Angiotensin II (AngII), the major effector of the renin angiotensin system, mediates kidney disease progression by signalling through AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) in order to identify potential AngII activity markers in the kidney. We utilized stable isotope labelling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by Selected Reaction Monitoring (SRM) assays. Both SILAC and SRM revealed the nuclear factor erythroid 2-related 2 (Nrf2) target protein, heme oxygenase-1 (HO-1) as the most significantly upregulated protein in response to AngII stimulation. AngII-dependent regulation of HO-1 gene and protein was further verified by qRT-PCR and ELISA in PTECs. In order to extend these in vitro observations, we utilized a systems biology approach. We thus overlaid a network of significantly enriched gene ontology (GO) terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five GO terms were enriched both in vitro and in vivo, and all included HO-1. Furthermore, four additional Nrf2 target proteins were functionally important in vitro and in vivo. We then studied HO-1 kidney expression and urinary excretion in AngII-treated wild type mice and mice with PTEC-specific AT-1R gene deletion. Deletion of the AT-1R gene in PTECs lowered both kidney expression and urine excretion of HO-1, confirming AngII/AT-1R mediated regulation of HO-1. In summary, our in vitro experiments identified novel molecular markers of AngII activity in PTECs and the animal studies demonstrated that these markers also reflect AngII activity in PTECs in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-01T21:22:36Z No. of bitstreams: 1 Nivea Farias Luz Papel da proteína hemeoxigenase....pdf: 1624634 bytes, checksum: 6f830105c144bc3c799743a490e5a511 (MD5) / Made available in DSpace on 2012-08-01T21:22:36Z (GMT). No. of bitstreams: 1 Nivea Farias Luz Papel da proteína hemeoxigenase....pdf: 1624634 bytes, checksum: 6f830105c144bc3c799743a490e5a511 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A leishmaniose visceral (LV) apresenta ampla distribuição geográfica e é fatal caso não seja tratada. As manifestações hematológicas são constantes na LV e em casos não tratados os pacientes evoluem à óbito por sangramento maciço ou anemia grave. Neste cenário, mecanismos ligados à hemólise, metabolismo do heme e atividade da enzima heme oxigenase podem estar envolvidos na imunopatogênese da LV, no entanto essa perspectiva ainda não foi explorada. A heme oxigenase (HO) tem importantes propriedades regulatórias e está envolvida em processos fisiológicos e patofisiológicos como citoproteção e inflamação. Apesar de sua sugestiva participação no contexto da infecção por Leishmania, uma rápida pesquisa no PubMed com as palavras heme oxigenase e Leishmania remete a somente três trabalhos até a presente data. Nesse projeto testaremos a hipótese de que a ativação da enzima heme oxigenase-1 (HO-1) favorece a infecção por Leishmania (L) chagasi, principal agente etiológico da LV humana no Brasil. Nossas observações nesse trabalho indicam que a enzima HO-1 é induzida em macrófagos durante a infecção por L. chagasi e que a indução farmacológica da HO-1, pela CoPP aumenta a carga parasitária de macrófagos infectados por L. chagasi e reduz a produção de mediadores pró-inflamatórios frente à estimulação por LPS, tais como TNF, NO, PGE2, MCP-1, IL-1β e IL-6. Além disso, a HO-1 favorece um ambiente anti-inflamatório onde prevalece a presença de IL-10 sobre a de TNF. Macrófagos derivados de medula óssea de camundongos deficientes no gene HO-1 tem menor carga parasitária, quando infectados por L. chagasi em comparação aos macrófagos de camundongos selvagens. Esses achados indicam um potencial deletério para a HO-1 na infecção por L. chagasi, bem como sugerem possíveis mecanismos envolvidos na imunopatogênese da LV. / Visceral leishmaniasis (VL) is a widespread disease and is fatal if left untreated. Hematological manifestations are common in VL and untreated patients evolve to death from massive bleeding and severe anemia. In this scenario, mechanisms related to hemolysis, heme metabolism and enzyme activity of heme oxygenase may be involved in the immunopathogenesis of VL. But that panorama has not been explored. Heme oxygenase (HO) has important regulatory properties and is involved in patho-physiological processes such as cytoprotection and inflammation. Despite HO participation in the context of Leishmania infection is suggestive, a quick search on PubMed with the words heme oxygenase and Leishmania refers to only three papers to date. This project will test the hypothesis that heme oxygenase- 1 (HO-1) activation favors Leishmania (L) chagasi, the main etiology agent of human VL in Brazil. Our observations indicate that HO-1 is induced in macrophages during L. chagasi infection and pharmacological induction of HO-1 by CoPP increases parasite load of infected macrophages and results in inhibition of TNF- α, IL-1β, IL-6, MCP-1, PGE2 and Nitrite levels upon LPS stimulation and simultaneously induced a higher IL-10/TNF-α ratio in peritoneal macrophages contributing to the anti inflammatory pathway that favors L. chagasi replication. Beyond this, we observed that bone marrow derived macrophages knockout to HO-1 gene have a significant low parasite load when infected by L. chagasi than their wild type counterparts. In summary, our findings suggest that this enzyme can play a deleterious role in VL and clarify one of the immunoregulatory mechanisms involved in VL.

A malária é uma doença causada pelo parasita do gênero Plasmodium e que foi responsável por cerca de 440.000 mortes em 2015. A síndrome do desconforto respiratório agudo (SDRA) é uma das principais complicações clínicas da malária. O modelo murino DBA/2 reproduz os sinais clínicos da SDRA observados em humanos, quando infectado com o Plasmodium berghei ANKA. Além disso, altos níveis da enzima heme oxigenase 1 (HO-1) foram observados em casos de malária cerebral e em SDRA em humanos. Os nossos dados indicam que os níveis da HO-1 estão aumentados em camundongos que desenvolvem SDRA associada à malária (SDRA-AM). Adicionalmente, a droga indutora de HO-1 (hemina) aumentou a sobrevivência e preveniu a SDRA-AM. Verificou-se também uma redução na permeabilidade pulmonar e nos níveis de VEGF, além de uma melhoria nos parâmetros respiratórios em animais tratados com hemina. Assim sendo, a indução da HO-1 antes do desenvolvimento da SDRA-AM é protetora e assim, a HO-1 pode ser um alvo de novos fármacos, como forma de prevenir o desenvolvimento da SDRA-AM em humanos. / Malaria is a serious disease, caused by the parasite of the genus Plasmodium, which was responsible to 440,000 deaths in 2015. Acute lung injury/ acute respiratory distress syndrome (ALI/ARDS) is one of the main clinical complications in severe malaria. The murine model DBA/2 reproduces the clinical signs of ALI/ARDS observed in humans, when infected with Plasmodium berghei ANKA. Additionally, high levels heme oxygenase 1 (HO-1) were reported in cases of cerebral malaria and in ALI/ARDS in humans. Our data have indicated that the HO-1 levels are increased in mice that develop malaria associated ALI/ARDS (MA-ALI/ARDS). Additionally, a HO-1 inducing drug (hemin) increased the survival rate and prevented mice from developing MA-ALI/ARDS in treated mice. Also, there was a decrease in the lung permeability and in lung VEGF levels, and an amelioration of respiratory parameters. Therefore, the induction of HO-1 before the development of MA-ALI/ARDS is protective, making this enzyme a possible target of new drugs to prevent the development of MA-ALI/ARDS in humans.

No description available.

Die Schwangerschaft ist ein komplexes Phänomen, bei dem es zu einer Interaktion zwischen dem mütterlichen Immunsystem und dem Fetus kommt. An der feto-maternalen Grenze kommt es zur Auslösung einer inflammatorischen Reaktion, die für eine normale Implantation und Schwangerschaft notwendig ist. Allerdings kann eine exzessive Entzündungsreaktion zu Schwangerschaftskomplikationen wie dem immunologisch vermittelten Sponatanabort füh-ren. Das zytoprotektive Enzym Hämoxygenase-1 (HO-1) spielt eine sehr wichtige Rolle bei der Kontrolle inflammatorischer Reaktionen. Inwiefern HO-1 für das Gelingen und Bestehen einer Schwangerschaft unabdingbar ist, wurde bisher nicht untersucht. Unsere Hypothese ist, dass HO-1 eine bedeutsame Rolle während der Schwangerschaft spielt. Die Beantwortung dieser wichtigen Frage ist deshalb Hauptziel dieser Dissertation. Es konnte gezeigt werden, dass eine spezifische Hochregulation des HO-1 Moleküls mittels Gentherapie in einem Mausmodell für Spontanabort zur signifikanten Reduktion der Abortra-te führte. Dieser protektive Effekt war mit einer erhöhten Th2/Th1 Zytokinen-Ratio und mit verminderter Apoptose assoziiert. Ein weiteres Teilziel dieser Arbeit bestand darin, die Rolle des HO-1 Moleküls während der Plazentation zu untersuchen. Dafür wurde eine Trophoblastenstammzelllinie benutzt, die in der Lage ist, zu Riesenzellen zu differenzieren. Die mittels Zinkprotoporphyrin (ZnPPIX) induzierte Expressionssuppression von HO-1 führte zur Verminderung der Überlebensrate von Trophoblastenstammzellen und zur Hemmung von deren Ausdifferenzierung in Trophoblastenriesenzellen. Um die Rolle des HO-1 Moleküls in anderen Schwangerschaftsprozessen zu untersuchen, wurden Hämoxygenase-1 defiziente (Hmox1-/-) Mäuse benutzt. Da die Verpaarung von Hmox1-/- Mäuse zu keinem erfolgreichen Abkömmling führt, war ein weiteres Teilziel dieser Arbeit gewesen, den zu Grunde liegenden Mechanismus aufzuklären. Es zeigte sich, dass Hmox1-/- Weibchen im Vergleich zu den Hmox1+/+ Weibchen weniger Oozyten produzieren. Auch konnten die Hmox1-/- Oozyten weniger erfolgreich als die Hmox1+/+ Oozyten fertili-ziert werden. Verschiedene Verpaarungsexperimente mit Hmox1+/+, Hmox1+/- und Hmox1-/- Mäusen ergaben einen indirekt proportionalen Zusammenhang zwischen HO-1 Expression und Aborthäufigkeit. Die hier gewonnenen Daten deuten daraufhin, dass HO-1 eine entscheidene Rolle in der Schwangerschaft spielt. Die gewonnenen Erkenntnisse tragen zum Verständnis der Pathologie des immunologisch vermittelten Spontaborts bei und können darüber hinaus helfen neue Be-handlungsstrategien gegen diese gefürchtete Schwangerschaftskomplikation zu entwickeln. / Mammalian pregnancy is a parabiotic union of two genetically different individuals, the fetus and the mother. At the feto-maternal interface, inflammatory processes can occur due to an immune reaction against alloantigens. It is known that some degree of systemic or uterine inflammation is necessary for both normal implantation and pregnancy. However, if this in-flammation becomes too excessive it can cause pregnancy complications such as abortion. Heme oxygenase-1 (HO-1), the enzyme responsible for the degradation of free heme, plays a key role in inflammatory processes. Viewing pregnancy mainly as an inflammatory process had led us to the idea that HO-1 may play an important role in pregnancy. Therefore, the main aim of this work was to analyze the role of HO-1 in the different processes related to preg-nancy by means of functional studies employing in vivo as well as in vitro models. First, we could show that a specific up-regulation of HO-1 in abortion-prone animals by means of an adenoviral vector is able to reduce the abortion rate. This HO-1 up-regulation improved pregnancy outcome by up-regulating the Th2/Th1 cytokines ratio and protecting tissues from apoptosis, suggesting an important role of HO-1 in pregnancy. In a second part of the work, we aimed to analyze the role of HO-1 in placentation. For that, a trophoblast stem cell line capable of differentiate into trophoblast giant cells was used. Inter-estingly, a down-regulation of HO-1 by means of ZnPPIX led to diminished survival of the trophoblast stem cells. Furthermore, these cells were unable to differentiate into trophoblast giant cells in the absence of HO-1, strongly suggesting a crucial role of HO-1 in placentation. Finally, a closer look into the role of HO-1 in pregnancy was performed by using heme oxy-genase-1 deficient mice (Hmox1-/- mice). Interestingly, Hmox1-/- females produce much less oocytes than wild type females. Analyses of the ovaries of both types of females showed dif-ferences in follicle development. Furthermore, when fertilized in vitro, a significant diminu-tion in the fertilization rate of Hmox1-/- oocytes when compared to Hmox1+/+ oocytes was found. Since the mating of Hmox1-/- mice does not yield progeny, we also aimed to clarify whether this is due to problems in the female, in the male or in both. For this, different mating combinations of mice partially or totally deficient in Hmox1 were performed. The analysis of the pregnancy outcome showed that, the less HO-1 in the combination, the higher the fetal rejection. In summary, a central role of HO-1 in different processes of reproduction could be demon-strated in this work which helps understanding the mechanisms behind pregnancy success.

Na lesão renal aguda, os mecanismos de defesa atuam como genes protetores, como a proteína heat shock 32 (HSP 32), também conhecida como heme oxigenase-1 (HO-1). A polimixina B (PmxB) é um antimicrobiano nefrotóxico. O objetivo deste estudo foi caracterizar a participação da enzima HO-1 na toxicidade da PmxB em células LLC-PK1. As células foram submetidas aos seguintes tratamentos: Controle (CTL- 0µM); Hemin (indutor de HO-1, 25µM); Hemin II (250M), Protoporfirina de zinco (ZnPP - inibidor de HO-1, 10M,); Nitro-L-arginina-metilester (L-NAME - inibidor de iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM de Hemin uma hora antes da PmxB); PmxB + ZnPP (10M de ZnPP uma hora antes da PmxB); PmxB + Hemin + L-NAME (25M de Hemin e 0,1mM de L-NAME uma hora antes da PmxB). Os grupos foram avaliados em 24 e 72 horas. Foram analisados os seguintes parâmetros: desidrogenase láctica (DHL), peroxidação lipídica (MDA), expressão gênica da HO-1 por RT-PCR, síntese protéica da HO-1 por imunofluorescência, óxido nítrico (NO) pelo método de Griess e expressão protéica da HO-1 e da iNOS por western blotting. Os resultados mostraram que a PmxB elevou o DHL com aumento dos níveis de MDA. O Hemin e a ZnPP elevaram as variáveis DHL, MDA e óxido nítrico (NO). O indutor de HO-1 incrementou a expressão protéica da HO-1 e da iNOS. A PmxB se confirmou como citotóxica e a HO-1 intensificou a lesão por mecanismos oxidativos. O efeito da HO-1 na lesão celular parece ser mediado pelo NO / In the acute kidney injury, the mechanisms of defense act as protector genes, as the protein heat shock 32 (HSP 32), also known as heme oxygenase-1 (HO-1). The polymyxin B (PmxB) is a nephrotoxic antimicrobial. The aim of this study was to distinguish the role of the HO-1 enzyme in the PmxB toxicity in LLC-PK1 cells. The cells were submitted to the following treatments: Control (CTL- 0µM); Hemin (inhibitor of HO-1, 25µM); Hemin II (250M), Zinc protoporphyrin (ZnPP - inhibitor of HO-1, 10M,); NG-nitro-L-arginine methyl ester (L-NAME - inhibitor of iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM of Hemin one hour before the PmxB); PmxB + ZnPP (10M of ZnPP one hour before the PmxB); PmxB + Hemin + L-NAME (25M of Hemin and 0,1mM of L-NAME one hour before the PmxB). All groups were evaluated in 24 and 72 hours. The following parameters were analysed: lactate dehydrogenase (LDH), lipid peroxidation (MDA), genic expression of HO-1 by RT-PCR, protein syntesis of HO-1 by immunofluorescence, nitric oxide (NO) by Griess method and protein expression of HO-1 and of iNOS by western blotting. The results showed that PmxB increased the LDH and the levels of MDA. Hemin and ZnPP also increased the LDH variables, MDA and nitric oxide (NO). The inducer of HO-1 improved the protein expression of HO-1 and of iNOS. The PmxB was confirmed as a cytotoxic and the HO-1 intensified the failure by oxidative mechanisms. The effect of HO-1 in the cell injury seemed to be mediated by NO

Les IgIV, une préparation thérapeutique d'IgG normales, sont utilisées dans le traitement de diverses maladies auto-immunes et inflammatoires. Les mécanismes par lesquels les IgIV exercent une activité anti-inflammatoire ne sont pas complètement compris. Elles interagissent avec de nombreux composants du système immunitaire et modulent leurs fonctions. Des études récentes ont rapporté que l'hème oxygénase-1 (HO-1) joue un rôle important dans la régulation de la réponse inflammatoire dans un certain nombre de pathologies. Plusieurs agents thérapeutiques exercent des effets anti-inflammatoires grâce à l'induction de l'HO-1. Etant donné le rôle commun anti-inflammatoire de l'HO-1 et des IgIV, j'ai étudié l'implication de l'HO-1 dans les mécanismes d'action des IgIV. J'ai montré que les effets des IgIV ne sont pas associés à l'induction de l'HO-1, que ce soit dans des cellules de l'immunité innée comme les monocytes, cellules dendritiques ou macrophages, ou dans les reins et foie de souris avec une encéphalomyélite auto-immune expérimentale traitées par les IgIV. Des données récentes dans des modèles expérimentaux suggèrent que les IgIV induisent la sécrétion d’IL-4 des basophiles en augmentant l’IL-33 des cellules innées SIGN-R1+. J’ai rapporté que les IgIV induisent directement l’activation de basophiles pré-stimulés avec l’IL-3 alors que contrairement au modèle murin, l’IL-33 n’est pas indispensable. L’activation des basophiles par les IgIV est associée à l’expression augmentée de CD69 et la sécrétion d’IL-4, d’IL-6 et d’IL-8. Ces fonctions sont médiées par les fragments F(ab’)2 qui se lient à des IgE membranaires et activent la voie Syk. / Intravenous immunoglobulin (IVIG), a therapeutic normal immunoglobulin G preparation, is used in the therapy of various autoimmune and inflammatory conditions. The mechanisms by which IVIG exerts anti-inflammatory effects are not completely understood. It interacts with numerous components of the immune system including dendritic cells, macrophages, T and B cells and modulates their functions. Recent studies have reported that heme oxygenase-1 (HO-1) pathway plays an important role in the regulation of inflammatory response in several pathologies. Several therapeutic agents exert anti-inflammatory effects via induction of HO-1. Therefore, in view of common anti-inflammatory role exerted by both HO-1 and IVIG, I investigated if mechanisms of IVIG implicate HO-1. I show that anti-inflammatory effects of IVIG were not associated with an induction of HO-1 either in innate cells such as monocytes, dendritic cells and macrophages or in the kidneys or liver of experimental autoimmune encephalomyelitis. Recent data in experimental models suggest that IVIG induces IL-4 in basophils by enhancing IL-33 in SIGN-R1+ innate cells. I reported that IVIG directly induces activation of IL-3-primed basophils while unlike mice IL-33 was dispensable. The activation of basophils by IVIG was associated with enhanced expression of CD69 and secretion of IL-4, IL-6 and IL-8. These functions of IVIG are mediated via F(ab’)2 fragments that bind to basophil surface IgE and activate Syk pathway.

QUEIROZ, Ismael Nilo Lino de. Caracterização estrutural preliminar e efeitos na inflamação da lectina da alga marinha verde Caulerpa cupressoides. 115 f. : Dissertação (Mestrado em Bioquímica) - Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará Fortaleza-CE, 2013. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-05-23T13:42:40Z No. of bitstreams: 1 2013_dis_inlqueiroz.pdf: 2778929 bytes, checksum: 1ecf927693bc8e91aabb5a746c3dfbce (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-05T19:19:39Z (GMT) No. of bitstreams: 1 2013_dis_inlqueiroz.pdf: 2778929 bytes, checksum: 1ecf927693bc8e91aabb5a746c3dfbce (MD5) / Made available in DSpace on 2016-07-05T19:19:39Z (GMT). No. of bitstreams: 1 2013_dis_inlqueiroz.pdf: 2778929 bytes, checksum: 1ecf927693bc8e91aabb5a746c3dfbce (MD5) Previous issue date: 2013 / Lectins seaweed have various pharmacological applications. This work was partially characterize the structure and evaluate the effects on classical models of inflammation lectin of the green seaweed Caulerpa cupressoides. The Caulerpa cupressoides lectin (CcL) was extracted with Tris-HCl 25 mM, pH 7.5 and isolated by ion exchange chromatography on DEAE-cellulose. Structural characterization showed a partial aminoterminal sequence with 31 amino acid residues, obtained according to the method of Edman degradation, while in nuclear magnetic resonance spectroscopy to the CcL 1H-NMR purified by Sephadex G-100 and DEAE-cellulose obtained by was demonstrated similarity between signals obtained in both spectra. The anti-inflammatory activity was evaluated in male Wistar rats (n = 6), using the model of paw edema induced by carrageenan (700 μg/paw), dextran (500 μg/paw), histamine (100 μg/paw), serotonin (20 μg/paw) or bradykinin (30 μg/paw). Groups of animals were treated with CcL (0.1, 1.0 or 10.0 mg/kg, i.v.) 30 min before the inflammatory stimulus. Was evaluated the involvement of CcL (1.0 mg/kg) towards heme oxygenase-1. Groups were used that were treated with inhibitor CcL linked mucin (8.0 mg/kg, i.v.) or only mucin (8.0 mg/kg, i.v.) and dexamethasone (1.0 mg/kg, s.c.) were used as controls. The effect edematogenic CcL was evaluated by applying the doses of 0.1, 1.0 or 10 mg/kg (i.pl.). In the trial of paw edema induced by carrageenan, CcL reduced edema formation was confirmed by determining tissue levels of myeloperoxidase. CcL (1.0 mg/kg) showed no mucin linked to anti-inflammatory effect on the paw edema induced by carrageenan, except in the first hour after stimulation. In dextran-induced edema, CcL also inhibited the osmotic swelling. Only the dose of 1.0 mg/kg was used in reducing histamine-induced edema by 40% the edema within 30 min CcL (1.0 mg/kg), however, did not reduce the edema induced by serotonin or bradykinin. Besides, in the histological analysis of tissue subplantar, CcL (1.0 and 10.0 mg/kg) was able to reduce cell migration. In the presence of ZnPP IX (3.0 mg/kg, s.c.), CcL has lost its ability to inhibit the carrageenan-induced edema, exerting its mechanism of action anti-inflammatory pathway through the involvement of HO-1. While in immunohistochemistry, CcL (10 mg/kg) reduced the expression of IL-1β, but intense staining occurred cytokines TNF-α and IL-6, and expression of HO-1 in the groups treated with the same dose of CcL. In relation the effect edematogenic, CcL was able to induce intense inflammatory process with dose-dependent effect. However, the induced edema at a dose of 10 mg/kg was CcL was inhibited by indomethacin, meclizine, pentoxifylline and dexamethasone. Therefore, when the CcL partially characterized presented in its aminoterminal sequence of 43% identity with the protein from unicellular green alga Chlamydomonas reinhardtii and showed anti-and pro-inflammatory therapeutic agent is considered a potential for future studies in inflammatory processes. / As lectinas de algas marinhas possuem várias aplicações farmacológicas. O objetivo deste trabalho foi caracterizar parcialmente a estrutura e avaliar os efeitos em modelos clássicos de inflamação da lectina da alga marinha verde Caulerpa cupressoides. A lectina de Caulerpa cupressoides (LCc) foi extraída com tampão Tris-HCl 25 mM, pH 7,5 e isolada por cromatografia de troca iônica em coluna de DEAE-celulose. A caracterização estrutural parcial apresentou uma sequência aminoterminal com 31 resíduos de aminoácidos, obtida de acordo com o método de degradação de Edman, enquanto que na espectroscopia de ressonância magnética nuclear unidimensional 1H para a LCc purificada por Sephadex G-100 e obtida por DEAE-celulose, foi demostrado uma similaridade entre os sinais obtidos em ambos os espectros. A atividade anti-inflamatória foi avaliada em ratos Wistar machos (n=6), utilizando o modelo de edema de pata induzidos por carragenana (700 μg/pata), dextrana (500 μg/pata), histamina (100 μg/pata), serotonina (20 μg/pata) ou bradicinina (30 μg/pata). Grupos de animais foram submetidos ao tratamento com LCc (0,1; 1,0 ou 10,0 mg/kg; i.v.), 30 min antes do estímulo inflamatório. Foi avaliado o envolvimento da LCc (1,0 mg/kg) na via da Heme oxigenase-1. Foram utilizados grupos que receberam tratamento com LCc ligada ao inibidor mucina (8,0 mg/kg; i.v.) ou somente mucina (8,0 mg/kg; i.v.) e dexametasona (1,0 mg/kg; s.c.) foram utilizados como controles. O efeito edematogênico de LCc foi avaliado aplicando as doses de 0,1; 1,0 ou 10 mg/kg (i.pl.). No ensaio de edema de pata induzido por carragenana, LCc reduziu a formação de edema sendo confirmado pela determinação dos níveis teciduais de mieloperoxidase. A LCc (1,0 mg/kg) ligada a mucina não apresentou efeito anti-inflamatório no edema de pata induzido por carragenana, exceto na primeira hora após o estímulo. No edema induzido por dextrana, LCc também inibiu o edema osmótico. Apenas a dose de 1,0 mg/kg foi utilizada no edema induzido por histamina reduzindo em 40% o edema no intervalo de 30 min LCc (1,0 mg/kg), entretanto, não reduziu a formação de edema induzido por serotonina ou bradicinina. Além disso, na análise histológica do tecido subplantar, LCc (1,0 e 10,0 mg/kg) foi capaz de reduzir a migração celular. Na presença de ZnPP IX (3,0 mg/kg; s.c.), LCc perdeu sua capacidade inibir o edema induzido por carragenana, exercendo seu mecanismo de ação anti-inflamatório através do envolvimento da via da HO-1. Enquanto que na imunohistoquímica, LCc (10 mg/kg) reduziu a expressão de IL-1β, porém ocorreu intensa marcação das citocinas TNF-α e IL-6, além da expressão de HO-1, nos grupos tratados com a mesma dose de LCc. Com relação ao efeito edematogênico, LCc foi capaz de induzir intenso processo inflamatório com efeito dose-dependente. No entanto, o edema induzido com a dose de 10 mg/kg de LCc foi foi inibido por indometacina, meclizina, pentoxifilina e dexametasona. Portanto, a LCc quando parcialmente caracterizada apresentou na sua sequência aminoterminal uma identidade de 43% com a proteína da alga verde unicelular Chlamydomonas reinhardtii e apresentou propriedades anti- e pró-inflamatórias, sendo considerada um agente terapêutico em potencial para estudos futuros nos processos inflamatórios.

Anorexia Nervosa (AN) and Bulimia Nervosa (BN) are diseases with considerable individual variation. Genetic background plays an important role in disease susceptibility and severity. To evaluate the relationship between certain genetic loci and diseases subtypes we genotyped and analysed evolution of selected clinical parameters. We investigated a group of 75 pacients with AN (1. study), 127 DSM-4 and ICD-10 diagnosed patients with AN and BN (2. study), and contributed to sample of 2907 AN patients in large GWAS study. Results from the 1st study support association of polymorphism -1438G/A in serotonine receptor 5-HT2A with AN and compare the results from other studies with metaanalyses. In next, polymorphism responsible for the serotonine neurotransmission (serotonine transporter 5-HTT, polymorphisms LPR and VNTR) the study shows different association trend of LPR with AN in Czech population compared to other studies. 5-HTT VNTR polymorphism had no observed association. The second study investigated the role of hemeoxygenase 1 (plays a pivotal role in metabolic stress protecting cells) in eating disorders, in interaction with enviromental stress. We investigated the usefulness of an aggregate measure of the risks of AN and BN that is based on genetic susceptibility loci and the added effect of...

Endothelial dysfunction is a pathologic state characterized by an altered equilibrium among vasodilatory and antithrombotic mediators and vasoconstrictive and prothrombotic mediators produced by the vascular endothelium. Multiple factors induce impaired production or increased consumption nitric oxide (NO), the key mediator of vascular homeostasis, produced by the nitric oxide synthase enzymes (NOS). Endothelial dysfunction represents one of the initial steps in the development of atherosclerosis, a chronic inflammatory disease of the vascular wall. The inducible enzyme heme oxygenase 1 (HO-1) represents one of the main cellular defense mechanisms against increased oxidative stress and decreased NO bioavailability accompanying endothelial dysfunction and atherosclerosis. We studied the genetic determinants of endothelial dysfunction and atherosclerosis by evaluating the association of the G894T endothelial NOS (eNOS) polymorphism and the HO-1 (GT)n promoter polymorphism with coronary artery atherosclerosis severity and risk profile and their evolution during hypolipidaemic treatment. In addition, we searched for genetic variations in exons 25 and 26 of eNOS gene, encoding the C-terminal part of the protein, deemed crucial for proper enzyme function and the 3'- untranslated region crucial for eNOS...