Toru Takano

Autoantibodies against four kinds of neurotransmitter receptors in psychiatric disorders

There is a hypothesis that autoimmune abnormalities in neurotransmitter receptors might cause some psychiatric disorders. Using a sensitive radioligand assay, we detected serum autoantibodies to recombinant human muscarinic cholinergic receptor 1 (CHRM1, 34.4%), mu-opioid receptor (OPRM1, 13.1%), 5-hydroxytryptamine receptor 1A (HTR1A, 7.4%), and dopamine receptor D2 (DRD2, 4.9%) in 122 psychiatric patients. Positive antibodies to CHRM1 were found in 34.1%, 34.9%, 33.3%, and 9.1% of patients with schizophrenic disorders (n=44), mood disorders (n=63), other psychiatric disorders (n=15) and autoimmune diseases (n=33), respectively. All three patients with neuroleptic maliganant syndrome had high activities of autoantibodies to CHRM1, OPRM1, and/or HTR1A. Our data suggest that autoimmunity to neurotransmitter receptors might be associated with the induction of psychiatric symptoms and have some relation to neuroleptic malignant syndrome.

High-throughput differential screening of mRNAs by serial analysis of gene expression: decreased expression of trefoil factor 3 mRNA in thyroid follicular carcinomas

To find mRNAs whose expression differs between thyroid follicular adenomas and carcinomas, a high-throughput analysis of mRNAs in these two tumours was performed. This method, named high-throughput differential screening by serial analysis of gene expression (HDSS), combines a modified method of serial analysis of gene expression (SAGE) and real-time quantitative reverse transcription polymerase chain reaction (RT–PCR). A total of 40 candidate tag sequences that showed extremely different expression levels between a follicular carcinoma and a follicular adenoma in the SAGE analysis were analysed by real-time quantitative RT–PCR, using RNAs from an additional four typical follicular carcinomas and adenomas. One sequence tag that represents trefoil factor 3 (TFF3) mRNA showed a clear difference in its expression level between adenomas and carcinomas. The expression levels of TFF3 mRNA in 48 follicular adenomas and 29 follicular carcinomas were measured by real-time quantitative RT–PCR using a specific probe for TFF3. They were significantly decreased in follicular carcinomas, especially in widely invasive types and those with evident metastases. These results indicate that the decreased expression of TFF3 mRNA is a marker of follicular carcinomas, especially those with a high risk of invasion or metastasis.

Differentiation of Follicular Thyroid Adenoma from Carcinoma by Means of Gene Expression Profiling with Adapter-Tagged Competitive Polymerase Chain Reaction

Objective: Since preoperative differentiation between follicular thyroid adenoma (FTA) and carcinoma (FTC) remains very difficult, the purpose of this study was to identify the genes differentially expressed in FTA and FTC in order to construct a diagnostic system based on such genes for differentiation of FTA and FTC. Methods:Gene expression profiles of 45 FTAs and 22 FTCs were analyzed by means of adapter-tagged competitive polymerase chain reaction (ATAC-PCR) with 2,516 genes (learning set). The genes differentially expressed in FTAs and FTCs were then used to construct a diagnostic system based on the weighted-voting algorithm. In addition, a validation study of this diagnostic system was conducted using 12 FTAs and 6 FTCs (validation set). Results: The diagnostic system for differentiation of FTA and FTC, constructed with the aid of the learning set samples, was based on 60 genes differentially expressed in FTA and FTC, which included several genes previously identified as overexpressed in FTC (DPP4, KRT19 and IGFBP3) or FTA (trefoil factor 3 and thyroid peroxidase).The leave-one-out cross-validation study showed that the accuracy of this diagnostic system was as high as 90% (sensitivity: 77.3% and specificity: 95.6%), and was confirmed by the validation study (diagnostic accuracy: 83.3%; 95% confidence interval: 62.8–95.4%, sensitivity: 66.7% and specificity: 91.2%). Conclusions: This diagnostic system using the ATAC-PCR assay is expected to be clinically useful for preoperative differentiation between FTA and FTC since ATAC-PCR can be used for the small amount of RNA obtained from fine needle aspiration biopsy.

Expression of nestin mRNA is a differentiation marker in thyroid tumors

Nestin is a maker that identifies stem cells in some adult tissues, and its expression is believed to relate to malignancy in cancer cells. In this study, we measured the expression levels of nestin mRNA in various kinds of thyroid tumor by the real-time quantitative reverse transcription-polymerase chain reaction. Unexpectedly, nestin mRNA was detected in almost all differentiated thyroid tumors and normal thyroid tissues, whereas extremely decreased expression was observed in anaplastic carcinomas, which are the most malignant of the thyroid follicular cell-derived tumors. These results suggest that nestin mRNA is a differentiation marker, and its expression does not relate to malignant characteristics in thyroid tumors.

Association of Seasonal Allergic Rhinitis is High in Graves' Disease and Low in Painless Thyroiditis

Hashimotos thyroiditis is thought to be a T-helper cell type 1 (TH1)-dependent disease, but it is not clear whether Graves disease is T-helper cell type 2 (TH2)-predominant or not. TH1-predominant diseases are infrequently and TH2-predominant diseases are frequently associated with allergic diseases. We examined the prevalence of seasonal allergic rhinitis to Japanese cedar pollen, a typical TH2-associated disease, in patients with Graves disease (n = 126), painless thyroiditis (n = 46) and Hashimotos thyroiditis (n = 88), and compared them to healthy controls (n = 766). Gender and age distribution were not different among patient groups and healthy controls, except for the higher age of patients with Hashimotos thyroiditis. The prevalence of seasonal allergic rhinitis was significantly high in patients with Graves disease (42.9%, p < 0.05) and low in patients with painless thyroiditis (13.0%, p < 0.01) but was not different in patients with Hashimotos thyroiditis (26.1%) compared to that of healthy controls (32.6%). When patients with painless thyroiditis were included in Hashimotos thyroiditis group, the prevalence of seasonal allergic rhinitis was 21.6% and significantly different from that of healthy controls (p < 0.05). These data indicate that Graves disease is TH2 predominant and painless thyroiditis is a TH1-predominant disease. Our findings suggest that the shift from TH2 toward TH1 immunogenesis may be important for achieving earlier remission of Graves disease.

Serum concentrations of androstenediol and androstenediol sulfate, and their relation to cytokine production during and after normal pregnancy.

Since it is known that androstenediol (ADIOL) has potent immunoregulatory effects, changes in ADIOL levels during and after pregnancy might affect the maternal immune system. We examined serum concentrations of ADIOL and androstenediol 3-sulfate (ADIOLS) together with IFN-gamma and IL-4 production levels during pregnancy and after delivery up to 10-11 months postpartum. The subjects were 73 normal pregnant, 76 normal postpartum, and 28 normal non-pregnant women. ADIOL and ADIOLS were measured using EIA and GC/MS, respectively. The cytokine levels in the supernatant of whole-blood cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin were measured using ELISA. ADIOL levels significantly decreased compared to non-pregnant levels in the first trimester (P < 0.05) and were reversed in the third trimester (P < 0.05). After pregnancy, ADIOL levels gradually declined, and a significant decrease was observed at 10-11 months postpartum (P < 0.05). ADIOLS levels were significantly lower in the third trimester (P < 0.05) and significantly higher at the first month postpartum (P < 0.001) compared to non-pregnant women. IFN-gamma and IL-4 levels decreased during pregnancy and subsequently increased postpartum. On the other hand, we found significant negative correlations between ADIOL concentrations and production levels of IFN-gamma (P < 0.05) or IL-4 (P < 0.05). These findings suggest that ADIOL may be involved in modifying the maternal immune response during and after pregnancy.

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.

Acquired activated protein C resistance is associated with IgG antibodies to protein S in patients with systemic lupus erythematosus

The objective of this study was to clarify the roles of anti-phospholipid antibodies (aPLs) in the pathogenesis of acquired activated protein C resistance (APC-R) in patients with systemic lupus erythematosus (SLE). We examined several aPLs levels (lupus anticoagulant, anti-cardiolipin antibodies, anti-beta2-glycoprotein I antibodies, anti-protein C antibodies, and anti-protein S antibodies), the APC-R test, and the factor V Leiden test in 85 SLE patients. Acquired APC-R, which was not found in any patient with the factor V Leiden mutation, was present in 26 (30.6%) of 85 patients, and confirmed that acquired APC-R was a significant risk factor for thromboembolic complications [odd ratio (OR), 3.36; 95% confidence interval (CI), 1.24-9.11]. Multivariate logistic analysis revealed that both LA and anti-PS strongly associated with the presence of APC-R, and that the correlation between anti-PS and APC-R was much stronger (OR, 46.7; 95%CI, 6.99-311) than that between LA and APC-R (OR, 11.3; 95%CI, 2.26-57.0). Furthermore, the mean value of APC sensitivity ratios was significantly lower in SLE patients with anti-PS (mean +/- SD, 1.68 +/- 0.37, p < 0.0001) than in those without anti-PS (2.23 +/- 0.40). These results suggest that acquired APC-R is most strongly attributable to functional interference of the APC pathway by anti-PS, which contribute to risk of thromboembolic complications.

Increase in Serum Levels of Autoantibodies after Attack of Seasonal Allergic Rhinitis in Patients with Graves’ Disease

Background: The prevalence of allergic disease is increasing worldwide, but its influence on the clinical course of autoimmune diseases is unknown. Objective: The purpose of this study was to assess the effect of seasonal allergic rhinitis on the clinical course of Graves’ disease, which has been considered a Th2-dominant autoimmune disease. Methods: Ten patients with Graves’ disease, who were considered to be in a state of remission or near remission, were serially examined for 18 months starting from August. Five of them had seasonal allergic rhinitis due to Japanese cedar pollen, and the remaining patients had no such allergic disorders. Peripheral eosinophil counts, serum concentrations of cedar-pollen-specific IgE, anti-TSH-receptor antibody, anti-thyroid-peroxidase antibody and antithyroglobulin antibody were assessed at 2- to 4-month intervals. Serum thyroid hormones and TSH levels were also measured to evaluate disease activity. Results: All patients with pollinosis had attacks of allergic rhinitis caused by cedar pollen in early March. Subsequently, peripheral eosinophil counts, pollen-specific IgE activity and serum levels of anti-thyroid-peroxidase and antithyroglobulin autoantibodies markedly increased. Serum levels of anti-TSH-receptor antibody increased in 3 patients in association with an increase in serum thyroid hormones but were always negative in 2 patients. The control patients without pollinosis showed no consistent change of these parameters. Conclusions: Seasonal allergic rhinitis aggravated the clinical course of Graves’ disease and induced an increase in serum antithyroid autoantibody concentrations as well as an increase in pollen-specific IgE concentration. These data suggest that environmental antigens induce not only local allergic reactions, but also stimulate thyroid immune reactions toward Th2 proliferation, and finally aggravate Th2-dependent autoimmune thyroid disease.

Genes that characterize T3-predominant Graves' thyroid tissues

OBJECTIVEn3,5,3-Triiodothyronine (T(3))-predominant Graves disease is characterized by the increasing volume of thyroid goiter resulting in poor prognosis. Although type 1 and type 2 iodothyronine deiodinases (DIO1 and DIO2 respectively) are known to be overexpressed in the thyroid tissues of T(3)-predominant Graves disease, the pathogenesis of this disease is still unclear. The aim of our study is to identify genes that characterize T(3)-predominant Graves disease tissue in order to clarify the molecular mechanism of this disease.nnnDESIGN AND METHODSnmRNAs from two thyroid tissues of both typical T(3)-predominant and common-type Graves disease were analyzed with DNA microarrays with probes for 28u200a869 genes. Genes identified to be differentially expressed between the two groups were further analyzed in the second and third screenings using 70 Graves thyroid tissues by real-time quantitative RT-PCR.nnnRESULTSnTwenty-three candidate genes were selected as being differentially expressed in the first screening with microarrays. Among these, seven genes, leucine-rich repeat neuronal 1 (LRRN1), bone morphogenetic protein 8a (BMP8A), N-cadherin (CDH2), phosphodiesterase 1A (PDE1A), creatine kinase mitochondrial 2 (CKMT2), integrin beta-3 (ITGB3), and protein tyrosine phosphatase non-receptor type 4 (PTPN4), were confirmed to be differentially expressed in DIO1 or DIO2 over- and underexpressing Graves tissues.nnnCONCLUSIONSnThese genes are related to the characteristics of T(3)-predominant Graves disease, such as high titer level of serum anti-TSH receptor antibody, high free T(3) to free thyroxine ratio, and a large goiter size. They might play a role in the pathogenesis of T(3)-predominant Graves disease.