Graduation date: 1994

Leukocyte transmigration is a critical step of the inflammatory process. In this project I have examined leukocyte responses to transmigration and endothelial responses to both chemical and mechanical stimuli which are known to be involved in leukocyte transmigration. My work has identified ~2500 differentially expressed genes following endothelial exposure to interleukin-1 beta (IL1β). Interestingly, IL1β induces up-regulation of claudin-1 and pre-b-cell colony enhancing factor and down-regulation of claudin-5 and occludin, which are all involved in maintaining endothelial cell-cell junctions. Analysis of endothelial cell (EC) transcriptional changes following neutrophil transmigration found few differentially expressed genes in comparison to IL1β treated ECs; indicating that the effects of transmigration on ECs are minimal in comparison to the global transcriptional changes induced by IL1β. Atherosclerosis, characterized by monocyte accumulation within the vessel lumen, is found in regions of flow reversal and low time averaged oscillatory shear stress. I have examined the effects of this type of shear stress on endothelial cell gene expression. My data indicates that most genes differentially expressed under these conditions are controlled by low average shear stress rather than flow reversal. These differentially expressed genes are involved in regulating the cell cycle and the immune response. My work shows that cell proliferation is increased following exposure to low steady shear stress or exposure to reversing oscillatory flow in comparison to high steady shear stress. Additionally monocyte adhesion is increased following exposure of ECs to reversing oscillatory flow. My work has also examined the impact of transmigration on monocyte gene expression. I have identified genes which are differentially expressed in monocytes by exposure to EC secretions, monocyte/EC contact, and diapedesis. I have also shown that freshly isolated human monocytes have reduced apoptosis following transmigration. Surprisingly, I also found that monocytes had reduced expression of anti-microbial peptides following transmigration. Overall my work identifies important endothelial and leukocyte transcriptional responses to the process of transmigration which extends from cytokine stimulation through diapedesis.

Thesis (Ph.D.)--Ohio University, November, 2005. / Title from PDF t.p. Includes bibliographical references (p. 165-167)

Made available in DSpace on 2014-06-11T19:30:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-02-02Bitstream added on 2014-06-13T21:01:16Z : No. of bitstreams: 1 arauco_lrr_dr_jabo.pdf: 644500 bytes, checksum: e57a53b05c75c737674e5912bc1fb10e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Foi avaliado o efeito do extrato hidroalcoólico de própolis (0,0; 0,2; 0,5; 1,0; e 1,5 %) misturado em ração comercial (45 % Proteína Bruta), em girinos de rã-touro. Foram utilizados 1400 girinos no estágio 26 da tabela de Gosner (1960), distribuídos em 20 tanques experimentais com 70 litros de água, na densidade de um girino por litro. O arraçoamento foi realizado quatro vezes ao dia. Para análise dos dados do ganho de peso, comprimento, sobrevivência, conversão alimentar, consumo de ração e metamorfose, foi utilizado um delineamento inteiramente casualizado com cinco tratamentos e quatro repetições. No final do experimento, foi colhido sangue do vaso caudal, de cinco girinos de cada repetição. A contagem diferencial de leucócitos foi realizada em extensões coradas pelo método de Rosenfeld (1947), em microscopia de luz. Foram contadas 100 células por lâmina. Para avaliar o efeito do extrato hidroalcoólico de própolis na porcentagem de leucócitos, usou-se um delineamento inteiramente casualizado com cinco tratamentos e três repetições. Para análise histológica no final do experimento foram sacrificados três girinos de cada repetição e retirada amostras do rim, fígado e intestino para lâminas histológicas. As amostras foram fixadas em formol, desidratadas em uma série de álcool, coradas com HE, analisadas e fotomicrografadas com fotomicroscópio Axiophot- Zeiss em microscópio óptico e medido com micrometro ocular a espessura do epitélio intestinal. Para análise estatística dos dados foi usado um delineamento inteiramente casualizado com cinco tratamentos e três repetições. A sobrevivência, consumo de ração, conversão alimentar e comprimento dos girinos, não foram influenciados pelo extrato hidroalcoólico de própolis. O ganho de peso foi influenciado significativamente (P < 0,05) observando-se... / The effect of the propolis hidroalcoolic extract was evaluated (0.0; 0.2; 0.5; 1.0; and 1.5 %) mixed to the commercial ration (45 % CP) in bullfrog tadpoles. 1,400 tadpoles were used at stage 26 (Gosner ,1960), distributed in twenty experimental tanks with 70 liters of water, in the density of one tadpole per liter. The feding was four times a day. For analysis of the data of the weight gain, length, survival, feed conversion, ration consumption and metamorphosis were used a completely randomized design with five treatments and four repetitions. In the end of the experiment, it was picked the blood of the vase flow, of five tadpoles of each repetition. The differential counting of leuccytes was accomplished in red-faced extensions by the method of Rosenfeld (1947), in light microscopia. To evaluate the effect of the propolis hidroalcoolic extract in the percentage of leucocytes, were used a completely randomized design with five treatments and three repetitions. They were counted 100 cells for sheet. For histological analysis in the end of the experiment three tadpoles of each repetition were sacrificed and samples of the kidney, liver and intestine were removed for you laminate histological. The samples were fastened in formol, dehydrated in one serializes of alcohol, red-faced with HE analyzed and fotomicrographed with fotomicroscópio Axioskop-Zeiss in optical and measured microscope with ocular micrometer the espessor of the intestinal epithelium. For statistical analysis of the data were used a completely randomized design with five treatments and three repetitions. The survival, ration consumption, feed conversion and length of the tadpoles, were not influenced by the propolis hidroalcoolic extract. The weight gain was influenced significantly (P< 0,05) being observed a worse in the tadpoles... (Complete abstract click electronic access below)

Submitted by LUANA CHIQUETTO PARACATÚ null (luanachiquetto@hotmail.com) on 2016-07-14T18:15:23Z No. of bitstreams: 1 TESE - Luana Chiquetto.pdf: 2823701 bytes, checksum: 584996ba0cd6c6754d03400de9c6de73 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-18T20:01:01Z (GMT) No. of bitstreams: 1 paracatu_lc_dr_arafcf.pdf: 2823701 bytes, checksum: 584996ba0cd6c6754d03400de9c6de73 (MD5) / Made available in DSpace on 2016-07-18T20:01:01Z (GMT). No. of bitstreams: 1 paracatu_lc_dr_arafcf.pdf: 2823701 bytes, checksum: 584996ba0cd6c6754d03400de9c6de73 (MD5) Previous issue date: 2016-06-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Inúmeras patologias têm a sua gênese e/ou progressão relacionadas à produção desregulada de intermediários oxidantes. O complexo multienzimático NADPH oxidase é um dos componentes de maior relevância neste contexto, pois é uma das principais fontes de ânion superóxido no organismo animal. Sendo expresso em inúmeros tecidos, incluindo leucócitos e células do tecido endotelial, o desenvolvimento de inibidores eficientes deste complexo enzimático poderá significar uma nova terapêutica para o tratamento de doenças inflamatórias crônicas. Com objetivo de explorar a relação entre a estrutura molecular, as propriedades químicas e atividades biológicas, utilizamos o éster fenetílico do ácido cafeico (CAPE) como inibidor do complexo enzimático NADPH oxidase e comparamos sua eficácia com o seu precursor ácido cafeico e os derivados, éster fenetílico do ácido cinâmico e o ácido clorogênico, correlacionando-os a respeito a sua hidrofobicidade, propriedades redox e inibição do complexo NADPH oxidase em leucócitos ativados. A hipótese seria de que um aumento da hidrofobicidade provocado pela esterificação do ácido cafeico poderia facilitar o seu acesso à membrana celular e assim alterar seu efeito como possível inibidor de NADPH Oxidase. Os resultados, em ensaios in vitro, mostraram que as alterações na hidrofobicidade não provocaram alterações significativas no potencial de oxidação e potencial antioxidantes dos compostos testados. Quando testados em leucócitos ativados (modelos ex vivo), a esterificação provocou uma melhora significativa na capacidade de inibição do complexo NADPH oxidase. Este potente efeito se propagou às EROs decorrentes de ânion superóxido e produzidas por leucócitos, como peróxido de hidrogênio e ácido hipocloroso, entretanto, sem alterar a capacidade fagocítica dos leucócitos. Os resultados deste estudo mostram que nos ensaios celulares o CAPE foi o composto mais potente em relação ao seu precursor ácido e ácido clorogênico, sendo significativamente mais efetivo na inibição da produção das EROs. Da mesma forma, CAPE foi o inibidor mais eficaz da expressão de TNF-α e IL-10 por Staphylococcus aureus células estimuladas. Em conclusão, a presença do grupo catecol e a maior hidrofobicidade, do CAPE, foram essenciais para os efeitos biológicos, confirmando nossa hipótese. Considerando-se o envolvimento da NADPH-oxidases na génese e progressão de doenças inflamatórias, CAPE deve ser considerada como uma droga anti-inflamatória promissora. / Several diseases have their genesis and / or progression related to unregulated production of oxidants intermediates. The multienzymatic complex NADPH oxidase is one of the most important components in this context because it is a major source of superoxide anion in animal organisms. It is expressed in numerous tissues, including leukocytes and endothelial tissue cells. Developing effective inhibitors of this enzyme complex may indicate a new therapy for the treatment of chronic inflammatory diseases. Several studies have described numerous anti-inflammatory properties attributed to caffeic acid phenethyl ester (CAPE), an active component found in propolis. In order to explore the relationship between the molecular structure, chemical properties and biological activities, we used CAPE to inhibit the enzyme complex NADPH oxidase and compare its efficacy with the its precursor caffeic acid and derivatives, phenethyl ester of cinnamic acid and chlorogenic acid, correlating them with regard to hydrophobicity, redox properties and inhibition of NADPH oxidase complex on activated leukocytes. The hypothesis was that an increase of hydrophobicity caused by the esterification of caffeic acid could facilitate access to cell membranes and thereby alter its effect as a possible inhibitor of NADPH oxidase. The results (in vitro), showed that the changes in hydrophobicity did not provoke significant changes in the oxidation potential and antiradical potency of the tested compounds. But when tested in activated leukocytes (ex vivo), the esterification caused a significant improvement in the ability to inhibit the NADPH oxidase complex. This potent inhibition effect resulted also in the blockage of production of hypochlorous acid, however, without altering the phagocytic ability of leukocytes. The results of this study show that in cellular assays, CAPE was the most potent compound in comparison to caffeic acid and chlorogenic acid, significantly more effective in inhibiting the production of ROS. Likewise, CAPE was the most effective inhibitor of expression of TNF-α and IL-10 in Staphylococcus aureus stimulated cells. In conclusion, the presence of the catechol moiety and the higher hydrophobicity of CAPE were essential for the biological effects. Considering the involvement of NADPH oxidases in the genesis and progression of inflammatory diseases, CAPE should be considered as a promising anti-inflammatory drug.

Orientadores: Fernando Ferreira Costa, Nicola Conran Zorzetto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T06:25:23Z (GMT). No. of bitstreams: 1 Canalli_AndreiaAverci_D.pdf: 1313778 bytes, checksum: 1832f98c252279d3722a62951dcfc9c0 (MD5) Previous issue date: 2008 / Resumo: A hemoglobina S (HbS) é decorrente de uma mutação de ponto que ocasiona a troca do ácido glutâmico pela valina na cadeia polipeptídica da globina beta. A polimerização da HbS desoxigenada (pouco solúvel), torna as hemácias menos flexíveis e favorece a impactação das mesmas na microcirculação com subseqüente obstrução da luz do endotélio e vaso-oclusão, seguida de lesões a orgãos alvos, responsáveis pela morbi-mortalidade associada a essa doença. Numerosas evidências sugerem que outros fatores participem da fisiopatologia da vaso-oclusão e do processo inflamatório crônico característico da anema falciforme (AF). A redução da biodisponibilidade do óxido nítrico (NO) pode ser um fator que favoreça a vaso-oclusão, uma vez que ele é um gás vasodilatador e pode inibir a adesão leucocitária. Nesse contexto, os objetivos do presente trabalho foram avaliar as propriedades adesivas dos neutrófilos de pacientes com AF e investigar o papel da via de sinalização do NO e dos nucleotídeos cíclicos (AMPc e GMPc) nos processos de vaso oclusão na AF. Os neutrófilos de pacientes com AF apresentaram maior adesão in vitro para ambos ligantes, fibronectina (FN) e molécula de adesão intercelular (ICAM-1), quando comparados à adesão de neutrófilos de controles. A co-incubação dos neutrófilos de pacientes com AF com agentes doadores de NO, nitroprussiato de sódio (SNP), dietilamina NANOato (DEANO) e com estimulador da guanilato ciclase solúvel (GCs), BAY 412272, diminuíram significativamente o aumento da adesão dos neutrófilos à FN e ao ICAM-1. O oxidiazolo [4,3-a]quinoxalin-1-one, um inibidor da GCs, reverteu a inibição da adesão provocada pelo SNP e DEANO à FN, indicando que a sinalização ependente de GMPc participa desse mecanismo. Os níveis intracelulares de GMPc foram significativamente maiores nos neutrófilos de pacientes que fizeram uso de terapias com hidroxiurea (HU). Em concordância com esses resultados, a adesão dos neutrófilos à FN e ao ICAM-1 foi significativamente menor em pacientes com AF em uso de HU se comparada com os neutrófilos de pacientes com AF sem terapia com HU. Adicionalmente, a co-incubação dos neutrófilos de pacientes com AF com KT5720 (um inibidor de PKA) reduziu a adesão basal enquanto que a co-incubação das células com KT5823 (inibidor de PKG) não alterou a adesão dos neutrófilos à FN. O estímulo dos neutrófilos de pacientes com AF com interleucina 8 (IL-8) aumentou significativamente a adesão à FN e esse aumento na adesão foi reduzido pela KT5720. Esses dados indicam que a regulação da sinalização da via dependente de AMPc tem um importante papel na alteração das propriedades adesivas nos pacientes com AF. Tal alteração pode ter implicação na fisiopatologia da AF e tanto a via PKA dependente de AMPc pode representar um alvo terapêutico para reduzir a alteração na função leucocitária, como agentes que estimulam a via de NO dependente de GMPc podem ter efeitos benéficos na função leucocitária desses pacientes / Abstract: Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease (SCD). Since nitric oxide (NO) bioavailability is decreased in SCD and NO may inhibit leukocyte adhesion, we determined whether stimulation of NO-signalling pathways can decrease the adhesive properties of neutrophils from SCD individuals (SCDneu). SCDneu presented greater adhesion, in vitro, to both fibronectin (FN) and ICAM-1 than control neutrophils; co-incubation of SCDneu with the NO-donor agents, sodium nitroprusside (SNP) and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly diminished increased adhesion to FN/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed SNP/DEANO-diminished adhesion to FN, implicating cGMP-dependent signalling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from SCD individuals on hydroxyurea (SCDHUneu) and, accordingly, SCDHUneu adhesion to FN/ICAM-1 was significantly lower than that of SCDneu. In addition, co-incubation of SCDneu with KT5720 (an inhibitor of PKA), but not KT5823 (inhibitor of PKG), abrogated increased basal SCDneu adhesion. Stimulation of SCDneu with IL-8 also significantly augmented SCDneu adhesion to FN and this increase in adhesion was abolished by KT5720. Data indicate that up-regulated cAMP signalling plays a significant role in altered adhesive properties in SCDneu. Such alterations may have significant implications for the pathophysiology of the disease and the cAMP-PKA pathway may represent a therapeutic target for the abrogation of altered leukocyte function. Agents that stimulate the NO/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these individuals / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

La maladie de Fabry est une maladie de surcharge lysosomale liée au chromosome X. Elle est causée par une mutation au niveau du gène GLA qui provoque un déficit de l’enzyme α-galactosidase A. Elle entraîne une accumulation de glycosphingolipides tels le globotriaosylcéramide (Gb3), le globotriaosylsphingosine (lyso-Gb3) et galabiosylcéramide (Ga2) ainsi que leurs isoformes/analogues respectifs au niveau des tissus et des liquides biologiques des patients. Les symptômes peuvent se présenter de manière très variable soit par de l’acroparesthésie, des troubles ophtalmologiques, des angiokératomes, des troubles cardiaques, rénaux ou encore neurologiques. Les connaissances au niveau physiopathologique de la maladie de Fabry sont à ce jour, toujours déficientes. Les biomarqueurs analysés ne permettent pas de prédire la sévérité et la progression de la maladie. Afin d’apporter des connaissances plus approfondies à ce niveau, l’objectif principal de cette étude était d’analyser les biomarqueurs au niveau cellulaire pour des patients atteints de la maladie. Les objectifs secondaires étaient: 1) de développer et de valider une méthode en spectrométrie de masse en tandem pour la quantification relative et simultanée de certains biomarqueurs susmentionnés dans les leucocytes, les lymphocytes B et les monocytes de patients Fabry et contrôles sains; 2) d’évaluer les biomarqueurs dans le total des leucocytes, les lymphocytes B, les monocytes, le plasma et l’urine chez les patients Fabry et les contrôles sains appariés selon le sexe et l’âge; et 3) d’établir des corrélations entre la quantité relative de ces biomarqueurs et le génotype des patients traités et non traités. Les résultats de cette étude démontrent qu’il n’y a pas de changements significatifs dans la distribution des groupes d’isoformes/analogues du Gb3 selon le type cellulaire pour les patients et les contrôles. La découverte d’isoformes/analogues méthylés du Gb3 suggère un processus de méthylation qui se produit directement au niveau des cellules sanguines et particulièrement pour les lymphocytes B et les monocytes. Des corrélations face à la quantité de biomarqueurs et le génotype ont été établies. Ces résultats pourraient permettre une meilleure compréhension des processus biochimiques reliés à l’accumulation du Gb3 dans les cellules sanguines.

Incorporation of N-acetyl neuraminic acid (NANA), or sialic acid, onto lipooligosaccharide (LOS) enhances the virulence of several bacterial species. In the present study, we assessed the effect of sialylation of Histophilus somni LOS on complement-mediated killing, binding of complement factor H (which converts C3b to inactive C3b (iC3b) and inhibit the alternative complement pathway) to the bacteria, complement activation by the LOS, and phagocytosis and killing of the bacteria by bovine polymorphonuclear leukocytes (PMN). Killing of H. somni by alternative complement pathway was measured by incubation of sialylated or non-sialylated H. somni with antibody-free precolostral calf serum (PCS) followed by viable plate count. A complement dose-dependent response to killing of non-sialylated H. somni by PCS was observed. However, sialylated H. somni were significantly (P = 0.001) more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated (P = 0.025) and consumed (P = 0.001) less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized sheep red blood cells or rabbit red blood cells, and by western blotting of C3 activation products. Sialylated H. somni bound more factor H than non-sialylated bacteria (determined by enzyme-linked immunosorbent assay) (P = 0.004), supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni inhibited both PMN phagocytosis of 3H-thymidine-labelled bacteria (P = 0.004) and intracellular killing of the bacteria (P = 0.0001), compared to non-sialylated bacteria. Therefore, sialylation of the LOS results in enhanced binding of complement factor H to the bacteria, resulting in diminished complement activation, resistance to complement-mediated lysis, and PMN phagocytosis and killing. / Master of Science

Yes / Sunburn is a commonly occurring acute inflammatory process, with dermal vasodilatation and leukocyte infiltration as central features. Ultraviolet (UV) B-induced hydrolysis of membrane phospholipids releases polyunsaturated fatty acids and their subsequent metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) may produce potent eicosanoid mediators modulating different stages of the inflammation. Our objective was to identify candidate eicosanoids formed during the sunburn reaction in relation to its clinical and histological course. We exposed skin of healthy humans (n=32) to UVB and for 72h examined (i) expression of pro- and anti-inflammatory eicosanoids using LC/ESI-MS/MS and (ii) immunohistochemical expression of COX-2, 12-LOX, 15-LOX and leucocyte markers, while (iii) quantifying clinical erythema. We show that vasodilatory prostaglandins (PG)E2, PGF2¿ and PGE3 accompany the erythema in the first 24-48h, associated with increased COX-2 expression at 24h. Novel, potent leukocyte chemoattractants 11-, 12- and 8-monohydroxy-eicosatetraenoic acid (-HETE) are elevated from 4-72h, in association with peak dermal neutrophil influx at 24h, and increased dermal CD3+ lymphocytes and 12- and 15-LOX expression from 24-72h. Anti-inflammatory metabolite 15-HETE shows later expression, peaking at 72h. Sunburn is characterized by overlapping phases of increases in COX products followed by LOX products that may regulate subsequent events and ultimately its resolution. / The Wellcome Trust

L'impact de la nutrition sur l'immunité intestinale de la truite arc-en-ciel est encore mal connu. C'est pourquoi cette thèse avait pour objectifs de mieux caractériser son système immunitaire intestinal et d'évaluer les possibilités de modulation de la réponse immune intestinale par l'ajout de nucléotides libres dans son alimentation. Nos résultats indiquent que les phagocytes intestinaux présentent une activité de phagocytose plus faible que ceux du rein antérieur. La cytotoxicité naturelle mesurée au niveau intestinal est deux fois plus élevée que celle du rein antérieur et cette observation est corrélée à une augmentation du transcrit codant NKEF (Natural Killer Enhancement Factor). Nous avons également montré que les lymphocytes intestinaux ne répondent pas à une stimulation mitogénique in vitro et que ceci n'est pas due à l'apoptose des cellules. Une forte expression des transcrits codant CD8a et CD3 a été détectée dans les leucocytes intestinaux, ce qui suggère une importante proportion de lymphocytes T exprimant la forme homodimérique aa de CD8 dans ce tissu. Enfin, nous avons montré que l'ajout de nucléotides libres à l'alimentation de truites saines stimule la prolifération spontanée ainsi que la phagocytose des leucocytes intestinaux in vitro. Par contre, aucune modulation de la cytotoxicité naturelle ou de l'expression des transcrits codant les marqueurs spécifiques des lymphocytes T et B et les cytokines inflammatoires n'a été observée. Il serait à présent intéressant de renouveler ces essais en utilisant des poissons infectés afin de pouvoir observer l'effet des nucléotides sur la réponse inflammatoire et sur la réponse spécifique / The impact of nutrition on rainbow trout intestinal immunity, a farmed fish with high economic value, remains unclear. Consequently, the objectives of this thesis were to better characterize the intestinal immune system of that fish and to determine if it is possible to modulate its intestinal immune response by dietary free nucleotides. Our results show that intestinal phagocytes are less activated by yeast cells but when they are activated they can ingest as many yeast cells as their head kidney (HK) counterparts. We noted that the natural cytotoxic activity of intestinal leukocytes is twice higher than the one of HK leukocytes. This natural cytotoxic activity is correlated with an increase of transcripts encoding the natural killer enhancement factor (NKEF). Intestinal leukocytes did not respond to an in vitro mitogenic stimulation. This lack of response is not due to apoptosis. We also observed a high expression of CD8a and CD3 transcripts in gut leukocytes, suggesting that the intestine could contain a high proportion of T cells expressing the aa homodimeric form of CD8. Finally, we observed that dietary free nucleotides stimulate the spontaneous proliferation and the phagocytic activity of intestinal leucocytes in vitro. However, they did not modulate natural cytotoxicity activity nor did they affect the amounts of transcripts encoding specific markers of T and B lymphocytes and inflammatory cytokines. In the future, it will be interesting to repeat these experiments using infected fish in order to study the effect of nucleotides on the inflammatory and specific immune responses