Development of a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry for human urine (RaDPi-U).

Drug screening tests are mandatory in the search for drugs in forensic biological samples, and immunological methods and mass spectrometry (e.g., gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry) are commonly used for that purpose. However, these methods have some drawbacks, and developing new screening methods is required. In this study, we develop a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry (PESI-MS/MS), which is an ambient ionization mass spectrometry method, for human urine, named RaDPi-U. RaDPi-U is carried out in three steps: (1) mixing urine with internal standard (IS) solution and ethanol, followed by vortexing; (2) pipetting the mixture onto a sample plate for PESI; and (3) rapid-fire analysis by PESI-MS/MS. RaDPi-U targets 40 forensically important drugs, which include illegal drugs, hypnotics, and psychoactive substances. The analytical results were obtained within 3 min because of the above-mentioned simple workflow of RaDPi-U. The calibration curves of each analyte were constructed using the IS method, and they were quantitatively valid, resulting in good linearity (0.972-0.999) with a satisfactory lower limit of detection and lower limit of quantitation (0.01-7.1 ng/mL and 0.02-21 ng/mL, respectively). Further, both trueness and precisions were 28% or less, demonstrating the high reliability and repeatability of the method. Finally, we applied RaDPi-U to three postmortem urine specimens and successfully detected different drugs in each urine sample. The practicality of the method is proven, and RaDPi-U will be a strong tool as a rapid-fire drug screening method not only in forensic toxicology but also in clinical toxicology.

Residue levels of organophosphate pesticides and dialkylphosphates in agricultural products in Japan.

High urinary levels of dialkylphosphates (DAPs), which are common structures of organophosphate pesticides (OPs), have been associated with several adverse health outcomes in human biomonitoring studies. Previous studies have indicated that dietary OP exposure and ingestion of environmentally degraded DAP, which is inactive with acetylcholinesterase, can lead to an increase in urinary DAP levels in the general population. However, the specific food sources contributing to the intake of OPs and DAPs have not been identified. In this study, we analyzed the levels of OPs and preformed DAPs in various food items. DAP levels were markedly high in certain fruits, such as persimmon, apple juice, kiwi, and mandarin. In contrast, only moderate levels of OPs were detected in these foods. Furthermore, the levels of OPs and DAPs were positively associated with vegetables, whereas no such association was observed in fruits. Increased consumption of certain fruits presumably leads to a marked increase in urinary DAP levels in individuals despite limited exposure to OPs, resulting in reduced reliability of urinary DAPs as a marker of OP exposure. Therefore, the possible effects of dietary habits and the resulting intake of preformed DAPs should be considered when interpreting biomonitoring data of urinary DAPs. Additionally, DAP levels in most organic foods were much lower than those in conventional foods, suggesting that the reduction in urinary DAPs by organic diet intervention may be mainly attributed to the reduced intake of preformed DAPs rather than reduced exposure to OPs. Therefore, urinary DAP levels may not be suitable indicators for evaluating ingested OP exposure.

Novel SPE purification approach using the direct adsorption of vaporised propionic acid in food for rapid HPLC determination.

Solid phase extraction (SPE) is a technique widely used in food analysis for the isolation of analytes. Herein, we proposed a novel application of SPE to extract vaporised propionic acid, a common preservative, from a heated sample solution. A sample was heated under acidified conditions and the resulting steam was directly passed through an SPE column to extract the propionic acid, followed by elution and HPLC analysis. Here, the extraction on the SPE column ensures direct capture of propionic acid. The results demonstrated excellent linearity (R2 greater than 0.999) and recoveries of 89.9%-97.6% with intra- and inter-day precisions lower than 3.9%. To the best of our knowledge, no study has investigated the applicability of SPE to an analyte vaporised in the headspace of food products. The proposed method is promising in its application to various volatile compounds and in the routine analysis of propionic acid in food.

[Internal Quality Control of Residual Pesticides Analysis Using Multiple Stable Isotope Labeled Compounds].

Internal quality control (IQC) is essential to ensure the reliability of the results of chemical analysis. In this study, we propose a novel method of IQC for multiresidue analysis of pesticides. A total of seven stable isotope labeled compounds (SILC) were added to analytical samples and were used to monitor and evaluate the quality of analytical results. In contrast to conventional IQC method in which only a limited number of control materials were analyzed to ensure the reliability of the results for an entire batch, the developed method can monitor the analytical quality of all the samples in the batch. It was shown that the developed method could achieve better performance than that of conventional method. Therefore, the developed method is considered to be promising for practical applications.(Received January 27, 2022; Accepted July 4, 2022).

Easily Operable Quantification Method of 21 Plant-Derived Alkaloids in Human Serum by Automatic Sample Preparation and Liquid Chromatography-Tandem Mass Spectrometry.

In this study, we developed an easily operable quantification method for 21 plant-derived alkaloids in human serum by automatic sample preparation and liquid chromatography-tandem mass spectrometry. We designed to perform parallel sample preparation by a developed apparatus, which increased sample throughput. We conducted an automatic sample preparation through de-proteinization with 0.1% formic acid in methanol and achieved recovery rates of 89-107% (2.0-14% RSD) for all targeted analytes, demonstrating its high repeatability. The method validation results were satisfactory as follows: the linearity (r 2) of each calibration curve ranged from 0.978 to 1.000; the inter- and intra-day accuracies were 89.0-125% and 82.1-110%, respectively; the inter- and intra-day precisions were below 13% and 10%, respectively. Additionally, the lower limits of detection and quantification were 0.0044-0.047 and 0.013-0.14 ng/mL, respectively. Finally, the developed method was applied to pseudo-protoveratrine A poisoning serum and pseudo-colchicine poisoning serum, which were prepared by diluting acute-poisoning mice serum with human serum. Our method successfully quantitated protoveratrine A (0.15-0.25 ng/mL) and colchicine (4.8-6.0 ng/mL). Thus, our method is essential for prompt clinical treatment and critical care on patient in acute intoxication cases caused by plant-derived alkaloids. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04212-5.

Immunomodulatory Cell Therapy Using αGalCer-Pulsed Dendritic Cells Ameliorates Heart Failure in a Murine Dilated Cardiomyopathy Model.

BACKGROUND: Dilated cardiomyopathy (DCM) is a life-threatening disease, resulting in refractory heart failure. An immune disorder underlies the pathophysiology associated with heart failure progression. Invariant natural killer T (iNKT) cell activation is a prospective therapeutic strategy for ischemic heart disease. However, its efficacy in nonischemic cardiomyopathy, such as DCM, remains to be elucidated, and the feasible modality for iNKT cell activation in humans is yet to be validated. METHODS: Dendritic cells isolated from human volunteers were pulsed with α-galactosylceramide ex vivo, which were used as α-galactosylceramide-pulsed dendritic cells (αGCDCs). We treated DCM mice harboring mutated troponin TΔK210/ΔK210 with αGCDCs and evaluated the efficacy of iNKT cell activation on heart failure in DCM mice. Furthermore, we investigated the molecular basis underlying its therapeutic effects in these mice and analyzed primary cardiac cells under iNKT cell-secreted cytokines. RESULTS: The number of iNKT cells in the spleens of DCM mice was reduced compared with that in wild-type mice, whereas αGCDC treatment activated iNKT cells, prolonged survival of DCM mice, and prevented decline in the left ventricular ejection fraction for 4 weeks, accompanied by suppressed interstitial fibrosis. Mechanistically, αGCDC treatment suppressed TGF (transforming growth factor)-ß signaling and expression of fibrotic genes and restored vasculature that was impaired in DCM hearts by upregulating angiopoietin 1 (Angpt1) expression. Consistently, IFNγ (interferon gamma) suppressed TGF-ß-induced Smad2/3 signaling and the expression of fibrotic genes in cardiac fibroblasts and upregulated Angpt1 expression in cardiomyocytes via Stat1. CONCLUSIONS: Immunomodulatory cell therapy with αGCDCs is a novel therapeutic strategy for heart failure in DCM.

Metabolomics and Data-Driven Bioinformatics Revealed Key Maternal Metabolites Related to Fetal Lethality via Di(2-ethylhexyl)phthalate Exposure in Pregnant Mice.

We performed serum metabolome analysis of di(2-ethylhexyl)phthalate (DEHP)-exposed and control pregnant mice. Pregnant mice (n = 5) were fed a DEHP-containing diet (0.1% or 0.2% DEHP) or a normal diet (control) from gestational days 0-18. After maternal exposure to 0.2% DEHP there were no surviving fetuses, indicating its strong fetal lethality. There were no significant differences in the numbers of fetuses and placentas between the 0.1% DEHP and control groups, although fetal viability differed significantly between them, suggesting that maternal exposure to 0.1% DEHP could inhibit fetal growth. Metabolomics successfully detected 169 metabolites in serum. Principal component analysis (PCA) demonstrated that the three groups were clearly separated on PCA score plots. The biological interpretation of PC1 was fetal lethality, whereas PC2 meant metabolic alteration of pregnant mice via DEHP exposure without fetal lethality. In particular, the first component was significantly correlated with fetal viability, demonstrating that maternal metabolome changes via DEHP exposure were strongly related to fetal lethality. Levels of some amino acids were significantly increased in the DEHP-exposed groups, whereas those of some fatty acids, nicotinic acid, and 1,5-anhydroglucitol were significantly decreased in the DEHP groups. DEHP-induced increases in glycine levels could cause fetal neurological disorders, and decreases in nicotinic acid could inhibit fetal growth. In addition, a machine-learning Random forest could determine 16 potential biomarkers of DEHP exposure, and data-driven network analysis revealed that nicotinic acid was the most influential hub metabolite in the metabolic network. These findings will be useful for understanding the effects of DEHP on the maternal metabolome in pregnancy and their relationship to fetal lethality.

Acceptance of Murine Islet Allografts Without Immunosuppression in Inguinal Subcutaneous White Adipose Tissue Pretreated With bFGF.

Prevention of immune rejection without immunosuppression is the ultimate goal of transplant immunobiology. One way to achieve this in cellular transplantation, such as with islet transplantation, is to create a favorable local environment at the transplant site. In the current study, we found that C57BL/6 mice with streptozotocin-induced diabetes remained normoglycemic for >1 year after transplantation of BALB/c islets without immunosuppression when the inguinal subcutaneous white adipose tissue (ISWAT) was the site of transplantation and when the site was pretreated with basic fibroblast growth factor. Mechanistically, mesenchymal stem cells (MSCs) expanded in the ISWAT after the treatment was found to produce transforming growth factor-ß (TGF-ß), and prevention of islet allograft rejection could be achieved by cotransplantation with syngeneic MSCs isolated from the ISWAT after the treatment, which was abolished by anti-TGF-ß antibody treatment. Importantly, TGF-ß-producing cells remained present at the site of cotransplantation up to the end of observation period at 240 days after transplantation. These findings indicate that prevention of islet allograft rejection without immunosuppression is feasible with the use of syngeneic TGF-ß-producing MSCs expanded in the ISWAT after the treatment with bFGF, providing a novel strategy for prevention of islet allograft rejection without immunosuppression.

A highly sensitive quantification method for 12 plant toxins in human serum using liquid chromatography tandem mass spectrometry with a quick solid-phase extraction technique.

We developed a highly sensitive quantification method using liquid chromatography tandem mass spectrometry (LC/MS/MS) for 12 plant toxins in human serum. In this paper, we selected lycorine, galanthamine, protoveratrine A, protoveratrine B, veratramine, veratridine, jervine, cyclopamine, cevadine, α-solanine, α-chaconine, and solanidine as targeted analytes. The ADME column was utilized for LC separation and a Monolithic SPE column (MonoSpin® C18) for analyte extraction. The total time for SPE clean-up and LC/MS/MS analysis was completed within 30 min. The method validation results were as follows: the linearity (r2) of each calibration curve was over 0.99; the inter- and intra-day accuracies were 92.7 %-116 % and 91.6 %-106 %, respectively; and the inter- and intra-day precisions were below 14 % and 11 %, respectively. Also, the lower limits of detection and quantification were 0.0071-0.15 and 0.022-0.46 ng/mL, respectively, indicating the method's high sensitivity. Finally, to confirm its feasibility, our method was applied to two model samples: (1) commercially available human serum and (2) pseudo poisoning serum via dilution of mouse serum with human serum. We were able to quantify α-chaconine at 0.84 ± 0.02 ng/mL in the serum (Case 1) and protoveratrine A at 0.15 ± 0.032 ng/mL in the pseudo poisoning serum (Case 2), demonstrating our method's practicality. This is the first time that the 12 plant toxins in human serum were simultaneously quantitated. Our method can investigate accidental poisonings involving toxic plants, enabling prompt decisions on patient treatment.

The protective function of invariant natural killer T cells in the relapse of experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) in mice provides a useful platform to study the pathogenesis and experimental therapeutics of human uveitis. One often used EAU model employs C57BL/6 (B6) mice sensitized with a peptide residue having 1 to 20 amino acids of human interphotoreceptor retinoid binding protein (hIRBP1-20). The model using the B6 background has permitted a liberal use of genetically engineered strains and has provided insights for understanding uveoretinitis. However, this is usually acute/monophasic and does not represent human uveoretinitis that is characterized as a chronic/recurrent disease. Several chronic/recurrent EAU models have been developed; of these, we employed administration of staphylococcal enterotoxin B (SEB) for relapse in the present study, and found that recurrence was induced at day 24 after primary immunization, which is thought to be the convalescent phase. We reported the activation of invariant natural killer T (iNKT)-cells upon primary immunization of the EAU model mice with the ligand RCAI-56, which was found to mitigate the disease in our previous study. Here, we first attempted to ameliorate EAU in the relapse model using a preventive regimen by activating iNKT cells at the same time relapse induction (day 24) or in a regimen after 3 days of relapse induction (day 27). The preventive as well as post-inductive regimens were successful in reducing histopathological scores by inhibiting the Ag-specific Th17-biased response. Collectively, activation of iNKT cells may be useful to mitigate the relapse response of EAU induced with SEB.

Residual Analysis of Aflatoxins in Spice by HPLC Coupled with Solid-Phase Dispersive Extraction and Solid-Phase Fluorescence Derivatization Method.

BACKGROUND: Aflatoxins (AFs) are carcinogenic mycotoxins. A simple, quick, and accurate method for the micro-analysis of AFs in foodstuffs, especially spices, is needed. OBJECTIVE: A sophisticated pretreatment method that combines solid-phase dispersive extraction (SPDE) and solid-phase fluorescence derivatization using immunoaffinity (IA) gel as the solid phase was developed to analyze AFs in spices simply, quickly, and sensitively by liquid chromatography with fluorescence detection. METHOD: White and black pepper samples were extracted with a mixed solution of methanol/water (4:1) and then diluted with 7% aqueous solution of Triton-X. The solution was subjected to cleanup by SPDE using IA gel. Trifluoroacetic acid was added to the IA gel for on-site solid-phase fluorescence derivatization. RESULTS: Chromatograms containing well-separated peaks and few interference peaks from contaminants were obtained. The method detection limit of AFs in white and black pepper was 0.15-0.29 ng/g. Repeatability and intermediate precision were <10% and <15%, respectively, and accuracy was 61.7-87.8%. In addition, inter-laboratory precision was <29% and mean recovery was 61.5-76.7%. A favorable z-score of |Z| ≦ 1 was obtained in seven laboratories, although one laboratory gave 2 < |Z| < 3. CONCLUSIONS: The validity, reliability, practicality, and robustness of the developed method were verified. HIGHLIGHTS: By using SPDE and solid-phase fluorescence derivatization in combination for AF analysis, fluorescence derivatization during cleanup was realized, leading to simplification of the pretreatment operation.

Determination of sterigmatocystin in foods in Japan: method validation and occurrence data.

A survey of the contamination of foods by sterigmatocystin (STC) was performed by an analytical method based on LC-MS/MS. STC was extracted from samples with acetonitrile/water (85/15, v/v) and then purified with immunoaffinity columns. The method was validated by a small-scale inter-laboratory study using spiked wheat samples. Mean recoveries of STC were 100.3% and 92.5% from two samples spiked at 0.5 and 5.0 µg/kg, respectively. A total of 583 samples were analysed between 2016 and 2018, and STC was detected in 19.9% of all samples at >0.05 µg/kg (limit of quantification). The foods that were contaminated by STC were wheat flour, Job's tears products, rye flour, rice, buckwheat flour, white sorghum, barley products, azuki bean and corn flour. STC was not found in beer or wine. The occurrence of STC in domestic wheat flour (44.4%), Job's tears products (41.7%) and rye flour (29.9%) accounted for the three highest values. The highest mean concentrations were obtained for Job's tears products (0.3 µg/kg) and rye flour (0.3 µg/kg). The maximum contamination level was present in a sample of rye flour (7.1 µg/kg). Although the contamination levels were low, these results indicate that STC frequently contaminates Japanese retail foods. A continuous survey is required to assess exposure to STC in Japan.

Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice.

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells.

A Novel Subcutaneous Site of Islet Transplantation Superior to the Liver.

BACKGROUND: Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently, the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2 to 3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved. METHODS: We developed a novel procedure of islet transplantation to the inguinal subcutaneous white adipose tissue (ISWAT) of mice and described functional and morphological characteristics of transplanted syngeneic islets. Also, it was determined whether islet allograft rejection in the ISWAT can be prevented by immunosuppressive agents. Furthermore, it was examined whether human islets function when grafted in this particular site of immune-deficient mice. RESULTS: In this site, transplanted islets are engrafted as clusters and function to reverse streptozotocin-induced diabetes in mice. Importantly, transplanted islets can be visualized by computed tomography and are easily retrievable, and allograft rejection is preventable by blockade of costimulatory signals. Of much importance, the efficiency of islet transplantation in this site is superior to the liver, in which hyperglycemia of diabetic recipient mice is ameliorated after transplantation of 200 syngeneic islets (the islet number yielded from 1 mouse pancreas) to the ISWAT but not to the liver. Furthermore, human islets transplanted in this particular site function to reverse diabetes in immune-deficient mice. CONCLUSIONS: Thus, the ISWAT is superior to the liver as the site of islet transplantation, which may lead to improved outcome of clinical islet transplantation.

Natural Killer T Cell-Targeted Immunotherapy Mediating Long-term Memory Responses and Strong Antitumor Activity.

Current tumor therapies, including immunotherapies, focus on passive eradication or at least reduction of the tumor mass. However, cancer patients quite often suffer from tumor relapse or metastasis after such treatments. To overcome these problems, we have developed a natural killer T (NKT) cell-targeted immunotherapy focusing on active engagement of the patient's immune system, but not directly targeting the tumor cells themselves. NKT cells express an invariant antigen receptor α chain encoded by Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans and recognize glycolipid ligand in conjunction with a monomorphic CD1d molecule. The NKT cells play a pivotal role in the orchestration of antitumor immune responses by mediating adjuvant effects that activate various antitumor effector cells of both innate and adaptive immune systems and also aid in establishing a long-term memory response. Here, we established NKT cell-targeted therapy using a newly discovered NKT cell glycolipid ligand, RK, which has a stronger capacity to stimulate both human and mouse NKT cells compared to previous NKT cell ligand. Moreover, RK mediates strong adjuvant effects in activating various effector cell types and establishes long-term memory responses, resulting in the continuous attack on the tumor that confers long-lasting and potent antitumor effects. Since the NKT cell ligand presented by the monomorphic CD1d can be used for all humans irrespective of HLA types, and also because NKT cell-targeted therapy does not directly target tumor cells, this therapy can potentially be applied to all cancer patients and any tumor types.

Alternative pathway for the development of Vα14+ NKT cells directly from CD4-CD8- thymocytes that bypasses the CD4+CD8+ stage.

Although invariant Vα14+ natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ double-positive (DP) thymocytes, the developmental origin of CD4-CD8- double-negative (DN) NKT cells still remains unresolved. Here we provide definitive genetic evidence obtained, through studies of mice with DP-stage-specific ablation of expression of the gene encoding the recombinase component RAG-2 (Rag2) and by a fate-mapping approach, that supports the proposal of the existence of an alternative developmental pathway through which a fraction of DN NKT cells with strong T-helper-type-1 (TH1)-biased and cytotoxic characteristics develop from late DN-stage thymocytes, bypassing the DP stage. These findings provide new insight into understanding of the development of NKT cells and propose a role for timing of expression of the invariant T cell antigen receptor in determining the functional properties of NKT cells.

Intradermal Delivery of Antigens Enhances Specific IgG and Diminishes IgE Production: Potential Use for Vaccination and Allergy Immunotherapy.

Skin is protected by a tough but flexible multilayered barrier and is a front line for immune responses against invading particles. For many years now, skin has been a tissue where certain vaccines are injected for the prevention of infectious disease, however, the detailed mechanisms of the skin immune response are not yet well understood. Using thin and small injection needles, we carefully injected OVA into a restricted region of mouse skin, i.e., intradermal (ID), and examined the antibody response in comparison with subcutaneous (SC) injection or epicutaneous patch administration of OVA. Epicutaneous patches induced a high IgE response against OVA, but IgG production was low. High IgG production was induced by both ID and SC injection, moreover, ID injection induced higher IgG production without any adjutants. Furthermore, OVA-specific IgE production was diminished by ID injection. We found that ID injection could efficiently stimulate skin resident DCs, drive Th1-biased conditions and diminish IgE production. The ID injection response was regulated by Langerin+ dermal DCs, because OVA was taken up mainly by these cells and, after transiently deleting them, the IgE response was no longer diminished and IgG1 production was enhanced. We also tested whether ID injection might be an effective allergy treatment by attempting to inhibit ongoing IgE production in mice with experimentally induced high serum IgE levels. Multiple ID injections of OVA were shown to prevent elevation of serum OVA-specific IgE after repeated allergen challenge. In contrast, SC OVA injection could only transiently inhibit the OVA-specific IgE production. These findings indicated that ID injection results in higher induction of antigen-specific IgG, and thus may be useful for vaccine delivery with little or no adjuvant components. Moreover, the observed diminishment of IgE and induction of Th1-biased immune responses suggest that ID may be a useful injection route for allergy immunotherapy.

Invariant natural killer T cells play dual roles in the development of experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) represents an experimental model for human endogenous uveitis, which is caused by Th1/Th17 cell-mediated inflammation. Natural killer T (NKT) cells recognize lipid antigens and produce large amounts of cytokines upon activation. To examine the role of NKT cells in the development of uveitis, EAU was elicited by immunization with a peptide from the human interphotoreceptor retinoid-binding protein (hIRBP1-20) in complete Freund's adjuvant and histopathology scores were evaluated in C57BL/6 (WT) and NKT cell-deficient mice. NKT cell-deficient mice developed more severe EAU pathology than WT mice. When WT mice were treated with ligands of the invariant subset of NKT cells (α-GalCer or RCAI-56), EAU was ameliorated in mice treated with RCAI-56 but not α-GalCer. IRBP-specific Th1/Th17 cytokines were reduced in RCAI-56-treated compared with vehicle-treated mice. Although the numbers of IRBP-specific T cells detected by hIRBP3-13/I-Ab tetramers in the spleen and the draining lymph node were the same for vehicle and RCAI-56 treatment groups, RORγt expression by tetramer-positive cells in RCAI-56-treated mice was lower than in control mice. Moreover, the eyes of RCAI-56-treated mice contained fewer IRBP-specific T cells compared with control mice. These results suggest that invariant NKT (iNKT) cells suppress the induction of Th17 cells and infiltration of IRBP-specific T cells into the eyes, thereby reducing ocular inflammation. However, in sharp contrast to the ameliorating effects of iNKT cell activation during the initiation phase of EAU, iNKT cell activation during the effector phase exacerbated disease pathology. Thus, we conclude that iNKT cells exhibit dual roles in the development of EAU.