Ulm, Univ., Diss., 2008.

La molécule d'adhésion intercellulaire-1 (ICAM-1, CD54) est une glycoprotéine membre de la superfamille des immunoglobulines exprimée à la surface d'une grande variété de types cellulaires. Sa fonction première est de lier les β2-intégrines LFA-1 et MAC-1 exprimées à la surface des lymphocytes et des macrophages, respectivement. Cette interaction permet à ces derniers d'atteindre le site de l'inflammation en procédant à leur liaison à l'endothélium vasculaire et à leur extravasation. L'expression de ICAM-1 fût par contre également associée à diverses pathologies telles que l'asthme, l'arthrite rhumatoïde, le diabète, le développement des plaques athérosclérotiques et le développement des métastases. Bien que l'expression constitutive de ICAM-1 soit relativement faible, elle peut être augmentée par de nombreux médiateurs de l'inflammation tels que le TNF-α et l'IFNγ. Ces signaux amplifient l'expression de ICAM-1 au niveau transcriptionnel par diverses voies de signalisation menant à l'activation de facteurs de transcription tels que NF-KB et Stati. Ces facteurs de transcription agissent en se liant aux multiples éléments de réponse présents sur le promoteur de ICAM-1. La capacité de ces facteurs de transcription à activer la transcription de ICAM-1 dépend de nombreuses interactions physiques et fonctionnelles dont la nature n'est pas toujours bien connue. Par ailleurs, les voies de signalisation menant à l'inactivation de la transcription de ICAM-1 demeurent également incertaines. Étant donné le rôle capital de ICAM-1 au sein du système immunitaire, il est essentiel d'approfondir nos connaissances sur les différentes étapes menant à l'activation tout comme l'inactivation de la transcription de ce gène. Nous avons donc étudié la relation entre deux familles de facteurs de transcription impliquées dans la régulation transcriptionnelle de ICAM-1, soit la famille STAT et la famille Ets. Nous avons non seulement découvert qu'il existe une coopération fonctionnelle entre ces deux familles, mais également que le facteur de transcription Stati interagit physiquement avec le facteur de transcription Etsl dans les cellules vivantes. Ensuite, nous avons étudié les effets du bpV(pic), un inhibiteur de tyrosine phosphatase, sur l'activation de la transcription de ICAM-1. Nous avons découvert que ce composé de peroxovanadium inactive les phosphatases responsables de l'inactivation de la voie JAK/STAT suite au traitement à l'IFNγ. Finalement, nous avons testé un nouveau protocole d'électroporation à double impulsion permettant une grande efficacité de transfection transitoire des cellules endothéliales à faible coût.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Introduction: The mechanism of cell activation by LPS in the absence of surface Toll-like receptor 4 (TLR4) is unclear. We hypothesize that ICAM-1 binds LPS on the cell surface, mediating cell activation independent of TLR4. Methods: The interaction between murine ICAM-1 and LPS was measured in a binding assay. Alveolar macrophages (AMs) isolated from TLR4 deficient mice were stimulated with LPS. Cell activation was measured by flow cytometry and cytokine production. The role of ICAM-1 in cell activation was determined by siRNA transfection. Results: Murine ICAM-1 binds LPS. TLR4 deficient AMs respond to LPS stimulation by upregulation of LPS binding sites, ICAM-1 expression and cytokine release. Cell activation is attenuated by treatment with polymyxin B and ICAM-1 gene silencing. Conclusions: ICAM-1 binds LPS and is important in TLR4-independent cell activation. Strategies devised to target ICAM-1 may have the potential to block the excessive inflammatory response seen in gram-negative sepsis.

Introduction: The mechanism of cell activation by LPS in the absence of surface Toll-like receptor 4 (TLR4) is unclear. We hypothesize that ICAM-1 binds LPS on the cell surface, mediating cell activation independent of TLR4. Methods: The interaction between murine ICAM-1 and LPS was measured in a binding assay. Alveolar macrophages (AMs) isolated from TLR4 deficient mice were stimulated with LPS. Cell activation was measured by flow cytometry and cytokine production. The role of ICAM-1 in cell activation was determined by siRNA transfection. Results: Murine ICAM-1 binds LPS. TLR4 deficient AMs respond to LPS stimulation by upregulation of LPS binding sites, ICAM-1 expression and cytokine release. Cell activation is attenuated by treatment with polymyxin B and ICAM-1 gene silencing. Conclusions: ICAM-1 binds LPS and is important in TLR4-independent cell activation. Strategies devised to target ICAM-1 may have the potential to block the excessive inflammatory response seen in gram-negative sepsis.

The blood-brain barrier (BBB) is located at the level of the cerebral microcapillaries, and functions to maintain environmental homeostasis by allowing the neurons access to the required nutrients and enabling the exchange of metabolic waste. BBB dysfunction has been observed in a number of pathophysiologic statres including peripheral inflammatory pain (Huber et al., 2001b). Using the lamda-carrageenan inflammatory pain (CIP) model, we observed alterations in the tight junction (TJ) proteins paralleled by an increase in BBB permeability to [14C] sucrose. The mechanisms by which these perturbations occurred remain to be elucidated. In the current study, we investigate the central mechanism for the BBB perturbations under CIP. It is our hypothesis that the modulations of the BBB under CIP, are mediated via a central signaling pathway. First, to investigate the involvement of neuronal input from pain activity on alterations in BBB, we developed a method for inhibiting the nociceptive input from the paw. Using a perineural injection of 0.75% bupivacaine into the right hind leg prior to CIP, we were able inhibit development thermal hyperalgesia induced by CIP, as tested by infrared heat stimulus, without effecting edema formation 1 h post CIP. Upon inhibition of nociception under CIP, there was an attenuation of both the changes in permeability and the changes in tight junction protein expression, with both returning to control levels. Next, we investigated intercellular adhesion molecule-1 (ICAM-1), a key signaling protein at the BBB, which in the presence of proinflammatory mediators, increases in expression leading to the activation of signaling pathways as well as morphological changes. We found a region specific increase in ICAM-1 mRNA and protein expression following CIP which directly correlated with increased expression of activated microglia. Finally, we investigated the influence activated microglia had on BBB permeability. Using an 0.150 mg intrathecal bolus injection of minocycline, a potent inhibitor of microglia activation (Klein and Cunha, 1995), we were able to inhibit the increased expression of activated microglia, and saw an attenuation of permeability to control levels. These findings suggest CIP induced BBB disruption is localized and has a central-mediated component independent of peripheral influence.

Résumé : Les maladies cardio-vasculaires (MCV) constituent l'une des principales causes de mortalité et de morbidité dans le monde. De nombreuses études épidémiologiques montrent que les niveaux des lipoprotéines de haute densité (HDL) sont inversement corrélés au risque des MCV. Les propriétés antiathérogènes des HDL sont attribuées, en partie, à l'action de l'enzyme Paraoxonase 1 (PON1) qui leur est associée exclusivement. Le rôle de la PON1 dans l’activité antioxydante des HDL a été largement étudié, alors que le mécanisme par lequel la PON1 exerce ses effets anti-inflammatoires reste à établir. La prévalence accrue des MCV avec l'augmentation de l'âge incite à rechercher certains facteurs qui pourraient expliquer cette tendance. Peu de travaux se sont intéressés à l'impact d'une réduction de la fonctionnalité de HDL dans le développement de l'athérosclérose et encore moins en présence d'un facteur de risque pour les MCV, tel que l'âge. À ce jour, plusieurs travaux ont mis en évidence une diminution de l'activité antioxydante de HDL et de la PON1 chez la personne âgée. Cependant, il n'existe pas de données sur l'effet de la réduction de l'activité de la PON1 sur l'activité anti-inflammatoire des HDL. Nos objectifs sont d'élucider le rôle de la PON1 dans les activités anti-inflammatoires des HDL et de déterminer l'effet de sa modulation sur l'activité athéroprotectrice des HDL chez les personnes âgées. Nos résultats montrent que la PON1 diminue significativement le niveau d'expression des molécules d'adhésion intercellulaire-1 (lCAM-1) au niveau des cellules endothéliales inhibant ainsi l'induction de l'inflammation en présence de phospholipides oxydés. Cette activité anti-inflammatoire de la PON1 est due à la fois à sa capacité de réduire l'oxydation des lipides (effet antioxydant) mais aussi à son activité phospholipase like, hydrolysant ainsi les lipides oxydés. Toutefois, cet effet anti-inflammatoire dépend de son association aux HDL. Nos résultats démontrent ainsi que l'effet anti-inflammatoire des HDL est dû en grande partie à la PON1. Cette activité des HDL diminue significativement au cours du vieillissement. Nos résultats montrent aussi que 12 semaines de consommation d'huile d'olive extra vierge (EVOO) augmentent significativement l'activité anti-inflammatoire des HDL. Cet effet est dû à une augmentation de l'activité de PON1 et d'une réduction du niveau de stress oxydant. Cet effet bénéfique de l'EVOO est plus significatif chez les personnes âgées que chez les personnes jeunes. Conclusions: Nos résultats apportent la démonstration de l'implication de la PON1 dans la régulation des activités antiathérogènes des HDL et proposent des mécanismes de l'implication de la PON1 dans la régulation de cette activité anti-inflammatoire des HDL. Aussi, nos résultats montrent l'importance de la diète de type méditerranéen dans la régulation des fonctions antiathérogènes des HDL, et plus particulièrement, en présence d'un facteur de risque pour les MCV, tel que le vieillissement. // Abstract : Cardiovascular diseases are one of the main causes of mortality and morbidity throughout the world. Several epidemiologic studies show that there is an inverse correlation between the levels of high density lipoproteins (HDL) and cardiovascular risk. The antiatherogenic properties of HDL are due, in part, to paraoxonase 1 (PON1) enzyme, which is associated exclusively to HDL. The role of PON1 in the antioxidant activity of HDL has been widely studied. However, the mechanism by which PON1 exerts its antiinflammatory effect remains to be determined. Increased prevalence of cardiovascular diseases and the increasing life span encourages investigations for certain predisposing factors that could explain this tendency. Few studies have focused on the impact of a reduction of HDL activity within the development of atherosclerosis during the aging process. Nowadays, several studies had demonstrated a reduction in the antioxidant activities of HDL and PON1 in the elderly population. However, there is no data of the effect of aging on the anti-inflamm atory activities of PON 1 and HDL. The main objectives of the present study were to explain the role of the PON1 in the anti-inflammatory properties of the HDL and to determine the effect of the modulation of PON1 activity on the atherprotective effect of HDL in the elderly population. Our results show that PON1 decreased significantly ICAM-1 expression within the endothelial cells, which inhibits the induction of inflammation in the presence of oxidized phospholipids. The anti-inflammatory activity of PON1 is due to its capacity to reduce lipid oxidation (antioxidant effect) and to its phospholipase-like activity, which consists of hydrolyzing oxidized lipids. Our results also show that 12 weeks of extra virgin oil (EVOO) increase significantly the anti-inflammatory activity of HDL. This effect is due principally to an improvement of the activity of PON1 and reduction of oxidative stress status. This beneficial effect of EVOO was more significant in the elderly than in young subjects. Conclusions: Our results demonstrate the role of the PON1 in the regulation of the antiatherogenic activities of HDL and suggest a mechanism for the implication of PON1 in the regulation of the anti-inflammatory activity of HDL. In addition, our results show the importance of the Mediterranean diet in the regulation of antiatherogenic function HDL, and particularly, in the presence of a risk factor for cardiovascular disease such as aging.

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. 75-89. Publié aussi en version électronique.

Thèse (de maîtrise)--Université Laval, 2003. / Bibliogr.: f. 75-98. Publié aussi en version électronique.

Submitted by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-06-14T18:24:22Z No. of bitstreams: 1 Publico-tese_noujaim.pdf: 2146495 bytes, checksum: b1240b89a7f24795fa03d7aeba0b14b5 (MD5) / Approved for entry into archive by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-06-14T18:26:55Z (GMT) No. of bitstreams: 1 Publico-tese_noujaim.pdf: 2146495 bytes, checksum: b1240b89a7f24795fa03d7aeba0b14b5 (MD5) / Made available in DSpace on 2016-06-14T18:26:55Z (GMT). No. of bitstreams: 1 Publico-tese_noujaim.pdf: 2146495 bytes, checksum: b1240b89a7f24795fa03d7aeba0b14b5 (MD5) Previous issue date: 2007 / Introdução: vários fatores estão associados à lesão de isquemia fria (IF) e referfusão quente (RQ) no transplante hepático (TxH), tais como infiltrado de neutrófilos e linfo-plasmocitário, liberação de citoquinas inflamatórias e apoptose. Porém, pouco se conhece sobre o papel da IF/RQ em enxertos esteatóticos. Objetivo: avaliar o papel da lesão de IF/RQ no TxH em humanos comparando enxertos esteatóticos e não esteatóticos. Métodos: entre maio/02 e março/07 foram realizadas 84 biópsias pós reperfusão (2hs após RQ) e 18 pré reperfusão, totalizando-se 84 TxH em 82 pacientes. As biópsias foram agrupadas em 5 grupos, de acordo com o grau de macro e microesteatose: GEL – leve (<30%), GEM – moderada (30-59%), GEG - grave (≥60%), GEA - sem esteatose, GPR-pré-reperfusão. Nas 102 biópsias foram analisadas: porcentagens de macro e microesteatose, graus de exudato de neutrófilos (0-3) e infiltrado linfo-plasmocitário portal (0-3), índices de apoptose (métodos de Túnel e Caspase- 3) e de ICAM-1. As esteatoses macro (n=49) e microvesicular (n=74) foram individualmente analisadas e classificadas em graus leve (G1), moderado (G2) e grave (G3) e ausente (G4). Resultados: o índice de apoptose (TUNEL) foi: GEL=0.262±0.111, GEM=0.278±0.113, GEG=0.244±0.117, GEA=0,275±0.094 e GPR=0.181±0.123, p-0.07. No grupo macroesteatose índice de apoptose (TUNEL) foi: G1=0.284± 0.106, G2+3=0.160±0.109, G4=0,275±0.094, p-0.05; e no grupo microesteatose, G1=0.222±0.123, G2+3=0.293±0.108, G4=0.275±0.094, p-0.049. O GEG expressou o ICAM-1 em 83% dos casos de forma difusa. Não existiu diferença estatística entre os grupos ao analisarmos os índices de apoptose (caspase-3) e ICAM-1. Conclusão: o GEG e o grupo macroesteatose (moderado e grave) apresentaram significante redução no índice de apoptose, enquanto o grupo microesteatose (moderado e grave), significante aumento. E o GEG apresentou expressão de ICAM-1 difusamente, podendo ser estes marcadores envolvidos na lesão de I/R hepática dos enxertos esteatóticos. / Introduction: many factors are responsable for ischemic/reperfusion (I/R) injury in liver transplant (LTx) as apoptosis. However, the role of I/R injury in steatotic grafts is still unclear. Mains: to analyze the role of I/R in LTx comparing steatotic vs non steatotic graft. Methods: between May/02 and march/07 we performed 84 liver biopsies (2hours) after arterial reperfusion of grafts. Overall we performed 84 LTx in 82 patients.The liver biopsies were divided in 5 groups according with degree of macro and microvesicular steatosis in mild (<30%)-MSG, moderate (30-59%)-MoSG, severe (≥60%) – SSG, absent stetatosis – ASG, before reperfusion – BRG. In 102 liver biopsies were analyzed: percentage of macro and micro steatosis, degree of neutrophils, apoptosis index (TUNEL and Caspase-3) and ICAM-1. Also were analyzed macro and micro steatosis alone and they were classified in different degree MILD (G1), moderate (G2), severe (G3) and absent (G4). Results: the apoptosis index (TUNEL) was: MSG=0.262±0.111, MoSG=0.278±0.113, SSG=0.244±0.117, ASG=0,275±0.094 e BPR=0.181±0.123, p-0.07. The macrosteatosis’ apoptosis index (TUNEL) was G1=0.284± 0.106, G2+3=0.160±0.109, G4=0,275±0.094, p-0.05; and in microsteatosis group - G1=0.222±0.123, G2+3=0.293±0.108, G4=0.275±0.094, p-0.049. There are no statistic difference among the groups when we analyzed apoptosis (caspase-3) and ICAM-1 index. Conclusion: the SSG and macrosteatosis (degree moderate and severe) presented significantly decrease of apoptosis index, probable because these cells died before start apoptotic process. However the microsteatosis (degree moderate and severe) increased apoptotic index associated with I/R injury.