Absence of stearoyl-CoA desaturase-1 (SCD1) in mice leads to chronic inflammation of the skin and increased susceptibility to atherosclerosis, while also increasing plasma inflammatory markers. A recent report suggested that SCD1 deficiency also increases disease severity in a mouse model of inflammatory bowel disease, induced by dextran sulfate sodium (DSS). However, SCD1-deficient mice are known to consume increased amounts of water, which would also be expected to increase the intake of DSS-treated water. The aim of this study was to determine the effect of SCD1 deficiency on DSS-induced acute colitis with DSS dosing adjusted to account for genotype differences in fluid consumption. Wild-type controls were treated with 3.5% DSS for 5 days to induce moderately severe colitis, while the concentration of DSS given to SCD1-deficient mice was lowered to 2.5% to control for increased fluid consumption. Colonic inflammation was assessed by clinical and histological scoring. Although SCD1-deficient mice consumed a total intake of DSS that was greater than that of wild-type controls, colonic inflammation, colon length and fecal blood were not altered by SCD1-deficiency in DSS-induced colitis, while diarrhea and total weight loss were modestly improved. Despite SCD1 deficiency leading to chronic inflammation of the skin and increased susceptibility to atherosclerosis, it does not accelerate inflammation in the DSS-induced model of acute colitis when DSS intake is controlled. These observations suggest that SCD1 deficiency does not play a significant role in colonic inflammation in this model. [The original version of this article, along with updated information and services is located on the World Wide Web at: http://dx.doi.org/10.1016/j.bbalip.2009.08.001]

Indiana University-Purdue University Indianapolis (IUPUI) / Nanoparticles (NPs) are an emerging technology in biomedical engineering with opportunities in diagnostics, imaging, and drug delivery. NPs can be prepared from a wide range of organic and/or inorganic materials. They can be fabricated to exhibit different characteristics for biomedical applications. The goal of this thesis was to develop NPs with tunable surface properties for selective cell targeting. Specifically, polyelectrolyte complexes composed of heparin (Hep, a growth factor binding glycosaminoglycan) and poly-L-lysine (PLL, a homopolymeric lysine) were prepared via a pulse sonication method. The Hep/PLL core NPs were further layered with additional Hep, tetrazine (Tz) modified Hep, or dextran sulfate (DS). The addition of Tz handle on Hep backbone permitted easy modification of NP surface with norbornene (NB) modified motifs/ligands, including inert poly(ethylene glycol) (PEG), cell adhesive peptides (e.g., RGD), and/or fluorescent marker. Both Hep and DS coated NPs could be readily internalized by J774A.1 monocytes/macrophages, whereas PEGylated NPs effectively reduced cellular uptake/recognition. The versatility of this NP system was further demonstrated by laying DS on the Hep/PLL NP surface. DS-coated NPs were recognized by J774A.1 cells more effectively. Furthermore, DS-layered NPs seemed to reduce IL-10 production on a per cell basis, suggesting that these NPs could be used to alter polarization of macrophages.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

Platelets play a vital role in processes of hemostasis and thrombosis under physiological and pathological conditions. Following vascular damage, platelets will accumulate and stably adhere to exposed subendothelial matrixes. The binding of platelet surface receptor Glycoprotein VI (GPVI) to exposed collagen initiates a signaling cascade, which culminates in platelet activation. Stimulation of GPVI pathways results in the generation of thromboxane and causes the platelets to secrete their granule contents. This generated thromboxane as well as constituents released from dense granules such as ADP, and serotonin, play an essential role in potentiating the platelet response through activation of other surface receptor mediated pathways. Importantly, downstream of all these separate pathways, kinases become activated and play a crucial role in phosphorylating their substrates to elicit critical cellular responses. Previously published studies have established the importance for myosin kinase in its role for phosphorylating the myosin light chain (MLC) downstream of ADP receptors. These studies have shown MLC phosphorylation occurs rapidly and is essential for shape change following the stimulation of ADP receptors. Technological advances in antibody development have resulted in the generation of commercially available phospho-specific antibodies for MLC phosphorylated on either threonine (Thr) 18 or serine (Ser) 19. These antibodies allowed us to revisit these prior studies and address whether phosphorylation on MLC (Ser) 19 would elicit one response while phosphorylation on (Thr) 18 may result in another functional response. Our result show, that MLC is phosphorylated rapidly on (Ser) 19 and plays an important role in shape change downstream of Gq pathways, while MLC (Thr) 18 phosphorylation occurs at a slower rate downstream of G12/13 pathways and contributes to platelet dense granule secretion. Protein kinase C's (PKC) are serine/threonine kinase, which become activated following the stimulation of many of the platelet surface receptors. PKCs are classified into three groups, classical (α, βI, βII, γ), novel (δ, ε, η, θ), and atypical (ζ, ι, λ, μ) based on their cofactor requirements for activation. The classical PKCs, which require diacylglycerol and calcium for their activation were investigated using the specific inhibitor Go6976. Much to our surprise, we demonstrated that downstream of GPVI pathways, Go6976 caused non-selective inhibition of Spleen tyrosine kinase (Syk) activity. This inhibition of Syk activity resulted in a concentration-dependent reduction in phosphorylation of downstream molecules Lat and PLCγ2 as well as platelet aggregation and secretion. Stimulation of surface receptors GPVI, CLEC-2, GPIb, and FcRIIa, all lead to the activation of tyrosine kinase pathways. The role for Syk in these pathways is essential and in the absence of its activity these pathways are completely shut down. We inadvertently discovered dextran sulfate (DxS) actives platelets. Our results show that DxS activates a Src-dependent pathway which does not utilize surface receptors GPVI, CLEC-2, GPIb, or FcRIIa. Platelets pretreated with Syk inhibitors OXSI-2 or Go6976 failed to cause αIIbβ3 activation in response to convulxin, however, platelets activated with DxS under the same conditions retained the ability to activate αIIbβ3. In response to DxS, platelet aggregation, intracellular calcium mobilization, and αIIbβ3 activation were significantly inhibited in platelets pre-treated with PI-3K inhibitors. Taken together these results for the first time establish a novel tyrosine kinase pathway in platelets that cause fibrinogen receptor activation in a PI-3K dependent manner without a role for Syk. In conclusion, we have evaluated the role of myosin light chain kinase, Syk, and PI-3 kinase downstream of platelet receptor-mediated pathways. We have examined the phosphorylation status of several of their effector molecules and have correlated these events with their functional responses in platelets. Here we have highlighted several roles for platelet kinases and their relative importance in regulating platelet functional outcomes. / Physiology

<p>Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity.</p><p>In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation<i> in vivo</i>. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF <i>in vitro</i> efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated <i>in vivo</i> in patients transplanted with isolated islets.</p><p>There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets <i>in vitro</i> and<i> in vivo</i>, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials. </p>

Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity. In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation in vivo. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF in vitro efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated in vivo in patients transplanted with isolated islets. There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets in vitro and in vivo, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials.

Made available in DSpace on 2016-08-17T18:39:47Z (GMT). No. of bitstreams: 1 5353.pdf: 2805182 bytes, checksum: dbd89699431c4bbf40d6959bb375df30 (MD5) Previous issue date: 2013-04-29 / Financiadora de Estudos e Projetos / The increasing interest in the employment of multipotent mesenchymal stromal cells (MSCs) to applications in tissue engineering and cell therapy is mainly attributed to their plasticity and regenerative capacity. The MSCs represent a revolution in the treatment of countless diseases and in the understanding of tissue repair mechanisms. Due to their low availability in the tissues (0.00001% to 0.0002%) as well as to the need of high doses for therapeutic applications (approximately 2 x 106 cells/kg patient), and to the inefficiency of traditional in vitro cultures (in monolayer) to meet the existing high demand to these applications, development of new technologies of ex vivo expansion on a large scale became indispensable. The use of spinner flasks-like bioreactors with microcarriers in suspension is an effective alternative system for this expansion. However, bibliographic report of cultures with this system have indicated the formation of large clusters of microcarriers with cells as a possible obstacle to obtain a greater cellular productivity. These clusters hamper the diffusion of nutrients, gases (especially oxigen), and reagents of indirect cellular quantification within them; and they harm the cell recovery at the end of culture. Alginate microspheres have become interesting to reduce the frequency of collisions between microcarriers, due to the fact that the microspheres do not adhere to cells nor to microcarries and can prevent the union of same. The dextran sulfate has been described in the literature as an inhibitor of cell-to-cell contact as well as of the encounter between microcarriers in culture, therefore, it too has become a promising option to avoid the formation of clusters of microcarriers. Thus, the aim of this work was to develop a methodology for the culture of MSCs in spinner flasks with microcarriers Cultispher-S for application in cell therapy and evaluate the influence of alginate microspheres and dextran sulfate on this culture. For this, the lineage hMSCTERT and 3 g/L Cultispher-S were held in 100 mL spinner containing &#945;-MEM culture medium (supplemented with 15% of bovine fetal serum, glucose and glutamine) at 37ºC, with pH control. The results showed that the addition of dextran sulfate (at 0.5% and 1%) and of 100% of alginate microspheres (in relation to the number of Cultspher-S particles) to the culture caused considerable cell death; and the control (without additives) as well as the addition of 50% of alginate microspheres promoted cell growth. The formation of clusters was observed in cultures; however, it was delayed with the addition of 50% of alginate microspheres. This occurrence allowed a cellular expansion factor of 50.4 times (with dilution factor correction) and of 13.1 times (without correction). The recovered cells after expansion kept their immunophenotypic characteristics. / O crescente interesse na utilização de células mesenquimais estromais multipotentes (CMMs) para aplicações na engenharia de tecidos e na terapia celular é atribuído principalmente à sua plasticidade e capacidade regenerativa. As CMMs representam uma revolução no tratamento de inúmeras doenças e no entendimento dos mecanismos de reparo tecidual. Sua baixa disponibilidade nos tecidos (de 0,00001 à 0,0002%) associada à necessidade de elevadas doses para as aplicações terapêuticas (aproximadamente 2 x 106 células/kg paciente) e à ineficiência dos cultivos tradicionais (em monocamada) in vitro para atender à alta demanda existente para essas aplicações, tornaram indispensável o desenvolvimento de novas tecnologias de expansão ex vivo em larga escala. A utilização de biorreatores tipo frascos spinner com microcarregadores em suspensão é um sistema alternativo eficaz para esta expansão. O relato bibliográfico de cultivos com esse sistema, no entanto, tem indicado a formação de grandes aglomerados de microcarregadores com células como um possível obstáculo na obtenção de maior produtividade celular. Tais aglomerados dificultam a difusão de nutrientes, gases (principalmente o oxigênio) e reagentes de quantificação celular indireta em seu interior; e prejudicam a recuperação das células no final do cultivo. Microesferas de alginato tornaram-se interessantes para diminuir a frequência de colisões entre os microcarregadores, pois não se aderem às células nem aos microcarregadores podendo evitar a união dos mesmos. O sulfato de dextrana foi descrito na literatura tanto como inibidor do contato célula-célula, quanto do encontro entre microcarregadores em cultivo, por isso também passou a ser uma opção promissora para evitar a formação de aglomerados de microcarregadores. Assim, os objetivos deste trabalho foram desenvolver uma metodologia de cultivo de CMMs em frasco spinner com microcarregadores Cultispher-S para aplicação em terapia celular e avaliar a influência de microesferas de alginato e do sulfato de dextrana nesse cultivo. Para isso, a linhagem hMSC-TERT e 3 g/L Cultispher-S foram mantidos em spinner de 100 mL contendo meio de cultura &#945;-MEM (suplementado com 15% de soro fetal bovino, glicose e glutamina), a 37ºC, com controle de pH. Os resultados mostraram que a adição de sulfato de dextrana (à 0,5% e 1%) e de 100% de microesferas de alginato (em relação ao número de partículas de Cultispher-S) ao cultivo provocaram morte celular considerável; e que o controle (sem aditivos), assim como a adição de 50% de microesferas de alginato promoveram crescimento celular. A formação de aglomerados foi observada nos cultivos, no entanto, ela foi atrasada com adição de 50% de microesferas de alginato. Esse fato permitiu a obtenção de um fator de expansão de 50,4 vezes (com a correção do fator de diluição) e de 13,1 vezes (sem a correção). As células recuperadas após a expansão mantiveram suas características imunofenotípicas.

As doenças inflamatórias intestinais (DII) são causadas por desequilíbrio entre as respostas imunes efetoras e reguladoras na mucosa intestinal e podem ser moduladas pelo eixo hipotálamo-hipófise-adrenal (HPA) por meio de interações neuroimunoendócrinas e secreção de cortisol. Embora os glicocorticóides (GC) sejam utilizados para tratar a DII, o cortisol produzido pelas glândulas supra-renais também está envolvido na resposta ao estresse, que pode levar a doenças inflamatórias descontroladas. Portanto, o objetivo deste trabalho é avaliar a participação do eixo HPA na modulação da resposta imune de mucosa intestinal. Para tal, camundongos C57BL/6 foram submetidos à remoção das glândulas adrenais seguida por indução de colite pela administração de água contendo 3% de dextran sulfato de sódio (DSS). Os resultados demonstraram que a ausência das adrenais levou à maior suscetibilidade à doença e mortalidade precoce, fenômeno que não foi prevenido pela reposição de GC. Os animais adrenalectomizados com colite apresentaram níveis significativamente menores de LPS, concomitantemente ao aumento de IL-6 no soro quando comparados aos camundongos não adrenalectomizados. Além disso, os animais adrenalectomizados apresentaram menor celularidade na lâmina própria (LP), menos áreas de erosão e menor escore histopatológico associado ao aumento de IFN-? e FasL, no intestino, sem produção local compensatória de corticosterona. Houve aumento na atividade das enzimas mieloperoxidase (MPO), N- acetilglicosaminidase (NAG) e eosinófilo-peroxidase (EPO) no intestino dos animais expostos ao DSS quando comparados ao grupo de camundongos controles saudáveis, independentemente da presença do eixo HPA intacto e o tratamento com GC nos animais adrenalectomizados levou à redução significativa da atividade de MPO. Também foi observado na LP dos camundongos adrenalectomizados aumento significativo na frequência de células dendríticas tolerogênicas CD11b+CD11c+CD103+, T auxiliares (CD3+CD4+), T citolíticas (CD3+CD8+) e NKT (CD3+CD49b+), além de redução significativa da população de células dendríticas pró-inflamatórias CD11b+CD11c+CD103-, leucócitos CD11b+ e linfócitos intra-epiteliais, de maneira dependente de GC. A ausência do eixo HPA intacto levou à diminuição de leucócitos totais no baço quando comparados ao grupo com colite, relacionada principalmente à redução significativa na frequência de células NKT (CD3+CD49b+), as quais foram restauradas nos camundongos tratados com GC exógenos. Durante a exposição ao DSS houve aumento de células Th2 e Th1 no baço dos camundongos não adrenalectomizados, enquanto que a remoção das adrenais levou a notável redução na população de células T CD4 produtoras de IL-4, IL-10, IFN-? ou IL-17, com aumento de células Th17 e diminuição significativa de células Th1 no baço dos camundongos adrenalectomizados e tratados com GC. De forma interessante, houve menor acúmulo de células T reguladoras juntamente à redução na intensidade média de fluorescência (MFI) de FOXP3 em células T CD4+CD25+ do baço dos camundongos adrenalectomizados expostos ao DSS, de maneira geral dependente de GC. Por fim, esta diminuição de mecanismos reguladores foi acompanhada de menor índice de proliferação e aumento de IL-10 no sobrenadante de cultura de esplenócitos de camundongos com o eixo HPA não ii funcional, indicando que a ausência de GC endógenos pode alterar significativamente a homeostase do sistema imunológico. Juntos, nossos resultados demonstram que o eixo HPA é importante na modulação da resposta imunológica durante a colite induzida experimentalmente / Inflammatory bowel diseases (IBD) are caused by imbalance between regulatory and effector immune responses in the intestinal mucosa and can be modulated by the hypothalamic-pituitary-adrenal (HPA) axis via neuroimmune endocrine interactions and secretion of cortisol. Although glucocorticoids (GC) are used to treat IBD, cortisol produced by the adrenals glands is also involved in the stress response, which can lead to uncontrolled inflammatory diseases. Therefore, the aim of this study was to evaluate the HPA axis in the modulation of the immune response of intestinal mucosa. C57BL/6 mice were subjected to removal of the adrenal glands followed by induction of colitis by administration of water containing 3% dextran sulfate sodium (DSS). The results showed that the absence of adrenals led to increased susceptibility to disease and early mortality, a phenomenon that was not prevented by GC replacement. Adrenalectomized animals exposed to DSS had significantly lower levels of LPS, concomitantly to increased IL-6 in the serum when compared to non-adrenalectomized mice. In addition, adrenalectomized animals had lower cellularity in the lamina propria (LP), less erosion areas and less histopathologic score associated with increased IFN-? and FasL in the intestine, without compensatory local production of corticosterone. There was an increase in the activity of the myeloperoxidase (MPO) enzyme, N- acetilglicosaminidase (NAG) and eosinophil-peroxidase (EPO) in the intestines of DSS-exposed animals when compared to the healthy control group of mice, regardless of the presence of intact HPA axis, while treatment with GC led to significantly reduced MPO activity. It was also observed in the LP of adrenalectomized mice significant increase in the frequency of tolerogenic dendritic cells CD11b+CD11c+CD103+, helper T (CD3+ CD4+), cytolytic T (CD3+ CD8+) and NKT (CD3+ CD49b+) besides significant reduction in the population of pro-inflammatory dendritic cells CD11c+ CD11b+ CD103-, leukocyte CD11b+ and intraepithelial lymphocytes, GC-dependent manner. The absence HPA intact carried decrease in total leukocytes in spleen when compared to the group with colitis, related mainly to significant reduction in the frequency of NKT cells (CD3+CD49b+), which were restored in the GC treated mice. During exposure to DSS there was increased Th2 and Th1 cells in the spleen of non-adrenalectomized mice, while the removal of the adrenals was associated to a marked reduction in the population of CD4 T cells producing IL-4, IL-10, IFN-? or IL-17 with increased Th17 cells and significant decrease in Th1 cells in the spleen of adrenalectomized mice treated with GC. Interestingly there was less accumulation of regulatory T cells together to a reduction in mean fluorescence intensity (MFI) of FOXP3 in CD4+CD25+ T cells in the spleen of mice exposed to DSS after adrenalectomy, most dependent on GC. Finally, the decline of regulatory mechanisms was accompanied by lower rates of proliferation and increased IL-10 in the supernatant culture of splenocytes of mice with disrupted HPA axis, indicating that the absence of endogenous GC altered significantly the homeostasis of the immune system. Together, our results demonstrate that the HPA axis is important in modulating the immune response during experimentally induced colitis

Introdução: Colite Ulcerativa (UC) é uma Doença Inflamatória Intestinal (DII) caracterizada por uma resposta imune exacerbada, com sintomas como diarreia, perda de peso e sangue nas fezes. Apesar dos medicamentos disponíveis, a remissão da doença nem sempre consegue ser alcançada e há a necessidade de terapias alternativas. A colite induzida por DSS (Dextran Sulfate Sodium) é um modelo animal utilizado na investigação de novas terapias por sua semelhança à UC humana. DSS provoca dano à barreira epitelial do cólon, induzindo uma resposta imune exacerbada; entretanto, o exato mecanismo não está totalmente esclarecido. O Ultrassom Terapêutico (TUS) foi utilizado para tratamento de injúria renal em modelo experimental, sua ação se dá através da estimulação do nervo vago (VN) e consequente ativação da via antiinflamatória colinérgica (CAIP). Uma vez que pacientes com DII podem exibir atividade disfuncional do VN, TUS pode ser investigado como terapia alternativa. Objetivos: Investigar temporalmente o perfil clínico, proteômico, histológico e imunológico da colite aguda induzida por DSS; e determinar os efeitos de TUS na colite induzida por DSS. Métodos: No primeiro estudo, a severidade da colite foi avaliada pela administração de DSS 1-3%, observando a resposta clínica e histológica. A análise temporal de DSS 3% incluiu uma avaliação proteômica e histológica do cólon, e a resposta imune celular no baço, linfonodo mesentérico (MLN) e cólon. No segundo estudo, utilizando o modelo de DSS 2%, TUS foi aplicado no abdômen dos animais e foram observados os sintomas clínicos, dano histológico, proteômica do cólon e respostas imunes celulares no baço, MLN e cólon. Animais esplenectomizados ou knockout para a7nAChR (marcador clássico para ativação de CAIP) foram utilizados. Resultados: No primeiro estudo, observou-se que a severidade da doença foi aumentada seguindo concentrações de 1-3% DSS. A análise temporal de DSS 3% demonstrou que os macrófagos (F4/80+) se apresentam como a primeira resposta celular, seguidos por células T CD25+, CD4+ e CD8+. A piora clínica da doença correspondeu ao aumento progressivo de fatores pró-inflamatórios e dano tecidual no cólon, exceto no dia 8. Foram observados menores níveis dos marcadores de células T CD25+, CD4+ e CD8+ no MLN e/ou baço, sugerindo a ocorrência de tropismo destas células para o intestino. No segundo estudo, a aplicação de TUS diminuiu a severidade da doença através da melhora de sintomas clínicos, danos teciduais e encurtamento do cólon. A proteômica do cólon demonstrou uma resposta anti-inflamatória durante a fase de injúria (D0-7), induzindo uma resolução acelerada da doença na fase de recuperação (D8-14). TUS diminuiu os níveis de células T CD8+ e normalizou os níveis de células T CD25+ no cólon. Animais esplenectomizados não demonstraram melhora clínica ou histológica, enquanto animais a7nAChR KO apresentaram piora da colite experimental. Além disso, TUS aumentou os níveis de células F4/80+a7nAChR+ no intestino de animais WT DSS 2%. Conclusão: Nossos resultados demonstram que a severidade da doença depende da concentração de DSS, relacionada com as respostas clínica, proteômica e imune no modelo animal de DSS 3%; e TUS diminuiu a severidade da colite induzida por DSS presumidamente pela da estimulação do VN e consequente ativação de CAIP através do baço. / Introduction: Ulcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) characterized by uncontrolled immune response, presenting with symptoms of diarrhea, weight loss and bloody stools. Despite available treatments, UC sustained remission is not achievable and there is still the need for alternative therapies. Dextran Sulfate Sodium (DSS)-induced colitis is a mouse model used to investigate novel therapies, since it closely mimics human UC. DSS damages the colonic epithelial barrier, leading to an exacerbated immune response. However, the exact mechanism is not totally understood. Previous studies showed the use of Therapeutic Ultrasound (TUS) to prevent kidney injury in mice through stimulation of the vagus nerve (VN) and activation of the cholinergic anti-inflammatory pathway (CAIP). Since IBD patients can present with dysfunctional VN activity, TUS could be studied as an alternative therapy. Objectives: To investigate the temporal clinical, proteomic, histological and cellular immune profiles of DSS-induced acute colitis; and to determine the effects of TUS directed toward the VN and spleen in the course of DSS-induced colitis. Methods: First, we analyzed DSS-induced colitis severity by administration of 1-3% DSS, observing the clinical course and histological damage. A time course analysis was performed at 3% DSS, including colon proteomics, colon histology and immune cell responses in the spleen, MLN (mesenteric lymph node) and colon. Next, utilizing 2% DSS in drinking water, we applied TUS over the mice abdomen and analyzed clinical symptoms, histological damage, colon proteomics and immune cell responses in the spleen, MLN and colon. Splenectomized and a7nAChR (key indicator of CAIP activation) KO animals were also used. Results: In the first study, we observed worsening of the disease when increasing DSS concentrations from 1 to 3%. Time course analysis of 3% DSS revealed macrophages to be the first responders, followed by CD25+, CD4+ and CD8+ T cells. Worsening of the disease corresponded to a progressive increase in pro-inflammatory colonic factors and histological damage, except at day 8. Lower levels of CD25+, CD4+ and CD8+ T cells in MLN and/or spleen suggest an immune cell tropism to the gut. In the second study, TUS attenuated DSS induced colitis through amelioration of clinical symptoms, histological damage and colon shortening. Proteomic colon analysis demonstrated an antiinflammatory profile during the injury phase (D0-7), whilst inducing an early resolution of the disease during the recovery phase (D8-14). TUS decreased CD8+ and normalized CD25+ T cell levels in the gut. Splenectomized animals demonstrated no improved clinical and pathological outcomes, and a7nAChR KO mice presented with worsening of the disease. Furthermore, there were increased levels of F4/80+a7nAChR+ cells in the colon of 2% DSS WT mice under TUS treatment. Conclusion: Our results demonstrate that the severity of colitis is dependent on DSS concentration, correlated with clinical, proteomic and cellular immune responses on 3% DSS; and TUS significantly improved DSS-induced acute colitis presumably through stimulation of the VN and consequent activation of CAIP through the spleen.

La consommation de polyphénols, micronutriments largement répandus dans lesaliments d’origine végétale, a été associée à la diminution du risque de développement denombreuses pathologies telles que maladies cardiovasculaires, maladies neurodégénérativesou cancers. Cet effet des polyphénols s’explique en partie par leurs propriétés antioxydanteset anti-inflammatoires. Du fait de leur faible absorption au niveau de l’intestin grêle, lespolyphénols sont présents en grande quantité dans le côlon où ils peuvent exercer cespropriétés. L’inflammation intestinale fait interagir le système immunitaire intestinal avecde nombreux facteurs environnementaux et est fréquemment associée à une augmentationdu stress oxydant via la production d’espèces réactives de l’oxygène par les cellulesimmunitaires. De nombreuses études ont montré, sur des modèles animaux d’inflammationintestinale, les effets protecteurs de certains polyphénols. La verveine odorante (Aloysiatriphylla (L’Hérit.) Britton) est une plante médicinale connue pour ses vertus thérapeutiquesdigestives et anti-spasmodiques et couramment consommée en infusion. L’infusé deverveine odorante contient de grandes quantités de polyphénols (acides phénoliquescomplexes et dérivés de flavones) et ses propriétés antioxydantes ont été mises en évidenceaussi bien in vitro qu’in vivo.L’objectif de cette thèse a donc été d'évaluer l’effet d’une consommation préventived’un infusé de verveine odorante à dose nutritionnelle (40 g/l et 4 g/l) sur le développementd’une inflammation intestinale modérée chez le rat. Des rats Wistar ont consommé commeboisson l’infusé de verveine seul pendant deux semaines puis associé à un agentinflammatoire, le sulfate de dextran sodique (DSS), à 4% pendant 7 ou 9 jours. L’effet de laverveine a été évalué sur différents paramètres cliniques (diarrhée, saignements rectaux,poids corporel), marqueurs de l’inflammation (longueur du côlon, score histologique,activité myéloperoxydase, cytokines) et du stress oxydant (peroxydation lipidique,glutathion, défenses antioxydantes enzymatiques). Les cellules immunitaires ont étéidentifiées dans le sang ainsi que dans les structures lymphoïdes secondaires par cytométrieen flux. Enfin l’étude du métabolisme des polyphénols en situation inflammatoire ou non aété initiée par l’analyse de l’excrétion urinaire des dérivés polyphénoliques.Lors d’une inflammation de 7 jours, la consommation préventive d’infusé deverveine à 40 g/l et 4 g/l retarde l’apparition de diarrhée et de saignements rectaux, limite larétraction du côlon et la diminution de la prise de poids des rats. Malgré l’absence d’effetsur l’activité myéloperoxydase, l’infusé à 40 g/l atténue les altérations histologiques de lamuqueuse colique induites par l’inflammation. L’infusé à 4 g/l stimule l’activité de lasuperoxyde dismutase et réduit la peroxydation lipidique. Les deux infusés modulent lespopulations de cellules immunitaires dans les structures lymphoïdes secondaires (ganglionsmésentériques et plaques de Peyer), en particulier les lymphocytes B et les lymphocytes Tcytotoxiques. L’excrétion urinaire des polyphénols de la verveine est faible et n'est pasaffectée par l'inflammation. Lors d’une inflammation de 9 jours, les deux infusés limitentl’augmentation d’activité de la myéloperoxydase. Seul l’infusé à 40 g/l limite la rétractiondu côlon, stimule l’activité de la glutathion réductase et diminue les taux d’IL-6 et deTNF-α. Ainsi, nous avons montré qu’une consommation préventive d’un infusé de verveineodorante offre des effets protecteurs lors de l’inflammation intestinale en agissant àdifférents niveaux. L’exploration des voies de signalisation impliquées pourrait permettre demieux comprendre les effets protecteurs de cette boisson de consommation courante. / Polyphenols are micronutrients widely distributed in foods of plant origin and theirconsumption has been associated with a decreased risk of various pathologies such ascardiovascular diseases, neurodegenerative diseases and cancer. This effect of polyphenolsis sustained by their antioxidative and anti-inflammatory properties. Due to their poorabsorption in the small intestine, high amounts of polyphenols reach the colon where theycan exert such properties. Intestinal inflammation results from an interaction between gutimmunity and various environmental factors and is frequently associated with an increase ofoxidative stress. Numerous studies have shown protective effects of polyphenols in animalcolitis models. Lemon verbena (Aloysia triphylla (L’Hérit.) Britton) is a medicinal herbknown for its digestive and antispasmodic properties and is widely consumed as an infusion.Lemon verbena infusion contains large amounts of polyphenols (complex phenolic acidsand flavone glycosides) and their antioxidative properties have been shown in vitro and invivo.The aim of the present thesis was to evaluate the effects of a preventive consumptionof lemon verbena infusion at nutritional doses (40 g/l and 4 g/l) on the development of amoderate colitis in the rat. Wistar rats ingested lemon verbena infusion alone as a drink fortwo weeks and then associated with the inflammatory agent dextran sulfate sodium (DSS) at4% for 7 or 9 days. Effects of lemon verbena were evaluated on several clinical parameters(diarrhoea, rectal bleeding, body weight), inflammatory markers (colon length, histologicalscore, myeloperoxidase activity, cytokines) and oxidative stress markers (lipid peroxidation,glutathione, antioxidative enzymatic defenses). Immune cells were identified in blood andgut associated lymphoid structures using flow cytometry. Moreover, the study of polyphenolmetabolism was initiated by the analysis of urinary polyphenol metabolites in healthy andcolitis rats.During a 7 days inflammation, the preventive consumption of lemon verbenainfusion at 40 g/l and 4 g/l delays apparition of diarrhoea and rectal bleeding, limits thecolon length reduction and the decrease of body weight gain. Despite no effect onmyeloperoxidase activity, the 40 g/l infusion attenuates colonic mucosa alterations due tothe colitis. The 4 g/l infusion increases superoxide dismutase activity and reduces lipidperoxidation. Both infusions modulate immune cell populations in gut associated lymphoidstructures (mesenteric lymph nodes and Peyer patches), especially B cells and cytotoxic Tcells. Urinary excretion of lemon verbena polyphenols is low and not modified byinflammation. During a 9 days inflammation, both lemon verbena infusions limitmyeloperoxidase increase. Only the 40 g/l infusion reduces colon retraction, increasesglutathione reductase activity and reduces colonic IL-6 and TNF-α levels.Thus, we have shown that the preventive consumption of a lemon verbena infusionprovided protection against intestinal inflammation at different levels. Exploration ofvarious signalling pathways could allow better insight into the protective effects of thiscommon beverage.