Le système RUSH, de l’anglais « Retention using selective hook » est un système développé récemment qui permet d'analyser et de quantifier en temps réel le transport d'une grande diversité de protéines. Le système RUSH permet, grâce à un excès de molécules de Streptavidine (Str.) dirigées dans différents compartiments cellulaires (appelées les hameçons), de retenir des protéines appelées les rapporteurs, comportant un biocapteur fluorescent tel que la GFP (« Green fluorescent protein ») fusionné avec un peptide SBP (« Streptavidin-binding peptide »). L’addition de biotine dans le milieu perturbe l’interaction entre SBP et la Streptavidine, libérant ainsi les rapporteurs de leur hameçon. Basé sur le système RUSH, nous avons établi une méthode de criblage pour identifier des agents pharmacologiques dotés de la capacité à induire la libération d’HMGB1 (« High Mobility Group Box 1 »). La translocation d’HMGB1 depuis le noyau vers le cytoplasme, ainsi que sa sécrétion ou libération passive dans l'espace extracellulaire à travers les membranes plasmiques perméabilisées, représente un signal de danger essentiel à l’activation du système immunitaire. Dans ce système RUSH modifié, une protéine de fusion du Str-NLS3 a été utilisée comme un hameçon nucléaire pour retenir la protéine chimère constituée d'HMGB1, SBP et GFP (HMGB1-SBP-GFP). Lorsque de la biotine est ajoutée en combinaison à des chimiothérapies inductrices de la mort cellulaire immunogène (ICD) telles que les anthracyclines, elle se lie de manière compétitive à Str-NLS3 et permet la libération et la translocation nucléo-cytoplasmique des rapporteurs HMGB1-SBP-GFP. Nous avons utilisé ce système pour des criblages à haut débit visant à identifier des agents induisant le relargage d’HMGB1. Les agents identifiés appartiennent à trois catégories différentes : les inducteurs connus de l’ICD, les inhibiteurs des microtubules et les modificateurs épigénétiques. Leur effet a été confirmé par des méthodes multiples de mesure de la quantité protéique d’HMGB1 nucléaire, cytoplasmique et extracellulaire dans des cellules humaines et murines in vitro ainsi que dans le plasma de souris. Nos données révèlent également que ces agents induisent la libération d’HMGB1 par des mécanismes distincts : arrêt du cycle cellulaire, acétylation des histones ou effets « on-target » par l'inhibition d’ADN méthyltransférase. Il serait alors intéressant d'étudier si les effets décrits ici peuvent contribuer aux effets immunostimulateurs des médicaments utilisés pour le traitement de cancers ou de maladies parasitaires.Le système RUSH permettant la synchronisation et la quantification de la sécrétion des protéines du réticulum endoplasmique (RE) vers l'appareil de Golgi, il permet de cribler un grand nombre de composés afin d’identifier des inhibiteurs des sécrétions candidates. Nous avons conçu et construit une lignée cellulaire humaine exprimant les chimères SBP-GFP sécrétables ainsi que les hameçons Str-KDEL ciblant l’ER ; la biotine permet donc la libération du rapporteur par les voies de sécrétion classiques. Nous avons identifié et validé plusieurs médicaments qui sont capables d’inhiber la sécrétion de protéines : les anti-angineux, les antidépresseurs, les anti-helminthiques, anti-psychotiques, anti-protozoaires, et agents immunosuppresseurs. Ces composés varient dans leur capacité à inhiber la synthèse des protéines et de compromettre la morphologie du RE ou l'intégrité du Golgi. Les données ont ensuite été soumises à une analyse bio-informatique et cette procédure a permis l'identification de quatre groupes en fonction de leur mode d'action. Cette partie démontre la faisabilité et l'utilité d'un nouvel essai de criblage phénotypique basé sur le système RUSH. Nous avons conçu des systèmes de HSC (« High Content Screening ») basés sur le système RUSH, qui ont permis l'identification d'agents pharmacologiques induisant la libération d’HMGB1, ainsi que des inhibiteurs de la sécrétion protéique. / The retention using selective hooks (RUSH) system allows withholding load cargoes with fluorescent biosensor such as green fluorescent proteins (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin (Str) molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the reporter from its hook. Based on the RUSH system, we developed a screening assay to identify pharmacological agents endowed with HMGB1 (high mobility group box 1) releasing capacities. The translocation of HMGB1 from the nucleus to the cytoplasm and its secretion or passive release through the permeabilized plasma membrane constitutes a major cellular danger signal. Extracellular HMGB1 can interact with specific pattern recognition receptors to stimulate pro-inflammatory and immunostimulatory pathways. In this modified RUSH system, a Str-NLS3 fusion protein was used as a nuclear hook to seize SBP fused with HMGB1 and GFP. When combined with biotin, which competitively binds to Stre-NLS3 to free the HMGB1-SBP-GFP, immunogenic cell death (ICD) inducers such as anthracyclines were able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. We used this system for high-content screenings (HCS) to identify HMGB1 releasing agents. Hits fell into three functional categories: known ICD inducers, microtubule inhibitors, and epigenetic modifiers. Their effective action was confirmed by multiple methods monitoring nuclear, cytoplasmic and extracellular HMGB1 pools, both in cultured human or murine cells, as well as in mouse plasma. These agents induced HMGB1 release through a whole set of distinct mechanisms, cell cycle arrest, histone acetylation, or on-target effect. It will be interesting to learn whether such effects may contribute to the immunostimulatory effects of drugs that are used to treat malignant disease or worm infection. For HCS of identification of pharmacological inhibitors of conventional protein secretion, we constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and Str-KDEL hook within the endoplasmic reticulum (ER) lumen, and biotin addition releases the reporter, ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant. We identified and validated a series of molecularly unrelated drugs including antianginal, antidepressant, anthelmintic, antipsychotic, antiprotozoal and immunosuppressive agents that inhibit protein secretion. These compounds vary in their capacity to suppress protein synthesis and to compromise ER morphology and Golgi integrity, as well as in the degree of reversibility of such effects. These data was then subjected to bioinformatics analysis including correlation analyses, non-supervised hierarchical clustering, and principal component analysis and led to the identification of 4 clusters of agents. We demonstrate the feasibility and utility of a novel RUSH-based phenotypic screening assay. In summary, we built HCS systems based on the improved RUSH sysytem for identification of agents that induce HMGB1 release or inhibit conventional protein secretion.

La plus grande sensibilité des souris TLR9-/- a révélé le rôle de ce récepteur dans l'infection par Leishmania major. Les cellules dendritiques (DCs) sont activées de manière TLR9-dépendante par l'ADN du L. major et d'autres Trypanosomatidae et non par l'ADN de vertébré. La nature de l'ADN capable d'activer le TLR9 reste controversée quant à la séquence/charpente de l'ADN et l'implication de cofacteurs se liant avec le TLR9 ou l'ADN. Nous avons démontré l'importance de la séquence d'ADN. Contrairement aux génomes de parasites, l'ADN de vertébré présente une contre-sélection des motifs activateurs du TLR9 au profit des motifs inhibiteurs. De plus, l'activation du TLR9 par l'ADN du parasite est augmentée en présence de la protéine HMGB1, qui se fixe mieux sur l'ADN de parasite que de vertébré. La maturation du TLR9 requiert un clivage protéolytique par des protéases endosomales, dont les cathepsines (Cat) B, S, L et l'asparagine endopeptidase (AEP) qui interviennent différemment dans les macrophages et les DCs. Après infection par L. major, nous avons montré que les souris AEP-/-, CatS-/- et CatL-/- ont une pathologie identique aux souris WT, ce qui peut être dû à la redondance de leur fonction. Etonnamment, les souris CatB-/- sont plus résistantes. Leurs lésions et la charge parasitaire dans les ganglions se résolvent plus rapidement, reflétant une réponse immune plus précoce et un contrôle plus rapide de la réaction inflammatoire.En conclusion, ces résultats contribuent à une meilleure compréhension des mécanismes permettant au TLR9 de discriminer entre l'ADN de pathogène et de vertébré et soulèvent le rôle non protecteur de la cathepsine B dans l'infection par L. major. / As TLR9-deficient mice are more sensitive to Leishmania major infection, we have shown previously that TLR9 receptor mediates this parasite infection. Dendritic cells (DCs) are activated by L. major and other Trypanosomatidae DNA and not by vertebrate DNA. There is an ongoing controversy concerning the properties of DNA required for TLR9 activation, regarding the DNA sequence or backbone or the implication of a cofactor interacting with TLR9 or DNA. We have established the importance of DNA sequences. In contrast to parasite genome, vertebrate genome have counter-selected stimulatory sequences and over-represented inhibitory motifs for TLR9. In addition, host proteins contribute to TLR9-dependent DC activation. HMGB1 enhances TLR9 activation only in the presence of L. major DNA and, surprisingly, HMGB1 binds more abundantly L. major than vertebrate DNA. TLR9 activation requires a proteolytic cleavage by endosomal proteases, as cathepsins (Cat) B, S and L and asparagine endopeptidase (AEP) that have a differential activity in macrophages and DCs. After L. major infection, we have showed that AEP-/-, CatS-/- and CatL-/- mice have a similar pathology than WT mice, likely due to their functionnally redundant activites. In contrast, CatB-/- mice are more resistant to the infection. Their lesion sizes and the parasite burdens in lymph nodes are significantly decreased, reflecting an earlier immune response and a more rapid control of the inflammatory response. In conclusion, our results bring further insights into how TLR9 discriminates between Trypanosomatidae and vertebrate DNA and reveal a non protective role of cathepsin B in L. major infection.

High mobility group box 1 (HMGB-1) protein is a non-histone chromosomal protein. As a DNA binding protein, HMGB-1 is involved in the maintenance of nucleosome structure, regulation of gene transcription and it is active in DNA recombination and repair. It has been known that HMGB-1 is a late mediator of endotoxemia and sepsis. HMGB-1 is released from activated macrophages, induces the release of other proinflammatory mediators, and mediates cell death when overexpressed. We speculated that the course of sepsis maybe different without the involvement of HMGB-1. The aims of this study are to investigate the role of HMGB-1 in mediating sepsis and to observe the effects by using RNAi to affect the production of HMGB-1. Lipopolysaccharide (LPS) was used to simulate sepsis in culture as well as stimulate the release of HMGB-1 from RAW 264.7 cells. Levels of HMGB-1 in the culture medium were subsequently measured by Western blot. Other proinflammatory cytokines (TNF-£\, IL-6 and TGF-£]) were measured by ELISA. HMGB-1 could not be detected in the culture medium in the absence of LPS stimuli, but after 0.5 £gg/ml LPS treatment HMGB-1 release could be detected. HMGB-1 the amount of released from LPS activated RAW 264.7 cells was in a time- and dose-dependent manner. The present study demonstrated that RNAi in the treatment of LPS-stimulated RAW264.7 cells resulted in the blockade of HMGB-1 and decreased LPS-induced inflammatory response. The results demonstrated that HMGB-1 plays a pivotal role in macrophage inflammatory responses by modulating the production of inflammatory mediators.

Asthma is a chronic disorder highlighted by intermittent airway inflammation and characterized by paroxysmal dyspnea and airway hyperresponsiveness (AHR). A key feature of severe asthma is the development of airway wall remodeling, which is thought to occur through repeated rounds of inflammation and tissue repair. Remodeling includes structural changes such as increased mass of airway smooth muscle (ASM), and excessive collagen deposition. ASM cells contribute to airway remodeling via the expression and secretion of extracellular matrix (ECM) proteins. This is particularly driven by inflammatory processes, which include mediators such as transforming growth factor (TGF)-β1 and damage associated molecular pattern (DAMP) proteins, such as high mobility group box 1 (HMGB1). HMGB1 is ubiquitously expressed as a non-histone DNA-binding protein that can regulate gene expression, but can also be released in response to stress to underpin inflammation and tissue repair. In this study we tested the hypothesis that extracellular HMGB1 induces signaling pathways that control responses linked to progression of airway inflammation, remodeling and hyperresponsiveness in human ASM cells. We used primary cultured ASM cells as well as hTERT-immortalized human ASM cells. With immunoblotting we demonstrate that exogenous HMGB1 (10 ng/mL) can induce rapid and sustained phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) that is comparable to that induced by a potent mitogen, platelet derived growth factor (PDGF-BB, 10 ng/mL). We also found that TGF-β1 (2.5 ng/mL) promotes the accumulation of secreted HMGB1 in culture medium in a time line concomitant with expression of ECM proteins, collagen and fibronectin, suggesting a role for HMGB1 in pro-fibrotic effects of TGF-β1. By lentiviral delivery, we induced stable expression of short hairpin RNA (shRNA) that silenced expression of endogenous HMGB1 or mammalian diaphanous 1 (mDia1), a cytoplasmic scaffold protein that is required for HMGB1-induced cell responses through one of its receptors, receptor for advanced glycation end products (RAGE). Immunoblot analyses revealed that silencing of mDia1 was associated with markedly decreased induction of p42/p44 MAPK phosphorylation by exogenous HMGB1. In HMGB1-silenced human ASM cells, we observed significantly reduced synthesis and secretion of collagen A1 and fibronectin in response to TGF-β1 (2.5 ng/mL, 0-48 hrs). However, exogenous HMGB1 was not sufficient to rescue ECM synthesis in response to TGF-β1 in HMGB1-silenced cells - this suggests that intracellular, but not necessarily secreted HMGB1, regulates ECM expression and secretion in response to TGF-β1. Consistent with this interpretation, exogenous HMGB1 alone was not sufficient to induce ECM synthesis or secretion in primary cultured ASM cells. In conclusion, we show that though in human ASM cells extracellular HMGB1 alone can activate MAPK signaling, likely via mDia1-dependent pathways involving RAGE. it is not capable of prompting ECM protein expression. Recombinanat exogenous HMGB1 does not appear to directly affect ECM synthesis, rather intracellular (nuclear) HMGB1 likely modulates activity of genes that are affected by TGF-β1. Overall, HMGB1 has potential to regulate tissue repair processes involving ASM through intracellular and extracellular mechanisms, thus our findings support further work to elucidate the role of HMGB1 in pathogenesis of obstructive airway disease.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

A sepse é um conjunto complexo de interações moleculares e celulares mediado pela estimulação de receptores celulares envolvidos na inflamação. O controle desta rede de sinalização é compensado pela resposta pró- e antiinflamatória do organismo. Os desequilíbrios inflamatórios induzidos alteram o estado fisiológico de diversos órgãos. No cérebro, observam-se diversas modificações que envolvem desde o aumento na permeabilidade da barreira hematoencefálica (BHE), morte de células neurais por apoptose ou necrose, redução do fluxo sanguíneo e aumento do estresse oxidativo, resultando no desenvolvimento do delirium associado à sepse. O delirium associado à sepse é um conjunto de disfunções neurológicas induzidas por uma resposta inflamatória sistêmica. Assim, é possível que diferentes mecanismos compensatórios estejam envolvidos durante a sepse. Este estudo objetivou analisar a influência do modelo de ligação cecal e punção (CLP), na excreção de ligantes do receptor para produtos finais de glicação avançada (RAGE) e as modificações ocasionadas no cérebro pela inflamação sistêmica decorrente da sepse. Nossos resultados demonstraram que os níveis séricos de TNF-α aumentaram após a indução da CLP, confirmando o estado inflamatório dos animais CLP, ocorrendo um aumento nos níveis de proteínas carboniladas, HSP70 e HMGB-1 no soro desses animais, indicando um aumento de estresse oxidativo nesses animais e liberação de proteínas associadas a manutenção e estabilização intracelular. Entretanto, encontramos uma redução no imunoconteúdo de destas proteínas no hipocampo e córtex, possivelmente por aumento nos níveis de necrose/lise celular ou por eliminação durante o período de disrupção da BHE. Assim, concluímos que durante a sepse ocorre uma aumento expressivo nos níveis séricos de proteínas associadas a resposta inflamatória crônica, além de alterações nos níveis destas proteínas relacionadas a proteção intracelular no hipocampo e córtex, que pode ter relação com as alterações observadas em pacientes e modelos animais. / Sepsis is a complex whole of molecular and cellular interactions mediated stimulation of cellular receptors involved in inflammation. The control of this system is compensated by pro and anti-inflammatory response in organism. The imbalance induced inflammatory alters the physiological state of different organs. In the brain, there are various modifications that involve from the increase in permeability of the blood-brain barrier (BBB), neural cell death by apoptosis or necrosis, reduced blood flow and increased oxidative stress, resulting in the development of sepsis-associated delirium. Sepsis-associated delirium is a whole of neurological dysfunctions induced a systemic inflammatory response. Thus, it is possible that different compensatory mechanisms are involved during sepsis. The aim of the study was to analyze the influence of the model cecal ligation and puncture (CLP), in the excretion of agonists for receptor advanced glycation end products (RAGE) and alterations in the brain caused by systemic inflammation caused by sepsis. Our results showed that serum levels of TNF-α increased after the induction of CLP, confirming the inflammatory status of CLP animals, which results in increased levels of carbonylated proteins, HSP70 and HMGB-1 in the serum of these animals, indicating an increase of stress oxidative these animals and release of proteins associated with maintaining and stabilizing intracellular. However, happened a reduction in immunocontent these proteins in the hippocampus and cortex, possibly by increased levels of necrosis/lysis cell or by removal during the period of disruption of the BBB. Therefore, concluded that during sepsis occurs a significant increase in serum proteins associated with chronic inflammatory response and changes in levels of proteins related to intracellular protection in the hippocampus and cortex, which may be related to the changes observed in patients and models animals.

A sepse é um conjunto complexo de interações moleculares e celulares mediado pela estimulação de receptores celulares envolvidos na inflamação. O controle desta rede de sinalização é compensado pela resposta pró- e antiinflamatória do organismo. Os desequilíbrios inflamatórios induzidos alteram o estado fisiológico de diversos órgãos. No cérebro, observam-se diversas modificações que envolvem desde o aumento na permeabilidade da barreira hematoencefálica (BHE), morte de células neurais por apoptose ou necrose, redução do fluxo sanguíneo e aumento do estresse oxidativo, resultando no desenvolvimento do delirium associado à sepse. O delirium associado à sepse é um conjunto de disfunções neurológicas induzidas por uma resposta inflamatória sistêmica. Assim, é possível que diferentes mecanismos compensatórios estejam envolvidos durante a sepse. Este estudo objetivou analisar a influência do modelo de ligação cecal e punção (CLP), na excreção de ligantes do receptor para produtos finais de glicação avançada (RAGE) e as modificações ocasionadas no cérebro pela inflamação sistêmica decorrente da sepse. Nossos resultados demonstraram que os níveis séricos de TNF-α aumentaram após a indução da CLP, confirmando o estado inflamatório dos animais CLP, ocorrendo um aumento nos níveis de proteínas carboniladas, HSP70 e HMGB-1 no soro desses animais, indicando um aumento de estresse oxidativo nesses animais e liberação de proteínas associadas a manutenção e estabilização intracelular. Entretanto, encontramos uma redução no imunoconteúdo de destas proteínas no hipocampo e córtex, possivelmente por aumento nos níveis de necrose/lise celular ou por eliminação durante o período de disrupção da BHE. Assim, concluímos que durante a sepse ocorre uma aumento expressivo nos níveis séricos de proteínas associadas a resposta inflamatória crônica, além de alterações nos níveis destas proteínas relacionadas a proteção intracelular no hipocampo e córtex, que pode ter relação com as alterações observadas em pacientes e modelos animais. / Sepsis is a complex whole of molecular and cellular interactions mediated stimulation of cellular receptors involved in inflammation. The control of this system is compensated by pro and anti-inflammatory response in organism. The imbalance induced inflammatory alters the physiological state of different organs. In the brain, there are various modifications that involve from the increase in permeability of the blood-brain barrier (BBB), neural cell death by apoptosis or necrosis, reduced blood flow and increased oxidative stress, resulting in the development of sepsis-associated delirium. Sepsis-associated delirium is a whole of neurological dysfunctions induced a systemic inflammatory response. Thus, it is possible that different compensatory mechanisms are involved during sepsis. The aim of the study was to analyze the influence of the model cecal ligation and puncture (CLP), in the excretion of agonists for receptor advanced glycation end products (RAGE) and alterations in the brain caused by systemic inflammation caused by sepsis. Our results showed that serum levels of TNF-α increased after the induction of CLP, confirming the inflammatory status of CLP animals, which results in increased levels of carbonylated proteins, HSP70 and HMGB-1 in the serum of these animals, indicating an increase of stress oxidative these animals and release of proteins associated with maintaining and stabilizing intracellular. However, happened a reduction in immunocontent these proteins in the hippocampus and cortex, possibly by increased levels of necrosis/lysis cell or by removal during the period of disruption of the BBB. Therefore, concluded that during sepsis occurs a significant increase in serum proteins associated with chronic inflammatory response and changes in levels of proteins related to intracellular protection in the hippocampus and cortex, which may be related to the changes observed in patients and models animals.

A sepse é um conjunto complexo de interações moleculares e celulares mediado pela estimulação de receptores celulares envolvidos na inflamação. O controle desta rede de sinalização é compensado pela resposta pró- e antiinflamatória do organismo. Os desequilíbrios inflamatórios induzidos alteram o estado fisiológico de diversos órgãos. No cérebro, observam-se diversas modificações que envolvem desde o aumento na permeabilidade da barreira hematoencefálica (BHE), morte de células neurais por apoptose ou necrose, redução do fluxo sanguíneo e aumento do estresse oxidativo, resultando no desenvolvimento do delirium associado à sepse. O delirium associado à sepse é um conjunto de disfunções neurológicas induzidas por uma resposta inflamatória sistêmica. Assim, é possível que diferentes mecanismos compensatórios estejam envolvidos durante a sepse. Este estudo objetivou analisar a influência do modelo de ligação cecal e punção (CLP), na excreção de ligantes do receptor para produtos finais de glicação avançada (RAGE) e as modificações ocasionadas no cérebro pela inflamação sistêmica decorrente da sepse. Nossos resultados demonstraram que os níveis séricos de TNF-α aumentaram após a indução da CLP, confirmando o estado inflamatório dos animais CLP, ocorrendo um aumento nos níveis de proteínas carboniladas, HSP70 e HMGB-1 no soro desses animais, indicando um aumento de estresse oxidativo nesses animais e liberação de proteínas associadas a manutenção e estabilização intracelular. Entretanto, encontramos uma redução no imunoconteúdo de destas proteínas no hipocampo e córtex, possivelmente por aumento nos níveis de necrose/lise celular ou por eliminação durante o período de disrupção da BHE. Assim, concluímos que durante a sepse ocorre uma aumento expressivo nos níveis séricos de proteínas associadas a resposta inflamatória crônica, além de alterações nos níveis destas proteínas relacionadas a proteção intracelular no hipocampo e córtex, que pode ter relação com as alterações observadas em pacientes e modelos animais. / Sepsis is a complex whole of molecular and cellular interactions mediated stimulation of cellular receptors involved in inflammation. The control of this system is compensated by pro and anti-inflammatory response in organism. The imbalance induced inflammatory alters the physiological state of different organs. In the brain, there are various modifications that involve from the increase in permeability of the blood-brain barrier (BBB), neural cell death by apoptosis or necrosis, reduced blood flow and increased oxidative stress, resulting in the development of sepsis-associated delirium. Sepsis-associated delirium is a whole of neurological dysfunctions induced a systemic inflammatory response. Thus, it is possible that different compensatory mechanisms are involved during sepsis. The aim of the study was to analyze the influence of the model cecal ligation and puncture (CLP), in the excretion of agonists for receptor advanced glycation end products (RAGE) and alterations in the brain caused by systemic inflammation caused by sepsis. Our results showed that serum levels of TNF-α increased after the induction of CLP, confirming the inflammatory status of CLP animals, which results in increased levels of carbonylated proteins, HSP70 and HMGB-1 in the serum of these animals, indicating an increase of stress oxidative these animals and release of proteins associated with maintaining and stabilizing intracellular. However, happened a reduction in immunocontent these proteins in the hippocampus and cortex, possibly by increased levels of necrosis/lysis cell or by removal during the period of disruption of the BBB. Therefore, concluded that during sepsis occurs a significant increase in serum proteins associated with chronic inflammatory response and changes in levels of proteins related to intracellular protection in the hippocampus and cortex, which may be related to the changes observed in patients and models animals.

Although the relationship between bone formation and the immune / inflammatory response is extremely important for the bone repair process, such interaction remains poorly understood. While an exaggerated immune / inflammatory response is associated with bone resorption, an ideal transient response of low magnitude is essential in the process. In this context, signaling mediated by Toll receptors (TLRs) and RAGE receptors plays a key role in initiating the immune / inflammatory response through the recognition of molecular patterns associated with damage (DAMPs). One of the major DAMPs recognized by TLR4 and RAGE is the HMGB1 protein. Once activated by HMGB1, these receptors are able to generate several important inflammatory mediators in the process of alveolar bone repair. Nevertheless, the mechanisms of "trigger" of the production of cytokines and inflammatory mediators with respect to the bone repair, nor the amount necessary, are not clear. In view of this, we aimed to investigate the influence of the HMGB1 ligand on the generation of immune / inflammatory response and on the bone repair subsequent to the extraction of the superior incisor of mice. For this, 40 mice were divided into two groups [Control Group - WT; Group GLY - treated with glycyrrhizin (HMGB1 inhibitor)] and analyzed for alveolar bone repair in the periods of 0, 7, 14, 21 days after exodontia. Samples were submitted to histological processing and analyzed under optical microscopy for histomorphometric characterization (qualitative and quantitative), and analyzed by MicroCt for description of bone structures. In the histomorphometric analysis, the results showed a higher clot density in the GLY group in the 14-day period (p <0.05); As well as more inflammatory cells in the period of 7 and 14 days compared to the control (p <0.05). As well, it presented higher density of fibroblasts in the periods of 7, 14 and 21 days and lower density of fibers in 7 days (p <0.05). The GLY group had lower vessel densities in the periods of 7, 14 and 21 days. (P <0.05). The GLY group presented lower osteoblasts and higher osteoclasts compared to the control group at 7 and 14 days (p <0.05). Regarding the MicroCt, the images of both groups were performed and did not present significant difference between the groups. Therefore, inhibiton of HMGB1 protein was not able to interfere in the kinetics of alveolar bone repair in mice, but showed differences in some components involved in alveolar bone repair kinetics. / Embora a relação entre a formação óssea e a resposta imune/inflamatória mostre-se extremamente importante para o processo de reparo ósseo, tal interação permanece pouco compreendida. Enquanto uma resposta imune/inflamatória exacerbada está associada à reabsorção óssea, uma resposta ideal transitória de baixa magnitude é essencial no processo. Nesse contexto, a sinalização mediada por receptores tipo Toll (TLRs) e receptores RAGE desempenha um papel fundamental no inicio da resposta imune/inflamatória através do reconhecimento de padrões moleculares associados a danos (DAMPs). Um dos principais DAMPs reconhecidos pelo TLR4 e RAGE é a proteína HMGB1. Uma vez ativados por HMGB1, estes receptores são capazes de gerar diversos mediadores inflamatórios importantes no processo de reparo ósseo alveolar. Ainda assim, não são claros os mecanismos de “trigger” da produção de citocinas e mediadores inflamatórios com relação ao reparo ósseo, nem a quantidade necessária. Visto isso tivemos como objetivo investigar a influencia do ligante HMGB1 na geração de resposta imune/inflamatória e no reparo ósseo subseqüente à extração do incisivo superior de camundongos. Para isso, foram utilizados 40 camundongos divididos em dois grupos [Grupo controle- WT; Grupo GLY – tratado com glicirrizina (inibidor de HMGB1)] e analisados quanto ao reparo ósseo alveolar nos períodos de 0, 7, 14, 21 dias após exodontia. Amostras foram submetidas ao processamento histológico e foram analisadas ao microscópio óptico para caracterização histomorfométrica (qualitativa e quantitativa), e analisadas por MicroCt para descrição de estruturas ósseas. Na análise histomorfométrica, os resultados mostram maior densidade de coágulo no grupo GLY no período de 14 dias (p<0,05); como também mais células inflamatórias no período de 7 e 14 dias comparado ao controle (p<0,05). Como também, apresentou maior densidade de fibroblastos nos períodos de 7, 14 e 21 dias e menor densidade de fibras em 7 dias ( p<0,05). O grupo GLY apresentou menores densidades de vasos nos períodos de 7, 14 e 21 dias. (p<0,05). Apensar de não demonstrar diferença singnificativa na formação óssea, o grupo GLY apresentou menores quantidades de osteoblastos e maiores de osteoclastos comparado ao grupo controle nos tempos de 7 e 14 dias (p<0,05). Quanto ao MicroCt, as imagens de ambos os grupos foram realizadas e não apresentou diferença significativa entre os grupos. Portanto, a inibição da proteína HMGB1 não foi capaz de interferir na cinética de reparo ósseo alveolar em camundongos, mas apresentaram diferenças em alguns componentes participantes da cinética do reparo ósseo alveolar. / Lagarto, SE