No description available.

Chronic kidney disease (CKD), a reduction in kidney function, has reached pandemic proportions and imposes a major healthcare burden worldwide. A hallmark of CKD is the accumulation of several chemical compounds, called uremic toxins, which inflict systemic and renal-specific damage. Of the known uremic toxins, kynurenine (Kyn) is known to be particularly vasculotoxic and is implicated in several complications of CKD. Indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes the first step in the metabolism of Tryptophan (Trp), regulates immune response to inflammatory cytokines in tissues. IDO plays a role in apoptosis and damage during acute kidney injury (AKI), a transient decrease in kidney function. During metabolism of Trp, IDO generates Kyn, a uremic solute, and therefore IDO may play a role in the brain and kidney damage due to accumulation of Kyn. The objective of the current study was to investigate the role and regulation of IDO in CKD pathology. Studies were performed to determine whether IDO is protective or pathologic and to find how IDO is regulated in the kidney during CKD. IDO in renopathology was examined using murine models of CKD. CKD was induced via a 0.2% adenine-supplemented diet (AD) model for 21 days. IDO regulation was examined using an Indoxyl Sulfate (IS)-specific solute model. Renal function in the IDO+/+ and IDO-/- AD mice was assessed through weekly measurement of blood urea nitrogen (BUN). H&E and Masson’s trichrome stains were used to assess percentages of glomerulosclerosis (GS) and immune infiltration (II), and combined interstitial fibrosis and tubular atrophy (IFTA) score in IDO+/+ and IDO-/- mice with and without CKD. IDO protein concentration in the kidneys of all mice with and without CKD and IDO+/+ IS mice was determined via immunoblotting. Patients with kidney disease suffer from neuropsychological disorders and neurocognitive decline. The effects of uremic solutes on the CNS was examined using immortalized human umbilical endothelial vein cells (HUVEC-TERT), in vitro. Cell proliferation and viability, in the presence of IS, were measured by BrdU and Alamar blue assays, respectively. In both IDO+/+ and IDO-/-, 21 days of AD results in significant deterioration of renal function. The average IFTA score and percentage of II in IDO-/- mice increased with AD compared to ND (p<0.05, p<0.001). IDO expression was seen sporadically in the glomeruli and walls of major vessels in the kidneys of 4d AD IDO+/+ mice, and in the tubules and vessel walls in the kidneys of 14d AD IDO+/+ mice. In IDO+/+ ND mice, endogenous IDO protein expression was undetectable at a signal intensity of 119.86 ± 268.01, whereas IDO+/+ AD mice showed a 370-fold higher level of IDO protein expression compared to IDO+/+ ND (p<0.001). IDO-/- AD IDO protein expression was 9.5-fold higher than in IDO-/- AD (p<0.05). IDO expression was found to be 58-fold higher in IDO+/+ mice with IS treatment (p<0.05). In the IS mice, non-significant trends toward decrease in cellular proliferation and viability with time were also observed (p=ns). IDO is upregulated at the protein level both in a CKD model and directly by the uremic solute, IS. IDO appears to be protective in the kidney during CKD, given the trend toward increased percentage of GS and II in IDO-/- compared to IDO+/+ mice with CKD, though there is little difference seen in total kidney IFTA. IDO upregulation is linked to increased apoptosis. Blocking uremic solute production would therefore prevent IDO protein upregulation and reduce apoptosis, alleviating renal damage during CKD.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21957号 / 医博第4499号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第17862号 / 薬博第793号 / 新制||薬||236(附属図書館) / 30682 / 京都大学大学院薬学研究科医療薬科学専攻 / (主査)教授 髙倉 喜信, 教授 橋田 充, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No description available.

Yes / Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss. / Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945).

A tolerância materno-fetaI continua a ser um intrigante enigma imunológico. Algumas teorias têm sido propostas sobre o estabelecimento deste estado, tais como a produção de moléculas solúveis como HLA-G (na gestação humana) por células fetais que inibiriam a atividade de células do sistema imune inato. Além da secreção de hormônios; liberação de citocinas e de alguns fatores pelo trofoblasto que induziriam alterações no micro-ambiente placentário favorecendo o sucesso da gestação. A tolerãncia materno-fetal parece ser resultar da interação de eventos específicos ou não da gestação. Neste contexto, a indoleamina 2,3-dioxigenase (IDO), uma enzima com transcrição induzida pelo INF-y, tem sua atividade relacionada com o estabelecimento de estado de tolerância materno frente a antígenos fetais em mulheres e em camundongas. A IDO catabolisa o triptofano, um aminoácido essencial, do micro-ambiente placentário. Desta forma, células T ativadas no tecido placentário não conseguem proliferar estacionando na fase G1 do ciclo celular, ma forma de regulação da resposta imune materna. O presente trabalho teve por objetivo analisar as concentrações de triptofano e seus produtos de catabolismo no tecido placentário bovino em gestações normais e de fetos donados. Homogenatos de tecidos placentários provenientes de gestações normais (n=29) e de fetos donados (n=5) foram analisados por cromatrografia líquida de alta performance (HPLC). O níveis de triptofano e quinurenina aumentaram com o avanço da idade gestacional; TRIPTOFANO: 479,33µM/L ±53,04ep; 745,87µM/L ±72,71ep; 983,39µM/L ±196,37ep no primeiro, segundo e terceiro trimestre da prenhez respectivamente; QUINURENINA: 15,13µM/L ±2,97ep; 25,26µM/L ±3,72ep; 52,77µM/L ±17,75ep no primeiro, segundo e terceiro trimestre da prenhez respectivamente. A razão quinurenina/triptofano (razão Q/T) não alterou durante a gestação (0.038 ±0.011ep, 0.035 ±0.005ep and 0.056 ±0,020ep; no primeiro, segundo e terceiro trimestre da prenhez respectivamente). Quando esses valores foram comparados à placentas provenientes de fetos clonados apresentaram-se menores, contudo sem diferenças estatísticas significantes. (0,031 ±0.003ep). Os valores de triptofano foram menores na gestação de clones (811,34µM/L ±232,14ep) bem como os encontrados de quinurenina (22,85µM/L ±4,09ep). Não houve diferenças estatisticamente significativas entre o terceiro trimestre gestacional de fetos normais quando comparados à gestação de clones neste contexto. O catabolismo de triptofano parece aumentar com o avanço da idade gestacional devido o aumento da concentração do aminoácido e de seus metabólitos (quinurenina), no entanto a razão Q/T não se alterou, sugerindo um aumento da atividade de IDO na placenta bovina para manutenção dos níveis de triptofano no micro-ambiente placentário. A presença de quinurenina na placenta bovina é um indicador da atividade da IDO neste tecido. / Maternal-fetal tolerance continues to be an intriguing immunological enigma. Some theories have been proposed about these phenomena such as the production of soluble molecules like HLA-G by fetal cells that would block some cells of the innate immune system and the secretion of cytokines, hormones and some factors by trophoblast cells that would induce changes in the placental microenvironment. Maternal tolerance is probably the consequence of a wide panel of mechanisms that may be or not pregnancy-specific. In this context, indoleamine 2, 3 dioxygenase (IDO), an inducible INFy enzyme, is a candidate protein to be involved in placental tolerance, as suggested in some reports on women pregnancy. IDO seems to catabolise the tryptophan, an essential amino acid necessary to cell proliferation. This way, activated T-cells in placental tis sue cannot proliferate due to starvation of tryptophan in milieu and these cells undergo apoptosis. This change may be one of the ways maternal-fetal tolerance occurs. Our aim was to evaluate the levels of tryptophan and its degradation products in normal and cloned bovine pregnancy, observing possible changes in tryptophan catabolism during this period. Homogenates from normal placental tissue from cows with normal (n=29) and cloned pregnancy (n=5) were analyzed by high performance liquid chromatograph. Levels of tryptophan and kinurenine reached their highest levels through of time of pregnancy; TRIPTOPHAN: 479,33µM/L ±53,04ste; 745,87µM/L ±72,71ste; 983,39µM/L ±196,37ste early. middle and late pregnancy respectively; KINURENINE: 15,13µM/L ±2,97ste; 25,26µM/L 3,72ste; 52,77µM/L ±17,75ste early. middle and late pregnancy respectively. The ratios of kynurenine to tryptophan does not increased during pregnancy (0.038 ±0.011ste, 0.035 ±0.005ste and 0.056 ±0,020ste; early, middle and late pregnancy respectively). When these values were compared to cloned pregnancy they showed lower values in these animais, however they were not statiscally significant (0,031 ±0.003ste). Tryptophan values were lower in cloned pregnancy (811,34µM/L ± ±232, 14ste) as well kinurenine (22,85µM/L ±4,09stde). There was no statiscal difference between normal late pregnancy and cloned pregnancy in this mater. The presence of kinurenine in bovine placental tissue is one indicator that IDO could be expressed in bovine placenta and indicate an IDO activity in this site.

A tolerância materno-fetaI continua a ser um intrigante enigma imunológico. Algumas teorias têm sido propostas sobre o estabelecimento deste estado, tais como a produção de moléculas solúveis como HLA-G (na gestação humana) por células fetais que inibiriam a atividade de células do sistema imune inato. Além da secreção de hormônios; liberação de citocinas e de alguns fatores pelo trofoblasto que induziriam alterações no micro-ambiente placentário favorecendo o sucesso da gestação. A tolerãncia materno-fetal parece ser resultar da interação de eventos específicos ou não da gestação. Neste contexto, a indoleamina 2,3-dioxigenase (IDO), uma enzima com transcrição induzida pelo INF-y, tem sua atividade relacionada com o estabelecimento de estado de tolerância materno frente a antígenos fetais em mulheres e em camundongas. A IDO catabolisa o triptofano, um aminoácido essencial, do micro-ambiente placentário. Desta forma, células T ativadas no tecido placentário não conseguem proliferar estacionando na fase G1 do ciclo celular, ma forma de regulação da resposta imune materna. O presente trabalho teve por objetivo analisar as concentrações de triptofano e seus produtos de catabolismo no tecido placentário bovino em gestações normais e de fetos donados. Homogenatos de tecidos placentários provenientes de gestações normais (n=29) e de fetos donados (n=5) foram analisados por cromatrografia líquida de alta performance (HPLC). O níveis de triptofano e quinurenina aumentaram com o avanço da idade gestacional; TRIPTOFANO: 479,33µM/L ±53,04ep; 745,87µM/L ±72,71ep; 983,39µM/L ±196,37ep no primeiro, segundo e terceiro trimestre da prenhez respectivamente; QUINURENINA: 15,13µM/L ±2,97ep; 25,26µM/L ±3,72ep; 52,77µM/L ±17,75ep no primeiro, segundo e terceiro trimestre da prenhez respectivamente. A razão quinurenina/triptofano (razão Q/T) não alterou durante a gestação (0.038 ±0.011ep, 0.035 ±0.005ep and 0.056 ±0,020ep; no primeiro, segundo e terceiro trimestre da prenhez respectivamente). Quando esses valores foram comparados à placentas provenientes de fetos clonados apresentaram-se menores, contudo sem diferenças estatísticas significantes. (0,031 ±0.003ep). Os valores de triptofano foram menores na gestação de clones (811,34µM/L ±232,14ep) bem como os encontrados de quinurenina (22,85µM/L ±4,09ep). Não houve diferenças estatisticamente significativas entre o terceiro trimestre gestacional de fetos normais quando comparados à gestação de clones neste contexto. O catabolismo de triptofano parece aumentar com o avanço da idade gestacional devido o aumento da concentração do aminoácido e de seus metabólitos (quinurenina), no entanto a razão Q/T não se alterou, sugerindo um aumento da atividade de IDO na placenta bovina para manutenção dos níveis de triptofano no micro-ambiente placentário. A presença de quinurenina na placenta bovina é um indicador da atividade da IDO neste tecido. / Maternal-fetal tolerance continues to be an intriguing immunological enigma. Some theories have been proposed about these phenomena such as the production of soluble molecules like HLA-G by fetal cells that would block some cells of the innate immune system and the secretion of cytokines, hormones and some factors by trophoblast cells that would induce changes in the placental microenvironment. Maternal tolerance is probably the consequence of a wide panel of mechanisms that may be or not pregnancy-specific. In this context, indoleamine 2, 3 dioxygenase (IDO), an inducible INFy enzyme, is a candidate protein to be involved in placental tolerance, as suggested in some reports on women pregnancy. IDO seems to catabolise the tryptophan, an essential amino acid necessary to cell proliferation. This way, activated T-cells in placental tis sue cannot proliferate due to starvation of tryptophan in milieu and these cells undergo apoptosis. This change may be one of the ways maternal-fetal tolerance occurs. Our aim was to evaluate the levels of tryptophan and its degradation products in normal and cloned bovine pregnancy, observing possible changes in tryptophan catabolism during this period. Homogenates from normal placental tissue from cows with normal (n=29) and cloned pregnancy (n=5) were analyzed by high performance liquid chromatograph. Levels of tryptophan and kinurenine reached their highest levels through of time of pregnancy; TRIPTOPHAN: 479,33µM/L ±53,04ste; 745,87µM/L ±72,71ste; 983,39µM/L ±196,37ste early. middle and late pregnancy respectively; KINURENINE: 15,13µM/L ±2,97ste; 25,26µM/L 3,72ste; 52,77µM/L ±17,75ste early. middle and late pregnancy respectively. The ratios of kynurenine to tryptophan does not increased during pregnancy (0.038 ±0.011ste, 0.035 ±0.005ste and 0.056 ±0,020ste; early, middle and late pregnancy respectively). When these values were compared to cloned pregnancy they showed lower values in these animais, however they were not statiscally significant (0,031 ±0.003ste). Tryptophan values were lower in cloned pregnancy (811,34µM/L ± ±232, 14ste) as well kinurenine (22,85µM/L ±4,09stde). There was no statiscal difference between normal late pregnancy and cloned pregnancy in this mater. The presence of kinurenine in bovine placental tissue is one indicator that IDO could be expressed in bovine placenta and indicate an IDO activity in this site.

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 Indoleamina 2,3-dioxigenase (IDO) é uma enzima que apresenta um importante papel na prevenção da rejeição fetal. A IDO demonstra efeitos na supressão da ativação de células T por catabolizar o aminoácido essencial Triptofano. Nós estudamos a expressão da IDO em cultura celulares de placenta com a adição individual de Estradiol, Progesterona, Interferon , Triptofano e 1-Metil-Triptofano com o uso de citometria de fluxo, imunocitoquímica e quantificação celular, imunofluorescência, peroxidação lipídica, análise das fases do ciclo celular, imunoblotting e PCR em placentas bovinas nos três trimestres gestacionais. A quantificação celular revelou que em bovinos a atividade da IDO aumenta com o avanço do período gestacional e, sua expressão varia de acordo com os fatores, sendo o mesmo padrão observado pela imunofluorescência. O Imunoblotting mostrou a presença de possíveis isoformas desta proteína na espécie bovina que ainda não foram identificadas. A investigação pela lipoperoxidação revelou que os hormônios modularam diferencialmente a produção de radicais peroxidados nos três terços de gestação e, possivelmente atuam como fatores indutores de proliferação. As células placentárias apresentaram padrões diferenciados de proliferação e apoptose ao longo da gestação, principalmente nos tratamentos com Estradiol e Progesterona, sendo também variantes com outros fatores em todos os grupos. Observou-se que a Progesterona atua na maturação das células placentárias independente da concentração utilizada. Em todos os grupos uma grande proporção de células apresentava-se em estado de quiescência (G1). No terceiro trimestre foi detectado um aumento no número de células em G2/M, indicando a parada da capacidade proliferativa ou de progressão no ciclo celular (\"arrest\"). Maior taxa de apoptose foi observada nos animais de segundo trimestre gestacional. Baseados nisso, podemos concluir que a IDO é suscetível ao controle pelos mecanismos testados, o que nos leva a formular hipóteses de implantação de possíveis mecanismos terapêuticos para a reprodução com a participação da IDO. / Indoleamina 2,3-dioxigenase (IDO) is an enzyme that plays an important role in preventing fetal rejection. IDO is related to the suppression of the T-cells activation due to the catabolism of essential amino acid Triptophan. We studied the expression of IDO in bovine placenta cell culture with individual supplementation of factors as Estrogen, Progesterone, Interferon , Triptophan and 1-Methyl-Triptophan. Evaluations were made by flow cytometry, immunocitochemistry and cellular quantification, lipidic peroxidation, cell cycle phases, imunoblotting, PCR and immunofluorescency in the three trimesters of pregnancy. Cellular quantification demonstrated that in bovines the activity of the IDO increases during pregnancy, and its expression is factor-dependent, which was also observed in immunofluorescency. Imunoblotting demonstrated the presence of possible unknown protein isoforms in bovine. Lipoperoxidation evaluation demonstrated that hormones distinguishingly modulated the production of peroxide radicals in the three trimesters of pregnancy and, possibly acting as inductive factors of proliferation. Placental cells demonstrated differentiated patterns of proliferation and apoptosis during the gestation, mainly in the presence of Estrogen and Progesterone, besides variant rates were also observed under other factors. Independent of the concentration, Progesterone influenced placental cells maturation. All groups presented great ratio of cells in quiescence state (G1). In third trimester, G2/M high rates indicated a pause in proliferative capacity or in cell cycle progression (arrest). High apoptosis rates were observed in animals at second pregnancy trimester. Based on the presented, we concluded that IDO is susceptible to be controlled by the applied-factors, what lead us to think about hypothetical therapeuthic mechnism for reproduction with the participation of IDO.