A platform for vaccine development has been developed at Georgia State University utilizing recombinant Salmonella typhimurium flagellin (FliC) fused to an antigen that can be overexpressed in Escherichia coli grown in a two-stage fermentation. The flagellin acts as an adjuvant to increase the immunopotency of the fused antigen. Flagellin is the ligand for Toll-like Receptor 5 (TLR5), a part of the innate immune system. Binding of the flagellin:antigen recombinant protein to TLR5 triggers a strong innate and adaptive immune response to the fused antigen leading to a potentially strong protective immunity to the antigen. Purification of the recombinant FliC fusion protein must meet rigorous criteria in order to be used as a vaccine. One of the major issues in purifying recombinant proteins expressed in a Gram-negative bacterium is the removal of endotoxin. Small amounts of endotoxin present in a vaccine can lead to serious complications, including death. Recombinant proteins are also expressed as either soluble or insoluble protein when over expressed in E. coli. Soluble proteins expressed by the bacterium are properly folded and biologically active, however removal of contaminants such as endotoxin, can be problematic. Insoluble protein is improperly folded and biologically inactive. The insoluble proteins aggregate into inclusion bodies with little or no contaminants associated with the protein, making purification easier. However, in order to restore the biological activity of the insoluble protein, it must first be solubilized and then refolded. This process is often expensive and time consuming, as there is currently no standardized method for protein refolding. In this study a purification method for the soluble protein of two FliC constructs, full-length FliC and FliC fused to a Marburg virus antigen, was evaluated for effectiveness in purification, removal of endotoxin and maintaining TLR5 activity. The proteins of interest were purified utilizing only the soluble protein containing the properly folded and biologically active recombinant protein. Utilizing methods for purification that take advantage of physical and chemical properties of the protein the recombinant proteins were purified and the level of endotoxin reduced to levels acceptable for use as a vaccine. The TLR5 activity of the soluble recombinant proteins was compared to recombinant protein that had been purified using a denaturing and refolding step. The soluble protein elicited a higher TLR5 response at a lower concentration of protein than the refolded protein. Purification of the soluble fraction also involved fewer step and less time than purification of both the soluble and insoluble protein.

All animals have evolved mechanisms to recognise and eliminate nonself in order to defend against invading pathogens and to prevent chimerism, the fusion between genetically distinct conspecifics. Like other metazoans, sponges are known to rely on sophisticated systems to maintain their self-integrity. As poriferans are also considered one of the most ancient extant metazoan phyla, they represent a critical comparative model for understanding the early evolution of immunity and self/nonself recognition in animals. The Toll-like receptor (TLR) signalling cascade plays a crucial role in immunity, and recent findings in the sponge Suberites domuncula suggest that its origin could predate eumetazoan cladogenesis. My genome and expressed sequence tag (EST) screens of the demosponge Amphimedon queenslandica detected homologues to most components of this pathway, supporting the notion that a primordial TLR signalling cascade emerged at the dawn of the Metazoa. The sponge also encodes a couple of putative TLR-related proteins (AmqIgTIRs) that consist of at least one extracellular immunoglobulin (Ig) and an intracellular Toll/Interleukin-1 receptor/resistance (TIR) domain. The presence of other unconventional TLRs in S. domuncula and in cnidarian representatives, implies that an ancestral TLR probably existed in the last common ancestor of all living metazoans, and independent duplication and divergence events led to the variety of forms observed in animals. Among the putative transcription factors present in Amphimedon, which are known to be activated by the TLR signalling cascade in other eumetazoans, I detected a single member of the Rel/nuclear factor-kappaB (NF-κB) family, AmqNF-κB, which is also the only Rel homology domain (RHD)-containing gene present in the sponge. This gene encodes a protein that is equipped with both a RHD and ankyrin (ANK) repeats, suggesting that the ancestral metazoan NF-κB was configured identically to contemporary vertebrate and sponge forms, and that the truncated NF-κB found in Nematostella vectensis resulted from the secondary loss of ANK. Aside from immunity, the Toll and TLR pathways contribute to a variety of biological processes in bilaterians, however their functions have only been investigated in detail in a limited number of metazoan model organisms. While studies have tested the immune role of various sponge genes, including components of the TLR cascade, no research has yet established whether they are also involved in development. Therefore, I investigated the expression of some of the immunity-related genes I isolated in Amphimedon in a developmental and immune context to shed light on the potential ancestral function(s) of the proteins they encode. Using in situ hybridisation, I demonstrate that AmqIgTIR2, AmqMyD88, AmqTollip, AmqPellino and AmqNF-ĸB are expressed during A. queenslandica early development. In contrast, the spatial and temporal expression of AmqIgTIR1 suggests it might encode a receptor that is specifically involved in the detection of metamorphic cues in larvae. A real-time quantitative PCR (qPCR) study performed on a pool of adult sponge cDNAs indicates that the expression levels of AmqIgTIR1, AmqIgTIR2, AmqMyD88 and AmqTollip are significantly affected by a nine-hour incubation in 50 µg/ml of lipopolysaccharide (LPS), and to a lesser extent by 105 colony forming units (cfu)/ml of live Vibrio harveyi. The gene expression of AmqIgTIR1 and AmqIgTIR2 suggests that they may encode proteins with antagonistic immunological functions. While AmqPellino and AmqNF-ĸB do not appear to be affected by LPS and Vibrio exposure, it is possible that these genes do not participate in the early immune response of poriferans. Together, my data indicate that the sponge genes surveyed might encode proteins that perform developmental, sensory and immunological functions, suggesting their roles could have also been multifaceted in the last common ancestor to all living metazoans. As is observed in other invertebrates, poriferans display an ontogenic shift in allorecognition; genetically different individuals can fuse during early development but, in most instances, not as adults. However, there is a limited understanding of the cellular organisation of sponge chimeras and the onset of poriferan allorecognition response. By following the fates of fluorescently tagged cells derived from genetically distinct Amphimedon larvae that are fused together at metamorphosis, I establish that there is a rapid ontogenic shift in the sponge allogeneic response about two weeks after the initiation of metamorphosis. Moreover, the molecular basis of the poriferan allorecognition system is possibly involved in creating differential cell affinities, which underlie the construction of the sponge body plan. Compatible with this scenario is the observation that cells from postlarvae that are allowed to develop for two weeks before contact do not fuse, and form a distinct boundary between genotypes. The molecules responsible for sponge cell reaggregation, the aggregation factors (AFs), have been proposed to drive the allorecognition response in poriferans. Notably, the Amphimedon genome encodes six putative AFs, of which five occur in a cluster. These findings indicate that the polymorphic variation observed in other poriferan AFs is probably the result of allelic variations of multiple genes belonging to the same family.

Dissertation (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 72-96.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 104 pages. Includes vita. Includes bibliographical references.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-11T14:30:43Z No. of bitstreams: 1 Cyntia Cajado de Souza.pdf: 4771962 bytes, checksum: bdc7d5b7181b8a72749dc6d7de91f2f3 (MD5) / Made available in DSpace on 2013-10-11T14:30:43Z (GMT). No. of bitstreams: 1 Cyntia Cajado de Souza.pdf: 4771962 bytes, checksum: bdc7d5b7181b8a72749dc6d7de91f2f3 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A anemia falciforme (HbSS) é uma doença genética com prevalência mundial elevada, caracterizada pela heterogeneidade clinica apresentando manifestações agudas e crônicas de caráter multifatorial, sendo caracterizada pela presença do estado inflamatório sistêmico. As alterações no sistema imune têm sido relacionadas a predisposição a infecções em pacientes HbSS, com produção exacerbada de anticorpos, alterações na função de leucócitos e na imunidade celular, entre outros. Estão inclusos nesta tese o conjunto de três manuscritos com o objetivo de investigar marcadores genéticos relacionados a inflamação em pacientes com HbSS, com ênfase para os polimorfismo e níveis séricos das citocinas TNFα -308G>A e IL-8 251A>T, para a provável influência dos polimorfismos gênicos TNFα-308G>A, FcRIIAH/R131, MPO-463G>A, TLR4 896A>G, e TLR9- 1237T>C na gravidade de infecções e complicações inflamatórias na HbSS, e ainda a expressão gênica dos receptores TLR2, TLR4, TLR5 e TLR9 em células dendríticas de pacientes HbSS. Nossos resultados mostraram que os níveis séricos elevados das citocinas TNFα e IL-8, bem como a presença do alelo mutante dos polimorfismos de TNFα e da IL-8 estudados estão associados com a gravidade clínica da doença. O estudo dos polimorfismos TLR9 -1237T>C e TLR4 896A>G mostraram associação com a ocorrência de infecção respiratória e acidente vascular encefálico (AVE), respectivamente, e o polimorfismo MPO-463G>A esteve associado com a ocorrência de infecções. Além disso, a presença concomitante do polimorfismo FcRIIA H/R131 com o MPO-463G>A, com o TLR4 896A>G, ou com o TNF-α influenciou a ocorrência de internações hospitalares, de sequestro esplênico, de AVE e do número de crises vaso-oclusivas. O estudo de novos marcadores de inflamação na anemia falciforme pode ajudar no entendimento da complexidade da fisiopatologia desta doença. Nossos dados enfatizam a identificação de novos biomarcadores genéticos e sua associação marcadores clássicos podem ser uma ferramenta importante para elucidar a diversidade fenotípica da HbSS. / Sickle cell anemia (HbSS) is a genetic disease with elevated worldwide distribution characterized by clinical heterogeneity and acute and chronic complication presenting systemic inflammatory state. The immune system changes are related to infection predisposition in HbSS, exacerbated antibody production, changes in leucocytes function and innate immunity. It was included in this thesis a set of three manuscripts that aim to investigate genetic markers related to inflammation in HbSS emphasizing polymorphisms and cytokine serum levels of TNFα -308G>A and IL-8 251A>T, to the influence of genetic polymorphisms of TNFα-308G>A, FcRIIAH/R131, MPO-463G>A, TLR4 896A>G and TLR9- 1237T>C in infection and inflammatory complication in HbSS and the gene expression of dendritic cell TLR2, TLR4, TLR5 and TLR9 receptors. Our results shows high serum levels and the presence of TNFα and IL-8 mutant allele were related to disease clinical severity. The study of TLR9 -1237T>C and TLR4 896A>G polymorphisms were associated to respiratory infection and encephalic vascular accident (EVA), respectively, and the polymorphism MPO-463G>A was associated to infection. The double presence of polymorphism FcRIIA H/R131 with MPO-463G>A or with TLR4 896A>G or with TNF-α influence the presence of hospitalization, spleen sequestration, EVA and vasooclusive crisis. The new sickle cell anemia inflammation markers study may help to highlight the disease physiopathology complexity. Our finds emphasizes new genetic and immunological biomarkers identification and its association with classical markers may be important to elucidate phenotypic HbSS diversity.

Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-20Bitstream added on 2014-06-13T20:29:44Z : No. of bitstreams: 1 pagliarone_ac_me_botib.pdf: 396538 bytes, checksum: 70c1ba1ab2097248edf4d300ec8ca0ae (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O estresse pode exercer atividades imunossupressoras, acarretando o desenvolvimento de doenças como câncer, inflamações crônicas, neurodegeneração, autoimunidade, além de maior susceptibilidade a infecções por microrganismos. Os recém-descobertos receptores Toll-like (TLRs) têm sido extensamente investigados recentemente por reconhecer microrganismos patogênicos, ativando, consequentemente, a resposta imune. Devido ao pouco conhecimento quanto ao papel da própolis no sistema imune desafiado pela ação do estresse, o objetivo deste trabalho foi verificar o possível efeito deste apiterápico sobre a produção de citocinas pró-inflamatórias (IL-1β e IL-6) e de padrão Th1 (IFN-γ e IL-2) e Th2 (IL-4 e IL-10), e sobre a expressão do receptor TLR-4 por células esplênicas de camundongos submetidos a estresse. Camundongos BALB/c foram divididos em 3 grupos: G1 (controle), G2 (estresse) e G3 (própolis + estresse). G2 foi submetido a estresse por imobilização por 3 dias consecutivos, e G3 foi tratado com própolis e submetido a estresse. Após o sacrifício, o sangue foi coletado para a dosagem de corticosterona, como indicador de estresse. O baço de cada animal foi extraído e culturas celulares foram estimuladas com LPS por 24h ou com Con A por 48 h para determinação da produção de citocinas. Parte do baço foi utilizada para a realização de PCR quantitativo em tempo real a fim de verificar a expressão gênica de TLR-4 pelas células esplênicas. Os grupos submetidos a estresse, tratados ou não com própolis, apresentaram aumento na concentração de corticosterona. Houve inibição na produção de IL-1β e IL-6 nestes mesmos grupos, em comparação ao controle. Não houve alterações na produção de IL-2 em nenhum dos grupos, enquanto que a de IFN-γ esteve inibida nos grupos submetidos a estresse, tratados ou não com própolis. Já a produção de IL-4... / Stress can exert immunossupressive activities, which lead to the development of diseases such as cancer, chronic inflammation, neurodegenerative dysfunctions, autoimmunity and a higher susceptibility to infectious microorganisms. Recently, Tolllike receptors (TLRs) have been widely investigated for recognizing pathogenic microorganisms and, as a consequence, activating the immune response. Since little is known about propolis effects on the immune system challenged by stress, the aim of this work was to verify the possible effect of this bee product on pro-inflammatory (IL- 1β and IL-6), Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines and on TLR-4 expression by spleen cells from stressed mice. BALB/c mice were divided into 3 groups: G1 (control), G2 (stress) and G3 (propolis + stress). G2 was submitted to restraint stress for 3 consecutive days and G3 was treated with propolis and immediately submitted to stress. After sacrifice, blood was collected for corticosterone determination as a stress indicator. Spleens of all groups were removed and cell cultures were stimulated with LPS for 24h or with Con A for 48 h to further cytokines determination. A portion of the spleens was used for quantitative PCR real time assay in order to verify TLR-4 gene expression. An increased corticosterone concentration was seen in stressed groups, treated or not with propolis. IL-1β and IL-6 production was inhibited in these same groups, comparing to control. No alterations were found in IL-2 production in the experimental groups, while IFN-γ was inhibited on stressed group even when treated with propolis. IL-4 production was inhibited on stressed mice, but propolis treatment was able to antagonize this inhibition. There were no alterations in IL-10 production between the experimental groups. TLR-4 expression was inhibited in stressed mice, but propolis treatment exerted... (Complete abstract click electronic access below)

Orientador: José Maurício Sforcin / Banca: Sandra Cordellini / Banca: Glenda Nicioli da Silva / Resumo: O estresse pode exercer atividades imunossupressoras, acarretando o desenvolvimento de doenças como câncer, inflamações crônicas, neurodegeneração, autoimunidade, além de maior susceptibilidade a infecções por microrganismos. Os recém-descobertos receptores Toll-like (TLRs) têm sido extensamente investigados recentemente por reconhecer microrganismos patogênicos, ativando, consequentemente, a resposta imune. Devido ao pouco conhecimento quanto ao papel da própolis no sistema imune desafiado pela ação do estresse, o objetivo deste trabalho foi verificar o possível efeito deste apiterápico sobre a produção de citocinas pró-inflamatórias (IL-1β e IL-6) e de padrão Th1 (IFN-γ e IL-2) e Th2 (IL-4 e IL-10), e sobre a expressão do receptor TLR-4 por células esplênicas de camundongos submetidos a estresse. Camundongos BALB/c foram divididos em 3 grupos: G1 (controle), G2 (estresse) e G3 (própolis + estresse). G2 foi submetido a estresse por imobilização por 3 dias consecutivos, e G3 foi tratado com própolis e submetido a estresse. Após o sacrifício, o sangue foi coletado para a dosagem de corticosterona, como indicador de estresse. O baço de cada animal foi extraído e culturas celulares foram estimuladas com LPS por 24h ou com Con A por 48 h para determinação da produção de citocinas. Parte do baço foi utilizada para a realização de PCR quantitativo em tempo real a fim de verificar a expressão gênica de TLR-4 pelas células esplênicas. Os grupos submetidos a estresse, tratados ou não com própolis, apresentaram aumento na concentração de corticosterona. Houve inibição na produção de IL-1β e IL-6 nestes mesmos grupos, em comparação ao controle. Não houve alterações na produção de IL-2 em nenhum dos grupos, enquanto que a de IFN-γ esteve inibida nos grupos submetidos a estresse, tratados ou não com própolis. Já a produção de IL-4... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: Stress can exert immunossupressive activities, which lead to the development of diseases such as cancer, chronic inflammation, neurodegenerative dysfunctions, autoimmunity and a higher susceptibility to infectious microorganisms. Recently, Tolllike receptors (TLRs) have been widely investigated for recognizing pathogenic microorganisms and, as a consequence, activating the immune response. Since little is known about propolis effects on the immune system challenged by stress, the aim of this work was to verify the possible effect of this bee product on pro-inflammatory (IL- 1β and IL-6), Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines and on TLR-4 expression by spleen cells from stressed mice. BALB/c mice were divided into 3 groups: G1 (control), G2 (stress) and G3 (propolis + stress). G2 was submitted to restraint stress for 3 consecutive days and G3 was treated with propolis and immediately submitted to stress. After sacrifice, blood was collected for corticosterone determination as a stress indicator. Spleens of all groups were removed and cell cultures were stimulated with LPS for 24h or with Con A for 48 h to further cytokines determination. A portion of the spleens was used for quantitative PCR real time assay in order to verify TLR-4 gene expression. An increased corticosterone concentration was seen in stressed groups, treated or not with propolis. IL-1β and IL-6 production was inhibited in these same groups, comparing to control. No alterations were found in IL-2 production in the experimental groups, while IFN-γ was inhibited on stressed group even when treated with propolis. IL-4 production was inhibited on stressed mice, but propolis treatment was able to antagonize this inhibition. There were no alterations in IL-10 production between the experimental groups. TLR-4 expression was inhibited in stressed mice, but propolis treatment exerted... (Complete abstract click electronic access below) / Mestre

Um papel importante foi atribuído ao receptor toll-like 4 (TLR4) no desenvolvimento da placa aterosclerótica. O TLR4 foi primeiramente descrito como um receptor para bactérias gram-negativas; posteriormente foi demonstrado que sua expressão está aumentada em placas ateroscleróticas e que pacientes que possuem um polimorfismo disfuncional do TLR4 são menos suscetíveis ao desenvolvimento dessa doença. Portanto, o objetivo desse estudo foi o de investigar, em um modelo experimental de aterosclerose, a influência da deleção do TLR4 na formação e morfologia da placa aterosclerótica, no perfil lipídico e em marcadores inflamatórios. Camundongos duplo knockout (DKO), deficientes no receptor de LDL e TLR4, foram gerados cruzando-se camundongos deficientes para o receptor de LDL (LDLrKO) com camundongos deficientes para o TLR4 (TLR4KO). Todos os grupos receberam dieta rica em gordura e colesterol por 12 semanas. As concentrações plasmáticas de colesterol e triglicérides foram medidas por ensaio colorimétrico. Cortes seriados da raiz aórtica foram corados com Oil red O e as áreas de lesão quantificadas por analisador de imagens. O colágeno foi medido por coloração de picrossirius. A formação de nitrotirosina e expressão de CD40L, MMP9 e iNOS nas placas foram feitas por imunohistoquímica. As comparações foram feitas por ANOVA com pós teste de Student Newman-Keuls. Os dados foram expressos como média ± EPM. Camundongos DKO desenvolveram placas menores que camundongos LDLrKO (117.6 ±1.4 vs 198.8 ± 3.3 104m2). Camundongos TLR4KO não formaram placa. As placas dos camundongos DKO apresentaram menor núcleo lipídico que as dos LDLrKO (76.2± 13.2 vs 161.7 ± 2.9 104m2). O colágeno ao redor do núcleo lipídico é maior nos camundongos DKO do que nos LDLrKO (24.9 ± 1.8 vs 16.5 ± 2.5 % da placa). A distribuição do colágeno nos camundongos DKO ocorre principalmente ao redor da placa, de forma mais organizada, enquanto que nos LDLrKO onde sua distribuição é mais difusa. As placas dos camundongos DKO apresentaram menor expressão de CD40L e iNOS do que as dos LDLrKO (13.1 ± 0.7 vs 18.5 ± 2.5 AU e 7.7 ± 0.9 vs 10.2 ± 0.4 AU, respectivamente). A expressão de MMP9 foi menor nas placas dos camundongos DKO do que as dos LDLrKO (2.99 ± 0.3 vs 1.99 ± 0.2 AU). A marcação para nitrotirosina foi maior nos camundongos LDLrKO quando comparada com as dos grupos DKO e TLR4KO (142.89 ± 208.5, 77.16 ± 227.7 e 71.73 ± 95.9 10m2, respectivamente). Todos esses resultados sugerem que o processo inflamatório é menor na ausência do TLR4. As concentrações plasmáticas de colesterol não foram diferentes entre os grupos LDLrKO e DKO mas os camundongos LDLrKO apresentaram concentrações plasmáticas de triglicérides maiores do que os camundongos DKO após a dieta (265.2 ± 27.6 vs 150.5 ± 8.8 mg/dL). O receptor toll-like 4 influencia na estrutura e formação da placa aterosclerótica independentemente dos níveis séricos de colesterol / A crucial role has been suggested for toll-like receptor 4 (TLR4) in atherosclerotic plaque formation and development. TLR4 was described primarily, as a receptor for gram-negative bacteria lipopolisacharide; later it was showed that its expression is increased in atherosclerotic plaques and patients that carries a TLR4 dysfunctional polymorphism are less susceptible to development of this disease. Therefore, the aim of this study was to investigate, in an experimental model of atherosclerosis, the influence of TLR4 deletion in atherosclerotic plaque formation and morphology, cholesterol profile and inflammatory markers. Double knockout mice (DKO), deficient in LDL receptor and TLR4, were generated by breeding LDL receptor knockout mice (LDLrKO) with TLR4 knockout mice (TLR4KO). All three experimental groups, LDLrKO, TLR4KO and DKO were fed a high fat-cholesterol diet for 12 weeks. Plasma cholesterol and triacylglicerol concentrations were measured by colorimetric assay. Cross sections of aortic sinus were stained with Oil red O and lesion areas were quantified by an image analyzer. Collagen content was measured by picrossirius staining. We also measured nitrotyrosine formation, CD40L, MMP9 and iNOS expression by immunohistochemistry. Comparisons were made by ANOVA followed by Student-Newman-Keuls post- test. Data are mean ± SEM. DKO mice developed smaller plaques than LDLrKO mice (117.6 ±1.4 vs 198.8 ± 3.3 104m2). TLR4KO mice developed no plaque. Plaques from DKO mice have also a smaller lipid core than the ones from LDLrKO mice (76.2± 13.2 vs 161.7 ± 2.9 104m2). Collagen content around the lipid core is higher in DKO mice compared to LDLrKO mice (24.9 ± 1.8 vs 16.5 ± 2.5 % of the whole plaque). Interestingly, collagen distribution in DKO mice seems to occur mainly on the plaque periphery, in a more organized manner, while in LDLrKO mice it is fuzzier, being present also inside the plaque. Plaques from DKO present lower expression of CD40L and iNOS than LDLrKO mice (13.1 ± 0.7 vs 18.5 ± 2.5 AU and 7.7 ± 0.9 vs 10.2 ± 0.4 AU, respectively). MMP9 expression is lower in DKO mice as compared to LDLrKO mice (2.99 ± 0.3 vs 1.99 ± 0.2 AU). Nitrotyrosine staining was higher in LDLrKO mice as compared to DKO and TLR4KO groups (142. 89 ± 208.5, 77.16 ± 227.7 and 71.73 ± 95.9 10m2, respectively). All together, these findings suggest that inflammatory process is milder in the absence of TLR4. Serum cholesterol were not different between LDLrKO and DKO mice but LDLrKO presented higher triacylglicerol serum levels after 12 weeks on high fat high cholesterol diet as compared to DKO mice (265.2 ± 27.6 vs 150.5 ± 8.8). Toll like receptor 4 influences atherosclerotic plaque formation and structure independently from serum cholesterol levels

Orientador: Mario Jose Abdalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T03:34:45Z (GMT). No. of bitstreams: 1 Tsukumo_DanielaMitiLemos_D.pdf: 7330041 bytes, checksum: 384133bd9ed3dd468d57225845733901 (MD5) Previous issue date: 2008 / Resumo: Estudos recentes demonstram que a obesidade está associada com a resistência à insulina e com um estado de inflamação crônica subclínica. Os ¿Toll-Like Receptors¿ (TLRs) têm um papel fundamental na ativação do sistema imune através do reconhecimento de antígenos de microorganismos. O TLR4 é um subtipo de TLRs que é ativado pelo lipopolissacarídeo de bactérias gram-negativas e por outros agonistas, como os ácidos graxos saturados. A ativação do TLR4 estimula vias inflamatórias relacionadas à indução de resistência à insulina, como a JNK e a IKKß. Neste estudo, demonstrou-se que camundongos C3H/HeJ, que apresentam uma mutação inativadora do TLR4, estão protegidos da obesidade e da resistência à insulina induzidas por dieta hiperlipídica. Além disso, músculos sóleos isolados de camundongos C3H/HeJ estão protegidos da resistência à insulina induzida por ácidos graxos. Camundongos C3H/HeJ tratados com dieta hiperlipídica (DH) apresentam um menor ganho de peso, maior tolerância à glicose e maior sensibilidade à insulina em relação aos controles em DH. A análise morfométrica do tecido adiposo evidenciou que os adipócitos dos camundongos C3H/HeJ em DH são 30% menores em relação aos adipócitos dos camundongos controle tratados com a mesma dieta. Foi evidenciada uma maior fosforilação em tirosina do IRS-1 e maior fosforilação da Akt, após estímulo com insulina, em músculo, tecido adiposo e fígado de camundongos C3H/HeJ tratados com DH em relação aos controles. Observou-se uma maior ativação da JNK, da IKKß e da iNOS em músculo, tecido adiposo e fígado de animais controle tratados com DH quando comparado com camundongos C3H/HeJ tratados com a mesma dieta. O tratamento com palmitato reduziu a captação de glicose e a síntese de glicogênio em 40-50% em músculo sóleo isolado de camundongos controle, mas este efeito não foi observado em músculo sóleo isolado de camundongos C3H/HeJ. Em resumo, o nosso estudo demonstra que a inativação do TLR4 previne a obesidade induzida por dieta, a ativação da IKKß, da JNK, a resistência à insulina em camundongos em DH, além da resistência à insulina induzida por palmitato em músculo isolado. O estudo sugere que o TLR4 tem um papel importante na interligação entre o sistema imune inato e a resistência à insulina, sendo um possível alvo terapêutico para a obesidade, resistência à insulina e diabetes mellitus tipo 2 / Abstract: Obesity is associated with insulin resistance and a state of abnormal inflammatory response. The Toll-like receptor 4 (TLR4) has an important role in inflammation and immunity and its expression has been reported in most tissues of the body, including the insulin-sensitive ones. Since it is activated by lipopolysaccharide (LPS) and saturated fatty acids, which are inducers of insulin resistance, TLR4 may be a candidate for participation in the cross-talk between inflammatory and metabolic signals. Here, we show that C3H/HeJ mice, which have a loss-of-function mutation in TLR4, are protected against the development of diet-induced obesity. In addition, these mice demonstrate decreased adiposity, increased oxygen consumption, a decreased respiratory exchange ratio, improved insulin sensitivity and enhanced insulin signaling capacity in adipose tissue, muscle and liver, as compared to control mice during high fat feeding. Moreover, in these tissues, control mice fed on a high fat diet show an increase in IKKß and JNK activity, which is prevented in C3H/HeJ mice. In isolated muscles from C3H/HeJ a protection from saturated fatty acid-induced insulin resistance is observed. Thus, TLR4 appears to be an important mediator of obesity and insulin resistance and a potential target for the therapy of these highly prevalent medical conditions / Doutorado / Ciencia Basica / Doutor em Clínica Médica

Orientador: Licio Augusto Velloso / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T09:35:56Z (GMT). No. of bitstreams: 1 Razolli_DanielaSoares_D.pdf: 6783117 bytes, checksum: a457ef1952b61a9b35b688c018c62b0a (MD5) Previous issue date: 2014 / Resumo: A ativação de TLR4 por ácidos graxos saturados presentes na dieta é um dos mais importantes mecanismos envolvidos no desenvolvimento da resistência à insulina. Entretanto, não está claro se este efeito decorre da ativação de TLR4 em células derivadas da medula óssea, em células de tecidos insulino-sensíveis, ou em ambos. O objetivo de nosso estudo foi investigar se a ausência ou presença de TLR4 exclusivamente em células derivadas da medula óssea (BM) é suficiente para modular a ação da insulina no fígado e no tecido adiposo de camundongos com obesidade induzida por dieta. Camundongos mutantes (TLR4-/-) ou selvagens (WT) para TLR4 foram irradiados em fonte de cobalto 60 e submetidos ao transplante de medula óssea (BMT). TLR4-/- receberam medula óssea de camundongos WT e vice-versa. Após a recuperação, os animais foram alimentados com dieta rica em gordura saturada ou dieta padrão para roedores por oito semanas. Foram avaliados ganho de peso, ingestão alimentar, gasto energético e marcadores de termogênese no tecido adiposo marrom (BAT). A sensibilidade à insulina foi analisada por ITT e pela fosforilação de AKT no fígado e tecido adiposo. O metabolismo de glicose foi avaliado por GTT e pela expressão relativa de PEPCK e G6PASE no fígado. Ainda, marcadores de inflamação foram avaliados no fígado e tecido adiposo por PCR em tempo real, e o acúmulo de gordura hepática foi analisado pela marcação por OilRed. Nossos resultados revelaram que em camundongos WT recebendo BM de doadores TLR4-/- ocorreu proteção contra o desenvolvimento de obesidade e resistência à insulina induzida por dieta. Em camundongos TLR4-/- recebendo BM de doadores WT houve perda da proteção contra a obesidade e resistência à insulina induzida por dieta. A transferência de fenótipo relativo a massa corporal foi acompanhada de modificação na eficiência energética e na expressão de UCP1 em tecido adiposo marrom. A transferência de fenótipo relativo a resistência à insulina foi acompanhado da modificação do padrão de ativação de transdução do sinal da insulina por meio de AKT em fígado e tecido adiposo. Particularmente no fígado, a presença de TLR4 funcional em células derivadas da BM foi fator determinante no desenvolvimento da esteatose. Por fim, a presença de TLR4 funcional em células derivadas da BM foi acompanhada da expressão de níveis elevados de citocinas pró-inflamatórias e nível reduzido de pelo menos uma citocina anti-inflamatória em fígado e tecido adiposo, sendo que a ausência de TLR4 nas células derivadas da BM resultou em fenótipo inverso. Desta forma, conclui-se que a presença ou ausência de TLR4 em células derivadas da BM tem papel determinante, sendo simultaneamente necessário e suficiente para o desenvolvimento do fenótipo de obesidade e resistência à insulina induzida por dieta / Abstract: Saturated fatty acid-induced activation of TLR4 is one of the most important mechanisms involved in the development of insulin resistance. However, it is not clear if this effect is due to activation of TLR4 present in bone marrow (BM)-derived cells, in cells present in tissues that are targets for insulin action, or in both. The aim of the present study was to investigate if a defective TLR4 signaling in bone-marrow (BM) derived cells is sufficient to modulate the whole-body insulin action. TLR4-mutant (TLR4-/-) and wild-type (WT) mice were irradiated in a cobalt 60 source and submitted to bone marrow transplantation (BMT). TLR4-/- received BM from WT and vice-versa. After recovery, mice were fed on a high fat diet (HFD) or standard chow for eight weeks. We evaluated body mass gain, food intake, energy expenditure and markers of thermogenesis in brown adipose tissue (BAT). Insulin sensitivity was analyzed by GTT, ITT and AKT phosphorylation in liver and adipose tissue. In addition, markers of inflammation were evaluated in both tissues by real-time PCR. WT submitted to BMT from TLR4-/- donors were protected from HFD-induced obesity and insulin resistance. In TLR4-/- submitted to BMT from WT donors this effect was blunted and mice gained weight and became insulin resistant. Phenotype transfer regarding body mass was accompanied by change in energy efficiency and expression of UCP1 in brown adipose tissue. Phenotype transfer regarding insulin resistance was accompanied by changes in insulin signal transduction through AKT in liver and adipose tissue. Particularly, in the liver, the presence of a functional TLR4 in BM-derived cells was a determinant factor for the development of steatosis. Finally, the presence of a functional TLR4 in BM-derived cells was accompanied by the increased expression of inflammatory cytokines and reduced expression of at least one anti-inflammatory cytokine in the liver and adipose tissue. Thus, we conclude that the presence or absence of TLR4 in BM-derived cells is both sufficient and necessary for the induction of obesity and insulin resistance induced by diet / Doutorado / Fisiopatologia Médica / Doutora em Ciências