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Objectives We sought to clarify the presence of radiographic thymus variants using a scoring system, and their association with clinical and immunological features in RA patients. Methods A total of 387 RA patients were randomly selected from all patients visiting our department who underwent chest CT scanning, with exclusion of patients with thymoma or thymic cyst, or age < 30 years. Thymus size and attenuation score in axial CT images were quantitatively interpreted and assessed. Associations between immunophenotype data and clinical and serological features were analysed in a subset of patients. Results Thymic enlargement was found in 76 (19.6%) patients, and a thymus attenuation score ≥ 2 was found in 50 (12.9%) patients. The score was significantly associated with antibodies to ACPA positivity. Thymic enlargement was significantly associated with the proportions of CD4+ effector memory T cells. Conclusion Radiographic thymus variants were frequently observed in RA patients and may reflect an abnormal immune response involved in the pathogenesis of RA.

In 2007, Human-induced pluripotent stem cells (iPSCs) were generated by transducing four genes (Oct3/4, Sox2, Klf4, c-Myc). Because iPSCs can differentiate into any types of cells in the body and have fewer ethical issues compared to embryonic stem (ES) cells, application of iPSCs for regenerative medicine has been actively examined. In fact, iPSCs have already been used for clinical applications, but at present, only autologous iPSC-derived grafts or HLA homozygous iPSC-derived grafts are being transplanted into patients following HLA matching. HLA is an important molecule that enables the immune system differentiates between self and non-self-components; thus, HLA mismatch is a major hurdle in the transplantation of iPSCs. To deliver inexpensive off-the-shelf iPSC-derived regenerative medicine products to more patients, it is necessary to generate universal iPSCs that can be transplanted regardless of the HLA haplotypes. The current strategy to generate universal iPSCs has two broad aims: deleting HLA expression and avoiding attacks from NK cells, which are caused by HLA deletion. Deletion of B2M and CIITA genes using the CRISPR/Cas9 system has been reported to suppress the expression of HLA class I and class II, respectively. Transduction of NK inhibitory ligands, such as HLA-E and CD47, has been used to avoid NK cell attacks. Most recently, the HLA-C retaining method has been used to generate semi-universal iPSCs. Twelve haplotypes of HLA-C retaining iPSCs can cover 95% of the global population. In future, studying which types of universal iPSCs are most effective for engraftment in various physiological conditions is necessary.

Introduction. While numerous disease-modifying anti-rheumatic drugs (DMARDs) have brought about a dramatic paradigm shift in the management of rheumatoid arthritis (RA), unmet needs remain, such as the small proportion of patients who achieve drug-free status. The aim of this study was to explore key molecules for remission at the T cell level, which are known to be deeply involved in RA pathogenesis, and investigate the disease course of patients who achieved molecular remission (MR). Methods. We enrolled a total of 46 patients with RA and 10 healthy controls (HCs). We performed gene expression profiling and selected remission signature genes in CD4+ T cells and CD8+ T cells from patients with RA using machine learning methods. In addition, we investigated the benefits of achieving MR on disease control. Results. We identified 9 and 23 genes that were associated with clinical remission in CD4+ and CD8+ T cells, respectively. Principal component analysis (PCA) demonstrated that their expression profiling was similar to those in HCs. For the remission signature genes in CD4+ T cells, the PCA result was reproduced using a validation cohort, indicating the robustness of these genes. A trend toward better disease control was observed during 12 months of follow-up in patients treated with tocilizumab in deep MR compared with those in non-deep MR, although the difference was not significant. Conclusion. We identified robust genes that represent remission status in CD4+ T cells using machine learning techniques. The current study will promote our understanding of the molecular mechanisms necessary to achieve deep remission during the management of RA. Trial registration: Not required.

Background. While numerous disease-modifying anti-rheumatic drugs (DMARDs) has brought about a dramatic paradigm shift in the management of rheumatoid arthritis (RA), unmet needs remain such as small proportion of achievement drug-free patients. The aim of this study is to explore key molecules for remission at T cell level, which is known to involve the pathogenesis of RA deeply, and investigate disease course of patients who achieved molecular remission (MR).Methods. We enrolled a total of 46 patients with RA and 10 healthy controls (HCs). We performed gene expression profiling and selected remission signature genes in CD4+ T cells and CD8+ T cells of patients with RA using machine learning methods. In addition, we investigated the benefit of achieving MR on disease control thereafter.Results. We identified 9 and 23 genes that were associated with clinical remission in CD4+ and CD8+ T cells. Principal component analysis (PCA) demonstrated expression profiling of them was similar with HCs. As to remission signature genes in CD4+ T cells, PCA result was reproduced using validation cohort, indicating their robustness. There was a trend towards better disease control during 12 months follow-up in patients treated with tocilizumab in deep MR than those in non-deep MR, although it was not significant.Conclusion. We identified robust genes which represented remission status in CD4+ T cells using machine learning techniques. The current study will promote our understandings about molecular mechanism to achieve deep remission in the management of RA.Trial registration: Not required.

Novel small molecules were synthesized and evaluated as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of inflammatory and autoimmune diseases. A hit compound, 1, was discovered by high-throughput screening of our compound library. The structure-activity relationship (SAR) study of compound 1 showed that the introduction of a chlorine group at the 3-position of 4-cyanophenyl moiety increased the potency and a 3-methylpentane-1,5-diamide linker is favorable for the activity. The carbazole moiety of 1 was also optimized; a quinazolinedione derivative 18i suppressed the increase of IL-17A mRNA level in the lymph node of a rat model of experimental autoimmune encephalomyelitis (EAE) upon oral administration. These results indicate that the novel quinazolinedione derivatives have great potential as orally available small-molecule RORγt inverse agonists for the treatment of Th17-driven autoimmune diseases. A U-shaped bioactive conformation of this chemotype with RORγt protein was also observed.

ObjectivesRheumatoid arthritis (RA) is an autoimmune disease accompanied by lymphocyte infiltration into joint synovium. While T cells are considered to be important for its pathogenesis, the features that are the most relevant to disease and how they change after treatment remain unclear. The aim of this study was to clarify the characteristics of T cells in RA, comprehensively.MethodsWe enrolled a total of 311 patients with RA and 73 healthy participants, and carefully classified them by disease state, constructed multiple cohorts and analysed clinical samples from them in a stepwise manner. We performed immunophenotyping with multiple evaluation axes, and two independent transcriptome analyses complementary to each other.ResultsWe identified that ‘effector memory-Tfh’ subset was specifically expanded in the peripheral blood (PB) of patients with RA in correlation with disease activity, and reverted after treatment. Besides, we revealed distinct features of T cells in synovial fluid (SF) that the expression of Tfh/Tph-related genes and pro-inflammatory cytokines and chemokines, includingCXCL13, were significantly enriched, whereas these phenotype were Th1-like. Finally, we identified specific pathways, such as mTORC1, IL-2-stat5, E2F, cell cycle and interferon-related genes, that were significantly enriched in SF, in particular, as well as PB of untreated patients with RA, and notably, these features reverted after treatment.ConclusionOur multi-dimensional investigation identified disease relevant T-cell subsets and gene signatures deeply involved in pathogenesis of RA. These findings could aid in our understanding of essential roles of T cells in RA and will facilitate to development better diagnostic and therapeutic interventions.

A series of novel phenylglycinamides as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists were discovered through optimization of a high-throughput screen hit 1. (R)-N-(2-((3,5-Difluoro-4-(trimethylsilyl)phenyl) amino)-1-(4-methoxyphenyl)-2-oxoethyl)-3-hydroxy-N-methylisoxazole-5-carboxamide (22) was identified as one of the best of these compounds. It displayed higher subtype selectivity and specificity over other nuclear receptors and demonstrated in vivo potency to suppress the transcriptional activity of RORγt in a mouse PD (pharmacodynamic) model upon oral administration.

Background The thymus, a primary lymphoid organ, plays a crucial role in immune system homeostasis [1,2]. Although several small-scale studies of the association between radiographic thymus alterations and serological features have been reported in systemic autoimmune diseases, information in patients with rheumatoid arthritis (RA) is limited. Objectives We conducted a large-scale cross-sectional analysis of radiographic thymus alterations and their association with clinical and immunological features in patients with RA. Methods RA patients were randomly selected from all patients who visited our department and underwent chest CT scan between January 2013 and December 2015. Patients with thymoma or thymic cyst and those aged less than 30 years were excluded. Thymic enlargement and thymus attenuation score in axial images of CT scans were quantitatively interpreted. We defined thymic enlargement as a thickness of more than 13 mm and graded the score by a four-point scale (score 0–3) according to previous studies [3,4]. Associations with immunophenotyping data of peripheral blood by flow cytometry and clinical and serological information were statistically analyzed in some available patients. Results 387 RA patients were enrolled. 78% were women and mean age was 65.2±12.3 years. Thymic enlargement was found in 76 (19.6%) patients. Thymus attenuation (score ≥2) was found in 154 (39.8%) patients. These findings were more frequent than in undiagnosed controls (11.3% ( P =0.078) and 22.5% ( P =0.017)). Importantly, radiographic thymus alterations in these RA patients, especially thymus attenuation score, were significantly associated with serological features such as serum level of CRP, ESR, IgG, RF-positivity or ACPA-positivity ( P =0.0003, 0.001, 0.0009, 0.005, and 0.0009 respectively). When we investigated the association with the proportion of 68 peripheral blood subpopulations in 83 RA patients, thymic enlargement was significantly associated with the proportions of CD45RA+CCR7+ naive CD4+T cell or CD45RO+CCR7-effector memory CD4+T cell ( P =0.04 and 0.009). Furthermore, thymus attenuation score was also significantly associated with the proportions of CD4+ naive T cell, CD4+effector memory T cell, CXCR5-CXCR3+CXCR6+CD4+Th1/Th17 cell, CD45RO-CCR7+CD95+CD4+ stem cell memory T cell, and CD19+ B cell ( P =0.03, 0.02, 0.04, 0.04, and 0.037 respectively). Conclusions Radiographic thymus alterations are frequent in RA patients and may reflect immunological features of autoantibody production or T cell differentiation and function. References Gorozny JJ, et al. Trends Immunol 2001;22:251–255. Seddon B, et al. Immunol Today 2000;21:94–99. Naidich P, et al. Lippincott-Raven 1999:57–73. Ackman JB, et al. Radiology 2013;268(1):245–253. Disclosure of Interest None declared

Adoptive T-cell immunotherapy is a promising approach to cancer therapy. Stem cell memory T (TSCM) cells have been proposed as a class of long-lived and highly proliferative memory T cells. CD8+ TSCM cells can be generated in vitro from naive CD8+ T cells via Wnt signalling; however, methods do not yet exist for inducing TSCM cells from activated or memory T cells. Here, we show a strategy for generating TSCM-like cells in vitro (iTSCM cells) from activated CD4+ and CD8+ T cells in mice and humans by coculturing with stromal cells that express a Notch ligand. iTSCM cells lose PD-1 and CTLA-4 expression, and produce a large number of tumour-specific effector cells after restimulation. This method could therefore be used to generate antigen-specific effector T cells for adoptive immunotherapy., Tumour-specific T cells can be expanded in vitro and adoptively transferred for therapy, but this strategy is limited by induction of short-lived T cell populations. Here the authors activate Notch signalling in cultured mouse or human T cells, resulting in the production of a long-lived stem cell memory T cell population that can fight tumours in mice.

BACKGROUND: Large number of rejuvenated antigen-specific T cells generated from iPS cells (iPSCs) may have a large impact on the T-cell immunotherapy field. We previously reported the generation of functional CD8 single positive (SP) cytotoxic lymphocytes (CTLs) from iPSCs (Minagawa etal. CellStemCell, 23: 850-858. (2018)), and a regenerative CTL-based immunotherapy is about to begin in clinical trials. However, these two-dimensional differentiation protocols using the OP9DL1 murine feeder cell line or DLL4 recombinant proteins could differentiate iPSCs into CD8SP CTLs but not into robust CD4SP helper T (Th) cells. It is clear that, we could better control immune reactions if we could produce each Th cell fraction. For example, we could enhance antitumor immunity if we could specifically induce Th1 cells to command cellular immunity. The drastic therapeutic effect of CD19 CAR modified T cells including both CD8SP CTLs and CD4SP Th cells is clearly based on the role of CD4SP Th cells in helping CD8SP CTLs prolong the therapeutic effect against B-cell malignancy. This led us to hypothesize that Th cell induction from iPSCs is essential for efficient immunotherapy. In this situation, a three-dimensional (3D) method called artificial thymic organoid (ATO) was reported to support robust differentiation of both CD4SP and CD8SP TCRαβ cells from primary hematopoietic stem and progenitor cells (Seet etal. NatureMethods, 14: 521-530. (2017)) and hematopoietic progenitor cells derived from ES cells and iPSCs (Montel-Hagen etal. CellStemCell, 24: 376-389. (2019)). Here, we evaluated the advantages and unsolved tasks of the 3D method to induce antigen-specific and functional CD4SP Th cell subsets from iPSCs. METHODS and RESULTS: By applying the ATO methods, we cultured mixed pellets of iPSC-derived hematopoietic progenitor cells (HPCs) and Notch ligand-expressing MS5 feeder cells on cell culture inserts for up to 9 to 12 weeks. Next, we analyzed differentiated T cells in the ATOs. We used iPSCs derived from antigen-specific Th cells containing HLA class II restricted TCR genes corresponding to an original Th cell clone. Most importantly, the ATO method supported robust differentiation of CD4SP T cells as expected even from these iPSCs. These CD4SP T cells showed high expression of Th-POK as a master regulator of Th cells, and high secretion ability of several key cytokines produced by Th cells-IL-2, IFN-γ, and IL-4. However, the majority of iPSC-derived CD4SP T cells showed simultaneous secretion of both IFN-γ and IL-4 unlike normal peripheral CD4SP Th cells. We tried to make the regenerated CD4SP T cells separately produce the Th1 (IFN-γ) and Th2 (IL-4) cytokines by optimizing culture conditions, but we failed to achieve separated Th1/2 differentiation. To understand the reason for the bipolarized cytokine production profile of regenerated CD4SP T cells, we checked the master regulator expression profile. We found that a majority of the CD4SP T cell population highly expressed T-bet, the master regulator of Th1. Contrary to our expectation, GATA3's expression levels were not high in most CD4SP T cells. And, GATA3 is master regulator not only of Th2 but of T cell differentiation itself, so we guessed knock out (KO) of GATA3 led to failure of differentiation into T cell. To "polarize" the CD4SP T cells to have Th1 or Th2 functions, we knocked out TBX21 (coding T-bet) or the Th2 "master cytokine" IL4 of undifferentiated iPSCs using CRISPR-Cas9 to obtain TBX21KO/KO iPSCs or IL4KO/KO iPSCs, respectively. Those iPSCs were successfully differentiated into HPCs, and ATOs were then prepared using these cells. After 9 to 12 weeks, mature CD4SP T cells and CD8SP T cells were observed in both ATOs with the same surface marker profile as T cells from wild type iPSCs, and TBX21KO/KO CD4SP T cells or IL4KO/KO CD4SP T cells from iPSCs with selective production of IL-4 or IFN-γ, respectively (Figure1). These results also suggested the potential utility of ATO-based invitro T cell differentiation from genome-edited iPSC for understanding of human developmental immunology. CONCLUSIONS: "Polarized" CD4SP Th cells were successfully obtained from master regulator or cytokine gene-knockout iPSCs in ATO-based invitro differentiation. We are now investigating the actual helper function of these "polarized" iPS-Th cells that could be induced by the target peptide on HLA Class II molecules of antigen presenting cells. Disclosures Shinohara: Takeda Pharmaceutical Company Limited: Employment. Koga:Takeda Pharmaceutical Company Limited: Employment. Kassai:Takeda Pharmaceutical Company Limited: Employment. Kiyoi:Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Perseus Proteomics Inc.: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding. Kaneko:KIRIN holdings Co.,Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Other: Scientific adviser, Research Funding; TERUMO Co., Ltd.: Research Funding; TOSOH Co., Ltd.: Research Funding; Thyas Co., Ltd.: Other: Founder, Shareholder, Chief Science Officer, Research Funding.

Background The aim of this study was to elucidate the function of circulating follicular helper T (Tfh) cell subsets in helping B cells in patients with active, untreated IgG4-related disease (IgG4-RD) and determine their relationship with disease activity. Methods Seventeen consecutive patients with active, untreated IgG4-RD, 20 with primary Sjögren syndrome (pSS), 5 with multicentric Castleman’s disease (MCD), and 12 healthy controls (HC) were enrolled. Tfh cell subset function was evaluated by co-culture with naïve B cells in vitro. Activated Tfh cell subsets were defined as a CCR7lowPD-1high subset among Tfh cell subsets. Disease activity was evaluated by IgG4-RD responder index (IgG4-RD RI) score. Results The number of Tfh2 cells was significantly higher in IgG4-RD compared to pSS, MCD, or HC, and correlated with serum IgG4 level or the number of plasmablasts. In vitro, Tfh2 cells more efficiently induced the differentiation of naïve B cells into plasmablasts compared to Tfh1 or Tfh17 cells. Of note, while IgG production in culture supernatants of Tfh2 cells was comparable between IgG4-RD and HC, IgG4 production was significantly higher with Tfh2 cells from patients with IgG4-RD than in those from HC. Accordingly, the IgG4:IgG ratio in culture supernatants was also significantly higher with Tfh2 cells from IgG4-RD compared to HC. Moreover, the number of activated Tfh2 cells was higher in IgG4-RD compared to pSS, MCD, or HC, and strongly correlated with IgG4-RD RI score in the baseline active phase. Particularly, the number of activated Tfh2 cells was associated with the number of affected organs and serum IgG4 level. Importantly, the number of activated Tfh2 cells was decreased after glucocorticoid treatment and paralleled disease improvement. Moreover, the number of activated Tfh1 cells was also increased in IgG4-RD compared to pSS, MCD, or HC, correlating with IgG4-RD RI score, but not with serum IgG4 level. Conclusions Tfh2 cells, but not Tfh1 or Tfh17 cells, induce the differentiation of naïve B cells into plasmablasts and enhanced production of IgG4 in patients with active, untreated IgG4-RD. Furthermore, activated Tfh2 cells reflect disease activity, suggesting the involvement of this T cell subset in the pathogenesis of IgG4-RD. Interestingly, the number of activated Tfh1 cells was also increased in IgG4-RD, correlating with disease activity but not with serum IgG4 level, suggesting the involvement of Tfh1 cells but not in the process of IgG4 production in patients with IgG4-RD. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1064-4) contains supplementary material, which is available to authorized users.

Background Scalp acupuncture (SA) therapy on strokes has been empirically established and widely used in clinics in China. The evidence from clinical studies suggests that SA produces significant benefits for some patients with stroke. Methods The effect of scalp acupuncture was studied using MRI for two different stroke models: spontaneously hypertensive stroke-prone (SHR-SP) rats and rats with transiently induced focal cerebral ischaemia by middle cerebral artery occlusion for 2 h (MCAO rats). Results Stroke onset in SHR-SP rats was characterised by a development of vasogenic oedema without any appearance of cytotoxic oedema. Scalp acupuncture reduced rapidly neurological dysfunction in SHR-SP rats and reduced the volume of the vasogenic oedema during the same period. In contrast, in MCAO rats, focal cerebral ischaemia caused an immediate development of cytotoxic oedema without any appearance of vasogenic oedema. Vasogenic oedema developed after reperfusion. Scalp acupuncture had no significant effects on the cytotoxic oedema, vasogenic oedema or neurological dysfunction of the MCAO rats within the time span examined. Conclusion Scalp acupuncture had a rapid and strong effect on neurological dysfunction only in the hypertensive stroke-model by reducing the vasogenic oedema. Our results suggest that, if there are similar underlying mechanisms in human strokes, scalp acupuncture may be more beneficial for patients with strokes of hypertension-caused vasogenic origin than ischaemic origin.

Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6), which mediates the activation of IkappaB kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here, we identified a novel zinc finger protein, ZCCHC11, that interacts with TIFA from phosphoproteins of a macrophage cell line, RAW 264.7, by using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database, we found a 200kDa full-length form (ZCCHC11L). ZCCHC11L was mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-kappaB activation. The N-terminal region (ZCCHC11S) including C2H2-type [corrected] Zn-finger motif was sufficient for suppression of NF-kappaB. We propose that ZCCHC11 is a unique TLR signal regulator, which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-kappaB.

Approximately 20% of human cancers are estimated to develop from chronic inflammation. Recently, the NF-kappaB pathway was shown to play an essential role in promoting inflammation-associated cancer, but the role of the JAK/STAT pathway, another important signaling pathway of proinflammatory cytokines, remains to be investigated. Suppressor of cytokine signaling-1 (SOCS1) acts as an important physiological regulator of cytokine responses, and silencing of the SOCS1 gene by DNA methylation has been found in several human cancers. Here, we demonstrated that SOCS1-deficient mice (SOCS1-/- Tg mice), in which SOCS1 expression was restored in T and B cells on a SOCS1-/- background, spontaneously developed colorectal carcinomas carrying nuclear beta-catenin accumulation and p53 mutations at 6 months of age. However, interferon (IFN)gamma-/- SOCS1-/- mice and SOCS1-/- Tg mice treated with anti-IFNgamma antibody did not develop such tumors. STAT3 and NF-kappaB activation was evident in SOCS1-/- Tg mice, but these were not sufficient for tumor development because these are also activated in IFNgamma-/- SOCS1-/- mice. However, colons of SOCS1-/- Tg mice, but not IFNgamma-/- SOCS1-/- mice, showed hyperactivation of STAT1, which resulted in the induction of carcinogenesis-related enzymes, cyclooxygenase-2 and inducible nitric oxide synthase. These data strongly suggest that SOCS1 is a unique antioncogene which prevents chronic inflammation-mediated carcinogenesis by regulation of the IFNgamma/STAT1 pathways.

Mitochondrial dysfunction has been implicated in the death of nigrostriatal dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated experimental models of Parkinson's disease (PD). Here we utilized proton magnetic resonance spectroscopy ((1)H MRS) to identify changes in energy metabolism in the striatum of MPTP-treated C57BL/6 mice. Remarkable increases in lactate/creatine (Lac/Cr) ratio were observed at 2 h and then quickly returned to about the basal level by 7 h after injection of MPTP. Neurochemical and Western blot analyses revealed that dopamine contents and protein levels of tyrosine hydroxylase and dopamine transporter in the striatum were profoundly decreased at 3 days after MPTP treatment. Pretreatment with deprenyl, a monoamine oxidase B inhibitor, or GBR-12909, a dopamine uptake inhibitor, almost completely attenuated both the increases in striatal Lac/Cr ratio and the subsequent loss of dopaminergic nerve terminals in MPTP-treated mice. The present study indicates that (1)H MRS is a sensitive measure of biochemical alterations of the brain in a mouse model of PD, and further shows that the increases in striatal Lac/Cr ratio induced by MPTP may be associated with mitochondrial energy crisis, followed by dopaminergic neurotoxicity.

Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2) skewing and lead to hypersensitivity to allergic diseases. To define the physiological roles of SOCS3 in T cells, we generated T cell–specific SOCS3 conditional knockout mice. We found that the mice lacking SOCS3 in T cells showed reduced immune responses not only to ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection. In vitro, SOCS3-deficient CD4+ T cells produced more transforming growth factor (TGF)-β1 and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like differentiation. We found that STAT3 positively regulates TGF-β1 promoter activity depending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-β1 promoter in SOCS3-deficient T cells than in control T cells. The activated STAT3 enhanced TGF-β1 and IL-10 expression in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these findings, we propose that SOCS3 regulates the production of the immunoregulatory cytokines TGF-β1 and IL-10 through modulating STAT3 activation.

To investigate the effects of cilostazol on the hemispheric ischemic lesion, we monitored the apparent diffusion coefficient (ADC) and T2 images by MRI techniques in comparison with histology at the terminal of and after 24-h reperfusion following 2-h occlusion of middle cerebral artery (MCA). The ADC values of tissue water and T2-weighted images were quantified by high field magnetic resonance. No significant difference was observed by ADC image among vehicle and cilostazol treatment groups when measured during MCA occlusion. Oral treatment with cilostazol 30 mg/kg two times at 5 min and 4 h significantly suppressed the hemispheric lesion area and volumes when detected by ADC, T2 images and histology, but 3 and 10 mg/kg cilostazol were without effect. Cilostazol (30 mg/kg) significantly reduced the increased cerebral water content at the ischemic hemisphere compared with vehicle group. In line with these results, the neurological deteriorations were much improved in the cilostazol-treated group. Taken together, it is concluded that post-treatment with cilostazol exerts a potent protective effect against cerebral infarct size by reducing the cytotoxic edema.

The orthodontic bonding characteristics were evaluated for the two types of orthodontic resin-modified glassionomer cements involving a chemical-activating cement (Fuji Ortho, GC) and a light-activating cement (Fuji Ortho LC, GC). Plastic, metal and ceramic brackets having individual irregularities for mechanical retention at their basal planes were bonded to tooth enamel with each of the cements. Their bond strengths were examined by means of shear and tensile tests and the fracture surfaces were also observed. The results obtained were as follows.1. Both the cements showed no or little bonding to plastic bracket.2. The bond strength between metal bracket and enamel was around 6 MPa in shear test and 5.5 MPa in tensile test. The fracture mode was cohesive failure in the cement, adhesive failure at the enamel-cement interface or their mixture.3. When ceramic bracket was used, the tensile bond strength was significantly (p

The purpose of this study was to evaluate disuse atrophy of skeletal muscle using a hind-limb suspension model, with special reference to energy metabolism. Twenty-four Sprague-Dawley rats were divided into four groups: control group (C), hind-limb suspended for 3 days (HS-3), for 7 days (HS-7) and for 14 days (HS-14). The gastrocnemius-plantaris-soleus (GPS) muscles in each group were subjected to the following measurements. After a 2-min rest, contraction of the GPS muscles was induced by electrical stimulation of the sciatic nerve at 0.25 Hz for 10 min, then the frequency was increased to 0.5 and 1.0 Hz every 10 min. During the stimulation, twitch forces were recorded by a strain gauge, and 31P-MRS was performed simultaneously. Maximum tension was measured at the muscle contraction induced at 0.25 Hz; the wet weight of the whole and each muscle in the GPS muscles was also measured. From the 31P-MR spectra during muscle contraction, the oxidative capacity was calculated and compared among the groups. The weights of the whole GPS muscles in C, HS-3, HS-7 and HS-14, were 2.66 +/- 0.09, 2.39 +/- 0.21, 2.34 +/- 0.21 and 2.18 +/- 0.14 (g) respectively. Thus, the muscle mass significantly decreased with time (p < 0.05). Among the GPS muscles, the decrease in weight of the soleus muscle was especially remarkable; in the HS-14 group its weight decreased to 60% of that in the C group. We evaluated maximum tension and oxidative capacity as the muscle function. The maximum tensions in C, HS-3, HS-7 and HS-14 were 519 +/- 43, 446 +/- 66, 450 +/- 23 and 465 +/- 29 (g), respectively. This was significantly greater in the C group than in any other groups, however there were no significant differences among the three HS groups. The oxidative capacity during muscle contraction in the C group was higher than in any HS group and it did not further decrease even if the suspension of the limbs was prolonged beyond 3 days. The present study showed that in disuse atrophy, muscle mass and muscle function did not change simultaneously. Thus, it is necessary to develop countermeasures to prevent muscle atrophy and muscle function deterioration independently.

We determined the effect of l-cis diltiazem, the enantiomer of diltiazem (d-cis isoform), on the energy metabolism of isolated guinea pig hearts during ischemia-reperfusion. We used 31P-NMR to measure the high-energy phosphate content and intracellular pH (pHi) during global ischemia for 30 min followed by reperfusion for 30 min. Before ischemia, the left ventricular developed pressure (LVDP) was reduced less by 10 μM l-cis diltiazem than by 3 μM diltiazem or 500 nM nifedipine. However, 10 μM l-cis diltiazem preserved the intracellular ATP content during ischemia and reperfusion, reduced the end-diastolic pressure increase during ischemia and reperfusion, and restored LVDP after reperfusion. Nifedipine at 50 nM, which reduced the LVDP more than 10 μM l-cis diltiazem, showed no cardioprotective effect. Ten micromolar l-cis diltiazem and 3 μM diltiazem, but neither 50 nor 500 nM nifedipine, reduced the pHi decrease that occurred 25 or 30 min after the onset of ischemia. Therefore, l-cis diltiazem has a cardioprotective effect on ischemic and reperfused myocardium and is less cardiodepressive than diltiazem and nifedipine. The effect of l-cis diltiazem during ischemia and reperfusion involves energy preservation, which is probably independent of its Ca2+-channel blocking action.

We studied the effects of denervation on the energy metabolism and peripheral circulation dynamics of rat hindlimb muscles during and after exercise. The sciatic nerves of male Wistar rats were cut to produce denervation. Energy metabolism was assessed by phosphorus-31 magnetic resonance spectroscopy (MRS), and circulation by fluorine-19 MRS. Exercise of rat hindlimb muscles was induced by electrical stimulation at 40 Hz. The inorganic phosphate (Pi) / ¿Pi + phosphocreatine (PCr)¿ ratio, an indicator of the energy level, was 0.795 immediately after denervation. The ratios 4 and 8 weeks after denervation were 0.870 and 0.853, respectively. The intracellular pH during the 4 min after initiation of stimulation was significantly reduced 4 and 8 weeks after denervation compared with the value immediately after denervation. The signal strength of the research perfluoro-carbon (FC-43; perfluorotributylamine) a measure of circulation dynamics, increased to 167% in controls during exercise, but an increase of only 134% was seen in rats 8 weeks after denervation. These results showed that the energy supply and circulation dynamics in denervated atrophic muscles decreased during exercise compared with findings in control muscles.

An anomalously large dielectric constant and a long relaxation time along the chain axis were first observed in the paraelectric phase of a ferroelectric copolymer of vinylidene fluoride and trifluoroethylene. Dielectric, X-ray scattering and shear sound velocity studies consistently show that the anomalous phenomena arise from the characteristic motion of a chain molecule of T G T G ′ chain conformation in the crystal: the chain in the paraelectric phase rotates independently of neighboring chains, and, in the chain, a T G T G ′ sequence, whose length increases around the Curie temperature, undergoes the flip-flop motion. The crystal in the paraelectric phase is a liquid crystal of 1D liquid and 2D solid.

Objective: We investigated if blockade of ATP-sensitive K + channels (K ATP ) abolishes the protective effect of ischemic preconditioning (IP) on myocardial metabolism and ischemia-induced reactive hyperemia (RH) in pigs. Methods: IP was elicited by a single cycle of 5 min occlusion and 5 min reperfusion of coronary artery, followed by 15 min of test ischemia and 120 min of reperfusion. Vehicle or the ATP-sensitive K + channels (K ATP ) blocker, glibenclamide (3 or 6 mg/kg; G3 or G6) was administered before IP (groups; IP, G3+IP, G6+IP). As respective controls, the same treatment was performed in groups without IP (groups; C, G3, G6). Tissue levels of ATP, creatine phosphate (CP) and intracellular pH (pHi) in the area at risk were measured by 31 P-nuclear magnetic resonance spectroscopy. RH after 5 min of preconditioning ischemia was assessed by regional myocardial blood flow. Results: ATP and pHi were preserved after 15 min of ischemia in the IP group [C/IP; ATP=57±4/76±10% of baseline, pHi=6.18±0.08/6.66±0.03, P Conclusions: Blockade of K ATP abolished the preconditioning effect on myocardial metabolism, and partially attenuated post-ischemic reactive hyperemia in pigs. These results indicate that K ATP activation might be involved in the mechanisms of these phenomena, reactive hyperemia is not sufficient to induce IP protection.

To elucidate the difference in the mechanisms for alkalization during ischemic acidosis between diabetic and non-diabetic hearts, intracellular pH (pHi) was measured by phosphorus-31 magnetic resonance spectroscopy. Diabetes was induced by the injection of streptozotocin. The accumulation of proton ion (DeltaH+) during 15 min global ischemia at 37 degreesC was calculated from pH i. There were no significant differences in DeltaH+ between diabetic (DM: 0. 54+/-0.03 micromol/l,n=6; mean+/-s.e.m.) and non-DM hearts (0.57+/-0.04, n=6), when perfused with bicarbonate buffer. However, perfusion with HEPES buffer revealed a significant increase of DeltaH+ in DM (0.85+/-0.07, n=5) compared with non-DM (0.61+/-0.06, n=5P0.05). On the contrary, the addition of a Na+/H+ exchange inhibitor (EIPA; 1 micromol/l) to bicarbonate buffer significantly increased DeltaH+ in non-DM (1.09+/-0.10, n=4) compared with DM (0.71+/-0.03, n=5P0.01). Perfusion with HEPES buffer and EIPA equally increased DeltaH+ in both groups (DM 1.13+/-0.13, n=4; non-DM 1.15+/-0.14, n=4). Thus, the activity of Na+/H+ exchanger during ischemic acidosis, assessed as the increase of DeltaH+ induced by addition of EIPA to bicarbonate buffer, was higher in non-DM (0.52) than DM (0.17). In contrast, the contribution of bicarbonate-dependent systems evaluated by the deference of DeltaH+ between the bicarbonate buffer and the HEPES buffer was markedly bigger in DM (0.31) than non-DM (0.04). These results indicate that Na+/H+ exchange is a major mechanism to compensate ischemic acidosis in non-DM hearts, whereas bicarbonate-dependent systems compensate the depressed activity of Na+/H+ exchange in DM.

We measured abdominal fat masses (intra-abdominal visceral fat summing retroperitoneal, mesenteric, and epididymal fat and subcutaneous fat) and analyzed abdominal fat distribution of Otsuka Long-Evans Tokushima Fatty (OLETF) rats with non-insulin-dependent diabetes mellitus and control strain Long-Evans Tokushima Otsuka (LETO) rats using magnetic resonance imaging. Intra-abdominal visceral and subcutaneous fat were highly correlated with body weight both in OLETF and in LETO rats. Both intra-abdominal visceral and subcutaneous fat of OLETF rats significantly accumulated compared with those of LETO rats. Intra-abdominal visceral fat mass correlated positively with subcutaneous fat mass, and the accumulation of intra-abdominal visceral fat mass was about 3.5 times that of subcutaneous fat. Thus, obesity of OLETF rats was characterized by marked accumulation of intra-abdominal visceral fat compared with that of subcutaneous fat. Body weight and abdominal fat of OLETF rats were closely correlated with the level of total plasma glucose measured by oral glucose tolerance test. However the ratio of intra-abdominal visceral to subcutaneous fat of OLETF rats was not correlated with the level of total plasma glucose.

Elastic, dielectric, and piezoelectric constant matrix elements of a “single crystalline’’ (SC) film of vinylidene fluoride trifluoroethylene copolymer, P(VDF/TrFE), in which the orthorhombic [001] and [110] axes of fully extended chain crystals are preferentially oriented parallel to the stretching axis and normal to the surface, respectively, were measured at temperatures ranging from 10 K to the Curie point (402–404 K) by using a piezoelectric resonance method. All of the electromechanical coupling factors (k31, k32, k33, k24, and k15) are larger than those of conventional lamellar crystalline films. Some of the matrix elements for a P(VDF/TrFE) single crystal are derived from the measured values of constant matrix elements for the SC film. Some features characteristic of the SC film are revealed. The SC film has a large Young’s modulus for the stretching direction (1/s11) (121 GPa at 10 K). The properties related to the molecular motions along the chain axis, such as 1/s11, shear stiffness constant c55, shear piezoelectric constant e15, etc., exhibit strong relaxations around 250 K. The origin of these relaxations in the crystalline phase is discussed.

RATIONALE AND OBJECTIVES The authors evaluate the changes in magnetic resonance (MR) relaxation times of rat skeletal muscles in vivo after nerve injury and during neural recovery, and determine the major determinants of relaxation times. MATERIALS Magnetic resonance relaxation times, blood volume, and water and fat content were examined after nerve injury and during recovery with time course. RESULTS Nerve injury led to longer T2 values compared with controls, but there were no significant changes in T1 values. After the initial prolongation of T2 after nerve injury, no changes were observed. Neural recovery resulted in a return of T2 values to normal. The time course of changes in blood volume was similar to that of changes in T2, and T2 values were correlated strongly with 19-fluorine-MR spectroscopy estimates of blood volume (r2 = 0.94). CONCLUSIONS T2 values may be useful to monitor recovery after nerve injury and may be related to the blood volume in skeletal muscle.

This study quantifies the myocardial glucose uptake and clarifies the pathway of augmented glucose uptake in myocardium reperfused after a brief period of ischemia (stunned myocardium). The glucose uptake rate was determined from the time course of the sugar phosphate (SP) resonance in rat myocardium (d[SP]/dt) with 31P nuclear magnetic resonance after the substitution of glucose with its analog 2-deoxyglucose. The d[SP]/dt in stunned myocardium [1.03 +/- 0.05 (SE) micromol x g wet wt(-1) x min(-1); n = 8] increased significantly compared with nonischemic control myocardium (0.18 +/- 0.03 micromol x g wet wt(-1) x min(-1); n = 8; P < 0.0001), reaching the maximal stimulatory uptake rate during exposure to insulin (1.05 +/- 0.04 micromol x g wet wt(-1) x min(-1); n = 8). Twenty minutes after reperfusion, the d[SP]/dt was still augmented (0.41 +/- 0.05 micromol x g wet wt(-1) x min(-1); n = 5; P < 0.05 vs. control myocardium). To elucidate further the mechanism of augmented glucose uptake, N6-(L-2-phenylisopropyl)-adenosine (PIA; 100 micromol/l), a potent blocker of the glucose transporter, was administered to stunned hearts and, as a control, to insulin-stimulated hearts. PIA significantly and comparably inhibited the increase in d[SP]/dt in stunned myocardium (0.36 +/- 0.07 micromol x g wet wt(-1) x min(-1); n = 4; P < 0.0001 vs. without PIA) and in insulin-stimulated myocardium (0.38 +/- 0.02 micromol x g wet wt(-1) x min(-1); n = 4; P < 0.0001 vs. without PIA). These results indicate that the augmented glucose uptake in stunned myocardium is maintained by the glucose transporter, the amount of which is almost equal to that which can be maximally recruited by insulin.

Background IgG4-related disease (IgG4-RD) is characterized by elevated serum IgG4 and increased circulating plasmablasts 1 . The high degree of somatic hypermutation and emergence of distinct plasmablast clones at disease relapse suggests the de novo recruitment of naive B cells into T-cell–dependent responses 1 . Follicular helper T (Tfh) cells play a major role in T-cell–dependent B cell responses, and their counterparts and subsets (Tfh1, Tfh2 or Tfh17 cells) have only been recognized in peripheral blood 2 . We previously reported that Tfh2 cells were increased in IgG4-RD 3 , although the functional role of Tfh2 cells remains to be elucidated. Objectives To elucidate the function of Tfh2 cells and involvement in the pathogenesis of active, untreated IgG4-RD. Methods Active, untreated IgG4-RD (n=17) were compared to healthy controls (HC) (n=12), patients with primary Sjogren syndrome (pSS) (n=20) and multicentric Castleman9s disease (MCD) (n=5). Tfh2 cells (CD3+CD4+CD45RA-CXCR5+CXCR3-CCR6-cells), activated Tfh2 cells (CCR7 lo PD-1 hi subset in Tfh2 cells) and plasmablasts (CD19+CD20-CD27+CD38+cells) were detected by flow cytometry. The function of Tfh2 cells was evaluated by co-culture with autologous naive B cells in vitro. Disease activity was evaluated by IgG4-RD Responder Index (IgG4-RD RI) score. Results The percentage of Tfh2 cells was significantly increased in IgG4-RD compared to pSS, MCD or HC, and correlated with serum IgG4 levels (ρ=0.7377, P =0.0011) or the percentage of plasmablasts (ρ=0.6127, P =0.0104). In vitro, Tfh2 cells induced a greater percentage of differentiation of naive B cells into plasmablasts in IgG4-RD and HC compared to Tfh1 and Tfh17 cells. Of note, while IgG production in culture supernatants of Tfh2 cells were comparable between IgG4-RD and HC, IgG4 production was significantly higher in IgG4-RD compared to HC. Accordingly, IgG4/IgG ratio in culture supernatants was also significantly higher only with Tfh2 cells from IgG4-RD. In addition, the percentage of activated Tfh2 cells was significantly increased in IgG4-RD (8.0 ± 1.6%) compared to pSS (1.4±0.2%, P P P P =0.0002). In particular, the percentage of activated Tfh2 cells associated with the number of affected organs (ρ=0.8152, P =0.0001), and decreased with glucocorticoid treatment in parallel with disease improvement. Of note, the percentage of activated Tfh2 cells was re-elevated at disease relapse. Conclusions Tfh2 cells, but not Tfh1 or Tfh17 cells, induce the differentiation of naive B cells into plasmablasts and IgG4 production in patients with active, untreated IgG4-RD. This increase in activated Tfh2 cells is linked to disease activity, suggesting that Tfh2 cells play a pivotal role in the pathogenesis of IgG4-RD. References J Allergy Clin Immunol. 2014;134:679–87. Immunity 2011;34:108–21. Arthritis Rheumatol. 2015;67:2476–81. Acknowledgement We thank the patients and healthy individuals who participated in this study. We thank Mr. Noriyasu Seki, Mr. Humitsugu Yamane, Mrs. Yoshiko Yogiashi and Ms. Yuki Otomo for their technical assistance. Disclosure of Interest None declared

The effects of addition of the 3rd component of thermotropic liquid crystal polymer (TLCP) on the mechanical properties and structure of Vectra A-reinforced poly(butylene terephthalate) (PBT) were studied. The 3rd TLCP was the copolymer based on p-hydroxybenzoate (PHB) and ethylene terephthalate, known as X-7G. PBT, Vectra A, and the third TLCP were blended in the molten state, the extrudate of which was cut and injection-molded. The mechanical properties of the blends were improved by the combination of the two kinds of TLCP. The dynamic viscoelastic analysis indicated that the thermal endurance of the ternary blends were enhanced when Vectra A was used as a major TLCP reinforcement. SEM revealed that both the binary and ternary blends were phase-separated, and the introduction of the 3rd component of TLCP was profitable for TLCP to form finer fibrils at the skin part of the sample. X-Ray diffraction analysis showed that the orientation of Vectra A fibril in the blend was raised by the addition of the 3rd TLCP, resulting in the improvement of the mechanical properties of the ternary blends. The conclusions obtained in this paper agreed with those reported in our previous paper.

Additive Effects on Thermotropic Liquid Crystal Polymer Alloys III. Effects of Third Component of Thermotropic Liquid Crystal Polymer on Mechanical Properties of Blends of Ekonol and Polyarylate

The effects of addition of the 3rd component of thermotropic liquid crystal polymer (TLCP) on the mechanical properties and structure of Vectra A-reinforced polycarbonate (PC) were studied. The 3rd TLCP was the copolymer of p-hydroxybenzoate (PHB) and ethyleneterephthalate, known as X-7G TLCP. PC, Vectra A, and a small amount of the 3rd TLCP were blended in the molten state, the extrudate of which was cut and injection-molded. The mechanical properties of the blends were improved dramatically by addition of a small amount of the 3rd TLCP component. SEM revealed that the blend systems were always phase-separated, even after injection-molded, and the fibrils of Vectra A in the injection-molded blends became finer and longer in the presence of the 3rd TLCP. FT-IR indicated that no special chemical reaction took place between the TLCP and PC. The dynamic viscoelastic analysis indicated that the addition of the 3rd TLCP component had almost no effect on the thermal endurance of the blends. X-Ray diffraction analysis showed that the orientation of Vectra A fibril in the blend became higher when the 3rd TLCP was added, which led to the great improvement of the mechanical properties. Based on the assumption of additivity law, the fibril modulus was evaluated and found to be in the same order as the orientation evaluation by X-ray diffraction. The thinning of fibrils by addition of the 3rd TLCP component was discussed based on the crystallization kinetics of rodlike molecules.

To prepare the molecular composite of poly(p-phenylenetere-phthalamide) (PPTA) and epoxy resin (Epoxy), N-grafted PPTA (PPTA-g-Epoxy) was synthesized from PPTA and Epoxy via metalation of PPTA in a solution of sodium methylsulfinylcarbanion in dimethylsulfoxide. The Characterization of PPTA-g-Epoxy was carried out by IR, TGA, DSC, and X-ray diffraction. Molecular composites were prepared from PPTA-g-Epoxy of more than 100% grafting of Epoxy, epoxy resin and triethylenetetramine as a hardener. The bending modulus of 3.0 GPa and bending strength of 164 MPa were obtained for the molecular composite, and found superior to those of epoxy resin by factors of 1.66 and 1.36, respectively. The reasons for low reinforcing effect are discussed.

1-Ethoxymethyl-5-fluorouracil (EM-FU) is a fluorinated pyrimidine derived from 5-FU, and 3-cyano-2,6-dihydroxypyridine (CNDP) is a chemical modulator which suppresses the catabolism of 5-FU by inhibiting dihydrouracil dehydrogenase in the liver. In this study, the metabolism of EM-FU and the suppression of 5-FU catabolism by CNDP were observed by in vivo 19F magnetic resonance spectroscopy in comparison with other similar drugs, because it is considered that the most effective mode of therapy using 5-FU is to suppress the catabolism of 5-FU in the liver and so to maintain for longer an effective blood level of 5-FU. The metabolism of EM-FU was very slow and the production of fluoro-beta-alanine was very low as compared to the case of tegafur. The catabolic suppression by CNDP was much stronger than that of uracil. Therefore co-administration of EM-FU and CNDP should suppress catabolism and maintain an effective blood level of 5-FU for a long period of time.

Poly[p-phenylene(2,5-di-p-carbonylphenylpyrazine)amide] (PPPA) was synthesized by the polycondensation of 2,5-di-p-chloroformylphenylpyrazine with p-phenylenediamine. Dicarboxylic acid was prepared by dimerization of p-cyanophenacyl bromide. Molecular characterization was carried out by IR and NMR. PPPA is soluble in sulfuric acid, forming liquid crystals. The temperature at the 10 wt% loss in TGA was 545°C. The fiber spinning was difficult due to the strong tendency to crystallization and rapid relaxation from liquid crystalline state. From the X-ray diffraction pattern of uniplanar oriented film of PPPA, approximate lattice constant was evaluated as a=7.94 A, b=5.23 A, and c=20.6 A (fiber axis). Fiber period agreed with the identity period for all trans conformation of PPPA. Tensile modulus along the molecular axis was estimated as 210–220 GPa by referring to the theoretical moduli of poly(p-phenyleneterephthalamide) and poly(p-phenylene).

Metabolism of tegafur in the rat liver was observed by in-vivo 19F magnetic resonance spectroscopy (MRS). After MRS observation, tegafur and q-fluorouracil (5-FU) in the liver were determined by a chromatographic method for comparison with the results of 19F-MRS. Rats were divided into 3 groups: 1) CCl4-induced liver injury group, 2) uracil combined group, 3) control group. Catabolism to fluoro-beta-alanine was suppressed in both the liver injury group and the uracil combined group. Low peaks of 5-FU and fluoronucleotides could be found only in the uracil combined group. The result of 19F MRS observation of each group was in agreement with the result of determination of tegafur and 5-FU by chromatography. This showed that substances which could be observed by 19F-MRS were in proportion to all intracellular fluoro-containing substances. 19F-MRS can provide direct information on the metabolism of fluoropyimidines non-invasively and it might be a useful aid in choosing suitable chemotherapy for patients.

We have previously developed a reproducible model of transient forebrain ischaemia in rats by bilateral carotid artery occlusion combined with temporary increase of ICP. With this model, reversibility of the energy metabolism and intracellular pH (pHi) was investigated by 31P-MRS during 120 min of recirculation in three groups of, respectively, 30, 60, and 120 min of ischaemia. With the induction of ischaemia, ATP and phosphocreatine (PCr) disappeared, and measurement of pHi showed severe acidosis in all rats. In the 30 min ischaemia group, both energy metabolism and pHi recovered almost completely. In the 60 min ischaemia group, ATP recovered to 74% of control values, but pHi showed full recovery. In the 120 min ischaemia group, ATP recovered to about 50% of control values, and recovery of pHi was variable. Showing logarithmical changes during recirculation in ATP and PCr, the rate of metabolic recovery was fast during 60 min of recirculation, but it decreased and reached plateau thereafter in all groups. Recovery of pHi was affected by ATP levels, and was precipitously accelerated as ATP levels exceeded 50% of pre-ischaemic values. These results suggest that prolongation of the duration of ischaemia limits the restoration of the energy state, and the quality of pHi recovery after cerebral ischaemia is affected by the degree of ATP recovery during 60 min of recirculation.

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production from monocytic cells. Enhanced expression of interleukin-10 (IL-10) has been suggested to be the mechanism of suppression. However, cAMP is still capable of suppressing production of the cytokines TNF-alpha and IL-12 in IL-10-deficient dendritic cells (DCs). Here, we demonstrated that the transcription factor c-Fos was responsible for the cAMP-mediated suppression of inflammatory cytokine production. c-Fos accumulated at high amounts in response to cAMP and lipopolysaccharide (LPS). Overexpression of c-Fos suppressed LPS-induced cytokine production, whereas cAMP-mediated suppression of TNF-alpha and IL-12 was impaired in Fos(-/-) DCs or in RAW264.7 cells treated with c-Fos siRNA. c-Fos physically interacted with p65 protein and reduced the recruitment of p65 to the Tnf promoter. Multiple sites of c-Fos were phosphorylated by the IKKbeta protein. Thus, we propose that c-Fos is a substrate of IKKbeta and is responsible for the immunosuppressive effect of cAMP.

An investigation was made of 5-fluorouracil metabolism in rat liver by 19F MRS. Fluoronucleotide synthesis increased markedly with liver injury and accelerated with uracil treatment, indicating that 19F MRS may serve as a new method for evaluating liver viability.

It has been shown that transforming growth factor beta1 (TGF-beta1) is critical in the generation of CD4(+)CD25(+)Foxp3(+)-inducible regulatory T cells (iTregs) from naïve CD4(+)T cells. However, in contrast to natural Tregs, TGF-beta1-induced iTregs rapidly lose both Foxp3 expression and suppression activity. We found that TGF-beta1-induced Foxp3 levels were maintained by the addition of the anti-interleukin 4 (IL-4) antibody or by STAT6 gene deletion. Thus, IL-4 is an important suppressor of Foxp3 induction, and T helper 2 development is a major cause for the disappearance of iTreg during long culture. Using promoter analysis in EL4 cells and primary T cells, we identified a silencer region containing a STAT6 binding site. STAT6 binding to this site reduced TGF-beta1-mediated Foxp3 promoter activation and chromatin modification. Retinoic acid has also been shown to suppress loss of Foxp3 induced by TGF-beta1. Retinoic acid in the presence of TGF-beta1 reduced STAT6 binding to the Foxp3 promoter and enhanced histone acetylation, thereby reverting the effect of IL-4. We propose that antagonistic agents for neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and the promotion of tolerance in Th2-dominated diseases such as allergy.

TGF-beta1 is a well-known immunosuppressive cytokine; however, little is known of the effect of TGF-beta1 on antigen-presenting cells (APCs). In this report, we investigated the molecular mechanisms of the suppressive effects of TGF-beta1 on APCs including dendritic cells and macrophages. Although TGF-beta1 did not greatly affect the activation of APCs, as assessed by the induction of IL-12 or the upregulation of CD40 in response to LPS, it strongly inhibited IFN-gamma-induced nitric oxide (NO) production from macrophages and dendritic cells. Using murine macrophage-like cell line RAW 264.7, we demonstrated that TGF-beta1 not only reduced the inducible NO synthase (iNOS) protein stability but also suppressed the iNOS gene transcription. We also found that TGF-beta1 directly inhibited IFN-gamma-induced STAT1 activation by reducing STAT1 tyrosine phosphorylation. The IFN-gamma Type I receptor (IFNGR1) was found to be associated with the TGF-beta1 Type I receptor (TGF-betaRI) and was phosphorylated by the TGF-betaRI. Reduced activation of STAT1 by TGF-beta1 was abrogated by the mutation in the IFNGR1 in which the serine residues of potential sites of phosphorylation by TGF-betaRI were replaced by alanine residues. Thus, multiple mechanisms are present for the TGF-beta1-mediated reduction of iNOS production, and we propose a novel mechanism for regulating inflammatory cytokine by an anti-inflammatory cytokine, TGF-beta1; i.e. suppression of IFN-gamma-induced STAT1 activation by an association of the IFNGR1 with the TGF-betaRI.

Mitochondrial dysfunction has been implicated in the death of nigrostriatal dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated experimental models of Parkinson's disease (PD). Here we utilized proton magnetic resonance spectroscopy ((1)H MRS) to identify changes in energy metabolism in the striatum of MPTP-treated C57BL/6 mice. Remarkable increases in lactate/creatine (Lac/Cr) ratio were observed at 2 h and then quickly returned to about the basal level by 7 h after injection of MPTP. Neurochemical and Western blot analyses revealed that dopamine contents and protein levels of tyrosine hydroxylase and dopamine transporter in the striatum were profoundly decreased at 3 days after MPTP treatment. Pretreatment with deprenyl, a monoamine oxidase B inhibitor, or GBR-12909, a dopamine uptake inhibitor, almost completely attenuated both the increases in striatal Lac/Cr ratio and the subsequent loss of dopaminergic nerve terminals in MPTP-treated mice. The present study indicates that (1)H MRS is a sensitive measure of biochemical alterations of the brain in a mouse model of PD, and further shows that the increases in striatal Lac/Cr ratio induced by MPTP may be associated with mitochondrial energy crisis, followed by dopaminergic neurotoxicity.

Background & Aims: A recent study has suggested that the methylation silencing of the suppressor of cytokine signaling-3 (SOCS3), a negative regulator of interleukin-6–related cytokines, could be involved in hepatocellular carcinoma (HCC). However, the roles of SOCS3 in hepatocellular carcinogenesis and hepatitis have not been established. We investigated the effect of deleting the SOCS3 gene on the development of hepatitis and HCC in hepatitis C virus–infected patients and mouse models. Methods: The expression of SOCS genes in HCC and non-HCC regions of patient samples was determined by real-time reverse-transcription polymerase chain reaction and immunoblotting. The conditional knockout approach in mice was used to determine the hepatocyte-specific roles of SOCS3. To generate a liver-specific deletion, floxed SOCS3 (SOCS3fl/fl) mice were crossed with albumin–Cre transgenic mice. Hepatitis and HCC were induced by administering concanavalin A and diethylnitrosamine, respectively. Results: SOCS3 expression was reduced in the HCC regions compared with the non-HCC regions. Carcinogen-induced hepatic tumor development was enhanced by deletion of the SOCS3 gene, which was associated with higher levels of the targets of signal transducers and activators of transcription (ie, B-cell lymphoma-XL, B-cell lymphoma-2, C-myelocytomatosis, cyclin D1, and vascular endothelial growth factor). In the concanavalin A–mediated hepatitis model, deletion of the SOCS3 gene in the hepatocytes protected against liver injury through suppression of interferon-γ signaling and induction of the antiapoptotic protein Bcl-XL. Conclusions: Deletion of the SOCS3 gene in hepatocytes promotes the activation of STAT3, resistance to apoptosis, and an acceleration of proliferation, resulting in enhanced hepatitis-induced hepatocarcinogenesis.