin-1 beta (IL-1<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" />) activates N-methyl-d-aspartate (NMDA)-receptors leading to prolonged excitotoxity, causing neuronal death and subsequently glial scarring. Cross-linked-hyaluronic acid gel and interleukin-1 receptor antagonist (IL-1RA) are believed to have a neuroprotective effect. The major aim of this study was to evaluate neuroprotection in the central neural system. Briefly, spinal cord slice cultures from mice (p9-12) were chemically injured with NMDA and treated with two hyaluronic acid-based gels with integrated, or added, IL1RA. RNA was extracted and transcripted to cDNA. The gene expression of Neuronal nuclear protein, <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" />-aktin and IL-1<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" /> were studies with RT-qPCR. Results showed that gel integrated with IL1RA had significant therapeutic effect, resembling undamaged cultures. Furthermore, axonal outgrowth was investigated in dorsal root ganglion (DRG) in which two preparations of the method were evaluated. Results demonstrated that changing medium every other day was more preferred, compared to adding 20 µl medium every day.  In conclusion, gels integrated with IL1RA have neuroprotective properties and in DRG preparations, medium should be changed every other day for optimal results.