Imunização de camundongos LDLr-/- com peptídeo mimético da LDL eletronegativa: uma potencial estratégia terapêutica para a ateroscleros / Immunization of LDLr - / - mice with electronegative LDL mimetic peptides: a potential therapeutic strategy for atherosclerosis

Introdução: A aterosclerose é uma doença inflamatória crônica decorrente de alterações na parede das artérias de médio e grande calibre e associadas a diversos fatores de risco, dentre os quais destaca-se as hiperlipidemias, ou seja, o aumento plasmático das lipoproteínas, mas também outras comorbidades, como a Síndrome Metabólica. Entre as lipoproteínas, a lipoproteína de baixa densidade (LDL) é de grande relevância na aterosclerose. Diferentes espécies de LDL modificada (LDLm) são originadas através de lipólise, glicação e proteólise, além da oxidação, variando em densidade e eletronegatividade, sendo melhor denominada LDL eletronegativa [LDL (-)]. Considerando as diferenças conformacionais entre a estrutura da ApoB-100 da LDL nativa e da LDL (-), em um estudo inicial, nosso grupo desenvolveu um anticorpo monoclonal (2C7) a partir da imunização de camundongos Balb/c com a LDL (-) humana. Em uma etapa seguinte foi mapeado o epítopo reconhecido pelo anticorpo monoclonal anti-LDL (-) através de phage display. O peptídeo ligante do anticorpo monoclonal anti-LDL (-) foi nomeado p2C7. Esse peptídeo não representa regiões da sequência linear da ApoB-100 humana, mas microdomínios conformacionais de epítopos da ApoB-100 da LDL (-), tornando-os candidatos para a imunomodulação da aterogênese. Portanto, investigar a imunomodulação induzida pelos peptídeo p2C7 miméticos da LDL (-), por representar um epítopo imunodominante da LDL (-), poderá abrir novas perspectivas terapêuticas futuras para a imunomodulação da aterosclerose. Objetivo: Avaliar a imunomodulação promovida pelo p2C7 in vivo, utilizando camundongos C57BL/6 LDLr -/- e amostras de plasma humano. Adicionalmente, no estágio (BEPE) realizado no Instituto Karolinska (dezembro de 2019 a março de 2021), investigou-se o imunometabolismo como mediador nas doenças cardiovasculares. Na parte II-A, estão descritos os resultados do estudo inicialmente proposto. Na parte II-B, apresenta-se os resultados que foram desenvolvidos posteriormente, com ampliação do escopo do projeto, abordando-se a inflamação vascular envolvida no aneurisma de aorta abdominal através de ferramentas de bioinformática. Na parte II-C, são apresentados os resultados do estudo do envolvimento da enzima indolamina 2,3 dioxigenase (IDO) na esteatohepatite não-alcoólica (NASH) e aterosclerose em camundongos ApoE-/- and ApoE/IDO/double-knockout. Metodologia: Foi avaliada a presença de anticorpos anti-p2C7 em amostras de plasma humano de indivíduos com ou sem síndrome metabólica. Realizamos a determinação de TNF circulante nas mesmas amostras e prosseguimos com regressões lineares associando os parâmetros inflamatórios com os níveis de anticorpos anti-p2C7. Camundongos C57BL/6 LDLr -/- foram imunizados com p2C7 e os adjuvantes Alum ou Montanide ISA 720, analisando-se os títulos de anticorpos contra p2C7 e LDL (-), a produção de citocinas (IL-10, IL-4, IL-2, IL-6, IFNγ, IL-17, TNFα) e células secretoras de anticorpos. Camundongos C57BL/6 LDLr -/- foram tolerizados contra os peptídeos mimotopos, com injeções intravenosas (veia caudal) e desafiados com a imunização contendo LDL (-) + Alum. Avaliou-se os títulos de anticorpos contra p2C7 e LDL (-) e a produção de citocinas (TNF-α, IFNγ, IL-12, IL-6, IL-10 e MCP-1). Os camundongos foram mantidos em dieta hipercolesterolêmica por 3 meses para formação da placa aterosclerótica. Após este período, os camundongos foram eutanasiados, avaliando-se a formação de placa aterosclerótica na artéria abdominal e arco aórtico, assim como a produção de citocinas (TNF-α, IFNγ, IL-12, IL-6, IL-10 e MCP-1). Camundongos C57BL/6 LDLr -/- foram imunizados com OVA-p2C7 e, após dieta hipercolesterolêmica de 3 meses para formação de placa aterosclerótica, foram avaliados os parâmetros inflamatórios e avaliada a captação de 18F-FDG no arco aórtico através de PET/CT. Resultados: A imunização com o p2C7 (livre) não foi capaz de induzir resposta humoral, não se observando títulos detectáveis de anticorpos reativos à p2C7 ou LDL (-) em nenhum camundongo imunizado, assim como não foram detectadas células secretoras de anticorpos específicos para a LDL (-). O grupo imunizado com Alum ou Montanide + p2C7 teve aumento significativo na produção de TNF- quando comparado com os demais grupos. O protocolo de tolerização foi realizado com sucesso, visto que os camundongos tolerizados apresentaram títulos de anticorpos inferiores aos controles para o epítopo utilizado. Apenas os camundongos tolerizados com o p2C7 apresentaram aumento significativo na produção de IL-6, IL-12, IL-10, TNF-α, IFNγ e MCP 1 após dieta hipercolesterolêmica. A imunização ativa com OVA-p2C7 foi capaz de reduzir a produção de TNF induzida pela dieta hipercolesterolêmica, assim como reduzir a captação de 18F-FDG. Conclusão: o epítopo p2C7 é altamente expresso na LDL (-) de pacientes com maior risco cardiovascular. Além disso, a imunização ativa com p2C7 também se mostra uma ferramenta promissora para prevenir e regular a inflamação causada pela LDL (-) no curso da aterosclerose

Introduction: Atherosclerosis is a chronic inflammatory disease resulting from changes in the wall of medium and large-caliber arteries and associated with several risk factors, among which hyperlipidemias stand out, ie, the increase in plasma lipoproteins, but also other comorbidities, such as Metabolic Syndrome. Among the lipoproteins, low-density lipoprotein (LDL) is of great relevance in atherosclerosis. Different isoforms of modified LDL (LDLm) are originated through lipolysis, glycation and proteolysis, in addition to oxidation, varying in density and electronegativity, being better called electronegative LDL [LDL (-)]. Considering the conformational differences between the ApoB-100 structure of native LDL and LDL (-), in an initial study, our group developed a monoclonal antibody (2C7) from the immunization of Balb/c mice with human LDL (-). In a next step, the epitope recognized by the anti-LDL monoclonal antibody (-) was mapped using phage display. The binding peptide of anti-LDL monoclonal antibodies (-) was named p2C7. This peptide does not represent linear sequence regions of human ApoB-100, but conformational microdomains of LDL (-) ApoB-100 epitopes, making them candidates for the immunomodulation of atherogenesis. Therefore, investigating the immunomodulation induced by p2C7 peptide mimetics of LDL (-) as it represents an immunodominant epitope of LDL (-) could open new future therapeutic perspectives for the immunomodulation of atherosclerosis. Objective: To evaluate the immunomodulation promoted by p2C7 in vivo, using C57BL/6 LDLr -/- mice, and human plasma samples. In addition, in the internship (BEPE), held at the Karolinska Institute (December 2019 to March 2021), immunometabolism as a mediator of Cardiovascular Diseases was studied. In part II-A, the results of the initially proposed study are described. In part II-B, the results that were developed later are presented, expanding the scope of the project, approaching the vascular inflammation involved in the abdominal aortic aneurysm through bioinformatics tools. In part II-C, the results of the study of the involvement of the enzyme indoleamine 2,3 dioxygenase (IDO) in non-alcoholic steatohepatitis (NASH) and atherosclerosis in ApoE-/- and ApoE/IDO/double mice are presented -knockout. Methodology: The presence of anti-p2C7 antibodies in human plasma samples with or without Metabolic Syndrome was evaluated. We measured circulating TNF in the same samples and proceeded with linear regressions associating inflammatory parameters with levels of anti-p2C7 antibodies. C57BL/6 LDLr -/- mice were immunized with p2C7 and the adjuvants Alum or Montanide ISA 720, analyzing the antibody titers against p2C7 and LDL (-), the production of cytokines (IL-10, IL-4, IL -2, IL-6, IFNγ, IL-17, TNFα) and antibody-secreting cells. C57BL/6 LDLr -/- mice were tolerized against mimotope peptides with intravenous injections (caudal vein) and challenged with immunization containing LDL (-) + Alum. Antibody titers against p2C7 and LDL (-) and cytokine production (TNF-α, IFNγ, IL-12, IL-6, IL-10 and MCP-1) were evaluated. The mice were kept on a hypercholesterolemic diet for 3 months for atherosclerotic plaque formation. After this period, the mice were euthanized, evaluating the formation of atherosclerotic plaque in the abdominal artery and aortic arch, as well as the production of cytokines (TNF-α, IFNγ, IL-12, IL-6, IL-10 and MCP -1). C57BL/6 LDLr -/- mice were immunized with OVA-p2C7 and, after a 3-month hypercholesterolemic diet for atherosclerotic plaque formation, inflammatory parameters were evaluated and 18F-FDG uptake was evaluated by PET/CT. Results: Immunization with p2C7 (free) was not able to induce a humoral response, with no detectable titers of antibodies reactive to p2C7 or LDL (-) being observed in any immunized mouse, as well as no detectable antibody-secreting cells for the LDL (-). The group immunized with Alum or Montanide + p2C7 had a significant increase in TNF-α production when compared to the other groups. The tolerance protocol was successfully performed, as the tolerized mice had lower antibody titers than controls for the epitope used. Only mice tolerated with p2C7 showed a significant increase in the production of IL-6, IL-12, IL-10, TNF-α, IFNγ and MCP 1 after a hypercholesterolemic diet. Active immunization with OVA-p2C7 was able to reduce TNF production induced by the hypercholesterolemic diet, as well as to reduce 18F-FDG uptake. Conclusion: the p2C7 epitope is highly expressed in LDL (-) of patients with higher cardiovascular risk. Furthermore, active immunization with p2C7 is also a promising tool to prevent and regulate inflammation caused by LDL (-) in the course of atherosclerosis

Papel da enzima Indoleamina 2, 3 dioxigenase (IDO1) na neuropatia hanseniana / Role of the enzyme Indoleamine 2, 3 dioxigenase (IDO1) in leprosy neuropathy

A hanseníase é uma doença infecciosa, desmielinizante, que pode levar à incapacidades e deformidades físicas permanentes. Dados anteriores demonstraram que Mycobacterium leprae, o agente etiológico da doença, consegue modular a biologia das células de Schwann. Um ponto ainda não elucidado é se o dano neural na hanseníase é causado diretamente pelo bacilo ou se é dependente do infiltrado inflamatório. Sabe-se que durante os episódios reacionais, o aumento de citocinas pró-inflamatórias, como IFN-γ, pode contribuir para o incremento do dano neural por um mecanismo ainda desconhecido e, uma correlação positiva entre concentrações séricas de TNF-α e desmielinização em pacientes com hanseníase já foi demonstrada. IFN-γ e TNF-α são citocinas capazes de induzir a expressão de Indoleamina 2,3 dioxigenase (IDO1), enzima chave na regulação da via das quinureninas, em diversos tipos celulares como monócitos, macrófagos e células dendríticas. Diversos estudos envolvendo patologias do sistema nervoso central apontam que as quinureninas produzidas pela degradação do triptofano podem ter uma função neurotóxica, mas pouco se sabe sobre o envolvimento de IDO1 e seus metabólitos na patogênese de doenças do sistema nervoso periférico. O presente estudo teve como objetivo avaliar a possível contribuição de IDO1 e seus metabólitos na neuropatia hanseniana. Inicialmente foi demonstrado que M. leprae aumenta a expressão de IDO1 em células de Schwann primárias e da linhagem ST88-14. No entanto, o bacilo não é capaz de aumentar a atividade de IDO1 em células de Schwann da linhagem ST88-14.

Foi observado que as citocinas pró-inflamatórias IFN-γ e TNF-α isoladamente não são capazes de induzir a expressão proteica de IDO1, mas aumentam significativamente sua atividade em células ST88-14. Em associação com M. leprae, IFN-γ exerce um forte estímulo na indução da atividade enzimática de IDO1. A infecção com M. leprae diminui a expressão gênica de Quinurenina aminotransferase II (AADAT), sugerindo que o mesmo possa estar direcionando a via para a produção de metabólitos neurotóxicos como o ácido quinolínico (QUINA), 3-hidroxiquinurenina (3-HK) e ácido 3-hidroxiantranílico (3HAA). A análise de viabilidade celular demonstrou que 3-HAA é capaz de induzir apoptose em células ST88-14 e, a análise por ELISA, dos sobrenadantes de 24h de cultura, demonstrou que M. leprae viável induz aumento na concentração do metabólito ácido quinurênico (KYNA) que é significativamente reduzida na presença de IFN-γ, enquanto IFN-γ aumenta significativamente a concentração de QUINA, principalmente quando associado à M. leprae morto. Foi observado um aumento na atividade de IDO1 em soro de pacientes com neuropatia hanseniana em relação a pacientes com outras neuropatias periféricas. Uma correlação positiva foi observada entre a atividade de IDO1 e a gravidade do dano neural, conforme avaliado pelo exame de eletroneuromiografia. Em conjunto, os dados apresentados sugerem o envolvimento da via das quinureninas no dano neural na hanseníase. (AU)

Neopterin Levels and Indoleamine 2,3-Dioxygenase Activity as Biomarkers of Immune System Activation and Childhood Allergic Diseases

BACKGROUND: Although Th2 immune activation is predominant in allergic diseases, neopterinlevels and indoleamine 2,3-dioxygenase (IDO)-1 activity (kynurenine:tryptophan ratio), which reflect Th1 immune activity, increase with interferon-gamma (IFN-γ) stimulation. We investigated neopterin, tryptophan, and kynurenine levels as biomarkersof the Th1 immune system activation and changes in IDO-1 activityin children with asthma, allergic rhinitis, and atopic dermatitis, as well as the relationship between these biomarkers and the total IgE level, age, and disease severity. METHODS: We divided 205 children (80 girls and 125 boys, four months to 17 years old) into four groups: controls, patients with asthma, patients with allergic rhinitis, and patients with atopic dermatitis. Peripheral venous blood samples were collected. Neopterin levels were determined by an enzyme immunoassay. Tryptophan and kynurenine levels were analyzed using HPLC. IDO-1 enzyme activity was calculated using tryptophan and kynurenine levels. IgE levels were measured. The Mann-Whitney U test, Kruskal-Wallis test, and Conover post-hoc method were used for statistical analysis. RESULTS: Neopterin, tryptophan, and kynurenine levels were higher and IgE levels and IDO-1 enzyme activity were lower in patients with asthma and allergic rhinitis than in controls (P < 0.05). Patients with atopic dermatitis showed higher neopterin, tryptophan, and kynurenine levels, higher IDO-1 activity, and lower IgE levels thancontrols (P < 0.05). CONCLUSIONS: The Th1/Th2 balance is disrupted in children with allergic diseases, concomitant with increased Th1-mediated immune response activation and reduced IgEproduction, which is promoted by Th2-type cytokines.

Inhibition of Indoleamine 2,3-Dioxygenase Enhances the Therapeutic Efficacy of Immunogenic Chemotherapeutics in Breast Cancer / 한국유방암학회지

PURPOSE: Breast cancer has become a major public health threat in the current society. Anthracycline doxorubicin (DOX) is a widely used drug in breast cancer chemotherapy. We aimed to investigate the immunogenic death of breast tumor cells caused by DOX, and detect the effects of combination of DOX and a small molecule inhibitor in tumor engrafted mouse model. METHODS: We used 4T1 breast cancer cells to examine the anthracycline DOX-mediated immunogenic death of breast tumor cells by assessing the calreticulin exposure and adenosine triphosphate and high mobility group box 1 release. Using 4T1 tumor cell-engrafted mouse model, we also detected the expression of indoleamine 2,3-dioxygenase (IDO) in tumor tissues after DOX treatment and further explored whether the specific small molecule IDO1 inhibitor NLG919 combined with DOX, can exhibit better therapeutic effects on breast cancer. RESULTS: DOX induced immunogenic cell death of murine breast cancer cells 4T1 as well as the upregulation of IDO1. We also found that treatment with NLG919 enhanced kynurenine inhibition in a dose-dependent manner. IDO1 inhibition reversed CD8+ T cell suppression mediated by IDO-expressing 4T1 murine breast cancer cells. Compared to the single agent or control, combination of DOX and NLG919 significantly inhibited the tumor growth, indicating that the 2 drugs exhibit synergistic effect. The combination therapy also increased the expression of transforming growth factor-β, while lowering the expressions of interleukin-12p70 and interferon-γ. CONCLUSION: Compared to single agent therapy, combination of NLG919 with DOX demonstrated better therapeutic effects in 4T1 murine breast tumor model. IDO inhibition by NLG919 enhanced the therapeutic efficacy of DOX in breast cancer, achieving synergistic effect.

Indolamina 2, 3-dioxigenase (IDO) em melanoma: regulação frente ao inibidor de BRAF e sua expressão na progressão da doença / Indoleamine 2, 3-dioxygenase (IDO) in melanoma: regulation against BRAF inhibitor and its expression in disease progression

Os inibidores de BRAF (iBRAFs) e de MEK (iMEK), inauguraram uma nova classe de medicamentos, a terapia direcionada, no combate ao melanoma metastático. Entretanto, os pacientes adquirem resistência ao tratamento em poucos meses. Além disso, a imunoterapia vem ganhando espaço no tratamento do câncer, incluindo o melanoma, porém, com alguns aspectos inexplorados. Dentro deste tema, a enzima IDO vem despertando um grande interesse pela participação nos mecanismos de imunotolerância, imunoescape e progressão tumoral. A IDO é responsável pelo consumo e depleção do triptofano, produzindo a quinurenina. Ela está presente em diversos tipos celulares, incluindo células do sistema imune e células tumorais. Este trabalho objetivou avaliar a expressão de IDO durante a progressão da doença - desde do nevo até o melanoma metastático e também avaliar a regulação de IDO induzido por IFN-γ após tratamento com iBRAF em linhagens parentais e resistentes ao iBRAF, buscando-se os mecanismos moleculares. Por fim, objetivou-se entender os efeitos do 1-metil-triptofano (1-MT), um inibidor de IDO, tanto na sua capacidade de inibir a atividade de IDO quanto na sua influência na capacidade clonogênica. O estudo de bioinformática sobre o repositório público GSE12391 mostrou que o nível de expressão gênica de IDO foi superior nos estágios mais avançado da doença. Além disso, todas amostras de melanoma primário de pacientes apresentaram a imunomarcação de IDO, enquanto que nenhuma amostra de nevo apresentou tal marcação. Adicionalmente, a ocorrência de IDO se deu nos infiltrados linfoides, em células mononucleares do sistema imune. Duas análises de bioinformática de expressão gênica demonstraram que a IDO estava expressa positivamente na fase de resistência ao iBRAF. Ademais, os resultados de expressão proteica mostraram que a inibição de via MAPK (tanto por iBRAF quanto por iMEK) conseguiu modular a expressão de IDO, sendo que a maioria das linhagens apresentou uma diminuição de IDO. A atividade de IDO, medida através da produção de quinurenina, por HPLC se mostrou em consonância com os resultados de expressão proteica, exceto pela linhagem WM164 que não apresentou atividade enzimática, embora a proteína estivesse presente. Por fim, o 1-MT conseguiu inibir de maneira eficiente a enzima IDO, bloqueando a produção de quinurenina. Além de que, o 1-MT reduziu a capacidade clonogênica de maneira dose-dependente. Portanto, conclui-se que a expressão de IDO é crescente conforme a progressão do melanoma, que a inibição da via MAPK regulou a expressão de IDO e que o 1-MT reduz a capacidade clonogênica, além da sua função primária de inibir IDO

BRAF and MEK inhibitors (BRAFi and MEKi) has launched a new class of medication, the target therapy, to combat metastatic melanoma. Nevertheless, patients acquired resistance to the treatment in few months. Additionally, immunotherapy has been gaining space in cancer treatment, including melanoma, but some aspects need to be explored. Inside this theme, IDO enzyme has called the attention due to its participation in the mechanisms of immune tolerance, scape and tumor progression. IDO is responsible for tryptophan consume e depletion, producing kynurenine. It is present in different cells, including cells from immune system and tumor cells. This work purposed evaluate IDO expression during disease progression - since nevus until metastatic melanoma and also, evaluate IFN-γ-induced IDO regulation after BRAFi treatment in parental and resistant melanoma cell lines, seeking the molecular mechanisms. Lastly, it was evaluated the effects of 1-methyltryptopahn (1-MT), an IDO inhibitor, by its ability to inhibit IDO and also by its influency on the clonogenic capability. Bioinformatic study performed on GSE12391 showed that gene expression level of IDO was superior in the most advanced stages of the disease. Additionally, all sample of patient's primary melanoma presented IDO immunostaining, whereas, no nevus samples presented such staining. Besides, IDO occurrence was in the lymphoid infiltrates, in mononuclear cells from immune system. Two bioinformatic analysis of gene expression demonstrated that IDO was differentially overexpressed during BRAFi resistance stage. Moreover, protein expression results presented that MAPK pathway inhibition (both by BRAFi and by MEKy) was able to modulate IDO expression, and most of the cell lines presented an IDO downregulation. IDO activity, measured through kynurenine production, by HPLC was consonant with protein expression results, except by WM164 cell line, which did not present enzymatic activity, albeit the protein was present. By the end, 1-MT could inhibit efficiently IDO enzyme, blocking kynurenine production. Furthermore, 1-MT reduced clonogenic capability in a dosedependent manner. Therefore, it was concluded that IDO expression increases along with melanoma progression, MAPK pathway inhibition regulated IDO expression and 1-MT reduced clonogenic capability, besides its primary function of IDO inhibitor

Overexpression of indoleamine 2,3-dioxygenase correlates with regulatory T cell phenotype in acute myeloid leukemia patients with normal karyotype

BACKGROUND: Production of immunosuppressive enzymes such as indoleamine 2,3-dioxygenase (IDO) is one of the strategies employed by hematologic malignancies, including acute myeloid leukemia (AML), to circumvent immune surveillance. Moreover, IDO has the ability to convert CD4+CD25− conventional T cells into regulatory T cells (Tregs). In this study, we evaluated the expression of IDO in cytogenetically normal acute myeloid leukemia (CN-AML) patients and its correlation with the Treg marker, FOXP3, as well as clinical and laboratory parameters. METHODS: Thirty-seven newly diagnosed CN-AML patients were enrolled in our study along with 22 healthy individuals. The expression of the IDO and FOXP3 genes was analyzed by SYBR Green real-time PCR. RESULTS: Both IDO and FOXP3 were highly upregulated in CN-AML patients compared to control groups (P=0.004 and P=0.031, respectively). A positive correlation was observed between IDO and FOXP3 expression among AML patients (r=0.512, P=0.001). Expression of IDO and FOXP3 showed no significant correlation with laboratory parameters such as white blood cell and platelet counts, hemoglobin levels, bone marrow blast percentage, gender, and FLT3 mutation status (P>0.05). CONCLUSION: Higher IDO expression in CN-AML patients may be associated with an increased Treg phenotype which may promote disease progression and lead to poor prognosis of CN-AML patients.

Feiji Recipe inhibits the growth of lung cancer by modulating T-cell immunity through indoleamine-2,3-dioxygenase pathway in an orthotopic implantation model / 中西医结合学报

<p><b>OBJECTIVE</b>Escape from the body's immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase (IDO) plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe.</p><p><b>METHODS</b>An orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein (2LL-EGFP) and 2LL-EGFP-IDO were investigated, and the apoptosis of T-cells was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high-performance liquid chromatography.</p><p><b>RESULTS</b>Compared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged (P = 0.0074), and the IDO protein level decreased (P = 0.0072); moreover, the percentages of CD4CD25 T-cells and Foxp3 T-cells were significantly decreased (P < 0.05). The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and regulatory T-cells.</p><p><b>CONCLUSION</b>The molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.</p>

Expressão de indoleamina 2,3-dioxigenase (IDO) e triptofano 2,3-dioxigenase(TDO) no ambiente cervicovaginal normal, na vaginose bacteriana e nas lesões cervicais associadas ao HPV / Expression of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) in normal cervicovaginal environment, bacterial vaginosis and cervical lesions associated with HPV

Neste estudo avaliamos o papel do metabolismo do triptofano (Trp) na homeostasia, na vaginose bacteriana e nas lesões cervicais associadas ao HPV. A importância do metabolismo do Trp se deve a sua ação na proliferação de microrganismos e de células do sistema imune. O consumo de triptofano tem sido identificado como uma forma de controlar o crescimento bacteriano limitando a infecção. Por outro lado, a oxidação de Trp produz quinurenina (QUIN), que tem papel chave na tolerância imunológica. A formação de QUIN se dá através das enzimas indoleamina 2,3-dioxigenase (IDO) e triptofano 2,3- dioxigenase (TDO). A mais estudada delas no âmbito das infecções/ imuno escape é a enzima IDO. Mais recentemente, tem-se dado ênfase ao papel da TDO no câncer. Nesta dissertação, o interesse foi avaliar a expressão da IDO no epitélio cervicovaginal de mulheres com vaginose bacteriana e de IDO e TDO em amostras cervicais de mulheres com diferentes graus de lesão cervical associada ao HPV. Foram incluídas 165 mulheres atendidas no CAISM/UNICAMP, as quais foram divididas em dois grupos: grupo caso composto por mulheres com lesão de baixo ou alto grau e carcinoma invasor (n=42) e grupo controle composto por mulheres com citologia oncológica normal, independente de apresentar infecção genital (n=123). IDO foi avaliada por imunocitoquímica em citologia em base líquida e IDO e TDO em biópsias cervicais. Mulheres com vaginose bacteriana apresentaram expressão aumentada de IDO em células escamosas em comparação às mulheres sem vaginose bacteriana (OR=7.41; IC 95%= 2.50 a 21.4; p <0.0001). No epitélio vaginal normal com ou sem infecção por HPV houve uma expressão leve de IDO em células escamosas. Na presença de lesões ou carcinoma, houve um aumento no número de células escamosas displásicas e de leucócitos IDO-positivos; aumento de IDO também pôde ser observada em culturas de pele organotípicas transduzidas com as oncoproteínas E6/ E7 do HPV16. Nas lesões cervicais, assim como visto para a IDO, a TDO esteve expressa em leucócitos, especialmente os infiltrados na região estromal e na parede dos vasos sanguíneos. A expressão basal de IDO no epitélio cervical normal e sua regulação positiva na infecção por HPV e lesões associadas sugerem a participação do metabolismo do Trp nos mecanismos imunossupressores envolvidos na doença. Embora o papel do IDO já tenha sido abordada anteriormente, até onde sabemos esta é a primeira evidência da expressão de TDO no epitélio vaginal, na neoplasia intraepitelial cervical e carcinoma de células escamosas. Ainda, em leucócitos, especialmente aqueles com morfologia típica de polimorfonucleares, parecem ser importantes fontes de IDO na cérvix uterina

In this study we evaluated the role of tryptophan (Trp) metabolism in cervix homeostasis, bacterial vaginosis and HPV-associated lesions. The importance of Trp metabolism is due to its action on microorganisms and immune cells. Tryptophan consumption has been identified as a way to controlling bacterial growth limiting infection. On the other hand, the oxidation of Trp produces kynurenine (Kyn) which plays a key role in immunological tolerance. The formation of Kyn occurs through the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). IDO is the most studied of them within the context of infections / immune escape. More recently, TDO has also been considered in studies of cancer progression. In this thesis, we were interested in cervicovaginal epithelium IDO expression in women with bacterial vaginosis and of IDO and TDO in cervical samples of women with different degrees of cervical lesion associated with HPV. A total of 165 women attended at CAISM/UNICAMP were divided into two groups: a case group composed of women with low or high grade lesions and invasive carcinoma (n = 42) and a control group composed of women with normal cytology, independent to present genital infection (n =123). IDO was evaluated by immunocytochemistry in liquid-based cytology and IDO and TDO in cervical biopsies. Women with bacterial vaginosis had increased IDO expression in squamous cells compared to women without bacterial vaginosis (OR = 7.41, 95% CI = 2.50- 21.74; p<0.0001). In normal vaginal epithelium with or without HPV infection there was a mild IDO expression in squamous cells. In the presence of cervical intraepithelial lesions or squamous cell carcinoma, there was an increase in the number of IDO-positive dysplastic squamous cells and leukocytes; increase in IDO can also be observed in organotypic skin cultures transduced with HPV-16 E6/E7 oncoproteins. In cervical lesions, as observed for IDO, TDO was expressed in leukocytes, especially infiltrates in the stromal region and in the wall of blood vessels. The basal expression of IDO in the normal cervical epithelium and its positive regulation in HPV infection and associated lesions suggests the participation of Trp metabolism in the immunosuppressive mechanisms involved in the disease. Although some previous data have already considered the role of IDO, as far as we know this is the first evidence of the participation of TDO in the vaginal epithelium, cervical intraepithelial neoplasia and squamous cell carcinoma. In addition, in leukocytes, especially those with a typical polymorphonuclear morphology, appear to be important sources of IDO in the uterine cervix

Caffeic acid phenethyl ester protects against photothrombotic cortical ischemic injury in mice

In this study, we aimed to investigate the neuroprotective effects of caffeic acid phenethyl ester (CAPE), an active component of propolis purified from honeybee hives, on photothrombotic cortical ischemic injury in mice. Permanent focal ischemia was achieved in the medial frontal and somatosensory cortices of anesthetized male C57BL/6 mice by irradiation of the skull with cold light laser in combination with systemic administration of rose bengal. The animals were treated with CAPE (0.5–5 mg/kg, i.p.) twice 1 and 6 h after ischemic insult. CAPE significantly reduced the infarct size as well as the expression of tumor necrosis factor-α, hypoxiainducible factor-1α, monocyte chemoattractant protein-1, interleukin-1α, and indoleamine 2,3-dioxygenase in the cerebral cortex ipsilateral to the photothrombosis. Moreover, it induced an increase in heme oxygenase-1 immunoreactivity and interleukin-10 expression. These results suggest that CAPE exerts a remarkable neuroprotective effect on ischemic brain injury via its anti-inflammatory properties, thereby providing a benefit to the therapy of cerebral infarction.

Expression and Prognostic Value of Indoleamine 2,3-dioxygenase in Pancreatic Cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Indoleamine 2,3-dioxygenase (IDO), an enzyme for tryptophan metabolism through the kynurenine pathway, exhibits an immunosuppressive effect and induces immune tolerance in tumor cells. The effects of IDO on pancreatic cancer are poorly understood. This study aimed to investigate the expression and prognostic significance of IDO in pancreatic cancer.</p><p><b>METHODS</b>We evaluated the protein expression of IDO in PANC-1, CFPAC-1, and BxPC-3 cell lines with or without 48 h treatment by 500 U/ml interferon-γ (IFN-γ). We performed immunohistochemical staining and Western blot analysis for IDO expression in both pancreatic cancer and normal pancreas tissues obtained from Chinese PLA General Hospital from July 2012 to December 2013. Survival analysis was performed to correlate IDO expression and histopathologic parameters with overall survival. The Kaplan-Meier method and Cox proportional hazards regression model were conducted.</p><p><b>RESULTS</b>PANC-1, CFPAC-1, and BxPC-3 cell lines expressed IDO at the protein level, and the relative expression amount increased after stimulation with 500 U/ml IFN-γ. Immunohistochemical analysis results revealed that high IDO expression was observed in 59% of pancreatic adenocarcinoma tissues. Compared with normal pancreatic tissues, pancreatic adenocarcinoma showed significantly higher IDO expression levels, especially among patients with high tumor node metastasis (TNM) stages (χ2 = 4.550, P = 0.030), poor histological differentiation (χ2 = 5.690, P = 0.017), and lymph node metastasis (χ2 = 4.340 P = 0.037). Kaplan-Meier survival curves showed that high IDO expression was correlated with low survival rates (hazard ratio [HR] = 0.49 P = 0.009). Multivariate analysis using Cox proportional hazards model indicated that lymph node metastasis (HR = 0.35 P = 0.010) and IDO expression (HR = 0.42 P = 0.020) were two independent prognostic predictors of pancreatic adenocarcinoma.</p><p><b>CONCLUSIONS</b>The study confirmed that high IDO expression in pancreatic adenocarcinoma was related to poor prognosis of patients. These findings provided evidence that IDO was involved in pancreatic adenocarcinoma progression and might serve as a relevant therapeutic target.</p>

Inhibitory Effect of Feiji Recipe on IDO Induced Immune Escape on the Murine Model of Lewis Lung Carcinoma / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Feiji Recipe (FR) intervening indoleamine 2,3-dioxygenase (IDO) induced immune escape on the murine model of Lewis lung carcinoma. Methods Totally 48 C57BL/6 mice inoculated with Lewis lung cancer cells transfected with human (enhanced green fluorescent protein,EGFP)-IDO gene were divided into four groups according to radom digit table, i.e., the model group (administered with normal saline by gastrogavage) , the Chinese medicine group (treated with FR Decoction at the daily dose of 100 mg/g by gastrogavage), the 1-methyl-D-trytaphan (1-MT) group (administered with 1-MT mixed liquor at the daily dose of 100 mg/kg by gastrogavage), and the Paclitaxel group (treated with Paclitaxel at the daily dose of 15 mg/kg by peritoneal injection), 12 in each group. The intervention was started from the 2nd day of modeling. The survival time was observed in 24 of them. Ratios of CD4+ CD25+ FoxP3+ regulatory T cells (Treg) in the spleen were detected in the rest 24 mice by flow cytometry respectively.</p><p><b>RESULTS</b>Compared with the model group, the survival time was significantly prolonged in the Chinese medicine group and the 1-MT group (P < 0.01); ratios of Treg cells remarkably decreased in the Chinese medicine group, the 1-MT group, and the Paclitaxel group (P < 0. 01). Compared with the Paclitaxel group, the survival time was significantly prolonged in the Chinese medicine group and the 1-MT group (P < 0.01); ratios of Treg cells decreased significantly in the 1-MT group (P < 0.05).</p><p><b>CONCLUSION</b>FR could inhibit the proliferation of lung cancer cells and immune eseape, improve the immune function, and prolong the survival of tumor-bearing mice.</p>

Desenvolvimento e validação de métodos bioanalíticos para o monitoramento dos produtos das vias metabólicas do triptofano em linhagem celular de glioma humano / Development and validation of bioanalytical methods for monitoring the product of the metabolic pathways of tryptophan in human glioma cell line

O metabolismo do triptofano (Trp) se dá pela via das quinureninas (QUIN), pela via serotoninérgica (SER) e pela via das aminas traço. A primeira gera QUIN e uma variedade de outros metabólitos secundários. Quando conduzida pela enzima indolamina 2,3 dioxigenase (IDO) contribui para os fenômenos de tolerância e imune escape de células tumorais; e quando conduzida pela triptofano 2,3 dioxigenase (TDO) no fígado, participa na síntese da niacina e NAD. A via SER leva à formação do neurotransmissor serotonina (SER), que pode gerar o hormônio melatonina (MEL), respectivamente e outros metabólitos biologicamente ativos. Outra via menos estudada, a via das aminas traço, produz produtos neuroativos. Dada a abrangência e importância das rotas metabólicas do Trp, nós desenvolvemos e validamos uma metodologia bioanalítica robusta, seletiva e sensível por cromatografia líquida de alta eficiência (HPLC), acoplado espectrometria de massas (MS) para a determinação simultânea do Trp e seus 15 metabólitos. Para tanto, escolhemos para a avaliação das três vias, linhagens de glioma humano. A escolha por este tipo celular deveu-se ao grande interesse de estudos de metabolismo de Trp em células tumorais, no qual células de glioma tem sido modelo. Nos ensaios com as células de glioma acompanhamos os efeitos de um indutor e inibidores da primeira etapa de metabolização do Trp pela via das quinureninas, ou seja, IFN-γ (indutor da IDO), 1-metiltriptofano (1-MT; inibidor competitivo da IDO) e 680C91 (inibidor seletivo da TDO). Pudemos observar o impacto que a indução ou a inibição do primeiro passo teve sobre os metabólitos subsequentes e as diferenças no metabolismo das duas linhagens estudadas, A172 e T98G. A linhagem T98G só tem atividade de IDO, enquanto que a A172 tem tanto atividade IDO quanto TDO. A indução por IFN-γ mostrou que essa citocina não só atua na formação da via QUIN, mas possui um impacto modesto nas demais rotas. Observamos também que a inibição do 1-MT mostrou seu impacto nos metabólitos invdividualmente, do que a simples relação Trp-QUIN. Contudo, nosso resultados nos permitiu mostrar pela primeira vez a descrição completa dessas vias, em especial nessas linhagens celulares, podendo supor estratégias terapêuticas nessas rotas que estão relacionadas a progressão ou não tumoral

The tryptophan metabolism (Trp) takes place by means of kynurenine (QUIN), by the serotonin pathway (SER) and by the pathway of trace amines synthesis. The first generates QUIN and a variety of other secondary metabolites. When driven by the enzyme indoleamine 2,3 dioxygenase (IDO) contributes to the phenomena of tolerance and immune escape of tumor cells; and when conducted by tryptophan 2,3 -dioxygenase (TDO) in the liver, participates in the niacin synthesis NAD. The SER pathway leads to the serotonin neurotransmitter (SER) formation, which can generate the hormone melatonin (MEL), respectively and other biologically active metabolites. Another less studied amines trace synthesis pathway produces neuroactive products. Given the scope and importance of Trp metabolic pathways,we developed and validated a robust, sensitive and selective bioanalytical method by high performance liquid chromatography (HPLC) coupled mass spectrometry (MS) for simultaneous determination of TRP and its 16 metabolites. Therefore, we chose to evaluate the three routes, glioma cell lines. The initial choice of this type of cell was due to the great interest in Trp metabolism studies in tumor cells, which glioma cells has been a model. In assays with glioma cells, we followed the effects of an inductor and inhibitors of the first stage of Trp metabolism, via the kynurenine pathway, or IFN -γ (IDO inducer) 1- methyltryptophane (1- MT; competitive IDO inhibitor) and 680C91 (selective TDO inhibitor). We could observe the first step induction or inhibition impact had over the further metabolites and the metabolism differences between the two studied strains, A172 and T98G. The T98G glioma cell has only IDO activity, while the A172 has both IDO and TDO activity as well. The IFN-γ indution showed that this cytokine not only acts in the formation of QUIN route, but has a modest impact on the others routes. Inhibition of IDO showed that the competitive inhibitor has activity in itself than a simple Trp-QUIN relationship. However, our results allow us to show the first time the complete description of these pathways, in particular, in these cell lines that can assume therapeutic strategies in these routes that are related or not with tumor progression

Influence of MicroRNA-382 on Biological Properties of Human Umbilical Cord-Derived Mesenchymal Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of microRNA-382 (miR-382) on the biological properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSC).</p><p><b>METHODS</b>The mimics and inhibitor of miR-382 were transfected into hUC-MSC with lipo2000. Inverted microscopy was used to observe the morphology change of hUC-MSC. The proliferation of hUC-MSC was detected by CCK-8. Oil red O and alizarin red staining were applied to assess the adipogenic and osteogenic differentiation of hUC-MSC. Cetylpyridinium chloride was used to the quantitative analysis of osteogenic differentiation. The expression of Runx2 and some cytokines were detected by RT-PCR.</p><p><b>RESULTS</b>miR-382 did not influence the morphology, proliferation and adipogenic differentiation of hUC-MSC miR-382 inhibited the expression of Runx2, thus could inhibit the osteogenesis of hUC-MSC, being confirmed by alizarin red stain; miR-382 could influence the expression of key cytokines secreted from hUC-MSC, such as IL-6, IDO1, G-CSF, M-CSF, GM-CSF.</p><p><b>CONCLUSION</b>miR-382 decreases the expression of Runx2 and inhibites the osteogenesis of hUC-MSC. In addition, it also affects the expression of some key cytokines secreted from hUC-MSC.</p>

Induction of Indoleamine 2,3-dioxygenase by Pre-treatment with Poly(I:C) May Enhance the Efficacy of MSC Treatment in DSS-induced Colitis

Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly owing to their immunosuppressive properties. The goal of the study was to determine whether TLR ligands can enhance the therapeutic efficacy of bone marrow-derived MSCs for the treatment of inflammatory bowel disease. Mice (C57BL6) were administered with 4% dextran sulfate sodium (DSS) in drinking water for 7 days and injected with MSCs on days 1 and 3 following DSS ingestion. Our results demonstrated that among various TLR ligands, MSCs treated with polyinosinic-polycytidylic acid [poly(I:C)], which is a TLR3 ligand, more profoundly induced IDO, which is a therapeutically relevant immunosuppressive factor, without any observable phenotype change in vitro. The poly(I:C)-treated MSCs attenuated the pathologic severity of DSS-induced murine colitis when injected i.p. but not i.v. In summary, preconditioning MSCs with poly(I:C) might improve their efficacy in treating DSS-induced colitis, and this effect at least partly depends on the enhancement of their immunosuppressive activity through increasing their production of IDO.

Research Progress on role of Abnormal Tryptophan Metabolism in Immune Thrombocytopenia / 中国实验血液学杂志

Immune thrombocytopenia (ITP) is a common acquired autoimmune hematological disorders. Platelet autoantibodies lead to the decrease of platelet production and (or) increase of its destruction. The latest researches showed that the abnormal tryptophan metabolism mediated by indoleamine-2, 3-dioxygenase(IDO) is related with the pathogenesis of ITP. The patients with ITP show less expression of IDO, reduction of Treg cells and increase of autoreactive T cells and autoantibodies. CTLA-4-Ig can improve the expression of IDO in the patients with ITP, which also can inhibit the proliferation and activation of self-reactive T cells. Thus, clarifying the abnormal tryptophan metabolism mediated by IDO may provide a new idea for improving the understand of the pathogenesis and treatment of ITP. This review focuses on reasearch progress of the tryptophan metabolism mediated by IDO and ITP.

Effect of gold nanoparticles on superoxide dismutase and indoleamine 2, 3-dioxygenase in various rat tissues.

Gold nanoparticles have diverse applications and are being used in food and cosmetic industry, for drug delivery and in the diagnosis and treatment of cancer. However there is a need to study their biochemical mode of action. In this study, in vivo effect of gold nanoparticles on the activities of the two antioxidant enzymes — superoxide dismutase (SOD) and indoleamine 2,3-dioxygenase (IDO) was investigated in various tissues of rats. Rats were injected with 20 μg/kg body wt of 20 nm gold nanoparticles for three consecutive days through intraperitoneal route. The animals were sacrificed by CO2 asphyxiation 24 h after the last dose of gold nanoparticles. Results showed that treatment with gold nanoparticles caused no significant change in SOD activity in most of the tissues, except kidneys. In kidneys, gold nanoparticles caused a significant increase in SOD activity, when compared to the activity in control rats. However, treatment with gold nanoparticles altered the expression pattern of SOD activity in various tissues. For example, in control rats highest SOD activity was demonstrated in heart and least in kidneys and spleen. But, in gold nanoparticles treated rats, maximum SOD activity was observed in liver and the lowest in spleen. Gold nanoparticles caused no significant change in IDO activity in the studied tissues.

Research on the suppressive effect on transplantation rejection by indoleamine 2, 3-dioxygenase / 中华外科杂志

<p><b>OBJECTIVE</b>To study the suppressive effect of indoleamine 2, 3-dioxygenase on transplantation rejection in mice heterotopic cardiac transplantation.</p><p><b>METHODS</b>Adenovirus vector containing IDO gene was used to infect donor (C57BL/6) DC to obtain IDO(+)DC. Mouse heterotopic cardiac transplantation models were established (C57BL/6-BALB/c) and the following groups were set up, including the control group, DC injection group, TC injection group, IDO(+)DC injection group and co-injection group of IDO(+)DC and TC, 12 donors and 12 recipients in each group.Survival time of the donor heart in every group was observed. Meanwhile, donor hearts were harvested 7 days post transplantation for different examinations, including pathological examination, mRNA expression of IDO through real-time PCR, IDO protein expression through Western blot. Peripheral blood of recipients was also harvested for CD3(+)T lymphocyte apoptosis rate examination through fluorescence-activated cell sorting.One-way ANOVA and Kaplan-Meier Survival Analysis were used for statistic analysis of IDO expression, CD3(+)T lymphocyte apoptosis rate and survival time of the donor heart respectively.</p><p><b>RESULTS</b>Cadiac allograft median survival time of each group were 7.0, 7.5, 11.0, 17.5, 24.0 days respectively. Compared with control and DC injection group, IDO(+)DC, TC and co-injection group significantly prolonged the survival time of donor hearts (t = 3.523-8.449, P < 0.01). Both IDO mRNA and protein expression showed significant increase(t = 5.974-16.176, P < 0.01). The CD3(+)T lymphocyte apoptosis rate was also significantly increased (t = 6.324-38.120, P < 0.01). Compared with IDO(+)DC or TC group alone, co-injection group significantly prolonged the survival time of the donor heart (t = 5.971 and 2.831, P < 0.05). Both IDO mRNA and protein expression showed significant increase (t = 2.853-15.194, P < 0.01).Furthermore, the CD3(+)T lymphocyte apoptosis rate was significantly increased as well (t = 26.069 and 7.643, P < 0.05).</p><p><b>CONCLUSIONS</b>Suppressive effect of co-injection of IDO(+)DC and TC is much more effective than administration of IDO(+)DC or TC alone, which suggests that IDO achieved immune suppressive effect through the pathway of tryptophan depletion and accumulation of TC.</p>

The tryptophan utilization concept in pregnancy

The decrease in maternal plasma total (free + albumin-bound) tryptophan (Trp) during the third pregnancy trimester is attributed to induction of indoleamine 2,3-dioxygenase (IDO). When measured, free [Trp] is increased because of albumin depletion and non-esterified fatty acid elevation. The Trp depletion concept in pregnancy is therefore not supported because of incorrect interpretation of changes in Trp disposition and also for not addressing mouse strain differences in Trp-related responses and potential inhibition of Trp transport by the IDO inhibitor 1-methyl tryptophan. Application of the Trp utilization concept in pregnancy offers several physiological advantages favoring fetal development and successful outcome, namely provision of Trp for fetal protein synthesis and growth, serotonin for signaling pathways, kynurenic acid for neuroprotection, quinolinic acid for NAD+ synthesis, and other kynurenines for suppression of T cell responses. An excessive increase in Trp availability could compromise pregnancy by undermining T cell suppression, e.g., in pre-eclampsia.

Heme-binding-mediated negative regulation of the tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) by IDO2

Indoleamine 2,3-dioxygenases (IDOs) are tryptophan-catabolizing enzymes with immunomodulatory functions. However, the biological role of IDO2 and its relationship with IDO1 are unknown. To assess the relationship between IDO2 and IDO1, we investigated the effects of co-expression of human (h) IDO2 on hIDO1 activity. Cells co-expressing hIDO1 and hIDO2 showed reduced tryptophan metabolic activity compared with those expressing hIDO1 only. In a proteomic analysis, hIDO1-expressing cells exhibited enhanced expression of proteins related to the cell cycle and amino acid metabolism, and decreased expression of proteins related to cell survival. However, cells co-expressing hIDO1 and hIDO2 showed enhanced expression of negative regulators of cell apoptosis compared with those expressing hIDO1 only. Co-expression of hIDO1 and hIDO2 rescued the cell death induced by tryptophan-depletion through hIDO1 activity. Cells expressing only hIDO2 exhibited no marked differences in proteome profiles or cell growth compared with mock-transfectants. Cellular tryptophan metabolic activity and cell death were restored by co-expressing the hIDO2 mutant substituting the histidine 360 residue for alanine. These results demonstrate that hIDO2 plays a novel role as a negative regulator of hIDO1 by competing for heme-binding with hIDO1, and provide information useful for development of therapeutic strategies to control cancer and immunological disorders that target IDO molecules.

The role of placental indoleamine 2,3-dioxygenase in human pregnancy

Munn et al. made a scientific observation of major biological importance. For the first time they showed that in the mammal the fetus does survive an immune attack mounted by the mother, and that the mechanism responsible for the survival depends on the fetus and placenta 'actively' defending itself from attack by maternal T cells by means of an enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42) dependent localised depletion of L-tryptophan. These findings raise critical questions for disease and its prevention during human pregnancy. Specifically, the role of this mechanism (discovered in mouse) in the human, and the extent to which defective activation of this process is responsible for major clinical diseases are unknown. Therefore some key facts about this enzyme expressed in the human placenta have been studied in order to test whether Munn et al.'s findings in mouse are met for human pregnancy. This short review attempts to describe our experimental work on human placental indoleamine 2,3-dioxygenase.