Masakazu Shinohara

Oral Anti-CD3 Antibody Treatment Induces Regulatory T Cells and Inhibits the Development of Atherosclerosis in Mice

Background— Accumulating evidence suggests that several subsets of regulatory T cells that actively mediate immunologic tolerance play crucial roles in atherogenesis. Recently, orally administered anti-CD3 monoclonal antibody has been shown as an inducer of novel regulatory T cells expressing latency-associated peptide (LAP) on their surface, which potently prevents systemic autoimmunity. In the present study, we hypothesized that oral anti-CD3 antibody treatment may inhibit atherosclerosis in mice. Methods and Results— Six-week-old apolipoprotein E–deficient mice on a standard diet were orally given anti-CD3 antibody or control immunoglobulin G on 5 consecutive days, and atherosclerosis was assessed at age 16 weeks. Oral administration of anti-CD3 antibody significantly reduced atherosclerotic lesion formation and accumulations of macrophages and CD4+ T cells in the plaques compared with controls. We observed a significant increase in LAP+ cells and CD25+Foxp3+ cells in the CD4+ T-cell population in anti-CD3–treated mice, in association with increased production of the antiinflammatory cytokine transforming growth factor-β and suppressed T-helper type 1 and type 2 immune responses. Neutralization of transforming growth factor-β in vivo abrogated the preventive effect of oral anti-CD3 antibody. Conclusions— Our findings indicate the atheroprotective role of oral anti-CD3 antibody treatment in mice via induction of a regulatory T-cell response. These findings suggest that oral immune modulation may represent an attractive therapeutic approach to atherosclerosis.

Oral Administration of an Active Form of Vitamin D3 (Calcitriol) Decreases Atherosclerosis in Mice by Inducing Regulatory T Cells and Immature Dendritic Cells With Tolerogenic Functions

Objective—To determine whether the administration of an active form of vitamin D3 (calcitriol) could prevent atherosclerosis through anti-inflammatory actions. Methods and Results—Recent clinical studies have shown that lack of vitamin D3 is a risk factor for cardiovascular events. Oral calcitriol administration decreased atherosclerotic lesions, macrophage accumulation, and CD4+ T-cell infiltration at the aortic sinus, when compared with the corresponding observations in control mice. We observed a significant increase in Foxp3+ regulatory T cells and a decrease in CD80+CD86+ dendritic cells (DCs) in the mesenteric lymph nodes, spleen, and atherosclerotic lesions in oral calcitriol-treated mice in association with increased interleukin 10 and decreased interleukin 12 mRNA expression. CD11c+ DCs from the calcitriol group showed reduced proliferative activity of T lymphocytes, suggesting the suppression of DC maturation. Neutralization of CD25 in vivo revealed that calcitriol inhibited atherosclerosis mainly in a regulatory T cell-dependent manner but also partly because of a decrease in DC maturation. Conclusion—Oral calcitriol treatment could prevent the development of atherosclerosis by changing the function or differentiation of DCs and regulatory T cells. These findings suggest that intestinal and systemic immune modulation by calcitriol may be a potentially valuable therapeutic approach against atherosclerosis.

A metabolomic approach to lung cancer

Lung cancer is one of the most common cancers in the world, but no good clinical markers that can be used to diagnose the disease at an early stage and predict its prognosis have been found. Therefore, the discovery of novel clinical markers is required. In this study, metabolomic analysis of lung cancer patients was performed using gas chromatography mass spectrometry. Serum samples from 29 healthy volunteers and 33 lung cancer patients with adenocarcinoma (n=12), squamous cell carcinoma (n=11), or small cell carcinoma (n=10) ranging from stage I to stage IV disease and lung tissue samples from 7 lung cancer patients including the tumor tissue and its surrounding normal tissue were used. A total of 58 metabolites (57 individual metabolites) were detected in serum, and 71 metabolites were detected in the lung tissue. The levels of 23 of the 58 serum metabolites were significantly changed in all lung cancer patients compared with healthy volunteers, and the levels of 48 of the 71 metabolites were significantly changed in the tumor tissue compared with the non-tumor tissue. Partial least squares discriminant analysis, which is a form of multiple classification analysis, was performed using the serum sample data, and metabolites that had characteristic alterations in each histological subtype and disease stage were determined. Our results demonstrate that changes in metabolite pattern are useful for assessing the clinical characteristics of lung cancer. Our results will hopefully lead to the establishment of novel diagnostic tools.

Serum metabolomics as a novel diagnostic approach for gastrointestinal cancer.

Conventional tumor markers are unsuitable for detecting carcinoma at an early stage and lack clinical efficacy and utility. In this study, we attempted to investigate the differences in serum metabolite profiles of gastrointestinal cancers and healthy volunteers using a metabolomic approach and searched for sensitive and specific metabolomic biomarker candidates. Human serum samples were obtained esophageal (n = 15), gastric (n = 11), and colorectal (n = 12) cancer patients and healthy volunteers (n = 12). A model for evaluating metabolomic biomarker candidates was constructed using multiple classification analysis, and the results were assessed with receiver operating characteristic curves. Among the 58 metabolites, the levels of nine, five and 12 metabolites were significantly changed in the esophageal, gastric and colorectal cancer patients, respectively, compared with the healthy volunteers. Multiple classification analysis revealed that the variations in the levels of malonic acid and L-serine largely contributed to the separation of esophageal cancer; gastric cancer was characterized by changes in the levels of 3-hydroxypropionic acid and pyruvic acid; and L-alanine, glucuronoic lactone and L-glutamine contributed to the separation of colorectal cancer. Our approach revealed that some metabolites are more sensitive for detecting gastrointestinal cancer than conventional biomarkers. Our study supports the potential of metabolomics as an early diagnostic tool for cancer.

GCMS‐based metabolomic study in mice with colitis induced by dextran sulfate sodium

Background: Metabolomics provides data about all the metabolic processes of a cell or organism. So far, the changes that occur in the levels of metabolites during the development of colitis have not been fully elucidated. Here we examined the changes of metabolite levels in the serum and colon tissue of colitis mice using gas chromatography mass spectrometry (GC/MS) with the aim of achieving a detailed understanding of the pathogenesis of inflammatory bowel disease (IBD). Methods: To induce colitis, C57BL/6J mice were administered 3.0% dextran sulfate sodium (DSS) in their drinking water for 5 days and were subsequently given drinking water alone. Results: A total of 77 and 92 metabolites were detected in serum and colon tissue, respectively, and among the metabolites the compositions of TCA cycle intermediates and amino acids changed depending on the degree of colitis. Then, partial least square discriminant analysis (PLS‐DA), a multiple classification analysis, showed distinct clustering and clear separation of the groups according to the degree of colitis. Furthermore, PLS‐DA loadings plots revealed that succinic acid, indole‐3‐acetic acid, glutamic acid, and glutamine were the main contributors to the separation of each stage of colitis. In addition, it was revealed that supplementation with glutamine, the level of which was significantly decreased in the acute phase of colonic inflammation, attenuated colitis induced by DSS. Conclusions: Our results suggest that metabolomics is capable of representing the various degrees of colitis, and our findings will aid in the discovery of therapeutic agents for IBD and other inflammatory disorders by metabolomic approaches. (Inflamm Bowel Dis 2011;)

Practical non-targeted gas chromatography/mass spectrometry-based metabolomics platform for metabolic phenotype analysis.

Gas chromatography coupled to mass spectrometry (GC/MS) is a core analytical method for metabolomics and has been used as a platform in non-targeted analysis, especially for hydrophilic metabolites. Non-targeted GC/MS-based metabolomics generally requires a high-throughput technology to handle a large volume of samples and an accumulated database (reference library) of the retention times and mass spectra of standard compounds for accurate peak identification. In this study, we provide a practical GC/MS platform and an auto peak identification technique that is not restricted to certain types of mass spectrometers. The platform utilizes a quadrupole mass spectrometer capable of high-speed scanning, resulting in greater output compared with Pegasus GC-time of flight (TOF)/MS, which has been an essential instrument for high-throughput experiments. Moreover, we show that our reference library is broadly applicable to other instruments; peak identification can be readily performed using the library without constructing a reference resource. The usefulness and versatility of our system are demonstrated by the analyses of three experimental metabolomics data sets, including standard mixtures and real biological samples.

A Specific Role for eNOS-Derived Reactive Oxygen Species in Atherosclerosis Progression

Objective—When the availability of tetrahydrobiopterin (BH4) is deficient, endothelial nitric oxide synthase (eNOS) produces superoxide rather than NO (uncoupled eNOS). We have shown that the atherosclerotic lesion size was augmented in apolipoprotein E–deficient (ApoE-KO) mice overexpressing eNOS because of the enhanced superoxide production. In this study, we addressed the specific importance of uncoupled eNOS in atherosclerosis, and the potential mechanistic role for specific versus nonspecific antioxidant strategies in restoring eNOS coupling. Methods and Results—We crossed mice overexpressing eNOS in the endothelium (eNOS-Tg) with mice overexpressing GTP-cyclohydrolase I (GCH), the rate-limiting enzyme in BH4 synthesis, to generate ApoE-KO/eNOS-Tg/GCH-Tg mice. As a comparison, ApoE-KO/eNOS-Tg mice were treated with vitamin C. Atherosclerotic lesion formation was increased in ApoE-KO/eNOS-Tg mice compared with ApoE-KO mice. GCH overexpression in ApoE-KO/eNOS-Tg/GCH-Tg mice increased vascular BH4 levels and reduced plaque area. This reduction was associated with decreased superoxide production from uncoupled eNOS. Vitamin C treatment failed to reduce atherosclerotic lesion size in ApoE-KO/eNOS-Tg mice, despite reducing overall vascular superoxide production. Conclusion—In contrast to vitamin C treatment, augmenting BH4 levels in the endothelium by GCH overexpression reduced the accelerated atherosclerotic lesion formation in ApoE-KO/eNOS-Tg mice, associated with a reduction of superoxide production from uncoupled eNOS.

Local Overexpression of Toll-Like Receptors at the Vessel Wall Induces Atherosclerotic Lesion Formation Synergism of TLR2 and TLR4

Objective—Atherosclerosis is now considered as a chronic inflammatory disease, and inflammation is closely related to immune systems, which consist of innate-immunity and adaptive-immunity. Recently, toll-like receptors (TLRs) have been identified as key components of innate-immunity. We examined the role of local expressions of TLRs at the vessel wall in atherosclerosis. Methods and Results—We transfected cDNA encoding human TLR2 and TLR4 into the carotid arterial vessel wall of rabbits fed high-cholesterol diets with the use of HVJ-liposome. The rabbits were transfected with (1) pCMV-&bgr;-gal, (2) empty vector, (3) TLR2, (4) TLR4, (5) TLR2+4. X-gal staining and immunohistochemical analysis showed that the transfected plasmids were mainly expressed in the media. Neither TLR2 nor TLR4 transfection induced significant augmentation of atherosclerosis. Transfection of TLR2- and TLR4-containing HVJ synergistically accelerated atherosclerosis and increased expressions of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and MCP-1. Moreover, transfection of TLR2 and TLR4 resulted in synergistic activation of NF-&kgr;B at the vessel wall in vivo, and in vascular smooth muscle cells in vitro. Conclusions—Expressions of both TLR2 and TLR4 at the vessel wall synergistically accelerated atherosclerosis. The present study revealed the role of TLRs expressed locally at the vessel wall in the early stage of atherosclerosis.

Highly sensitive and rapid profiling method for carotenoids and their epoxidized products using supercritical fluid chromatography coupled with electrospray ionization-triple quadrupole mass spectrometry.

Epoxy carotenoids, which are products of carotenoid oxidation, are potential oxidative stress markers. However, it is difficult to profile epoxy carotenoids owing to their small amount and difficulty in their separation from hydroxy carotenoids. In this study, a high-performance analytical system based on supercritical fluid chromatography (SFC) coupled with tandem mass spectrometry (MS/MS) was developed for the simultaneous analysis of carotenoids and epoxy carotenoids. SFC is an effective separation technique for hydrophobic compounds, by which major carotenoids in human serum and their epoxidation products can be analyzed within 20 min. The use of MS/MS increased the sensitivity; the detection limit for each carotenoid was of the sub-fmol order. When the constructed method was applied to biological samples such as human serum and low-density lipoprotein (LDL), the precise detection of the target carotenoids was disturbed by several isomers. However, highly selective detection of epoxy carotenoids was performed by targeting product ions that were generated with a structure-specific neutral loss of 80Da. Furthermore, the sample volume needed for the analysis was only 0.1ml for the serum, indicating the efficiency of this system in performing small-scale analyses. Using the analytical system developed in this study, highly sensitive and selective analysis of epoxy carotenoids could be performed in a short time. These features show the usefulness of this system in application to screening analysis of carotenoid profiles that are easily modified by oxidative stress.

Augmentation of Vascular Remodeling by Uncoupled Endothelial Nitric Oxide Synthase in a Mouse Model of Diabetes Mellitus

Objective—Diabetes mellitus is associated with increased oxidative stress, which induces oxidation of tetrahydrobiopterin (BH4) in vessel wall. Without enough BH4, eNOS is uncoupled to L-arginine and produces superoxide rather than NO. We examined the role of uncoupled eNOS in vascular remodeling in diabetes. Methods and Results—Diabetes mellitus was produced by streptozotocin in C57BL/6J mice. Under stable hyperglycemia, the common carotid artery was ligated, and neointimal formation was examined 4 weeks later. In diabetic mice, the neointimal area was dramatically augmented. This augmentation was associated with increased aortic superoxide formation, reduced aortic BH4/dihydrobiopterin (BH2) ratio, and decreased plasma nitrite and nitrate (NOx) levels compared with nondiabetic mice. Chronic BH4 treatment (10 mg/kg/d) reduced the neointimal area in association with suppressed superoxide production and inflammatory changes in vessels. BH4/BH2 ratio in vessel wall was preserved, and plasma NOx levels increased. Furthermore, in the presence of diabetes, overexpression of bovine eNOS resulted in augmentation of neointimal area, accompanied by increased superoxide production in the endothelium. Conclusions—In diabetes, increased oxidative stress by uncoupled NOSs, particularly eNOS, causes augmentation of vascular remodeling. These findings indicate restoration of eNOS coupling has an atheroprotective benefit in diabetes.