"A thesis submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy". / Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007. / Bibliography: p. 243-277. / Introduction -- A convenient synthesis of 30HKG -- Facile synthesis of the UV filter compounds 30HKyn and AHBG -- Synthesis, identification and quantification of novel human lens metabolites -- Modification of bovine lens protein with UV filters and related metabolites -- Effect of UV light on UV filter-treated lens proteins -- Conclusions and future directions. / The kynurenine-based UV filters are unstable under physiological conditions and undergo side chain deamination, resulting in α,β-unsaturated carbonyl compounds. These compounds can react with free or protein bound nucleophiles in the lens via Michael addition. The key sites of the UV filters kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) modification in human lenses include cysteine (Cys), and to a lesser extent, lysine (Lys) and histidine (His) residues. Recent in vivo studies have revealed that 3-hydroxykynurenine-O-β-D-glucoside (3OHKG) binds to Cys residues of lens crystallins in older normal human lenses. As a result of this binding, human lens proteins become progressively modified by UV filters in an age-dependent manner, contributing to changes that occur with the development of age-related nuclear (ARN) cataract. Upon exposure to UV light, free UV filters are poor photosensitisers, however the role of protein-bound species is less clear. It has been recently demonstrated that Kyn, when bound to lens proteins, becomes more susceptible to photo-oxidation by UV light. Therefore, the investigation of 3OHKG binding to lens proteins, and the effect of UV light on proteins modified with 3OHKG and 3OHKyn, were major aims of this study. As a result of the role of these compounds as UV filters and their possible involvement in ARN cataract formation, it is crucial to understand the nature, concentration and modes of action of the UV filters and their metabolites present in the human lenses. Therefore, an additional aim was to investigate human lenses for the presence of novel kynurenine-based human lens metabolites and examine their reactivity.--As 3OHKG is not commercially available, to conduct protein binding studies, an initial aim of this study was to synthesise 3OHKG (Chapter 2). Through the expansion and optimisation of a literature procedure, 3OHKG was successfully synthesised using commercially available and inexpensive reagents, and applying green chemistry principles, where toxic and corrosive reagents were replaced with benign reagents and solvent-free and microwave chemistry was used. A detailed investigation of different reaction conditions was also conducted, resulting in either the improvement of reaction yields or reaction time compared to the literature method. Applying the same synthetic strategy, and using key precursors from the synthesis of 3OHKG, the UV filters 3OHKyn and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid-O-β-D-glucoside (AHBG), were also successfully synthesised (Chapter 3). / Chapter 4 describes the investigation of both normal and cataractous human lenses in an attempt to identify novel human lens metabolites derived from deaminated Kyn and 3OHKyn (Chapter 4, Part A). Initially, 4-(2-aminophenyl)-4-oxobutanoic acid (AHA), glutathionyl-kynurenine (GSH-Kyn), kynurenine yellow (Kyn yellow), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid (AHB), glutathionyl-3-hydroxykynurenine (GSH-3OHKyn) and 3-hydroxykynurenine yellow (3OHKyn yellow) were synthesised and human lenses were examined for their presence. AHA and AHB were synthesised from similar precursors to those used in the synthesis of 3OHKG, while the GSH adducts and yellow compounds were synthesised from Kyn and 3OHKyn via base induced deamination. Following isolation and structural elucidation, AHA, AHB and GSH-Kyn were confirmed as novel human lens metabolites. They were quantified in low pmol/mg lens (dry mass) levels in normal and cataractous lenses of all ages, while GSH-3OHKyn, Kyn yellow and 3OHKyn yellow were not detected. In contrast to AHA, the lens metabolites AHB, GSH-Kyn and GSH-3OHKyn were found to be unstable at physiological pH. The spectral properties of these compounds suggest that they may act as UV filters. --Chapter 4 (Part B) also describes the identification and characterisation of a novel human lens UV filter, cysteinyl-3-hydroxykynurenine -O-β-D-glucoside (Cys-3OHKG). An authentic standard was synthesised via Michael addition of cysteine to deaminated 3OHKG. Cys-3OHKG was detected in low pmol/mg lens (dry mass) levels in normal lenses only after the 5th decade of life and was absent in cataractous lenses. Cys-3OHKG showed rapid decomposition at physiological pH. / Chapter 5 describes the identification and quantification of amino acids involved in covalent binding of 3OHKG to lens proteins. Model studies with bovine lens proteins and 3OHKG at pH 7.2 and 9.5 were undertaken. The amino acid adducts were identified via total synthesis and spectral analysis, and subsequently quantified upon acid hydrolysis of the modified lens proteins. Under both pH conditions, 3OHKG was found to react with lens proteins predominantly via Cys residues with low levels of binding also detected at Lys residues. Comparative studies with Kyn (pH 9.5) and 3OHKyn (pH 7.2 and 9.5) resulted in modified lens proteins at Cys residues, with only minor modification at Lys residues at pH 9.5. The extent of modification was found to be significantly higher at pH 9.5 in all cases. His adducts were not identified. 3OHKG-, Kyn- and 3OHKyn-modified lens proteins were found to be coloured and fluorescent, resembling those of aged and ARN cataractous lenses. In contrast, AHB and AHA, which can not form α,β-unsaturated carbonyl compounds, resulted in non-covalent modification of lens proteins. AHB may contribute to lens colouration and fluorescence as further reactions of this material yielded species that have similar characteristics to those identified from 3OHKyn modification. These species are postulated to arise via auto-oxidation of the o-aminophenol moiety present in both 3OHKyn and AHB.--In Chapter 6, the potential roles of 3OHKG and 3OHKyn, and the related species AHA and AHB, in generating reactive oxygen species and protein damage following illumination with UV light was examined. The UV filter compounds were examined in both their free and protein-bound forms. Kyn-modified proteins were used as a positive control. Exposure of these compounds to UV light (λ 305-385 nm) has been shown to generate H2O2 and protein-bound peroxides in a time-dependent manner, with shorter wavelengths generating more peroxides. The yields of peroxides were observed to be highly dependent on the nature of the UV filter compound and whether these species were free or protein bound, with much higher levels being detected with the bound species. Thus, protein-bound 3OHKyn yielded higher levels of peroxide than 3OHKG, with these levels, in turn, higher than for the free UV filter compounds. AHB-treated lens proteins resulted in formation of low but statistically significant levels of peroxides, while AHA-treated lens proteins resulted in insignificant peroxide formation. The consequences of these photochemical reactions have been examined by quantifying protein-bound tyrosine oxidation products (3,4-dihydroxyphenylalanine [DOPA], di-tyrosine [di-Tyr]) and protein cross-linking. 3OHKG-modified proteins gave elevated levels of di-Tyr, but not DOPA, whereas 3OHKyn-modified protein gave the inverse. DOPA formation was observed to be independent of illumination and most likely arose via o-aminophenol auto-oxidation. AHB- and AHA-treated lens proteins resulted in statistically insignificant di-Tyr formation, while a light independent increase in DOPA was observed for both samples. Both reducible (disulfide) and non-reducible cross-links were detected in modified proteins following illumination. These linkages were present at lower levels in modified, but non-illuminated proteins, and absent from unmodified protein samples. / This work has provided an optimised synthetic procedure for 3OHKG and other lens metabolites (Chapters 2 and 3). Four novel lens metabolites have been identified and quantified in normal and cataractous human lenses (Chapter 4). Subsequent experiments, described in Chapter 5, identified the major covalent binding sites of 3OHKG to lens proteins, while AHA and AHB showed non-covalent binding. Further work described in Chapter 6 showed that protein-bound 3OHKG, Kyn and 3OHKyn were better photosensitisers of oxidative damage than in their unbound state. Together, this research has provided strong evidence that post-translational modifications of lens proteins by kynurenine-based metabolites and their interaction with UV light appear, at least in part, responsible for the age-dependent colouration of human lenses and an elevated level of oxidative stress in older lenses. These processes may contribute to the progression of ARN cataract. / Mode of access: World Wide Web. / xxxix, 308 p. ill. (some col.)

A enzima indolamina 2,3-dioxigenase (IDO) é responsável pela degradação de triptofano (TRP) pela via das quinureninas. O aumento da expressão de IDO é a grande responsável pela resposta de tolerância imunológica e pode ser induzida por IFN-γ e lipopolissacarídeos (LPS). É conhecido que a IDO participa do mecanismo de imuno escape de células tumorais, sendo relatada como marcadora de progressão tumoral. Desta forma, a inibição da atividade da IDO para a restauração da imunidade anti-tumoral do hospedeiro tem sido considerada uma estratégia para a terapêutica antineoplásica. N,N-dimetiltriptamina (DMT) e triptamina (TRY) são compostos indólicos de origem endógena, provenientes de uma rota paralela de metabolismo do triptofano - a rota das triptaminas, que ocorre principalmente no sistema nervoso central. O presente estudo teve como objetivo avaliar se DMT e TRY modulariam a atividade de IDO. Os resultados obtidos demonstraram ações inibitórias para ambos os compostos e as cinéticas enzimáticas revelaram Ki de 506µM para o DMT e de 156µM para a TRY, com perfis inibitórios característicos de inibidores não-competitivos clássicos. A atividade inibitória foi também observada sobre a enzima IDO expressa constitutivamente, ou induzida por IFN-γ, em células da linhagem de glioblastoma humano A172. Nesta mesma linhagem, em estudo paralelo do grupo, foi avaliada a influência de DMT e TRY sobre a expressão gênica da enzima IDO. Concluímos que nas células a ação inibitória dos compostos avaliados é exercida diretamente sobre sua atividade enzimática, sem redução de sua transcrição. Os resultados deste estudo também serviram como base para estudos da atividade inibitória da DMT e TRY em sistema de co-cultura das células A172 com células mononucleares humanas, onde foi observada redução significativa da atividade enzimática e da proliferação das células tumorais. Em conclusão mostramos a possibilidade de que a via das triptaminas, através de seus metabólitos, possa contribuir com a regulação endógena da via das quinureninas e que o DMT e a TRY deveriam ser avaliados como candidatos a inibidores farmacológicos da enzima IDO e/ou como protótipos para a síntese de novos inibidores. / Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme via the kynurenine pathway. Increased IDO expression is largely responsible for the immune tolerance response and can be induced by IFN-γ and lipopolysaccharide (LPS). It is known that IDO participates in the mechanism of tumor immune tolerance and have been reported as a tumor progression marker. Thus, to restore the anti-tumor immunity of the host, IDO inhibition has been considered as a strategy for anticancer therapy. N, N-dimethyltryptamine (DMT) and tryptamine (TRY) are endogenous indolic compounds originated from a parallel route of tryptophan metabolism - the tryptamines route, which occurs mainly in the central nervous system. This study aimed to assess whether DMT and TRY modulate the IDO activity. The results showed inhibitory actions for both compounds and enzyme kinetics revealed Ki = 506µM to DMT and Ki = 156µM to TRY, with inhibitory profiles characteristic of classical non-competitive inhibitors. The inhibitory activity was also observed on the IDO constitutively expressed or induced by IFN-γ in A172 human glioblastom cell line. Using the same line, in parallel group study, we evaluated the influence of DMT and TRY on the IDO gene expression. We conclude that in cells the evaluated compounds inhibitory action is exerted directly on its enzymatic activity without reduction of its transcription. The results of this study also served as the basis for studies of DMT and TRY inhibitory activity in A172 cells with human mononuclear cells co-culture system, in which was observed significant reduction in enzyme activity and tumor cells proliferation. In conclusion we show the possibility that the tryptamines route, through its metabolites, may contribute to quinurenines pathway endogenous regulation and that DMT and TRY should be evaluated as IDO pharmacological inhibitors candidates and / or as prototypes to new inhibitors synthesis.

Triptofano (TRP), um amino ácido essencial, é metabolizado por duas vias principais, a via das quinureninas e a via serotonérgica. Em ambas as vias há a possibilidade de formação de compostos ativos no sistema imune que se caracterizam pelas ações imunossupressoras e indutoras de tolerância. Na via serotonérgica há a formação de serotonina (5-HT) e em alguns tecidos de melatonina (MEL). Este composto pode ainda ser oxidado por ação de peroxidases aos seus produtos de abertura de anel indólico o AFMK (N1-acetil-n2-formil-5-metoxiquinuramina) e AMK (N1-acetil-5-metoxiquinuramina). Já na via das quinureninas, o TRP é diretamente metabolizado à N-formilquinurenina (NFK) e este é rapidamente deformilado a quinurenina (QUIN). Neste projeto avaliamos qual o efeito do choque endotóxico induzido por injeção endovenosa de LPS (1 mg/kg) sobre a biodisponibilidade de TRP e formação de seu metabólito QUIN. Este estudo foi realizado em condições controle e na vigência de sobrecarga de TRP (administração subcutânea de 0,8 mg/kg). Utilizamos ratos machos Wistar com 30 dias separados em quatro grupos: GI (controle), GII (LPS), GIII (TRP) e GIV (TRP+LPS). TRP (0,8 mg/Kg) foi injetado por via subcutânea nos tempos 0 e 2 horas. Quando injetado, LPS (1 mg/kg) foi administrado por via intravenosa no tempo 2 horas. Após 1 hora da última administração, sangue e cérebro foram coletados. O cérebro foi seccionado em três regiões: cerebelo, córtex e mesencéfalo, os quais foram processados para obtenção de homogenatos. Tanto os homogenatos quanto o soro foram tratados com acetona para extração de TRP e seus metabólitos. A análise destes compostos foi realizada por cromatografia líquida de alta eficiência (HPLC). A administração de TRP elevou significativamente a sua concentração no soro e no SNC. Quando da administração de LPS no grupo que já havia recebido sobrecarga de TRP (GIV) houve uma marcada elevação de TRP e de QUIN séricos e das regiões do SNC, especialmente na região do córtex. Concluímos que na vigência de choque endotóxico há um aumento da biodisponibilidade de TRP, tanto no soro como no SNC e que há um aumento da metabolização deste pela rota das quinureninas, possivelmente via IDO. Estes resultados contribuem para a compreensão da toxicidade de TRP, especialmente relevante no caso em que haja um choque endotóxico concomitante e evidencia o córtex como uma região mais susceptível para os efeitos tóxicos do TRP. / Tryptophan (TRP) is an essential amino acid, metabolized by two main paths; the kynurenine and the serotonergic pathways. In both, there is the possibility of generation of biologic active compounds, especially on the immune system leading to immunosuppression and tolerance. In the serotonergic path there is the formation of serotonine (5-HT) and in some tissues of melatonine (MEL). The latter can be oxidized by the action of peroxidases to its indole ring opening product AFMK (N1-acetil-n2-formil-5-methoxikynuramine) and AMK (N1-acethyl-5-methoxykynuramine). In the kynurenine path, TRP is metabolized to N-formylkynurenine (NFK) that is deformilated to kynurenine (KYN). In this study we evaluated the effect of a endotoxic skock induced by an intravenous injection of LPS (1 mg/kg) on the bioavailability of TRP and formation of KYN. This study was carried out in control conditions and on TRP overload (subcutaneous administration of 0,8 mg/Kg). One month old male Wistar rats were divide in four groups: GI(control), GII(LPS), GIII(TRP) and GIV (TRP+LPS). TRP (0,8 mg/kg) was subcutaneously injected at zero and 2h times. When injected, LPS (1mg/kg) was intravenously administered at 2 h. After one hour from the last administration, blood and brain were collected. Brain is separated in cerebellum, midbrain and cortex and was lysed for the preparation of homogenates. Both, serum and homogenates were extracted in acetone; TRP and KYN were analyzed by HPLC. TRP overload caused a significant increase in its concentration in serum and brain. When LPS was administered in conjunction with TRP overload (GIV) there was a remarkable increase in TRP and KYN in serum and brain, especially in cortex. Our conclusion is that in the bioavailability of TRP, in serum and in brain, and its metabolization to kynurenine is increased by inflammation. IDO is probably involved in this condition. Our results contribute to the knowledge of TRP toxicity, particularly with a concomitant inflammation and demonstrate the cortex as a region of more susceptibility to TRP toxicity.

P. aeruginosa est un pathogène opportuniste capable d'infecter un large spectre d'hôtes. Elle possède un vaste arsenal de facteurs de virulence. Le système de sécrétion de type III (SSTT) est un facteur de virulence majeur dont la régulation est complexe pour permettre une adaptation la plus précise possible de la bactérie au cours de l'infection. Nous nous sommes intéressés à déterminer le rôle potentiel de nouveaux acteurs de l'adaptation de P.aeruginosa au cours de l'infection. La porine OprF qui représente la protéine la plus abondante de la membrane externe de P. aeruginosa lui permettrait d'évaluer l'état d'activation du système immunitaire de son hôte afin d'adapter sa virulence. Chez P. aeruginosa, le tryptophane est le précurseur des kynurenines qui sont également produites par l'hôte à partir du tryptophane et qui, dans ce dernier contexte, sont des immunomodulateurs. Peu ou pas d'études ont été réalisées pour mettre en œuvre un éventuel rôle d'immunomodulation ou dans la virulence des kynurénines bactériennes. Dans un premier temps, nous nous sommes intéressés à un signal anciennement découvert au laboratoire et qui réprime l'expression du SSTT à haute densité bactérienne. Nous avons montré que ce signal exerce une régulation post-transcriptionnelle en plus d'une inhibition de la transcription des gènes du SSTT. Le métabolisme du tryptophane et de l'anthranilate semble être au cœur de ce processus de régulation. En inactivant des voies du catabolisme du tryptophane, nous avons montré que la production de ce signal dépend partiellement de la voie des kynurénines mais ne dépend pas ni des voies classiques du quorum sensing ni de l'opéron phnAB, impliqué dans la synthèse de l'anthranilate. Cependant, la voie des phénazines pourrait être impliquée dans la production de ce signal. Par CLHP couplée à la spectrométrie de masse, nous avons pu séparer des espèces moléculaires réprimant le SSTT et qui sont contenues dans ce signal, mais l'identification précise nécessite plus d'investigations. Dans un second temps, nous nous sommes intéressés aux kynurénines produites par la bactérie. Nous avons confirmé que P. aeruginosa produit des kynurénines et le gène kynA est le gène clé de la voie de synthèse de ces métabolites. En utilisant des fusions transcriptionnnelles, nous avons montré que le tryptophane et la kynurénine régulent positivement la production des kynurénines en agissant sur l'expression des gènes clés. D'autres parts, nous avons remarqué que la bactérie module l'activité de la voie métabolique des kynurénines issue du tryptophane en fonction de son état de croissance. Nous avons montré qu'au cours du dialogue interrègne bactérie/hôte, la voie des kynurénines de P. aeruginosa est stimulée par certains composants du système immunitaire. Grâce à un modèle d'infection pulmonaire aiguë, nous avons prouvé que les kynurénines produites par la bactérie sont importantes pour sa virulence. Selon notre hypothèse les kynurénines pourraient avoir une action sur la réponse immune, mais cela reste à déterminer. Dans un troisième temps, nous nous somme focalisés sur la porine OprF. Nous avons montré que la mutation ∆oprF est à l'origine d'une altération de la production mais vraisemblablement pas de la sécrétion des exotoxines du SSTT. Un ligand connu d'OprF, l'interféron gamma, module la voie des kynurénines. OprF pourrait donc avoir un rôle central dans les différents aspects de la régulation de la virulence. Nous avons donc produit des anticorps monoclonaux anti-OprF. Ces derniers se sont révélés capables de reconnaître spécifiquement la protéine OprF. Afin de vérifier l'efficacité de ces anticorps, des expériences de neutralisation de la bactérie in vitro puis in vivo seront réalisées. Mots clés : Pseudomonas aeruginosa, Système de Sécrétion de Type III, régulation, catabolisme du tryptophane, kynurénines, OprF. / P. aeruginosa is an opportunistic pathogen capable of infecting a wide host range. It possesses a large arsenal of virulence factors. The type III secretion system (TTSS) is a major virulence factor whose regulation is complex to allow the most accurate adaptation of the bacteria during infection. We were interested to determine the potential role of new actors in the adaptation of P. aeruginosa during infection. OprF represents the most abundant protein of the outer membrane of P. aeruginosa. This protein allows bacteria to assess the activation status of the host's immune system to adapt its virulence. In P. aeruginosa, tryptophan is the precursor of kynurenines that are also produced by the host from tryptophan and in the latter context, are immunomodulators. Little or no studies have been done to determine a possible role of bacterial kynurenines in immune modulation or virulence. Initially, we were interested in a signal previously discovered in the laboratory and which suppresses the expression of TTSS at high bacterial density. We have shown that this signal exerts a post-transcriptional regulation in addition to inhibition of TTSS genes transcription. The metabolism of tryptophan and anthranilate appears to be at the heart of this regulatory process. By inactivating pathways of tryptophan catabolism, we showed that production of this signal depends partly on the kynurenines pathway but does not depend neither classical ways of quorum sensing or phnAB operon involved in the synthesis of anthranilate. However, the phenazines pathway could be involved in the production of this signal. By HPLC coupled with mass spectrometry, we were able to separate molecular species suppressing the TTSS and which are contained in this signal, but accurate identification requires further investigation. In a second time, we were interested to kynurenines produced by the bacterium. We confirmed that P. aeruginosa produces kynurenines and KynA is the key gene in the synthesis of these metabolites. We showed that tryptophan and kynurenine upregulate the production of kynurenines by acting on the expression of key genes. Other shares, we found that the bacterium modulates the activity of the kynurenines pathway depending on its state of growth. We showed that during the dialogue bacteria / host, the pathway of kynurenines in P. aeruginosa is stimulated by certain immune system components. With an acute lung infection model, we proved that kynurenines produced by the bacterium are important to its virulence. We hypothesized that the kynurenines could have an effect on the immune response, but this remains to be determined. In a third time, we focused on the protein OprF. We showed that mutation ΔoprF is causing an alteration in production but probably not the secretion of TTSS exotoxins. One known ligand of OprF is the gamma interferon. It modulates the pathway of kynurenines. OprF could therefore have a central role in various aspects of the regulation of virulence. So, we produced monoclonal anti-OprF which recognizes specifically the protein OprF. To verify the effectiveness of these antibodies, neutralization experiments of the bacteria in vitro and in vivo will be realized.

Made available in DSpace on 2014-12-17T15:18:09Z (GMT). No. of bitstreams: 1 LeonamGC.pdf: 218692 bytes, checksum: 002840894937241c14e673ee875d7524 (MD5) Previous issue date: 2008-03-18 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Activation of the kynurenine (KYN) pathway (KP) by modulators of immune system has been observed during several neurological diseases. Here we assessed the association of chemo-/cytokine levels with the concentration of KP metabolites in cerebrospinal fluid (CSF) and plasma samples from patients with bacterial meningitis (BM). All samples were collected from 42 patients diagnosed with acute bacterial meningitis (ABM), aseptic meningitis, tuberculous meningitis and patients without infection neurological disorders. CSF and plasma concentration of metabolites from the KP was assessed by high pressure liquid chromatography (HPLC) and cytokines and chemokines by Bio-plex 200 suspension array system. Concentrations of the KP metabolites KYN and kynurenic acid (KYNA) were significantly higher in CSF of patients with ABM compared to other groups. Tryptophan (TRP), anthranilic acid (AA), 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) did not show statistical significance, although some of them presented a good accumulation during ABM. The expression of TNF-alpha, IL-6, IL-1beta, IFN-gamma, IL-10, IL-1 receptor antagonist (IL-1Ra), MIP-1alpha, MIP-1beta, MCP-1 and G-CSF was about 100-fold higher in CSF from ABM patients than other infected groups. In all CSF and plasma samples, the concentration of IL-2, IL-12(p70), IL-4, IL-8 and GM-CSF was not significant. ABM still showed significant concentrations of IL-6, IL-10, IL-1Ra and MCP-1 in plasma samples. Based on the comparison of KP metabolites concentrations between plasma and CSF samples we conclude that the activation of the tryptophan pathway upon BM occurs within the brain. This increase in KP metabolites is most due to activation of the KP by molecules as IFN-gamma and TNF-alpha in response to infection. / A ativa??o da rota da quinurenina por moduladores do sistema imune tem sido observada durante diversas doen?as neurol?gicas. Neste trabalho foi avaliado a associa??o entre os n?veis de citocinas e quimiocinas com a concentra??o dos metab?litos da rota da quinurenina ( Kynurenine pathway KP) em amostras de l?quor e plasma de pacientes com meningite bacteriana. Todas as amostras foram coletadas de 42 pacientes hospitalizados com o diagn?stico de meningites bacteriana aguda, tubercul?sica, ass?ptica e pacientes cujo diagn?stico excluiu infec??o no sistema nervoso central. As concentra??es dos metab?litos da quinurenina foram avaliadas pela cromatografia l?quida de alta press?o enquanto os n?veis de citocinas e quimiocinas foram medidos pelo Bio-plex 200 suspension array system. Concentra??es de quinurenina e ?cido quinur?nico foram significativamente mais elevadas no l?quor de pacientes com meningite bacteriana aguda do que nos outros grupos. Ainda no l?quor, os n?veis de triptofano, ?cido antran?lico, 3-hidroxiquinurenina e ?cido 3-hidroxiantran?lico n?o mostraram signific?ncia estat?stica, embora alguns deles apresentaram um bom ac?mulo durante meningite bacteriana aguda. No l?quor dos pacientes com meningite bacteriana foram observados n?veis mais elevados de TNF-alfa, IL-6, IL-1beta, IFN-gama, IL-10, IL-1Ra, MIP-1alpha, MIP-1beta, MCP-1 e G-CSF do que os outros grupos infectados. Em todas as amostras, as concentra??es de IL-2, IL-12(p70), IL-4, IL-8 e GM-CSF n?o variaram significativamente. No grupo com meningite bacteriana aguda foram tamb?m observadas elevadas concentra??es de IL-6, IL-10, IL-1Ra e MCP-1 no plasma. Baseado na compara??o das concentra??es dos metab?litos da via quinurenina no l?quor e no plasma, conclui-se que a ativa??o desta via metab?lica durante a meningite ocorre primordialmente dentro do c?rebro. O aumento na concentra??o dos metab?litos da quinurenina durante a infec??o deve-se ativa??o da enzima idoleamina 2,3 dioxigenase por prote?nas da resposta imune hospedeira principalmente pelo IFN-gama, IL1beta e TNF-alfa.

Triptofano (TRP), um amino ácido essencial, é metabolizado por duas vias principais, a via das quinureninas e a via serotonérgica. Em ambas as vias há a possibilidade de formação de compostos ativos no sistema imune que se caracterizam pelas ações imunossupressoras e indutoras de tolerância. Na via serotonérgica há a formação de serotonina (5-HT) e em alguns tecidos de melatonina (MEL). Este composto pode ainda ser oxidado por ação de peroxidases aos seus produtos de abertura de anel indólico o AFMK (N1-acetil-n2-formil-5-metoxiquinuramina) e AMK (N1-acetil-5-metoxiquinuramina). Já na via das quinureninas, o TRP é diretamente metabolizado à N-formilquinurenina (NFK) e este é rapidamente deformilado a quinurenina (QUIN). Neste projeto avaliamos qual o efeito do choque endotóxico induzido por injeção endovenosa de LPS (1 mg/kg) sobre a biodisponibilidade de TRP e formação de seu metabólito QUIN. Este estudo foi realizado em condições controle e na vigência de sobrecarga de TRP (administração subcutânea de 0,8 mg/kg). Utilizamos ratos machos Wistar com 30 dias separados em quatro grupos: GI (controle), GII (LPS), GIII (TRP) e GIV (TRP+LPS). TRP (0,8 mg/Kg) foi injetado por via subcutânea nos tempos 0 e 2 horas. Quando injetado, LPS (1 mg/kg) foi administrado por via intravenosa no tempo 2 horas. Após 1 hora da última administração, sangue e cérebro foram coletados. O cérebro foi seccionado em três regiões: cerebelo, córtex e mesencéfalo, os quais foram processados para obtenção de homogenatos. Tanto os homogenatos quanto o soro foram tratados com acetona para extração de TRP e seus metabólitos. A análise destes compostos foi realizada por cromatografia líquida de alta eficiência (HPLC). A administração de TRP elevou significativamente a sua concentração no soro e no SNC. Quando da administração de LPS no grupo que já havia recebido sobrecarga de TRP (GIV) houve uma marcada elevação de TRP e de QUIN séricos e das regiões do SNC, especialmente na região do córtex. Concluímos que na vigência de choque endotóxico há um aumento da biodisponibilidade de TRP, tanto no soro como no SNC e que há um aumento da metabolização deste pela rota das quinureninas, possivelmente via IDO. Estes resultados contribuem para a compreensão da toxicidade de TRP, especialmente relevante no caso em que haja um choque endotóxico concomitante e evidencia o córtex como uma região mais susceptível para os efeitos tóxicos do TRP. / Tryptophan (TRP) is an essential amino acid, metabolized by two main paths; the kynurenine and the serotonergic pathways. In both, there is the possibility of generation of biologic active compounds, especially on the immune system leading to immunosuppression and tolerance. In the serotonergic path there is the formation of serotonine (5-HT) and in some tissues of melatonine (MEL). The latter can be oxidized by the action of peroxidases to its indole ring opening product AFMK (N1-acetil-n2-formil-5-methoxikynuramine) and AMK (N1-acethyl-5-methoxykynuramine). In the kynurenine path, TRP is metabolized to N-formylkynurenine (NFK) that is deformilated to kynurenine (KYN). In this study we evaluated the effect of a endotoxic skock induced by an intravenous injection of LPS (1 mg/kg) on the bioavailability of TRP and formation of KYN. This study was carried out in control conditions and on TRP overload (subcutaneous administration of 0,8 mg/Kg). One month old male Wistar rats were divide in four groups: GI(control), GII(LPS), GIII(TRP) and GIV (TRP+LPS). TRP (0,8 mg/kg) was subcutaneously injected at zero and 2h times. When injected, LPS (1mg/kg) was intravenously administered at 2 h. After one hour from the last administration, blood and brain were collected. Brain is separated in cerebellum, midbrain and cortex and was lysed for the preparation of homogenates. Both, serum and homogenates were extracted in acetone; TRP and KYN were analyzed by HPLC. TRP overload caused a significant increase in its concentration in serum and brain. When LPS was administered in conjunction with TRP overload (GIV) there was a remarkable increase in TRP and KYN in serum and brain, especially in cortex. Our conclusion is that in the bioavailability of TRP, in serum and in brain, and its metabolization to kynurenine is increased by inflammation. IDO is probably involved in this condition. Our results contribute to the knowledge of TRP toxicity, particularly with a concomitant inflammation and demonstrate the cortex as a region of more susceptibility to TRP toxicity.

Tryptophan is metabolised along the kynurenine pathway under the influence of tryptophan 2,3 dioxygenase and indoleamine 2,3 dioxygenase. Quinolinic acid and kynurenine, two neuroactive metabolites of the kynurenine pathway are, in chronic renal failure patients, considered as uraemic toxins. Related research is generally hampered by the non-availability of relevant analytical techniques. The primary aim of this study was, therefore, to develop and validate suitable methods for the determination of tryptophan, kynurenine and quinolinic acid. The second aim was to quantify the levels of these substances in the blood of chronic renal failure patients on renal replacement therapies and to compare the levels of haemodialysis patients to those on peritoneal dialysis. Patients’ quality of life was investigated relative to disturbances in tryptophan metabolism. Gas chromatography coupled to mass spectrometry (GC-MS) gave the best results for the analysis of tryptophan, kynurenine and quinolinic acid. A Hewlett Packard HP GC 6890 series gas chromatographer was coupled to a MS 5973 series mass spectrometer. Analytes were separated on a DB-5MS column with a nominal length of 30 metres, a diameter of 250.0 µm and film thickness of 0.10 µm. Helium was used as carrier gas, and the chromatographic analysis run time 12.5 minutes. The validation results were within the acceptance criteria for newly developed methods. The linear calibration curves constructed for all of the analytes gave r2 correlation coefficients >0.99. Other validation data such as precision, bias, accuracy and stability all fell within acceptable validation limits. In the study on chronic renal failure patients significant differences were seen between patients and controls. Tryptophan levels were 5.34 SD 5.04 µM for the haemodialysis group, 6.73 SD 3.18 µM for the peritoneal dialysis group and 28.4 SD 4.31 µM for the control group. Kynurenine levels were 4.7 SD 1.9 µM for the haemodialysis group, 2.9 SD 2.0 µM for the peritoneal dialysis group and 2.1 SD 0.6 µM for the control group. Quinolinic acid levels were 4.9 SD 2.0 µM for the haemodialysis group, 2.8 SD 2.0 µM for the peritoneal dialysis group and 0.3 SD 0.1 µM for the control group. Tryptophan was lower for the total patient group than for controls with significantly lower levels for haemodialysis versus control (p<0.05) and peritoneal dialysis versus control (p<0.05). Kynurenine levels were higher in the total patient group with significantly higher levels for the haemodialysis versus control group (p=0.0001). The patient groups had higher quinolinic acid levels with significantly higher levels for the haemodialysis versus control (p<0.05) and peritoneal dialysis versus control (p<0.05) groups. This study was the first to determine the three substances simultaneously in both haemodialysis and peritoneal dialysis patients. The study showed significant tryptophan depletion, as well as kynurenine and quinolinic acid accumulation for both groups. No significant differences were found between the patient groups other than higher kynurenine levels in the haemodialysis group. The quality of life (SF-36) was largely similar in the two patient groups. This decrease in the quality of life strongly correlated with the degree of tryptophan depletion. / Dissertation (MSc)--University of Pretoria, 2008. / Chemical Pathology / unrestricted

Amino acid availability plays an important role in modulating the activity of T-cells. One of the pathways employed by T-cells to sense nutrient levels is the “mammalian target of rapamycin” (mTOR) pathway that is inhibited in response to nutrient depletion. Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme along the tryptophan catabolising kynurenine pathway. T-cells are very sensitive to lack of this essential amino acid in their microenvironment and this confers strong immunomodulatory properties to cells expressing active IDO. It therefore has a significant physiological role as a homeostatic mechanism used in mammalian organisms to dampen excessive activation of the immune system but is also used as an immune evasion mechanism by many cancers. In this study, we investigated the IDO inhibitory properties and mechanism of action of the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) that potentially forms a negative feedback loop in the kynurenine pathway. We studied the molecule in enzymatic assays, in live cells and discovered that it inhibits IDO in an indirect way via the formation of hydrogen peroxide. Secondly, we looked at the effects of tryptophan and its metabolites on T-cell proliferation and mTOR activity, and discovered a metabolite that inhibits T-cell proliferation. Lastly we examined mechanisms of T-cell suppression employed by myeloid derived suppressor cells (MDSCs), focusing on their ability to deplete amino acids from their microenvironment. We were able to exclude tryptophan consumption as a suppressive mechanism and established that by manipulating extracellular concentrations of several amino acids other than arginine and cysteine – that are known to be utilised by MDSCs - we were able to reduce their inhibitory properties. In summary, we have described in detail how 3-HAA inhibits IDO in in vitro assays, outlined how some tryptophan metabolites can inhibit T-cell proliferation, and clarified aspects of suppressive mechanism employed by MDSCs.

A artrite reumatoide (AR) é a artropatia inflamatória mais prevalente no mundo, de etiologia multifatorial e fenótipos heterogêneos. Busca-se, além de definir fatores etiológicos, compreender as interações entre mecanismos envolvidos na fisiopatologia da AR. Entre estes, fatores genéticos, tanto genes do antígeno leucocitário humano (HLA), especialmente a presença do epítopo compartilhado (Shared epitope - SE) do HLA-DRB1, como genes não-HLA, e fatores ambientais e epigenéticos têm sido associados à doença. Assim, a identificação de novos fatores relacionados à etiopatogenia da AR e suas possíveis associações com características clínicas motivaram esse estudo. Um estudo caso-controle foi desenhado e dividido em duas etapas. Para a primeira etapa, foi obtido plasma de 18 indivíduos de AR e 18 voluntários saudáveis de Ribeirão Preto, no qual foram identificados quinurenina (Kyn), Trp, serotonina (5-HT) e taxa Kyn/Trp (KTR) por cromatografia líquida de ultra-eficiência (CLUE) acoplada a espectrômetro de massas sequencial (CLUE-DAD-EM/EM). Na segunda etapa, de estudo genético, uma coorte formada por 328 indivíduos com AR e por 234 voluntários saudáveis de Ribeirão Preto e de Porto Alegre foi avaliada quanto ao polimorfismo do gene da enzima indoleamine 2,3-dioxigenase 1 (IDO1). Foram obtidos dados clínicos e epidemiológicos e coletadas amostras de sangue periférico para extração de DNA pelo método de salting-out. Em seguida, tipificação HLA e reação em cadeia de polimerase (RCP) das variantes da IDO1, rs7820268, rs3739319, rs61753677, rs35059413, rs35099072 e rs9298586, foram realizadas. A positividade para fator reumatoide (FR) em indivíduos com AR foi associada ao tabagismo (p= 0.0002) e ao SE (p < 0.0001), e para anticorpo antipeptídeo citrulinado cíclico (anti-CCP), associado ao SE (p < 0.0001). Quando combinadas a presença de SE e a de tabagismo, houve associação estatisticamente significante para FR (p < 0.001) e para anti-CCP (p = 0.03). Foram observadas menores concentrações plasmáticas de 5-HT em indivíduos com AR quando comparados a voluntários saudáveis (p =0.006), mas sem diferença para níveis de Trp, Kyn e KTR. Para estes, diferenças apareceram quando avaliados subgrupos. Em indivíduos com AR sem tratamento com drogas modificadoras do curso da doença (DMCDs), os valores plasmáticos de Trp foram menores quando comparados aos em terapia (p = 0.0016), enquanto em pacientes com AR tabagistas os valores de Kyn e KTR foram menores que em pacientes não tabagistas (p = 0.039 e p = 0.032, respectivamente). Não foram identificadas associações estatisticamente significantes entre as variantes genéticas estudadas e o risco de desenvolver AR, nem entre os polimorfismos da IDO1 estudados e a concentração plasmática de Trp, Kyn e 5-HT e KTR. Este estudo não identificou relação das variantes do gene da IDO1 com suscetibilidade para AR. Assim, novos estudos são necessários para que possam ser explicadas as associações encontradas na via das Kyns e na 5-HT em etiopatogenia da AR. / Rheumatoid arthritis (RA) is the most prevalent inflammatory arthropathy in the world, with multifactorial etiology and heterogeneous phenotypes. Besides defining etiological factors, it is sought to understand the interactions between mechanisms in RA pathophysiology. About these, genetic factors, both human leucocity antigen (HLA) genes, especially the HLA-DRB1 Shared epitope (SE) presence, and not-HLA genes, and environmental and epigenetics factors have been associated with the disease. Therefore, the aim of this study was to identify new possible associations between RA clinical features and its etiopathogenesis. A case-control study was designed and it was divides in two phases. The first phase, it was obtained plasma of 18 RA patients and 18 healthy controls from Ribeirão Preto to identify the kynurenine (Kyn), Trp and serotonin (5-HT) concentrations and Kyn/Trp ratio (KTR) by ultra-high performance liquid chromatography coupled to sequential mass spectrometer. The second phase was a genetic study that evaluated a cohort of 328 RA patients and 234 healthy volunteers from Ribeirão Preto and Porto Alegre about the indoleamine 2,3-dioxygenase 1 (IDO1) gene polymorphism. Clinical and epidemiological data were obtained and peripheral blood samples were collected to DNA extraction by salting-out method. Then, HLA typification and polymerase chain reaction to identify IDO1 genetic variants rs7820268, rs3739319, rs61753677, rs35059413, rs35099072 and rs9298586 were performed. Rheumatoid factor (RF) positivity was associated to smoking (p = 0.0002) and SE (p < 0.0001), and cyclic citrullinated peptide autoantibodies (anti-CCP) positivity was associated to SE (p < 0.0001). When SE presence and smoking were combined, there was statistically significant association to RF (p < 0.001) and anti-CCP (p = 0.03). We observed lower plasma 5-HT concentrations in RA patients than in healthy volunteers (p = 0.006), but no significant difference to Trp, Kyn and KTR levels. For these, differences were observed when subgroups were evaluated. In RA patients not using disease modifying antirheumatic drugs (DMARDs) the plasma Trp levels were lower than RApatients using DMARDs, while the plasma Kyn concentrations and KTR in smokers RA patients were lower than nonsmokers RA patients (p = 0.039 and p = 0.032 respectively). We did not indetify statistically significant associations neither between studied genetic variants and risk to develop RA nor between IDO1 polymorphisms and plasma Trp, Kyn, 5HT concentrations and KTR. This study did not identify relation between IDO1 genetic variants with susceptibility to RA. Therefore, new studies are necessary to explain the searched associations between Kyns pathway and 5-HT in RA etiopathogenenesis.

A artrite reumatoide (AR) é a artropatia inflamatória mais prevalente no mundo, de etiologia multifatorial e fenótipos heterogêneos. Busca-se, além de definir fatores etiológicos, compreender as interações entre mecanismos envolvidos na fisiopatologia da AR. Entre estes, fatores genéticos, tanto genes do antígeno leucocitário humano (HLA), especialmente a presença do epítopo compartilhado (Shared epitope - SE) do HLA-DRB1, como genes não-HLA, e fatores ambientais e epigenéticos têm sido associados à doença. Assim, a identificação de novos fatores relacionados à etiopatogenia da AR e suas possíveis associações com características clínicas motivaram esse estudo. Um estudo caso-controle foi desenhado e dividido em duas etapas. Para a primeira etapa, foi obtido plasma de 18 indivíduos de AR e 18 voluntários saudáveis de Ribeirão Preto, no qual foram identificados quinurenina (Kyn), Trp, serotonina (5-HT) e taxa Kyn/Trp (KTR) por cromatografia líquida de ultra-eficiência (CLUE) acoplada a espectrômetro de massas sequencial (CLUE-DAD-EM/EM). Na segunda etapa, de estudo genético, uma coorte formada por 328 indivíduos com AR e por 234 voluntários saudáveis de Ribeirão Preto e de Porto Alegre foi avaliada quanto ao polimorfismo do gene da enzima indoleamine 2,3-dioxigenase 1 (IDO1). Foram obtidos dados clínicos e epidemiológicos e coletadas amostras de sangue periférico para extração de DNA pelo método de salting-out. Em seguida, tipificação HLA e reação em cadeia de polimerase (RCP) das variantes da IDO1, rs7820268, rs3739319, rs61753677, rs35059413, rs35099072 e rs9298586, foram realizadas. A positividade para fator reumatoide (FR) em indivíduos com AR foi associada ao tabagismo (p= 0.0002) e ao SE (p < 0.0001), e para anticorpo antipeptídeo citrulinado cíclico (anti-CCP), associado ao SE (p < 0.0001). Quando combinadas a presença de SE e a de tabagismo, houve associação estatisticamente significante para FR (p < 0.001) e para anti-CCP (p = 0.03). Foram observadas menores concentrações plasmáticas de 5-HT em indivíduos com AR quando comparados a voluntários saudáveis (p =0.006), mas sem diferença para níveis de Trp, Kyn e KTR. Para estes, diferenças apareceram quando avaliados subgrupos. Em indivíduos com AR sem tratamento com drogas modificadoras do curso da doença (DMCDs), os valores plasmáticos de Trp foram menores quando comparados aos em terapia (p = 0.0016), enquanto em pacientes com AR tabagistas os valores de Kyn e KTR foram menores que em pacientes não tabagistas (p = 0.039 e p = 0.032, respectivamente). Não foram identificadas associações estatisticamente significantes entre as variantes genéticas estudadas e o risco de desenvolver AR, nem entre os polimorfismos da IDO1 estudados e a concentração plasmática de Trp, Kyn e 5-HT e KTR. Este estudo não identificou relação das variantes do gene da IDO1 com suscetibilidade para AR. Assim, novos estudos são necessários para que possam ser explicadas as associações encontradas na via das Kyns e na 5-HT em etiopatogenia da AR. / Rheumatoid arthritis (RA) is the most prevalent inflammatory arthropathy in the world, with multifactorial etiology and heterogeneous phenotypes. Besides defining etiological factors, it is sought to understand the interactions between mechanisms in RA pathophysiology. About these, genetic factors, both human leucocity antigen (HLA) genes, especially the HLA-DRB1 Shared epitope (SE) presence, and not-HLA genes, and environmental and epigenetics factors have been associated with the disease. Therefore, the aim of this study was to identify new possible associations between RA clinical features and its etiopathogenesis. A case-control study was designed and it was divides in two phases. The first phase, it was obtained plasma of 18 RA patients and 18 healthy controls from Ribeirão Preto to identify the kynurenine (Kyn), Trp and serotonin (5-HT) concentrations and Kyn/Trp ratio (KTR) by ultra-high performance liquid chromatography coupled to sequential mass spectrometer. The second phase was a genetic study that evaluated a cohort of 328 RA patients and 234 healthy volunteers from Ribeirão Preto and Porto Alegre about the indoleamine 2,3-dioxygenase 1 (IDO1) gene polymorphism. Clinical and epidemiological data were obtained and peripheral blood samples were collected to DNA extraction by salting-out method. Then, HLA typification and polymerase chain reaction to identify IDO1 genetic variants rs7820268, rs3739319, rs61753677, rs35059413, rs35099072 and rs9298586 were performed. Rheumatoid factor (RF) positivity was associated to smoking (p = 0.0002) and SE (p < 0.0001), and cyclic citrullinated peptide autoantibodies (anti-CCP) positivity was associated to SE (p < 0.0001). When SE presence and smoking were combined, there was statistically significant association to RF (p < 0.001) and anti-CCP (p = 0.03). We observed lower plasma 5-HT concentrations in RA patients than in healthy volunteers (p = 0.006), but no significant difference to Trp, Kyn and KTR levels. For these, differences were observed when subgroups were evaluated. In RA patients not using disease modifying antirheumatic drugs (DMARDs) the plasma Trp levels were lower than RApatients using DMARDs, while the plasma Kyn concentrations and KTR in smokers RA patients were lower than nonsmokers RA patients (p = 0.039 and p = 0.032 respectively). We did not indetify statistically significant associations neither between studied genetic variants and risk to develop RA nor between IDO1 polymorphisms and plasma Trp, Kyn, 5HT concentrations and KTR. This study did not identify relation between IDO1 genetic variants with susceptibility to RA. Therefore, new studies are necessary to explain the searched associations between Kyns pathway and 5-HT in RA etiopathogenenesis.