RESUMO
Phenalenone derivatives sourced from fungi are polyketides that have attracted significant interest because of their diverse chemical structures and potential bioactivities. As part of our ongoing quest to discover novel natural products with biological properties from diverse natural resources, three unreported phenalenone derivatives (1-3), named ent-12-methoxyisoherqueinone (1), (-)-scleroamide (2), and (+)-scleroamide (3), together with four known phenalenone derivatives, ent-atrovenetinone (4), isoherqueinone (5), herqueinone (6), and ent-peniciherquinone (7) were isolated from the Hawaiian soil fungus Penicillium herquei FT729, collected on the Big Island, Hawaii. Compounds 2 and 3 were enantiomers, which were separated using a chiral-phase HPLC column, which provided optically pure compounds 2 and 3. The structures of the novel compounds were established by extensive spectroscopic analyses, including 1D and 2D NMR and high-resolution ESIMS. Their absolute configurations were determined using quantum chemical electronic circular dichroism (ECD) calculations. The inhibitory activity of the isolated compounds (1-7) against indoleamine 2,3-dioxygenase 1 (IDO1) was assessed. Compounds 1, 5-7 inhibited IDO1, with IC50 values of 32.59, 36.86, 19.05, and 24.18 µM, respectively. These findings demonstrated that the phenalenone derivatives 1 and 5-7, as IDO1 inhibitors, are promising anticancer immunotherapeutic agents.
Assuntos
Antineoplásicos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Penicillium , Fenalenos/farmacologia , Microbiologia do Solo , Antineoplásicos/química , Havaí , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Concentração Inibidora 50 , Fenalenos/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Photodynamic therapy (PDT) may elicit antitumor immune response in addition to killing cancer cells. However, PDT as a monotherapy often fails to induce a strong immunity. Immune checkpoint inhibitors, which selectively block regulatory axes, may be used in combination with PDT to improve treatment outcomes. Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme and an important meditator of tumor immune escape. Combination therapy with PDT and IDO-targeted immune checkpoint blockage is promising but has been seldom been explored. METHODS: Herein we report a composite nanoparticle that allows for simultaneous delivery of photosensitizer and IDO inhibitor. Briefly, we separately load ZnF16Pc, a photosensitizer, and NLG919, an indoleamine 2,3-dioxygenase (IDO) inhibitor, into ferritin and poly(lactide-co-glycolic)-block-poly(ethylene glycol) (PEG-PLGA) nanoparticles; we then conjugate these two compartments to form a composite nanoparticle referred to as PPF NPs. We tested combination treatment with PPF NPs first in vitro and then in vivo in B16F10-tumor bearing C57/BL6 mice. RESULTS: Our results showed that PPF NPs can efficiently encapsulate both ZnF16Pc and NLG919. In vivo studies found that the combination treatment led to significantly improved tumor suppression and animal survival. Moreover, the treatment increased tumor infiltration of CD8+ T cells, while reducing frequencies of MDSCs and Tregs. 30% of the animals showed complete tumor eradication, and they successfully rejected a second tumor inoculation. Overall, our studies introduce a unique composite nanoplatform that allows for co-delivery of photosensitizer and IDO inhibitor with minimal inter-species interference, which is ideal for combination therapy.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Imunoterapia/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Nanoconjugados/uso terapêutico , Nanopartículas/uso terapêutico , Fotoquimioterapia/métodos , Animais , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Inibidores Enzimáticos/farmacologia , Ferritinas , Humanos , Imidazóis , Isoindóis , Camundongos , Células Supressoras Mieloides , Nanoconjugados/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Fármacos Fotossensibilizantes/uso terapêuticoRESUMO
Background: Tryptophan metabolism via the kynurenine pathway is considered the link between the immune and endocrine systems. Dysregulation of serotonergic transmission can stem from the direct influence of interferon-α on the activity of serotonergic receptors 5-HT1A and 5-HT2A, and from its indirect effect on tryptophan metabolism. Induction of the kynurenine pathway increases the concentration of neurotoxic kynurenine metabolites, and the activity of kynurenine derivatives is linked to the onset of depression. The aim of our study was to evaluate the relationships between depressive symptoms and kynurenine, tryptophan, anthranilic acid and kynurenic acid concentrations, indolamine 2,3-dioxygenase (IDO) activity and tryptophan availability to the brain. Methods: The study followed a prospective longitudinal cohort design. We evaluated 101 patients with chronic hepatitis C who were treated with pegylated interferon-α2a, and 40 controls who were awaiting treatment. We evaluated the relationships between total score on the Montgomery-Åsberg Depression Rating Scale and kynurenine, tryptophan, anthranilic acid and kynurenic acid concentrations, IDO activity and tryptophan availability to the brain. A logistic regression model was adapted for the diagnosis of major depressive disorder at each time point, taking into account changes in parameters of the kynurenine pathway between a given time point and the baseline measurement. Results: Of the treated patients, 44% fulfilled the criteria for major depressive disorder at least once during the 24 weeks of treatment. Anthranilic acid concentrations were significantly increased compared to baseline for all time points except week 2. Tryptophan availability showed a significant decrease (ß = -0.09, p = 0.01) only in week 12 of treatment. Over time, kynurenine, tryptophan and anthranilic acid concentrations, as well as IDO activity and tryptophan availability to the brain, were significantly associated with total score on the Montgomery-Åsberg Depression Rating Scale. A logistic regression model revealed that participants with decreased tryptophan availability to the brain at 12 weeks of treatment and participants with increased anthranilic acid concentrations at week 24 of treatment were at increased risk for diagnosis of major depressive disorder (odds ratios 2.92 and 3.59, respectively). Limitations: This study had an open-label design in a population receiving naturalistic treatment. Conclusion: The present study provides the first direct evidence of the role of anthranilic acid in the pathogenesis of inflammation-induced major depressive disorder during treatment for hepatitis C with pegylated interferon-α2a.
Assuntos
Antivirais/farmacologia , Depressão , Transtorno Depressivo Maior , Hepatite C Crônica/tratamento farmacológico , Fatores Imunológicos/farmacologia , Interferon-alfa/farmacologia , Polietilenoglicóis/farmacologia , Ribavirina/farmacocinética , ortoaminobenzoatos/metabolismo , Adulto , Antivirais/efeitos adversos , Estudos Transversais , Depressão/imunologia , Depressão/metabolismo , Depressão/fisiopatologia , Transtorno Depressivo Maior/imunologia , Transtorno Depressivo Maior/metabolismo , Transtorno Depressivo Maior/fisiopatologia , Feminino , Humanos , Fatores Imunológicos/efeitos adversos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon-alfa/efeitos adversos , Ácido Cinurênico/metabolismo , Cinurenina/efeitos dos fármacos , Cinurenina/metabolismo , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis/efeitos adversos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/farmacologia , Ribavirina/efeitos adversos , Triptofano/efeitos dos fármacos , Triptofano/metabolismo , ortoaminobenzoatos/sangueRESUMO
Conversion of tryptophan to N-formylkynurenine is the first and rate-limiting step of the tryptophan metabolic pathway (i.e., the kynurenine pathway). This conversion is catalyzed by three enzyme isoforms: indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), and tryptophan 2,3-dioxygenase (TDO). As this pathway generates numerous metabolites that are involved in various pathological conditions, IDOs and TDO represent important targets for therapeutic intervention. This pathway has especially drawn attention due to its importance in tumor resistance. Over the last decade, a large number of IDO and TDO inhibitors have been developed, many of which have entered clinical trials. Here, detailed structural comparisons of these three enzymes (with emphasis on their active sites), their involvement in cellular signaling, and their role(s) in pathological conditions are discussed. Furthermore, the most important recent inhibitors described in papers and patents and involved in clinical trials are reviewed, with a focus on both selective and multiple inhibitors. A short overview of the biochemical and cellular assays used for inhibitory potency evaluation is also presented. This review summarizes recent advances on IDO and TDO as potential drug targets, and provides the key features and perspectives for further research and development of potent inhibitors of the kynurenine pathway.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase , Neoplasias , Triptofano Oxigenase , Ensaios Clínicos como Assunto , Previsões , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Triptofano Oxigenase/efeitos dos fármacosRESUMO
l-tryptophan induces IDO (indoleamine 2,3-dioxygenase) 1-dependent vasodilation. IDO1 is expressed in placental endothelial cells and downregulated in preeclampsia. Hypothesizing that this may contribute to diminished placental perfusion, we studied l-tryptophan-induced vasodilation in healthy and early-onset preeclampsia placental arteries, focusing on placental kynurenine pathway alterations. Despite IDO1 downregulation, kynurenine pathway metabolite concentrations (measured with ultra-performance liquid chromatography-tandem mass spectrometry) were unaltered in preeclamptic versus healthy placentas. Most likely, this is due to enhanced l-tryptophan uptake, evidenced by increased l-tryptophan levels in preeclamptic placentas. Ex vivo perfused cotyledons from healthy and preeclamptic placentas released similar amounts of l-tryptophan and kynurenine pathway metabolites into the circulations. This release was not altered by adding l-tryptophan in the maternal circulation, suggesting that l-tryptophan metabolites act intracellularly. Maternally applied l-tryptophan did appear in the fetal circulation, confirming placental passage of this essential amino acid. After in vitro incubation of placental arteries with IDO1-upregulating cytokines interferon-γ and tumor necrosis factor-α, l-tryptophan induced vasodilation. This vasodilation was attenuated by both IDO1 and nitric oxide (NO) synthase inhibitors. Despite IDO1 downregulation, l-tryptophan-induced relaxation was enhanced in preeclamptic versus healthy placental arteries. However, cytokine stimulation additionally upregulated the LAT (l-type amino acid transporter) 1 in preeclamptic placental arteries only. Vasodilation to the lipophilic, transporter independent ethyl ester of l-tryptophan was reduced in preeclamptic versus healthy placental arteries, in agreement with reduced IDO1 expression. In conclusion, l-tryptophan induces IDO1- and NO-dependent relaxation in placental arteries, which is determined by l-tryptophan uptake rather than IDO1 expression. Increased l-tryptophan uptake might compensate for reduced IDO1 expression in preeclamptic placentas.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Placenta/irrigação sanguínea , Pré-Eclâmpsia/fisiopatologia , Triptofano/farmacologia , Vasodilatação/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Adulto , Artérias/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Citocinas/farmacologia , Indução Enzimática/efeitos dos fármacos , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/metabolismo , Troca Materno-Fetal , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Placenta/efeitos dos fármacos , Placenta/metabolismo , Pré-Eclâmpsia/enzimologia , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Triptofano/análogos & derivadosRESUMO
AIM: To test the hypothesis that the antidepressant-like effect of omega-3 polyunsaturated fatty acids is related to the Indoleamine-2,3-Dioxygenase (IDO) inhibition. METHODS: Animals were supplemented for 50 days with 3.0 g/kg of Fish Oil (FO) or received water (Control group - C), via gavage. At the end of this period, both groups were injected with LPS 24 h before the modified forced swim test (MFST) and the open field. To assess the possible involvement of IDO in the FO effects, we performed two independent experiments, using two IDO inhibitors: the direct inhibitor 1-methyl-DL-tryptophan (1-MT) and the anti-inflammatory drug minocycline (MINO), administered 23 h, 5 h and 1 h before the tests. After the tests, the animals' hippocampi were removed for quantification of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) by HPLC, and for IDO expression by western blot. RESULTS: LPS induced a depressive-like state in the animals, and this effect was blocked by 1-MT, MINO and FO. Regardless of IDO inhibition, FO supplemented animals displayed an antidepressant-like response by increasing swimming and decreasing immobility frequencies in the MFST when compared to the control group. The immune challenge induced an over-expression of IDO and reduced hippocampal 5-HT levels, both of which were reversed by MINO and FO. CONCLUSION: FO induced a pronounced antidepressant-like effect and prevented LPS-induced depressive-like behavior, and this effect was related to decreased IDO expression and increased 5-HT levels in the hippocampus.
Assuntos
Anti-Inflamatórios/farmacologia , Antidepressivos/farmacologia , Depressão/metabolismo , Depressão/prevenção & controle , Óleos de Peixe/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase , Minociclina/farmacologia , Serotonina/metabolismo , Triptofano/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Antidepressivos/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Depressão/induzido quimicamente , Suplementos Nutricionais , Ácidos Graxos Ômega-3/farmacologia , Óleos de Peixe/administração & dosagem , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Minociclina/administração & dosagem , Ratos , Ratos Wistar , Triptofano/administração & dosagemRESUMO
Chlamydia trachomatis infections are the most prevalent sexually transmitted infections with potentially debilitating sequelae, such as infertility. Mouse models are generally used for vaccine development, to study the immune response and histopathology associated with Chlamydia infection. An important question regarding murine models is the in vivo identification of murine host genes responsible for the elimination of the murine and human Chlamydia strains. RNA sequencing of the Chlamydia muridarum infected BALB/c lung transcriptome revealed that several genes with direct antichlamydial functions were induced at the tissue level, including the already described and novel members of the murine interferon-inducible GTPase family, the CXCL chemokines CXCL9, CXCL11, immunoresponsive gene 1, nitric oxide synthase-2 (iNOS), and lipocalin-2. Indoleamine 2,3-dioxygenase 1-2 (IDO1-2) previously described potent antichlamydial host enzymes were also highly expressed in the infected murine lungs. This finding was novel, since IDO was considered as a unique human antichlamydial defense gene. Besides a lower level of epithelial cell positivity, immunohistochemistry showed that IDO1-2 proteins were expressed prominently in macrophages. Detection of the tryptophan degradation product kynurenine and the impact of IDO inhibition on Chlamydia muridarum growth proved that the IDO1-2 proteins were functionally active. IDO1-2 activity also increased in Chlamydia muridarum infected C57BL/6 lung tissues, indicating that this phenomenon is not mouse strain specific. Our study shows that the murine antichlamydial response includes a variety of highly up-regulated defense genes in vivo. Among these genes the antichlamydial effectors IDO1-2 were identified. The potential impact of murine IDO1-2 expression on Chlamydia propagation needs further investigation.
Assuntos
Infecções por Chlamydia/metabolismo , Chlamydia muridarum/efeitos dos fármacos , Chlamydia muridarum/metabolismo , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Pulmão/metabolismo , Animais , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Infecções por Chlamydia/genética , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina , Lipocalina-2/genética , Lipocalina-2/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Transcriptoma , Triptofano/análogos & derivados , Triptofano/antagonistas & inibidores , Triptofano/metabolismoRESUMO
Major depression and anxiety are highly incapacitating psychiatric disorders often present simultaneously, and the causal relationship between these disorders and inflammation are under extensive investigation. The treatment for this comorbidity still relies on drugs acting on the serotonergic neurotransmission, but the modulation of immune-inflammatory pathways has attained an increasing interest in the drug discovery. We have previously demonstrated that the selenoorganic compound 3-[(4-chlorophenyl)selanyl]-1-methyl-1H-indole (CMI) possess antioxidant, anti-inflammatory, antinociceptive and antidepressant-like effect in mice. Considering these pharmacological properties and the structural similarities between tryptophan, serotonin and CMI, the aim of the present study was to investigate whether CMI ameliorates depression- and anxiogenic-like behavior induced by lipopolysaccharide (LPS) in Swiss male mice by modulating the serotonergic system and reducing neuroinflammation. The administration of CMI (1â¯mg/kg, i.g) reversed the behavioral deficits induced by LPS (0.83â¯mg/kg, i.p) in the tail suspension test, splash test and elevated plus maze. The pre-treatment of mice with WAY100635 (5-HT1A receptor antagonist), ketanserin (5-HT2A/2C receptor antagonist) and ondansetron (5-HT3 receptor antagonist) prevented the antidepressant- and anxiolytic-like effect elicited by CMI treatment after the LPS challenge. The administration of CMI also counteracted the increased expression of pro-inflammatory cytokines and indoleamine 2,3-dioxygenase (IDO) in the prefrontal cortex and hippocampus of mice challenged with LPS. Additionally, a molecular docking analysis showed that CMI binds to the active site of the serotonin transporter and IDO. These findings suggest that CMI reversed behavioral and biochemical alterations in the depression-anxiety comorbidity induced by LPS, possibly by modulation of neuroinflammatory mediators and the serotonergic system.
Assuntos
Ansiolíticos/farmacologia , Antidepressivos/farmacologia , Ansiedade/tratamento farmacológico , Comportamento Animal/efeitos dos fármacos , Depressão/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indóis/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Compostos de Selênio/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Animais , Ansiedade/induzido quimicamente , Ansiedade/imunologia , Depressão/induzido quimicamente , Depressão/imunologia , Modelos Animais de Doenças , Lipopolissacarídeos , Masculino , Camundongos , Simulação de Acoplamento MolecularRESUMO
BACKGROUND: Over-expression and over-activation of immunosuppressive enzyme indoleamine 2, 3 -dioxygenase 1 (IDO1) is a key mechanism of cancer immune escape. However, the regulation of IDO1 has not been fully studied. The relation between hydrogen sulfide (H2S) and IDO1 is unclear. METHODS: The influences of endogenous and exogenous H2S on the expression of IDO1, iNOS and NF-κB and STAT3 signaling proteins were investigated using qPCR or western blot, and the production of nitric oxide (NO) was analyzed by nitrate/nitrite assay in Cse-/- mice and MCF-7 and SGC-7901 cells. The effect of H2S on IDO1 activity was investigated by HPLC and in-vitro enzymatic assay. The effect of H2S on tryptophan metabolism was tested by luciferase reporter assay in MCF-7 and SGC-7901 cells. The correlation between H2S-generating enzyme CSE and IDO1 was investigated by immunostaining and heatmaps analysis in clinical specimens and tissue arrays of hepatocellular carcinoma (HCC) patients. The immunotherapeutic effects of H2S on H22 HCC-bearing mice were investigated. RESULTS: Using Cse-/- mice, we found that H2S deficiency increased IDO1 expression and activity, stimulated NF-κB and STAT3 pathways and decreased the expression of NO-generating enzyme Inos. Using IDO1-expressing MCF-7 and SGC-7901 cells, we found that exogenous H2S inhibited IDO1 expression by blocking STAT3 and NF-κB pathways, and decreased IDO1 activity via H2S/NO crosstalk, and combinedly decreased the tryptophan metabolism. The negative correlation between H2S-generating enzyme CSE and IDO1 was further validated in clinical specimens and tissue arrays of HCC patients. Additionally, H2S donors effectively restricted the tumor development in H22 HCC-bearing mice via downregulating IDO1 expression, inducing T-effector cells and inhibiting MDSCs. CONCLUSIONS: Thus, H2S, as a novel negative regulator of IDO1, shows encouraging antitumor immunotherapeutic effects and represents a novel therapeutic target in cancer therapy.
Assuntos
Carcinoma Hepatocelular/enzimologia , Sulfeto de Hidrogênio/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Neoplasias Hepáticas/enzimologia , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Imunoterapia/métodos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Células Tumorais CultivadasRESUMO
Marine plants and animals have omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). EPA is required for biological processes, but humans are unable to synthesize them and must be obtained from dietary sources. EPA has been used as an antitumor agent but the molecular mechanisms for the regulation of tumor microenvironment immunity by EPA are still unknown. The indoleamine 2,3-dioxygenase 1 (IDO) catalyzes conversion of tryptophan to kynurenine to induce immune evasion in tumor microenvironment. In this study, EPA inhibited the expression of IDO via downregulation of protein kinase B (Akt)/mammalian targets of rapamycin (mTOR) signaling pathway in tumor cells. Meanwhile, a significant decrease in kynurenine levels and increase in T cell survival were observed after tumor cells treated with EPA. The results demonstrated that EPA can activate host antitumor immunity by inhibiting tumor IDO expression. Therefore, our finding suggests that EPA can be enormous potential for cancer immunotherapy.
Assuntos
Ácido Eicosapentaenoico/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Animais , Regulação para Baixo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Linfócitos T , Células Tumorais CultivadasRESUMO
×Ö¹ndoleamine-2,3-dioxygenase (IDO) plays a role in immune regulation. Increased IDO activity was reported in systemic lupus erythematosus (SLE). We investigated the effects of the tolerogenic peptide hCDR1, shown to ameliorate lupus manifestations, on IDO gene expression. mRNA was prepared from splenocytes of hCDR1- treated SLE-afflicted (NZBxNZW)F1 mice, from blood samples of lupus patients, collected before and after their in vivo treatment with hCDR1 and from peripheral blood mononuclear cells (PBMC) of patients incubated with hCDR1. IDO gene expression was determined by real-time RT-PCR. hCDR1 significantly down-regulated IDO expression in SLE-affected mice and in lupus patients (treated in vivo and in vitro). No effects were observed in healthy donors or following treatment with a control peptide. Diminished IDO gene expression was associated with hCDR1 beneficial effects. Our results suggest that the hCDR1-induced FOXP3 expressing regulatory T cells in lupus are not driven by IDO but rather by other hCDR1 regulated pathways.
Assuntos
Anticorpos Monoclonais/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/genética , Fragmentos de Peptídeos/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Adulto , Idoso , Animais , Anticorpos Monoclonais/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Regulação para Baixo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Baço/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Both depression and cancer are related to a dysregulation of inflammatory and immune pathways. Indeed, depression is associated with increased expression of interferon-γ, interleukin-1ß, and tumor necrosis factor α (TNF-α). In contrast, reductions of the activity of major histocompatibility complex protein molecules - class I and class II and natural killer cells are also observed. Similarly, cancers present elevated levels of TNF-α, reduced major histocompatibility complex class I and II, and natural killer cells. Indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of the tryptophan catabolite (TRYCAT) pathway, is induced by interferon-γ, interleukin-6, TNF-α, and oxidative stress. IDO catabolizes tryptophan, the amino acid precursor of serotonin and melatonin, to the metabolites collectively called TRYCATs. TRYCAT pathway activation is accompanied by downregulation of immune cell proliferation, function, and survival. The increase in IDO activity in tumor microenvironments is related to tumor cell escape from immune surveillance. Despite the evidence of inflammatory mechanisms underlying cancer and depression, it is important to emphasize that both diseases are heterogeneous and, as such, inflammatory mechanisms may not be relevant to all patients. Thus, the purpose of this review is to examine whether detrimental TRYCATs - synthesis of which increases in depression and cancer - are a pathophysiological link between the two diseases, and whether IDO is a potential pharmacological target for the treatment of the comorbid depression and cancer.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Triptofano/metabolismo , Triptofano/fisiologia , Animais , Depressão/imunologia , Depressão/metabolismo , Depressão/fisiopatologia , Transtorno Depressivo/fisiopatologia , Humanos , Imunidade/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indóis , Inflamação , Neoplasias/imunologia , Neoplasias/fisiopatologia , Estresse Oxidativo , Serotonina , Transdução de Sinais/fisiologiaRESUMO
Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.
Assuntos
Adjuvantes Imunológicos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Fibrose Cística/genética , Citocinas/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Timosina/análogos & derivados , Animais , Autofagia/efeitos dos fármacos , Western Blotting , Linhagem Celular , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Inflamação , Camundongos , Camundongos Endogâmicos CFTR , Técnicas de Patch-Clamp , Estabilidade Proteica/efeitos dos fármacos , Células RAW 264.7 , Mucosa Respiratória/citologia , Timalfasina , Timosina/farmacologia , Ubiquitina Tiolesterase/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Type 1 diabetes (T1D) originates from autoimmune ß-cell destruction. IMT504 is an immunomodulatory oligonucleotide that increases mesenchymal stem cell cloning capacity and reverts toxic diabetes in rats. Here, we evaluated long-term (20 doses) and short-term (2-6 doses) effects of IMT504 (20 mg·kg(-1)·day(-1) sc) in an immunodependent diabetes model: multiple low-dose streptozotocin-injected BALB/c mice (40 mg·kg(-1)·day(-1) ip for 5 consecutive days). We determined blood glucose, glucose tolerance, serum insulin, islet morphology, islet infiltration, serum cytokines, progenitor cell markers, immunomodulatory proteins, proliferation, apoptosis, and islet gene expression. IMT504 reduced glycemia, induced ß-cell recovery, and impaired islet infiltration. IMT504 induced early blood glucose decrease and infiltration inhibition, increased ß-cell proliferation and decreased apoptosis, increased islet indoleamine 2,3-dioxygenase (IDO) expression, and increased serum tumor necrosis factor and interleukin-6 (IL-6). IMT504 affected islet gene expression; preproinsulin-2, proglucagon, somatostatin, nestin, regenerating gene-1, and C-X-C motif ligand-1 cytokine (Cxcl1) increased in islets from diabetic mice and were decreased by IMT504. IMT504 downregulated platelet endothelial cell adhesion molecule-1 (Pecam1) in islets from control and diabetic mice, whereas it increased regenerating gene-2 (Reg2) in islets of diabetic mice. The IMT504-induced increase in IL-6 and islet IDO expression and decreased islet Pecam1 and Cxcl1 mRNA expression could participate in keeping leukocyte infiltration at bay, whereas upregulation of Reg2 may mediate ß-cell regeneration. We conclude that IMT504 effectively reversed immunodependent diabetes in mice. Corroboration of these effects in a model of autoimmune diabetes more similar to human T1D could provide promising results for the treatment of this disease.
Assuntos
Glicemia/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , RNA Mensageiro/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL1/efeitos dos fármacos , Quimiocina CXCL1/genética , Citocinas/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Modelos Animais de Doenças , Teste de Tolerância a Glucose , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-6/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Litostatina/efeitos dos fármacos , Litostatina/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nestina/efeitos dos fármacos , Nestina/genética , Proteínas Associadas a Pancreatite , Molécula-1 de Adesão Celular Endotelial a Plaquetas/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Proglucagon/efeitos dos fármacos , Proglucagon/genética , Precursores de Proteínas/efeitos dos fármacos , Precursores de Proteínas/genética , Proteínas/efeitos dos fármacos , Proteínas/genética , RNA Mensageiro/metabolismo , Somatostatina/efeitos dos fármacos , Somatostatina/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transcriptoma/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by a progressive cognitive decline along with various neuropsychiatric symptoms, including depression and anxiety. Increasing evidence has been proposed the activation of the tryptophan-degrading indoleamine-2,3-dyoxigenase (IDO), the rate-limiting enzyme of kynurerine pathway (KP), as a pathogenic factor of amyloid-beta (Aß)-related inflammation in AD. In the current study, the effects of an intracerebroventricular (i.c.v.) injection of Aß1-42 peptide (400pmol/mice; 3µl/site) on the regulation of KP biomarkers (IDO activity, tryptophan and kynurerine levels) and the impact of Aß1-42 on neurotrophic factors levels were investigated as potential mechanisms linking neuroinflammation to cognitive/emotional disturbances in mice. Our results demonstrated that Aß1-42 induced memory impairment in the object recognition test. Aß1-42 also induced emotional alterations, such as depressive and anxiety-like behaviors, as evaluated in the tail suspension and elevated-plus maze tests, respectively. We observed an increase in levels of proinflammatory cytokines in the Aß1-42-treated mice, which led to an increase in IDO activity in the prefrontal cortex (PFC) and the hippocampus (HC). The IDO activation subsequently increased kynurerine production and the kynurenine/tryptophan ratio and decreased the levels of neurotrophic factors in the PFC and HC, which contributed to Aß-associated behavioral disturbances. The inhibition of IDO activation by IDO inhibitor 1-methyltryptophan (1-MT), prevented the development of behavioral and neurochemical alterations. These data demonstrate that brain IDO activation plays a key role in mediating the memory and emotional disturbances in an experimental model based on Aß-induced neuroinflammation.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Ansiedade/induzido quimicamente , Comportamento Animal/efeitos dos fármacos , Depressão/induzido quimicamente , Hipocampo/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/induzido quimicamente , Cinurenina/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Fatores de Crescimento Neural/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Triptofano/efeitos dos fármacos , Peptídeos beta-Amiloides/administração & dosagem , Animais , Modelos Animais de Doenças , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Injeções Intraventriculares , Camundongos , Fragmentos de Peptídeos/administração & dosagem , Reconhecimento Psicológico/efeitos dos fármacosRESUMO
Although T cells expressing CD19-specific chimeric antigen receptors (CARs) are a promising new therapy for B-cell malignancies, objective responses are observed at lower frequencies in patients with lymphoma than in those with acute B-cell leukemia. We postulated that the tumor microenvironment suppresses CAR-expressing T cells (CARTs) through the activity of indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme that converts tryptophan into metabolites that inhibit T -: cell activity. To investigate the effects of tumor IDO on CD19-CART therapy, we used a xenograft lymphoma model expressing IDO as a transgene. CD19-CARTs inhibited IDO-negative tumor growth but had no effect on IDO-positive tumors. An IDO inhibitor (1-methyl-tryptophan) restored IDO-positive tumor control. Moreover, tryptophan metabolites inhibited interleukin (IL)-2-, IL-7-, and IL-15-dependent expansion of CARTs; diminished their proliferation, cytotoxicity, and cytokine secretion in vitro in response to CD19 recognition; and increased their apoptosis. Inhibition of CD19-CARTs was not mitigated by the incorporation of costimulatory domains, such as 4-1BB, into the CD19-CAR. Finally, we found that fludarabine and cyclophosphamide, frequently used before CART administration, downregulated IDO expression in lymphoma cells and improved the antitumor activity of CD19-CART in vivo. Because tumor IDO inhibits CD19-CARTs, antagonizing this enzyme may benefit CD19-CART therapy.
Assuntos
Antígenos CD19/imunologia , Imunoterapia/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Linfoma/enzimologia , Linfoma/imunologia , Linfócitos T/imunologia , Animais , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Ciclofosfamida/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Citometria de Fluxo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão , Linfócitos T/efeitos dos fármacos , Vidarabina/análogos & derivados , Vidarabina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: There is an urgent need for improved vaccines to protect against tuberculosis. The currently available vaccine Bacille Calmette-Guerin (BCG) has varying immunogenicity and efficacy across different populations for reasons not clearly understood. MVA85A is a modified vaccinia virus expressing antigen 85A from Mycobacterium tuberculosis which has been in clinical development since 2002 as a candidate vaccine to boost BCG-induced protection. A recent efficacy trial in South African infants failed to demonstrate enhancement of protection over BCG alone. The immunogenicity was lower than that seen in UK trials. The enzyme Indoleamine 2,3-dioxygenase (IDO) catalyses the first and rate-limiting step in the breakdown of the essential amino acid tryptophan. T cells are dependent on tryptophan and IDO activity suppresses T-cell proliferation and function. METHODS: Using samples collected during phase I trials with MVA85A across the UK and South Africa we have investigated the relationship between vaccine immunogenicity and IDO using IFN-γ ELISPOT, qPCR and liquid chromatography mass spectrometry. RESULTS: We demonstrate an IFN-γ dependent increase in IDO mRNA expression in peripheral blood mononuclear cells (PBMC) following MVA85A vaccination in UK subjects. IDO mRNA correlates positively with the IFN-γ ELISPOT response indicating that vaccine specific induction of IDO in PBMC is unlikely to limit the development of vaccine specific immunity. IDO activity in the serum of volunteers from the UK and South Africa was also assessed. There was no change in serum IDO activity following MVA85A vaccination. However, we observed higher baseline IDO activity in South African volunteers when compared to UK volunteers. In both UK and South African serum samples, baseline IDO activity negatively correlated with vaccine-specific IFN-γ responses, suggesting that IDO activity may impair the generation of a CD4+ T cell memory response. CONCLUSIONS: Baseline IDO activity was higher in South African volunteers when compared to UK volunteers, which may represent a potential mechanism for the observed variation in vaccine immunogenicity in South African and UK populations and may have important implications for future vaccination strategies. TRIAL REGISTRATION: Trials are registered at ClinicalTrials.gov; UK cohort NCT00427830, UK LTBI cohort NCT00456183, South African cohort NCT00460590, South African LTBI cohort NCT00480558.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , RNA Mensageiro/metabolismo , Vacinas contra a Tuberculose/farmacologia , Tuberculose/prevenção & controle , Adulto , Vacina BCG , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , ELISPOT , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/sangue , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , África do Sul , Reino Unido , Vacinação , Vacinas de DNA , Adulto JovemRESUMO
BACKGROUND: The aim of this prospective study was to gain a more comprehensive picture of the biopsychosocial effects of interferon-α (IFN-α) treatment of patients with chronic hepatitis C (HCV). The predictors of depressive development and changes in health-related quality of life, life satisfaction and cognitive ability were measured with the inclusion of the social context. Furthermore, the effects of IFN-α treatment on indoleamine 2,3-dioxygenase, the level of tryptophan supply in the brain, the development of neurotoxic kynurenine metabolites and the thyroid glands were investigated. Therefore, for the first time the conditions for the development of depressive episodes in HCV patients treated with IFN-α were examined over the entire period of treatment as well as 3 months later, applying a holistic biopsychosocial model. METHOD: Psychiatric and biological assessments were carried out at 6 different times: before, during (at 1, 3, 6 and 9 months) and after the end of IFN-α treatment. RESULTS: During IFN-α treatment 22 (53.7%) of 41 patients fulfilled the criteria for a treatment-related depressive disorder at least once during treatment. Contributing factors are tryptophan depletion (tryptophan to competing amino acids quotient), increased neurotoxic challenge (kynurenine to kynurenic acid quotient), less social support, female gender, preexisting psychiatric vulnerability, means of transmission, low financial security, impaired sexual satisfaction, small circle of friends, impaired physical role, strong body pain, low general health and vitality, reduced social functioning, impaired mental health and impaired emotional role. CONCLUSIONS: The awareness of relevant risk factors of IFN-α treatment-induced depression is essential to develop preventative treatment strategies.
Assuntos
Transtorno Depressivo/induzido quimicamente , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/efeitos adversos , Qualidade de Vida , Adulto , Análise de Variância , Transtorno Depressivo/metabolismo , Transtorno Depressivo/psicologia , Feminino , Hepatite C Crônica/metabolismo , Hepatite C Crônica/psicologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Entrevista Psicológica , Ácido Cinurênico/metabolismo , Cinurenina/metabolismo , Masculino , Modelos Teóricos , Testes Neuropsicológicos/estatística & dados numéricos , Satisfação Pessoal , Estudos Prospectivos , Escalas de Graduação Psiquiátrica/estatística & dados numéricos , Fatores de Risco , Fatores Sexuais , Apoio Social , Inquéritos e Questionários , Testes de Função Tireóidea , Triptofano/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-oxidizing enzyme with immune-inhibitory effects. The aim of this study was to investigate the expression of IDO by lipopolysaccharide (LPS), a component of gram-negative bacteria, in human periodontal ligament (PDL) cells. MATERIAL AND METHODS: Human PDL cells and gingival fibroblasts (GFs) were prepared from explants of human PDLs and from gingival tissues of clinically healthy donors, respectively. Real-time RT-PCR, western blotting and the IDO enzyme assay were performed to determine the expression of IDO following LPS treatment of cells. LPS was injected into mice tail veins to evaluate the effects of LPS in vivo in the maxillary first molar. Immunofluorescence staining and histological analysis were followed to localize IDO in mouse PDL. RESULTS: The level of expression of IDO mRNA in primary human PDL cells after LPS treatment was increased in a dose-dependent manner, reaching a peak 8 h after LPS treatment. The expression and activities of IDO protein were significantly increased in comparison with those of the control. In addition, the increased production of kynurenine in culture medium was observed 72 h after LPS treatment. In the immunofluorescence findings, stronger immunoreactivities were shown in PDL than in gingival tissues in the maxillae. In accordance with the immunofluorescence findings, LPS treatment induced a strong up-regulation of IDO mRNA in human PDL cells, whereas human GFs showed only a weak response to LPS. CONCLUSION: These results clearly show that IDO was induced by LPS in primary human PDL cells, suggesting that PDL might be involved in the regulation of oral inflammatory disease.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Ligamento Periodontal/enzimologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Escherichia coli , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Imunofluorescência , Gengiva/citologia , Gengiva/efeitos dos fármacos , Gengiva/enzimologia , Humanos , Interleucina-1beta/efeitos dos fármacos , Cinurenina/análise , Cinurenina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Fatores de Tempo , Regulação para CimaRESUMO
We have previously shown that pre- and post-transplant infusions of donor splenocytes treated with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI-SPs) provide permanent donor-specific protection of islet allografts. The efficacy of donor ECDI-SPs in protecting vascularized cardiac allografts and mechanism(s) of protection are unknown. In this study, we show that infusions of ECDI-SPs significantly prolong cardiac allograft survival concomitant with an impressive accumulation of CD11b(+) IDO(+) cells in the cardiac allograft, and that the presence of this population is dependent on Gr1(+) cells. Consequently, depletion of Gr1(+) cells or inhibition of indoleamine 2,3 dioxygenase (IDO) activity abrogates graft protection by ECDI-SPs infusions. In addition, T cells from ECDI-SPs treated recipients secrete high levels of interleukin 10 and interleukin 13 upon in vitro restimulation, which are also dampened in recipients treated with the IDO inhibitor. Furthermore, combination of donor ECDI-SPs with a short course of rapamycin provides indefinite cardiac allograft survival in 100% of the recipients. These findings reveal a novel mechanism of donor ECDI-SPs in inducing cardiac transplant tolerance and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.