As metaloproteinases são abundantes em venenos de serpentes. Estas enzimas são homólogas às de mamíferos, encontradas em níveis elevados em inflamações articulares. Neste estudo avaliou-se a capacidade da BaP1, induzir: i) a inflamação e incapacitação articulares e a participação do TNF-<font face=\"symbol\">a e PGE2 nesses efeitos e ii) a ativação de macrófagos em cultura e a natureza de sua interação com estas células. A BaP1 induziu aumento da permeabilidade vascular, liberação de TNF-<font face=\"symbol\">a, MMP-9 e PGE2 e acúmulo de leucócitos na cavidade articular e tecido sinovial. Ainda, induziu dor articular. O pré-tratamento dos animais com indometacina ou anti-TNF-<font face=\"symbol\">a reduziu a dor e o influxo leucocitário, induzidos pela BaP1. A BaP1 induziu a expressão de COX-2 e de TNF-<font face=\"symbol\">a e a liberação desta citocina, em macrófagos isolados. Nestas células detectou-se a internalização da BaP1. Em conclusão, a BaP1 induz inflamação e nocicepção articulares, dependentes de TNF-<font face=\"symbol\">a e PGE2. A COX-2 deve estar envolvida na liberação de PGE2 e os macrófagos são alvos importantes para as ações dessa metaloproteinase. / Metalloproteinases are major enzymes in snake venoms showing high grade of homology with mammal matrix metalloproteinases, present in high levels in inflamed joints. In this study we examined the ability of BaP1, to induce: i) inflammation and hypernociception in rat articular joints and participation of TNF-<font face=\"symbol\">a and PGE2 in these effects, and ii) activation of cultured macrophages. BaP1 increased vascular permeability, induced release of TNF-<font face=\"symbol\">a, PGE2 and pro-MMP-9 in joint cavities, and leucocyte influx into joint cavities and synovial tissues. Moreover, BaP1 induced articular hypernociception. Treatment of animals with indomethacin or antiserum anti-TNF-<font face=\"symbol\">a significantly reduced hypernociception and leukocyte influx induced by BaP1. Incubation of macrophages with BaP1 caused expression of TNF-<font face=\"symbol\">a and COX-2 as well as TNF-<font face=\"symbol\">a release. In conclusion, BaP1 induces inflammation and hypernociception in articular joints. These effects are dependent on PGE2 and TNF-<font face=\"symbol\">a. COX-2 may contribute for BaP1-induced PGE2 release and macrophages are key targets for BaP1 induced effects.

Assim como visto nos vertebrado, os invertebrados também possuem um sistema imune bastante complexo, apresentando respostas humorais e celulares. Esta última é a principal forma de defesa, envolvendo células amebóides móveis capazes de isolar e/ou eliminar material estranho. Em equinodermos, as células envolvidas nestas respostas são categorizadas como celomócitos, uma denominação genérica que engloba vários tipos celulares encontrados nas cavidades corporais e no tecido conjuntivo. Entretanto, além da função imune, são reportados como tendo outros papéis diversos, como excreção, digestão, transporte e estocagem de nutrientes e a síntese e deposição de fibras de colágeno e da matriz extracelular Não existe consenso sobre quais os papéis específicos desempenhados por estas células num organismo saudável e nem diante de um processo inflamatório. Assim, o presente trabalho foi realizado para caracterizar o processo inflamatório no espinho de Eucidaris tribuloides causado por sabinella troglodytes, um molusco ectoparasita no espinho. A primeira parte do trabalho traz a caracterização das células da cavidade celomática. Para investigá-las foi realizada uma abordagem integrada onde os celomócitos foram descritos por meio da utilização de células vivas, citoquímica e microscopia eletrônica de transmissão. Foram encontrados sete tipos celulares, sendo um novo e dois pouco conhecidos. Com esta abordagem inicial, foi possível obter as ferramentas necessárias para investigar o processo inflamatório. Para estudar o processo inflamatório no espinho de E. tribuloides, utilizou-se uma abordagem estrutural, combinando histologia, microscopia eletrônica de varredura e microtomografia computadorizada, assim como celular/molecular. Os dados indicam que a inflamação causada pelo molusco parece ser um evento local, que altera tanto a matriz orgânica quanto a calcária, mas parece não se propagar para a cavidade celomática do hospedeiro / The invertebrates, as well as vertebrates, also have a very complex immune system, presenting humoral and cellular responses. The latter is their main form of defense, involving mobile amoeboid cells capable of isolating and/or eliminating foreign material. In Echinodermata, the cells involved in these responses are categorized as coelomocytes. This is a generic term that encompasses various cell types found in the body cavities and connective tissue. However, in addition to immune function, they are reported as having various other roles, such as excretion, digestion, transport and nutrient storage and the synthesis and deposition of collagen fibers and extracellular matrix. There is no consensus on what the specific role played by each one of these cells in healthy individuals and the knowledge about the inflammatory process is much worse. Thus, the present study was performed to characterize the inflammatory process in the spine of Eucidaris tribuloides caused by Sabinella troglodytes, a spine ectoparasites gastropod. The first part of the study provides the characterization of the cells of the coelomic cavity. To investigate them, an integrated approach was used, where the coelomocytes were described through the use of living cells, immunocytochemistry and transmission electron microscopy. Seven cell types were found: one new and two poorly known. This initial approach enables us to obtain the necessary tools to investigate the inflammatory process. To study the inflammatory process in the spine of E. tribuloides, we used a structural approach, combining histology, scanning electron microscopy and computed microtomography, and the cellular/molecular one. The data indicate that inflammation caused by snail appears to be a local event, which alters both the organic and calcareous matrix, but does not seem to be reflected within the coelomic cavity of the host

Disease progression in Multiple Sclerosis (MS), an autoimmune disease of the CNS, is widely accepted to be due to persistent myelin loss (demyelination) coinciding with lost nerve cells and nerve fibres (neuroaxonal loss). Current treatments are immunomodulatory and do not address the neuroaxonal or demyelinating pathology of the disease. It is hypothesised that a lack of growth factors within the CNS may result in the failure of remyelination. Therefore, biologics such as recombinant therapeutic proteins used for gene therapy offer a promising therapeutic intervention to the progressive stages of the disease. However, due to the short half-lives of these therapeutics and their pleiotropic effects, there is cause for concern over their safety and efficacy. Using LAP technology (the fusion of the therapeutic protein with the latent associated peptide [LAP] of TGFβ), the half-life of the therapeutic protein can be increased and can be targeted to sites of inflammation and disease. This study aimed to investigate the potential neuroprotective, remyelinating and anti-inflammatory effects of latent versions of the growth factors erythropoietin (EPO), insulin-like growth factor 1 (IGF1) and transforming growth factor beta (TGF) respectively. Firstly, using molecular cloning techniques, these growth factors were individually fused and linked to the LAP of TGF via a matrix metalloproteinase (MMP) cleavage site resulting in three latent growth factors. Secondly, these latent growth factors were shown to be expressed, and to be biologically active in vitro when released by MMP cleavage. Finally, syngeneic fibroblasts were engineered to express the latent growth factors. It was found that, in CREAE, the fibroblasts engineered to produce latent TGF significantly reduced the disease clinical score as compared to controls whilst latent EPO produced by transduced fibroblasts failed to exert a statistically significant effect on disease progression. Nonetheless, this study demonstrates the feasibility of the latency platform technology to generate latent therapeutics with the ability to act as an intervention to disease progression in MS.

Inflammation has incompletely characterized effects on cardiopulmonary vascular reactivity. Sepsis is a major inflammatory disease characterized by two main vasomotor complications, generalized vasodilation with hyporesponsiveness to vasoconstrictors and pulmonary hypertension. The main aim of this study is to examine the molecular mechanisms involved in cardiopulmonary vascular reactivity changes induced by the powerful inflammatory stimulus lipopolysaccharide (LPS). Pulmonary and aortic rings from male Wistar rats (250-300g) were isolated and incubated for 20 h in culture medium (DMEM+10% FBS) with or without LPS (E. coli O55:B5, 10 μg.ml⁻¹). The effect of organ culture and LPS type, concentration and incubation time in addition to tissue contraction to endothelin-1 (ET-1), phenylephrine, 80 mM KCl, and U46619; and relaxation responses to ACh, sodium nitroprusside (SNP), 8-pCPT-cGMP, BAY 41-2272, T-0156, nifedipine, SKF-96365, Ro-31-8425, and Y-27632 were measured by standard organ bath techniques. Nitric oxide (NO) production was measured by the Griess method and SNP-induced cGMP production was measured by ELISA. mRNAs expression levels of eNOS, iNOS, ET-1, ETA and ETB were measured by qRT-PCR and the expression levels of PKC, sGCα₁, sGCβ₁ and PDE5 and phosphorylation of MLC₂₀, ROKα, CPI-17 and MYPT1 were measured by immunoblotting. The effect of endothelium removal, indomethacin, trolox, external Ca²⁺ removal, 1400W, ODQ, glibenclamide, iberiotoxin and cycloheximide in addition to changes in intracellular Ca²⁺ ([Ca²⁺]i) in aortic vascular smooth muscle cells (VSMCs) induced by ET-1 were also measured. LPS selectively induced vascular hyporeactivity to different vasoconstrictors in rat aorta but not in the pulmonary artery, which is not due to organ culturing and is not affected by changing the LPS type, but is enhanced by increasing LPS concentration or the incubation time. This aortic hypocontractility to ET-1 is largely mediated by NO-independent activation of sGC and depends on external Ca²⁺ influx through non-VOCCs, but not on ET-1 receptor expression or Ca²⁺ sensitization. In addition, this aortic hyporeactivity to ET-1 is dependent on protein synthesis. The pulmonary artery is not affected because LPS induces a desensitization of the sGC/cGMP dependent pathway by decreasing protein expression levels of sGCβ₁, and hence sGC activity, and increasing PDE5 activity. Neither the endothelium, cyclooxygenase, reactive oxygen species nor K⁺ channels are involved in these LPS effects. Therefore, it is likely that both Ca²⁺ homeostasis and the sGC/cGMP pathway play important roles in vasomotor complications in sepsis. sGC and/or PDE5-selective inhibitors, together with manipulating VSMC [Ca²⁺]i, could be important in controlling systemic and pulmonary vasomotor complications in sepsis.

The overall aim of the work presented in this thesis was to further characterise aspects of metabolic and immune system cross-talk in human subcutaneous adipose tissue, with a particular emphasis on the potential role of T-lymphocytes in adipose tissue dysfunction and insulin resistance. Chapter 3 characterised macrophage and T-lymphocyte populations residing in adipose tissue from lean through to class I obese men. This work demonstrated that T-lymphocytes display increased activation with increased adiposity and that potential compensatory mechanisms may be present to help counteract adipose tissue inflammation. In Chapter 4, the same participants were exposed to a meal-based stimulus in order to examine the postprandial metabolic and inflammatory responses in blood and adipose tissue. Despite increased glucose and insulin responses in blood with obesity, there were no differences in inflammatory cytokine gene expression responses in adipose tissue. This suggests that mechanisms may be present to limit or dampen inflammatory output from adipose tissue after feeding in individuals with modestly increased adiposity. Chapter 5 examined metabolic and immune system changes to 50 % calorie restriction for 3 days, resulting in reduced serum leptin which was temporally associated with a reduction in blood T-lymphocyte activation. In adipose tissue, however, leptin gene expression/secretion was not reduced and neither was resident T-lymphocyte activation, indicating that there may be local tissue-specific responses of immune cells to caloric restriction. Chapter 6 characterised differences between obese individuals with either normal or impaired glucose tolerance, and their respective responses to 10 days of diet and activity modification. Overall, this thesis highlights key differences in properties of T-lymphocyte populations with increasing levels of adiposity and insulin resistance together with responses in adipose tissue and the immune system in times of feeding, severe calorie restriction and glucose lowering diet and activity.

Orientador: Geovanni Dantas Cassali / Resumo: As neoplasias mamárias constituem os tumores com maior incidência em cadelas. Dentre os fatores que contribuem para o desenvolvimento desta e de outras neoplasias, o microambiente tumoral inflamatório desempenha um papel crucial. Vários estudos relataram papéis importantes para linfócitos, macrófagos, plasmócitos, neutrófilos, eosinófilos e mastócitos neste contexto. No presente estudo, o objetivo foi avaliar a densidade de células inflamatórias e área de fibrose tumoral e sua relação com tumores mamários caninos com diferentes características histológicas e clínicas (tumor misto benigno, carcinoma em tumor misto, carcinoma sólido e carcinoma tubular). A análise de células inflamatórias e área de fibrose tumoral foram realizadas por meio de técnicas histoquímicas, enquanto a identificação de linfócitos Tregs foram realizadas por imuno-histoquímica. A análise estatística das densidades de células inflamatórias e áreas fibrose tumoral e sua relação com os tipos histológicos revelou diferença significativa para as plasmócitos (p = 0,035), neutrófilos (p = 0,0113), macrófagos (p = 0,0047) e fibrose tumoral (p = 0,05). Os dados encontrados sugerem associações entre alto número de neutrófilos e neoplasias mamárias agressivas, entre altas densidades de plasmócitos, macrófagos e células CD8+ e entre menor densidade de células CD4+ e neoplasias menos agressivas. Maiores áreas de fibrose tumoral mostraram relação com neoplasias mamárias caninas mais agressivas. / Abstract: Mammary neoplasias constitute the tumors with higher incidence in bitches. Among the factors that contribute for the development of this and other neoplasias, the inflammatory tumor microenvironment plays a crucial role. Several studies reported important roles for lymphocytes, macrophages, plasma cells, neutrophils, eosinophils and mast cells in this context. In the present study, the goal was to evaluate density of inflammatory cells and area of tumor fibrosis and their relation with canine mammary tumors with different histologic and clinical presentation (benign mixed tumor, carcinoma in mixed tumor, solid carcinoma and tubular carcinoma) Counting and staining of inflammatory cells and tumor fibrosis were performed through histochemistry, while counting and staining of Tregs were performed through immunohistochemistry. Statistical analysis of the densities of inflammatory cells and tumor fibrosis related to histologic types revealed significant difference for plasma cells (p=0,035), neutrophils (p=0,0113), macrophages (p=0,0047), and tumor fibrosis (p=0,05). The found data suggest associations between high number of neutrophils and agressive mammary neoplasias, between high densities of plasma cells, macrophages and CD8+ cells and between low density of CD4+ cells and less aggressive neoplasias. Higher areas of tumor fibrosis showed relation with more aggressive canine mammary neoplasias. / Mestre

Orientador: Irma de Godoy / Banca: José Antonio Baddini Martinez / Banca: Maria do Patrocínio Tenório Nunes / Banca: Aparecida Yooko Outa Angeleli / Banca: Alexandrina Sartori / Resumo: A doença pulmonar obstrutiva crônica (DPOC) está associada a processo inflamatório; entretanto, a definição de quais marcadores participam do processo inflamatório local e sistêmico, e suas correlações com o tabagismo e com a intensidade de obstrução das vias aéreas, ainda não está esclarecida. Avaliar simultaneamente a inflamação no sangue periférico e nas vias aéreas de tabagistas com e sem DPOC e avaliar a associação com o estado de tabagismo atual e com a gravidade da doença. Foram avaliados 52 pacientes com DPOC moderada a muito grave com idade média de 63,6±8,9 anos e VEF1(%) 50,6±17,1 e 20 indivíduos tabagistas com idade média de 49,1±6,2 anos e VEF1(%) 106,5±15,8. Todos os indivíduos da pesquisa foram submetidos à avaliação clínica, espirometria pré e pós-broncodilatador, oximetria de pulso, avaliação nutricional, teste de caminhada de seis minutos (TC-6), indução e coleta de escarro e coleta de sangue para exames bioquímicos e dosagem dos seguintes mediadores inflamatórios: TNF-a, IL-6, IL-8, IL-10 e PCR. Os tabagistas sem obstrução das vias aéreas apresentaram maiores níveis séricos de TNF-a quando comparados aos pacientes com DPOC. Além disso, entre os pacientes com DPOC, aqueles que eram tabagistas ativos apresentaram elevação da concentração sérica de TNF-a em relação aos ex-tabagistas. A IL-6 e a PCR estavam elevadas nos pacientes com DPOC comparados aos tabagistas sem obstrução das vias aéreas. No escarro induzido os pacientes com DPOC apresentaram maiores níveis do TNF-a e da IL-10 em relação aos tabagistas. Em relação à gravidade da doença, houve aumento do TNF-a sérico nos pacientes com doença moderada em relação aos com DPOC grave ou muito grave, e elevação do TNF-a e da IL-10 no escarro induzido dos pacientes ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chronic obstructive pulmonary disease ( COPD) is an inflammatory disease; however, the markers that characterize the localized and systemic inflammation and the... (Complete abstract click electronic access below) / Doutor

OBJECTIVE: Tumor necrosis factor-alpha (TNF-α) is a multifunctional proinflammatory cytokine that plays a critical role in mediating inflammatory and immunological responses. TNF-α has been shown to elicit both beneficial and detrimental biological effects by acting through its two receptors, TNFR1/p55 and TNFR2/p75. Previous studies from this laboratory have shown that TNF-TNFR2/p75 signaling plays a critical role in ischemia-induced neovascularization in muscle and heart tissues. However, the role of TNF-TNFR2/p75 signaling in ischemia induced inflammation and muscle regeneration remains to be characterized. METHODS: To evaluate ischemia induced inflammation responses, young wild type (WT) and young TNFR2/p75 knockout (p75KO) mice were subjected to unilateral hind limb ischemia (HLI) surgery. Operated hind limb tissue samples were collected at 1, 3, 7, and 10 days post-HLI surgery and studied for neutrophil (myeloperoxidase-1 positive cells) and macrophage (F4/80 positive cells) infiltration as well as satellite-cell activation (neural cell adhesion molecule positive cells) at each time point. To determine possible synergistically negative roles of tissue aging and the absence of TNFR2/p75 in either the tissue or bone marrow (BM), two chimeric BM transplantation (BMT) models were generated where young Green Fluorescent Protein (GFP) positive (+) p75KO and WT BM-derived cells were transplanted into adult p75KO mice. HLI surgery was performed one month post-BMT, after confirming complete engraftment of the recipient BM with GFP donor cells. Operated hind limb tissue samples were evaluated up to 28 days post-surgery to examine proliferation and apoptosis of BM-derived cells in ischemic tissue. RESULTS: Ischemia induced significant and long-lasting inflammation associated with a considerable decrease in satellite-cell activation in p75KO muscle tissue 1-10 days post-HLI surgery. For the BMT studies, in adult p75KO with the WT-BMT, proliferative (Ki67+) cells were detected only by day 28 and were exclusively GFP (+), suggesting delayed contribution of young WT-BM cell to adult p75KO ischemic tissue recovery. No GFP (+) young p75KO BM cells survived in adult p75KO tissue. CONCLUSION: The data demonstrate that: (1) ischemia-induced recovery in skeletal muscle tissue is impaired in young p75KO mice; (2) inflammatory responses are significantly increased and long-lasting in p75KO mice; (3) in the absence of TNFR2/p75 signaling, satellite-cell activation is affected in p75KO mice; (4) during post-ischemic recovery, tissue aging combined with decreased/absent TNFR2/p75 signaling may have synergistically negative roles on survival and proliferation in the damaged tissue.

Submitted by Eunice Novais (enovais@uepg.br) on 2018-07-24T14:58:19Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Mariane A Sanson.pdf: 3058378 bytes, checksum: 43bb48ea0be645db69444ec684b43bac (MD5) / Made available in DSpace on 2018-07-24T14:58:19Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Mariane A Sanson.pdf: 3058378 bytes, checksum: 43bb48ea0be645db69444ec684b43bac (MD5) Previous issue date: 2016-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Compostos de ouro já apresentam propriedades anti-inflamatórias estabelecidas pela literatura, além de importante potencial antioxidante. A utilização do ouro na forma nanoparticulada é uma aposta promissora para o tratamento de condições inflamatórias, como a doença periodontal, pelo seu mecanismo de ação. Apesar da biocompatibilidade estabelecida do ouro, é importante atentar-se para a possibilidade de efeito tóxico do ouro na forma nanoparticulada, devido a sua maior facilidade de penetrar nos tecidos e atravessar membranas, em resposta principalmente ao seu tamanho e concentração de ouro presente. O objetivo deste estudo foi a caracterização das nanopartículas de ouro (AuNPs) de 10, 20 e 30 nm utilizadas no trabalho, bem como testar seu efeito tópico em modelos de inflamação aguda local in vivo (edema de orelha induzido por TPA e edema de pata induzido por LPS) e verificar sua citotoxicidade em cultura de fibroblastos (3T3) e macrófagos (RAW 264.7) murinos após 12, 24 e 48h pelos ensaios de MTT, VN e teste de exclusão do azul de tripan. Para a caracterização das AuNPs foram realizados os testes de espectroscopia no ultravioleta-visível (Uv-Vis), difratometria de raios-x, absorbância atômica, potencial zeta e microscopia eletrônica de varredura por canhão de emissão de campo elétrico (FEG). Os resultados dos testes de caracterização demonstraram que as nanopartículas apresentavam-se estáveis e realmente com os diâmetros de 10, 20 e 30 nm, como proposto pelo método de síntese, estando adequadas para a utilização nos testes subsequentes. No modelo de edema de orelha induzido por TPA as formulações de AuNPs em creme a 0,1% reduziu a formação do edema em 47,76%, enquanto a formulação em creme a 0,06% reduziu o edema em 33,72% após 6h. Após 24h todas as formulações testadas apresentaram redução da formação do edema, apresentando resultados sem diferença estatística em comparação ao grupo tratado com a dexametasona. No modelo de edema de pata induzido por LPS a formulação de AuNPs em creme a 0,1% foi capaz de reduzir a formação do edema em 84,46% e a formulação em creme a 0,6% reduziu em 86,57%, também sem apresentar diferença estatística em comparação ao grupo tratado com dexametasona. Quanto aos testes de viabilidade celular, os métodos do MTT e VN não foram capazes de obter resultados confiáveis, porém o teste de exclusão do azul de tripan indicou ausência de citotoxicidade das AuNPs em 12h nas culturas de fibroblastos e macrófagos. Após 24h de incubação, a maior concentração (35 mg/L) das dispersões de AuNPs nos 3 diâmetros testados reduziu a viabilidade de macrófagos, e os fibroblastos obtiveram seus valores de viabilidade celular diminuídos pela maioria das dispersões de AuNPs testadas. Após 48h de incubação, todas as concentrações e diâmetros testados das dispersões de AuNPs foram capazes de diminuir drasticamente as porcentagens de viabilidade celular tanto em fibroblastos quanto em macrófagos. / Gold compounds already demonstrated anti-inflammatory properties in previous studies, in addition to important antioxidant potential. The use of gold in nanoparticle form is promising for the treatment of inflammatory conditions such as periodontal disease, especially for its mechanism of action. Although the established gold biocompatibility, it is important to pay attention to the possible toxic effect of gold on nanoparticulate form, for its ease of penetration through the tissues and membranes, mainly in response to its size and gold concentration. The study purpose was the physical characterization of gold nanoparticles (AuNPs) of 10, 20 and 30 nm used in the experiments, in addition to test their topical effect in models of acute inflammation in vivo (ear edema TPA-induced and paw edema LPS-induced) and verify its cytotoxicity in fibroblasts and macrophages mice cell cultures after 12, 24 e 48 hours by MTT and Neutral Red assay and exclusion of trypan blue test. To characterize the AuNPs were performed the ultraviolet-visible spectroscopy, x-ray diffraction, atomic absorbance, zeta potential and Field Emission Gun Scanning Electron Microscopy (FEG). The characterization tests results shown that the nanoparticles are stable and truly presented the diameters of 10, 20 and 30 nm, as proposed by synthesis method, and so are suitable for use in subsequent experiments. In the ear edema TPA-induced model AuNPs cream formulation at 0.1% reduced edema formation at 47.76%, while the cream formulation at 0.06% reduced 33.72% of edema after 6 hours. After 24 hours all formulations tested showed a reduction of edema formation, presenting results with no statistical difference compared to the group treated with dexamethasone. In LPS-induced paw edema model the AuNPs cream formulation at 0.1% was able to reduce edema formation in 84.46% and cream formulation at 0.6% decreased 86.57%, also without showing statistical difference compared to the group treated with dexamethasone. In viability tests, the MTT and Neutral Red methods were not able to obtain reliable results, but exclusion trypan blue test indicated no cytotoxicity of AuNPs after 12h in fibroblasts and macrophages cell cultures. After 24h incubation, the dispersions of AuNPs in 3 diameters at the highest concentration (35 mg/L) reduced the macrophages viability and fibroblast viability decreased with most of the tested AuNPs dispersions. After 48h all tested AuNPs dispersions concentrations and diameters were able to reduce the percentages of fibroblasts and macrophages cell viability.

Several successful pathogens evade host defenses resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory oral bone loss and systemic inflammation manifested as atherosclerosis. The pathogenic mechanisms contributing to P. gingivalis evasion of host immunity and chronic inflammation are not well defined. P. gingivalis evades host immunity at Toll-like receptor (TLR)-4 through expression of an atypical lipopolysaccharide (LPS) that contains lipid A species that exhibit TLR4 agonist or antagonist activity or fail to activate TLR4. By utilizing a series of P. gingivalis lipid A mutants we demonstrated that expression of antagonist lipid A structures resulted in weak induction of proinflammatory mediators. Moreover, expression of antagonist lipid A failed to activate the inflammasome, which correlated with increased bacterial survival in macrophages. Oral infection of atherosclerotic prone apolipoprotein E (ApoE) deficient mice with the antagonist lipid A strain resulted in vascular inflammation characterized by macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain expressing exclusively agonist lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11 dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE deficient mice infected with the agonist lipid A strain exhibited diminished vascular inflammation. Notably, the ability of P. gingivalis to induce local inflammatory oral bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. We next investigated the role of TLRs and lipid A on bacterial trafficking by the autophagic pathway. Originally characterized as a cell autonomous pathway for recycling damaged organelles and proteins, autophagy is now recognized to play a critical role in innate defense and release of the proinflammatory cytokine interleukin (IL)-1β. We demonstrated that P. gingivalis suppresses the autophagic pathway in macrophages for pathogen survival and intercepts autophagy-mediated IL-1β release. P. gingivalis-mediated suppression of autophagy was independent of lipid A expression but partially dependent on TLR2 signaling. Collectively, our results indicate that P. gingivalis evasion of innate immunity plays a role in chronic inflammation.