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Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3'UTR with two regions of AID-dependent Ago2-binding sites (5'- and 3'dABs). Top1 3'UTR knockout (3'UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3'UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3'UTR-mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5'- and 3'dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3'UTRKO cells to wild-type cells whereas it does not impact on 3'UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3'UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation.

HTLV-1-associated myelopathy (HAM/TSP) is a chronic and progressive inflammatory disease of the central nervous system. The aim of our study was to identify genetic determinants related to the onset of HAM/TSP in the Japanese population. We conducted a genome-wide association study comprising 753 HAM/TSP patients and 899 asymptomatic HTLV-1 carriers. We also performed comprehensive genotyping of HLA-A , -B , -C , -DPB1 , -DQB1 , and -DRB1 genes using next-generation sequencing technology for 651 HAM/TSP patients and 804 carriers. A strong association was observed in HLA class I ( P = 1.54 × 10 -9 ) and class II ( P = 1.21 × 10 -8 ) loci with HAM/TSP. Association analysis using HLA genotyping results showed that HLA-C * 07:02 ( P = 2.61 × 10 -5 ), HLA-B * 07:02 ( P = 4.97 × 10 -10 ), HLA-DRB1 * 01:01 ( P = 1.15 × 10 -9 ) and HLA-DQB1 * 05:01 ( P = 2.30 × 10 -9 ) were associated with disease risk, while HLA-B * 40:06 ( P = 3.03 × 10 -5 ), HLA-DRB1 * 15:01 ( P = 1.06 × 10 -5 ) and HLA-DQB1 * 06:02 ( P = 1.78 × 10 -6 ) worked protectively. Logistic regression analysis identified amino acid position 7 in the G-BETA domain of HLA-DRB1 as strongly associated with HAM/TSP ( P = 9.52 × 10 -10 ); individuals homozygous for leucine had an associated increased risk of HAM/TSP (odds ratio, 9.57), and proline was protective (odds ratio, 0.65). Both associations were independent of the known risk associated with proviral load. DRB1-GB-7-Leu was not significantly associated with proviral load. We have identified DRB1-GB-7-Leu as a genetic risk factor for HAM/TSP development independent of proviral load. This suggests that the amino acid residue may serve as a specific marker to identify the risk of HAM/TSP even without knowledge of proviral load. In light of its allele frequency worldwide, this biomarker will likely prove useful in HTLV-1 endemic areas across the globe., Competing Interests: Competing interest statement: S.K. and F.M. are board members of GenoConcierge Kyoto Inc. S.K., M. Shimizu, and F.M. have patents pending for HLA typing software, primer sets for PCR amplification of HLA genes and its experimental protocol, and a risk marker of DRB1-GB-7 for HAM/TSP development., (Copyright © 2021 the Author(s). Published by PNAS.)

Takayasu arteritis (TAK) is a systemic vasculitis with severe complications that affects the aorta and its large branches. HLA-B*52 is an established susceptibility locus to TAK. To date, there are still only a limited number of reports concerning non-HLA susceptibility loci to TAK. We conducted a genome-wide association study (GWAS) and a follow-up study in a total of 633 TAK cases and 5,928 controls. A total of 510,879 SNPs were genotyped, and 5,875,450 SNPs were imputed together with HLA-B*52. Functional annotation of significant loci, enhancer enrichment, and pathway analyses were conducted. We identified four unreported significant loci, namely rs2322599, rs103294, rs17133698, and rs1713450, in PTK2B , LILRA3 / LILRB2 , DUSP22 , and KLHL33 , respectively. Two additional significant loci unreported in non-European GWAS were identified, namely HSPA6 / FCGR3A and chr21q.22. We found that a single variant associated with the expression of MICB , a ligand for natural killer (NK) cell receptor, could explain the entire association with the HLA-B region. Rs2322599 is strongly associated with the expression of PTK2B Rs103294 risk allele in LILRA3 / LILRB2 is known to be a tagging SNP for the deletion of LILRA3 , a soluble receptor of HLA class I molecules. We found a significant epistasis effect between HLA-B*52 and rs103294 ( P = 1.2 × 10 -3 ). Enhancer enrichment analysis and pathway analysis suggested the involvement of NK cells ( P = 8.8 × 10 -5 , enhancer enrichment). In conclusion, four unreported TAK susceptibility loci and an epistasis effect between LILRA3 and HLA-B*52 were identified. HLA and non-HLA regions suggested a critical role for NK cells in TAK., Competing Interests: Conflict of interest statement: Editor Tasuku Honjo and several authors are affiliated with Kyoto University but do not have any active collaborations.

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.

Background: A previous study revealed the association between susceptibility to Takayasu arteritis (TAK) and a single nucleotide polymorphism (SNP) rs6871626 located in IL12B, which encodes interleukin (IL)-12p40, a common component of IL-12p70 and IL-23. We investigated the expression of these cytokines in patients with TAK, stratifying them into those with or without the risk allele at the rs6871626 SNP., Methods: Plasma levels of IL-12p40, IL-12p70, and IL-23 were quantified in 44 patients with TAK and 19 healthy controls (HCs) by enzyme-linked immunosorbent assays. Monocytes were obtained from 20 patients with TAK and 14 HCs, treated with interferon-γ (IFN-γ) and lipopolysaccharide, and then supernatant cytokines were quantified. In addition, the ratio of IFN-γ + or IL-17A + cells to CD4 + T cells was measured by flow cytometric analysis of peripheral blood mononuclear cells., Results: The levels of plasma IL-12p40, plasma IL-12p70, and supernatant IL-12p70 were significantly higher in patients with TAK than in HCs, whereas there were no significant differences in the levels of plasma IL-23, supernatant IL-23, or supernatant IL-12p40. The levels of plasma IL-12p70, supernatant IL-12p40, and supernatant IL-12p70 were significantly higher in patients with the risk allele than in those without. The ratio of CD4 + IFN-γ + cells was significantly higher in patients with the risk allele, whereas CD4 + IL-17A + cells showed no differences., Conclusions: The rs6871626 SNP in IL12B may influence the increased expression of IL-12p40 and IL-12p70. These enhanced cytokines might play roles in the pathophysiology of TAK.

The accurate typing of human leukocyte antigen (HLA) alleles is critical for a variety of medical applications, such as genomic studies of multifactorial diseases, including immune system and inflammation-related disorders, and donor selection in organ transplantation and regenerative medicine. Here, we developed a new algorithm for determining HLA alleles using next-generation sequencing (NGS) results. The method consists of constructing an extensive dictionary of HLA alleles, precise mapping of the NGS reads, and calculating a score based on weighted read counts to select the most suitable pair of alleles. The developed algorithm compares the score of all allele pairs, taking into account variation not only in the domain for antigen presentation (G-DOMAIN), but also outside this domain. Using this method, HLA alleles could be determined with 6-digit precision. We showed that our method was more accurate than other NGS-based methods and revealed limitations of the conventional HLA typing technologies. Furthermore, we determined the complete genomic sequence of an HLA-A-like-pseudogene when we assembled NGS reads that had caused arguable typing, and found its identity with HLA-Y*02:01. The accuracy of the HLA-A allele typing was improved after the HLA-Y*02:01 sequence was included in the HLA allele dictionary., (© 2017 The Authors. **Human Mutation published by Wiley Periodicals, Inc.)

Objectives: Systemic sclerosis (SSc) is an autoimmune disease characterised by skin and systemic fibrosis culminating in organ damage. Previous genetic studies including genome-wide association studies (GWAS) have identified 12 susceptibility loci satisfying genome-wide significance. Transethnic meta-analyses have successfully expanded the list of susceptibility genes and deepened biological insights for other autoimmune diseases., Methods: We performed transethnic meta-analysis of GWAS in the Japanese and European populations, followed by a two-staged replication study comprising a total of 4436 cases and 14 751 controls. Associations between significant single nuclear polymorphisms (SNPs) and neighbouring genes were evaluated. Enrichment analysis of H3K4Me3, a representative histone mark for active promoter was conducted with an expanded list of SSc susceptibility genes., Results: We identified two significant SNP in two loci, GSDMA and PRDM1 , both of which are related to immune functions and associated with other autoimmune diseases (p=1.4×10 -10 and 6.6×10 -10 , respectively). GSDMA also showed a significant association with limited cutaneous SSc. We also replicated the associations of previously reported loci including a non-GWAS locus, TNFAIP3. PRDM1 encodes BLIMP1, a transcription factor regulating T-cell proliferation and plasma cell differentiation. The top SNP in GSDMA was a missense variant and correlated with gene expression of neighbouring genes, and this could explain the association in this locus. We found different human leukocyte antigen (HLA) association patterns between the two populations. Enrichment analysis suggested the importance of CD4-naïve primary T cell., Conclusions: GSDMA and PRDM1 are associated with SSc. These findings provide enhanced insight into the genetic and biological basis of SSc., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: Pulmonary arterial hypertension (PAH) is a severe lung disease with only few effective treatments available. Familial cases of PAH are usually recognized as an autosomal dominant disease, but incomplete penetrance of the disease makes it difficult to identify pathogenic variants in accordance with a Mendelian pattern of inheritance., Methods: To elucidate the complex genetic basis of PAH, we obtained whole exome- or genome-sequencing data of 17 subjects from 9 families with heritable PAH and applied gene-based association analysis with 9 index patients and 300 PAH-free controls., Results: A burden of rare variants in BMPR2 significantly contributed to the risk of the disease (p = 6.0 × 10 -8 ). Eight of nine families carried four previously reported single nucleotide variants and four novel insertion/deletion variants in the gene. One of the novel variants was a large 6.5 kilobase-deletion. In the remaining one family, the patient carried a pathogenic variant in a member of potassium channels, KCNK3, which was the first replicative finding of channelopathy in an Asian population., Conclusions: The variety of rare pathogenic variants suggests that gene-based association analysis using genome-wide sequencing data from increased number of samples is essential to tracing the genetic heterogeneity and developing an appropriate panel for genetic testing.

Whole-genome and -exome resequencing using next-generation sequencers is a powerful approach for identifying genomic variations that are associated with diseases. However, systematic strategies for prioritizing causative variants from many candidates to explain the disease phenotype are still far from being established, because the population-specific frequency spectrum of genetic variation has not been characterized. Here, we have collected exomic genetic variation from 1208 Japanese individuals through a collaborative effort, and aggregated the data into a prevailing catalog. In total, we identified 156 622 previously unreported variants. The allele frequencies for the majority (88.8%) were lower than 0.5% in allele frequency and predicted to be functionally deleterious. In addition, we have constructed a Japanese-specific major allele reference genome by which the number of unique mapping of the short reads in our data has increased 0.045% on average. Our results illustrate the importance of constructing an ethnicity-specific reference genome for identifying rare variants. All the collected data were centralized to a newly developed database to serve as useful resources for exploring pathogenic variations. Public access to the database is available at http://www.genome.med.kyoto-u.ac.jp/SnpDB/.

Background: Though the dosimetric criteria for the gastrointestinal tract were met, late gastrointestinal toxicity was seen in several cases. Therefore, we thought that it was caused by the positional variation of gastrointestine surrounding pancreatic cancer because of peristalsis., Method: They were confirmed by CT image regularly. And we evaluated that how much the difference of matching methods for correcting the positional variation influenced dose distribution., Result: The fiducial markers could follow the position of pancreatic cancer and the duodenum. But it could reproduce the dose distribution to pancreatic cancer and the duodenum., Discussion: In proton therapy, the reproducible improvement of the duodenum position did not make the dose of the duodenum same as planning dose because the matching of fiducial markers made the positional relations between beam compensator and the duodenum change., Conclusion: The fiducial markers are useful for correcting the position of pancreatic cancer and the duodenum. But in proton therapy, it could not reproduce the dose distribution to pancreatic cancer and the duodenum.

Introduction: Although susceptibility genes for anti-citrullinated peptide/protein antibodies (ACPA)-positive rheumatoid arthritis (RA) have been successfully discovered by genome-wide association studies (GWAS), little is known about the genetic background of ACPA-negative RA. We intended to elucidate genetic background of ACPA-negative RA., Method: We performed a meta-analysis of GWAS comprising 670 ACPA-negative RA and 16,891 controls for 1,948,138 markers, followed by a replication study of the top 35 single nucleotide polymorphisms (SNPs) using 916 cases and 3,764 controls. Inverse-variance method was applied to assess overall effects. To assess overlap of susceptibility loci between ACPA-positive and -negative RA, odds ratios (ORs) of the 21 susceptibility markers to RA in Japanese were compared between the two subsets. In addition, SNPs were stratified by the p-values in GWAS meta-analysis for either ACPA-positive RA or ACPA-negative RA to address the question whether weakly-associated genes were also shared. The correlations between ACPA-positive RA and the subpopulations of ACPA-negative RA (rheumatoid factor (RF)-positive and RF-negative subsets) were also addressed., Results: Rs6904716 in LEMD2 of the human leukocyte antigen (HLA) locus showed a borderline association with ACPA-negative RA (overall p = 5.7 × 10(-8)), followed by rs6986423 in CSMD1 (p = 2.4 × 10(-6)) and rs17727339 in FCRL3 (p = 1.4 × 10(-5)). ACPA-negative RA showed significant correlations of ORs with ACPA-positive RA for the 21 susceptibility SNPs and non-HLA SNPs with p-values far from significance. These significant correlations with ACPA-positive RA were true for ACPA-negative RF-positive and ACPA-negative RF-negative RA. On the contrary, positive correlations were not observed between the ACPA-negative two subpopulations., Conclusion: Many of the susceptibility loci were shared between ACPA-positive and -negative RA.

Objective: While antinuclear antibodies (ANAs) are observed in healthy populations as well as in patients with autoimmune diseases such as systemic lupus erythematosus (SLE), the detailed genetic background of ANAs has remained unclear. We undertook this study to identify the genetic determinants of ANAs in the general population in order to elucidate the underlying mechanisms of ANA production and to distinguish disease susceptibility genes from ANA production genes., Methods: A total of 9,575 Japanese volunteers were registered, and their ANA levels were quantified using indirect immunofluorescence to analyze correlates of ANA positivity. Genetic studies were performed using 7,148 of the 9,575 subjects. We performed a genome-wide association study using 3,185 subjects genotyped for 303,506 single-nucleotide polymorphisms (SNPs), followed by a replication study of 3,963 subjects. HLA-DRB1 and HLA-DQB1 alleles were imputed, and associations between ANA positivity and the SNPs or the HLA alleles associated with SLE were analyzed., Results: Female sex and old age were associated with ANA positivity, except for the nucleolar pattern. The T allele of rs2395185 in the HLA locus, which was in moderate linkage disequilibrium with HLA-DRB1*0405, was significantly associated with ANA positivity (P = 1.3 × 10(-11) ). The T allele of rs2395185 displayed increasing effects on the frequency of speckled and homogeneous patterns (P = 7.5 × 10(-12) and P = 2.2 × 10(-11) , respectively) but decreasing effects on the frequency of the nucleolar pattern (P = 0.0045). The 7 SNPs and 4 HLA-DRB1 alleles associated with SLE did not display strong associations with ANA positivity., Conclusion: SNP rs2395185 linked with HLA-DRB1*0405 is a genetic determinant of ANA production in the Japanese population. Overlapping of loci for susceptibility to SLE and to ANA positivity was limited. The nucleolar pattern showed different associations from other staining patterns, both with correlates of ANA positivity and with the HLA locus., (Copyright © 2014 by the American College of Rheumatology.)

NOD/LtSzscid/IL-2Rγ(-/-) (NSG) mice have advantages in establishing humanized mouse models. However, transferring human PBMCs into these mice often causes lethal GVH disease. In this study, we discovered an improved method for the engraftment of normal or pathological human PBMCs into NSG mice and examined the subsequent induction of specific immune responses. We sequentially transferred human CD4+ memory T (Tm) and B cells obtained from PBMCs of healthy adults or patients with autoimmune diseases into NSG mice. Removing naïve CD4+ T cells from the transferred PBMCs allowed successful engraftment without lethal GVH disease. The transferred Tm cells were found to reside mainly in the spleen and the lymphoid nodules, where they expressed MHC class II molecules and produced cytokines, including IL-21. Surprisingly, the transferred B cells were also well maintained in the lymphoid organs, underwent de novo class-switch recombination, and secreted all isotypes of human Igs at significant levels. Moreover, transferring patient-derived Tm and B cells resulted in sustained production of IgM-rheumatoid factor and antiaminoacyl transfer RNA synthetase Abs in these mice. These results suggest that transfer of Tm and B cells derived from human PBMCs into NSG mice could be a useful method for the study of human autoimmune mechanisms., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Prostate-specific antigen (PSA) is a useful marker for prostate cancer (PCa) and is widely used for screening of PCa. Previous studies have shown that genetic components influence the levels of PSA, and some of these genetic components would lead to better diagnostic sensitivity and specificity to PCa. However, genetic studies for PSA from Asian countries are limited. Our aim was to identify genetic components influencing PSA levels in the Japanese population using genome-wide association study (GWAS) and to analyse whether genetic components would lead to better screening abilities of PCa., Methods: We performed a GWAS comprising 1086 male subjects using 303 283 single nucleotide proteins, followed by a replication study of 1302 subjects. PSA levels were quantified by chemiluminescence immunoassay method. Quantitative linear regression analysis was performed to assess genetic components of PSA levels. A total of 413 subjects with prostate biopsies were analysed to examine whether genetic determinants would improve diagnostic ability., Results: Rs16856139 in SLC45A3, the same region as the previous Chinese study, showed an overall significant association with PSA levels (p=2.4×10(-11)) along with rs1058205 in KLK3. In silico analysis revealed significant association between rs16856139 and expression of SLC45A3. Genetic scores of PSA showed a dose-dependent decrease of area under curve (AUC) of PCa and successfully subgrouped the individuals with significantly different AUC (p≤0.0097)., Conclusions: Rs16856139, associated with the expression of SLC45A3, is significantly associated with the levels of PSA in the Japanese population. Classification of subjects based on PSA genetic determinants would improve screening ability of PSA to detect PCa., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Interstitial lung disease (ILD) is a common complication and sometimes a prognostic factor of connective tissue diseases (CTDs) in humans. However, suitable animal model of severe CTD-associated ILD (CTD-ILD) has been limited. In this study, we showed that zymosan-treated SKG mice developed not only arthritis but also chronic-progressive ILD with high mortality over several months. The pathological and clinical features of ILD in zymosan-treated SKG mice were similar to that of human severe CTD-ILD. ILD in this mouse was characterized by massive infiltration of Th17 cells, GM-CSF-producing CD4(+) T cells, and CD11b(+) Gr1(+) neutrophils with fibrosis. Naive SKG T cells were skewed to differentiate into GM-CSF-producing cells, and GM-CSF secreted by T cells enhanced IL-6 and IL-1β production by macrophages, which in turn enhanced differentiation of IL-17A- and/or GM-CSF-producing T cells and infiltration of neutrophils into lung. Neutralization of GM-CSF completely blocked the development of this ILD, and the blocking of IL-6 signaling resulted in partial prevention of it, whereas neutralization of IL-17A did not. In contrast, the progression of arthritis was inhibited by the neutralization of GM-CSF and slightly by the neutralization of IL-17A, but not by the blocking of IL-6 signaling. These data suggested zymosan-treated SKG mice could be a useful mouse model of severe CTD-ILD, and GM-CSF, rather than IL-17A or IL-6, contributed to the development of ILD in zymosan-treated SKG mice, indicating that neutralization of GM-CSF would be a useful therapeutic strategy for severe CTD-ILD., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Takayasu arteritis (TAK) is an autoimmune systemic vasculitis of unknown etiology. Although previous studies have revealed that HLA-B*52:01 has an effect on TAK susceptibility, no other genetic determinants have been established so far. Here, we performed genome scanning of 167 TAK cases and 663 healthy controls via Illumina Infinium Human Exome BeadChip arrays, followed by a replication study consisting of 212 TAK cases and 1,322 controls. As a result, we found that the IL12B region on chromosome 5 (rs6871626, overall p = 1.7 × 10(-13), OR = 1.75, 95% CI 1.42-2.16) and the MLX region on chromosome 17 (rs665268, overall p = 5.2 × 10(-7), OR = 1.50, 95% CI 1.28-1.76) as well as the HLA-B region (rs9263739, a proxy of HLA-B*52:01, overall p = 2.8 × 10(-21), OR = 2.44, 95% CI 2.03-2.93) exhibited significant associations. A significant synergistic effect of rs6871626 and rs9263739 was found with a relative excess risk of 3.45, attributable proportion of 0.58, and synergy index of 3.24 (p ≤ 0.00028) in addition to a suggestive synergistic effect between rs665268 and rs926379 (p ≤ 0.027). We also found that rs6871626 showed a significant association with clinical manifestations of TAK, including increased risk and severity of aortic regurgitation, a representative severe complication of TAK. Detection of these susceptibility loci will provide new insights to the basic mechanisms of TAK pathogenesis. Our findings indicate that IL12B plays a fundamental role on the pathophysiology of TAK in combination with HLA-B(∗)52:01 and that common autoimmune mechanisms underlie the pathology of TAK and other autoimmune disorders such as psoriasis and inflammatory bowel diseases in which IL12B is involved as a genetic predisposing factor., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Follistatin-related protein (FRP)/follistatin-like 1 (FSTL1) has multi-specific binding nature especially with TGF-β superfamily proteins, and thereby modulates organ development. However, its function in immune systems remains unclear. Previously, we reported FRP interacts with CD14, which is known to mediate toll-like receptor 4 (TLR4) signaling. Here, we investigated whether FRP activates TLR4 signaling. Recombinant FRP induced interleukin 6 or interleukin 8 production from target cells in a CD14- and TLR4-dependent manner. Moreover, similar to lipopolysaccharide (LPS), FRP induced tolerance to the second LPS stimulation. FRP has the function of evoking innate immune responses as one of the endogenous TLR4 agonists., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10(-4) by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10(-8), OR 1.23, 95% CI: 1.14-1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease.

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA.

Purpose: Proton therapy is a sophisticated treatment modality for prostate cancer. We investigated how physiologic factors affected the distribution of autoactivation as detected by positron emission tomography (PET) after proton beam therapy., Methods and Materials: Autoactivation was evaluated in 59 patients treated with a 210-MeV proton beam. Data acquisition for autoactivation by PET started 5 minutes after proton irradiation to assess activation. In the first 29 patients, five regions of interest were evaluated: planning target volume (PTV) center, urinary bladder inside the PTV, urinary bladder outside the PTV, rectum (outside the PTV), and contralateral femoral bone head (outside the PTV). In the remaining 30 patients, urine activity was measured directly. In a phantom study autoactivation and its diffusion after proton beam irradiation were evaluated with water or an ice block., Results: Mean activities calculated by use of PET were 629.3 Bq in the PTV center, 555.6 Bq in the urinary bladder inside the PTV, 332.5 Bq in the urinary bladder outside the PTV, 88.4 Bq in the rectum, and 23.7 Bq in the femoral bone head (p < 0.001). Mean urine activity was 679.4 Bq, recorded 10 minutes after therapy completion, and the half-life for urine autoactivation was 4.5 minutes., Conclusions: Urine is a major diffusion mediator of autoactivation after proton beam therapy. Our results indicate that physiologic factors can influence PET images of autoactivation in the context of proton beam therapy verification.

Objective: Although interleukin-17 (IL-17)-producing gamma/delta T cells were reported to play pathogenic roles in collagen-induced arthritis (CIA), their characteristics remain unknown. The aim of this study was to clarify whether gamma/delta T cells or CD4+ T cells are the predominant IL-17-producing cells, and to determine what stimulates gamma/delta T cells to secret IL-17 in mice with CIA. The involvement of IL-17-producing gamma/delta T cells in SKG mice with autoimmune arthritis and patients with rheumatoid arthritis (RA) was also investigated., Methods: IL-17-producing cells in the affected joints of mice with CIA were counted by intracellular cytokine staining during 6 distinct disease phases, and these cells were stimulated with various combinations of cytokines or specific antigens to determine the signaling requirements. Similar studies were performed using SKG mice with arthritis and patients with RA., Results: Gamma/delta T cells were the predominant population in IL-17-producing cells in the swollen joints of mice with CIA, and the absolute numbers of these cells increased in parallel with disease activity. IL-17-producing gamma/delta T cells expressed CC chemokine receptor 6, were maintained by IL-23 but not by type II collagen in vitro, and were induced antigen independently in vivo. Furthermore, IL-17 production by gamma/delta T cells was induced by IL-1beta plus IL-23 independently of T cell receptor. In contrast to what was observed in mice with CIA, IL-17-producing gamma/delta T cells were nearly absent in the affected joints of SKG mice and patients with RA, and Th1 cells were predominant in the joints of patients with RA., Conclusion: Gamma/delta T cells were antigen independently stimulated by inflammation at affected joints and produced enhanced amounts of IL-17 to exacerbate arthritis in mice with CIA but not in SKG mice with arthritis or patients with RA.

Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC-characteristic genes--including nine transcription factor genes--using DNA microarray and real-time RT-PCR analyses: Most of the MSC-characteristic genes were down-regulated 24 h after incubation with osteogenesis-, chondrogenesis- or adipogenesis-induction medium, or 48-72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the self-renewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic- and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules--including GATA6, TRPC4, FLG and TGM2--revealed that MSC-like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC-characteristic transcription factors are involved in MSC stemness regulation.

Particle radiotherapy using proton and heavy ion beams has shown improved clinical results and is a promising cancer therapy which is expected to gradually spread in Japan. There are, however, no special regulations for radiotherapy treatment facilities. They have been operated under the same safety regulations as for a research facility using a research accelerator. Significantly high-energy radiation is necessary for particle radiotherapy compared with conventional radiation therapy. The treatment facility, therefore, should have a large accelerator, which is installed in a room with a thick shield wall. Data on radiation protection for such high energy medical facilities is fragmentary and insufficient. In this study, we examined the necessity of other regulations for the safe operation of medical facilities for particle radiotherapy. First, we measured activation levels of the therapeutic devices and of patients. Next the safety level of the medical facility was evaluated from the viewpoint of radiation protection. We have confirmed the facilities can be safely operated by present regulations given in the Law Concerning Prevention from Radiation Hazards due to Radiation Isotopes, etc. or the Law for Health Protection and Medical Care.

A nano-electrospray ionization (nanoESI) emitter for analysis of a biological solution was developed by packing a nanoESI needle with two types of resins for desalting and preconcentration of target molecules. Determination of secreted histamine and serotonin molecules in cell culture buffers was demonstrated using 5-methyltryptamine as internal standard. The results showed good linearity of target signals in the concentration range from 0.25 to 50.0 ng/mL of histamine or serotonin. These molecules were monitored to be secreted by A23187 (calcium ionophore) stimulant in rat peritoneal mast cells. Using a combination of a video-microscope and a mass spectrometer, we could visualize exocytotic moments and analyze secreted molecules by mass spectrometry simultaneously. Time-dependent release of histamine and serotonin from activated mast cells showed that significant production of these molecules occurred and reached a maximal level at 15 min for serotonin and at 30 min for histamine, respectively. These results showed that this method allows the direct and timely analysis of secreted molecules in biological responses., (Copyright 2007 John Wiley & Sons, Ltd.)

Unlabelled: We isolated and expanded BMSCs from human alveolar/jaw bone at a high success rate (70%). These cells had potent osteogenic potential in vitro and in vivo, although their chondrogenic and adipogenic potential was less than that of iliac cells., Introduction: Human bone marrow stromal cells (BMSCs) have osteogenic, chondrogenic, and adipogenic potential, but marrow aspiration from iliac crest is an invasive procedure. Alveolar BMSCs may be more useful for regenerative medicine, because the marrow can be aspirated from alveolar bone with minimal pain., Materials and Methods: In this study, alveolar bone marrow samples were obtained from 41 patients, 6-66 years of age, during the course of oral surgery. BMSCs were seeded and maintained in culture with 10% FBS and basic fibroblast growth factor. In addition, BMSCs were induced to differentiate into osteoblasts, chondrocytes, or adipocytes in appropriate medium., Results and Conclusion: From a small volume (0.1-3 ml) of aspirates, alveolar BMSCs expanded at a success ratio of 29/41 (70%). The success rate decreased with increasing donor age, perhaps because of age-dependent decreases in the number and proliferative capacity of BMSCs. The expanded BMSCs differentiated into osteoblasts under osteogenic conditions in 21-28 days: the mRNA levels of osteocalcin, osteopontin, and bone sialoprotein, along with the calcium level, in alveolar BMSC cultures were similar to those in iliac cultures. However, unlike iliac BMSC, alveolar BMSC showed poor chondrogenic or adipogenic potential, and similar differences were observed between canine alveolar and iliac BMSCs. Subsequently, human alveolar BMSCs attached to beta-tricalcium phosphate were transplanted into immunodeficient mice. In transplants, new bone formed with osteoblasts and osteocytes that expressed human vimentin, human osteocalcin, and human GAPDH. These findings suggest that BMSCs have distinctive features depending on their in vivo location and that alveolar BMSCs will be useful in cell therapy for bone diseases.

We investigated the biological effect of combining carbon-beam and X-ray in vitro. The results showed that when we employed Gray equivalent as the indication of therapeutic dose, the effects could be explained with simple additive way in the treatment plan. This fact provides important information about the combined therapy of carbon-beam and X-ray.

MALDI-TOF mass spectrometry was used to detect intracellular molecules from a single intact cell on monolayers of other cells. Intracellular molecules, e.g., histamine, were gradually increased in a mouse bone marrow-derived mast cell by a maturation process. A single cell was captured by a microsuction pipette, and the mass spectra of intracellular histamine were measured directly. Finally, the time course of the intracellular molecular contents and the maturation stage from a single cell were estimated.