T-bet over-expression regulates aryl hydrocarbon receptor-mediated T helper type 17 differentiation through an interferon (IFN)γ-independent pathway.

Various transcription factors are also known to enhance or suppress T helper type 17 (Th17) differentiation. We have shown previously that the development of collagen-induced arthritis was suppressed in T-bet transgenic (T-bet Tg) mice, and T-bet seemed to suppress Th17 differentiation through an interferon (IFN)-γ-independent pathway, although the precise mechanism remains to be clarified. The present study was designed to investigate further the mechanisms involved in the regulation of Th17 differentiation by T-bet over-expression, and we found the new relationship between T-bet and aryl hydrocarbon receptor (AHR). Both T-bet Tg mice and IFN-γ-/- -over-expressing T-bet (T-bet Tg/IFN-γ-/- ) mice showed inhibition of retinoic acid-related orphan receptor (ROR)γt expression and IL-17 production by CD4+ T cells cultured under conditions that promote Th-17 differentiation, and decreased IL-6 receptor (IL-6R) expression and signal transducer and activator of transcription-3 (STAT-3) phosphorylation in CD4+ T cells. The mRNA expression of ahr and rorc were suppressed in CD4+ T cells cultured under Th-17 conditions from T-bet Tg mice and T-bet Tg/IFN-γ-/- mice. CD4+ T cells of wild-type (WT) and IFN-γ-/- mice transduced with T-bet-expressing retrovirus also showed inhibition of IL-17 production, whereas T-bet transduction had no effect on IL-6R expression and STAT-3 phosphorylation. Interestingly, the mRNA expression of ahr and rorc were suppressed in CD4+ T cells with T-bet transduction cultured under Th17 conditions. The enhancement of interleukin (IL)-17 production from CD4+ T cells by the addition of AHR ligand with Th17 conditions was cancelled by T-bet over-expression. Our findings suggest that T-bet over-expression-induced suppression of Th17 differentiation is mediated through IFN-γ-independent AHR suppression.

RORγt antagonist suppresses M3 muscarinic acetylcholine receptor-induced Sjögren's syndrome-like sialadenitis.

We showed recently that M3 muscarinic acetylcholine receptor (M3R)-reactive CD3+ T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid-related orphan receptor-gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R-/- ) mice immunized with murine M3R peptide mixture were inoculated into recombination-activating gene 1 knockout (Rag-1-/- ) mice (M3R-/- →Rag-1-/- ) with MIS. Immunized M3R-/- mice (pretransfer treatment) and M3R-/- →Rag-1-/- mice (post-transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide-stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme-linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4+ T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)-γ and interleukin (IL)-17 production significantly compared with phosphate-buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4+ T cells rather than CD11c+ cells. Post-transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN-γ and IL-17 production (P < 0·05). In vitro, A213 suppressed IFN-γ and IL-17 production from M3R-stimulated splenocytes and CD4+ T cells of immunized M3R-/- mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL-17 production from Th17 differentiated CD4+ T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS-like sialadenitis through suppression of IL-17 and IFN-γ production by M3R-specific T cells.

The regulatory role of interferon-γ producing gamma delta T cells via the suppression of T helper 17 cell activity in bleomycin-induced pulmonary fibrosis.

Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with poor prognosis and high mortality. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of pulmonary γδT cells in IP. In wild-type (WT) mice exposed to bleomycin, pulmonary γδT cells were expanded and produced large amounts of interferon (IFN)-γ and interleukin (IL)-17A. Histological and biochemical analyses showed that bleomycin-induced IP was more severe in T cell receptor (TCR-δ-deficient (TCRδ(-/-) ) mice than WT mice. In TCRδ(-/-) mice, pulmonary IL-17A(+) CD4(+) Τ cells expanded at days 7 and 14 after bleomycin exposure. In TCRδ(-/-) mice infused with γδT cells from WT mice, the number of pulmonary IL-17A(+) CD4(+) T cells was lower than in TCRδ(-/-) mice. The examination of IL-17A(-/-) TCRδ(-/-) mice indicated that γδT cells suppressed pulmonary fibrosis through the suppression of IL-17A(+) CD4(+) T cells. The differentiation of T helper (Th)17 cells was determined in vitro, and CD4(+) cells isolated from TCRδ(-/-) mice showed normal differentiation of Th17 cells compared with WT mice. Th17 cell differentiation was suppressed in the presence of IFN-γ producing γδT cells in vitro. Pulmonary fibrosis was attenuated by IFN-γ-producing γδT cells through the suppression of pulmonary IL-17A(+) CD4(+) T cells. These results suggested that pulmonary γδT cells seem to play a regulatory role in the development of bleomycin-induced IP mouse model via the suppression of IL-17A production.

Optimizing the Aging Brain: The BEAD Study on the Ethics of Dementia Prevention.

Dementia has lately undergone a profound reconceptualization. Long conceived of as an unpreventable process of mental deterioration, current evidence shows that it can be prevented in at least one in three cases intervening on a specified set of factors. Issues of justice and equity loom large on the implementation of dementia prevention, from a global health perspective. Our project thus embraces emerging evidence about dementia risk factors and their uneven distribution nationally and globally by specifically focusing on the situated aspects of dementia prevention. The aim of the BEAD study (Optimizing the Aging Brain? Situating Ethical Aspects in Dementia Prevention) is to dissect the ethical and clinical assumptions of this novel understanding of dementia, and to analyze how such new discourse on dementia prevention plays out in three countries: Canada, Germany and Switzerland. This study adopts a multi-perspective, comparative, qualitative approach, combining stakeholder interviews with different kinds of focused ethnographies, elaborating on conceptual, ethical, and social aspects of what we would like to call the "new dementia". By situating the paradigmatic shifts in Alzheimer's and dementia research within current aging cultures and contemporary social policies, we aim to initiate a debate about the often implicit unresolved social, ethical, and political implications and preconditions of the medical understanding and handling of cognitive disorders.

Manufacturing carbon storage sintered body using microwave-selective and high-speed heating techniques.

Microwave sintering of fly ash samples with large amounts of unburned carbon and CaCO3 was examined in this study. To this end, CaCO3 was mixed with fly ash sintered body to fix CO2. The decomposition of CaCO3 was observed when the raw material was heated to 1000 °C using microwave irradiation; however, a sintered body containing aragonite was obtained when the raw material was heated to 1000 °C with added water. Further, carbides in the fly ash could be selectively heated by controlling the microwave irradiation. The microwave magnetic field created a temperature gradient of 100 °C in a narrow region of 2.7 µm or less in the sintered body, and it helped suppress the CaCO3 decomposition in the mixture during sintering. By storing water in the gas phase before spreading, CaCO3, which is difficult to sinter using conventional heating, can be sintered without decomposing.

Activation of natural killer T cells by α-carba-GalCer (RCAI-56), a novel synthetic glycolipid ligand, suppresses murine collagen-induced arthritis.

Alpha-carba-GalCer (RCAI-56), a novel synthetic analogue of α-galactosylceramide (α-GalCer), stimulates invariant natural killer T (NK T) cells to produce interferon (IFN)-γ. IFN-γ exhibits immunoregulatory properties in autoimmune diseases by suppressing T helper (Th)-17 cell differentiation and inducing regulatory T cells and apoptosis of autoreactive T cells. Here, we investigated the protective effects of α-carba-GalCer on collagen-induced arthritis (CIA) in mice. First, we confirmed that α-carba-GalCer selectively induced IFN-γ in CIA-susceptible DBA/1 mice in vivo. Then, DBA/1 mice were immunized with bovine type II collagen (CII) and α-carba-GalCer. The incidence and clinical score of CIA were significantly lower in α-carba-GalCer-treated mice. Anti-IFN-γ antibodies abolished the beneficial effects of α-carba-GalCer, suggesting that α-carba-GalCer ameliorated CIA in an IFN-γ-dependent manner. Treatment with α-carba-GalCer reduced anti-CII antibody production [immunoglobulin (Ig)G and IgG2a] and CII-reactive interleukin (IL)-17 production by draining lymph node (DLN) cells, did not induce apoptosis or regulatory T cells, and significantly increased the ratio of the percentage of IFN-γ-producing T cells to IL-17-producing T cells (Th1/Th17 ratio). Moreover, the gene expression levels of IL-6 and IL-23p19, Th17-related cytokines, were reduced significantly in mice treated with α-carba-GalCer. In addition, we observed higher IFN-γ production by NK T cells in α-carba-GalCer-treated mice in the initial phase of CIA. These findings indicate that α-carba-GalCer polarizes the T cell response toward Th1 and suppresses Th17 differentiation or activation, suggesting that α-carba-GalCer, a novel NK T cell ligand, can potentially provide protection against Th17-mediated autoimmune arthritis by enhancing the Th1 response.

Inhibition of transforming growth factor-beta signalling attenuates interleukin (IL)-18 plus IL-2-induced interstitial lung disease in mice.

Interstitial lung disease (ILD) is an intractable disease induced by various factors in humans. However, there is no universally effective treatment for ILD. In this study, we investigated the role of transforming growth factor (TGF)-beta signalling in the pathogenesis of ILD by using model mice. Injection of interleukin (IL)-18 plus IL-2 in C57BL6 (B6) mice resulted in acute ILD by infiltration of natural killer (NK) cells and a significant increase of TGF-beta mRNA in the lung. To examine the pathogenetic role of TGF-beta in ILD mice, we used SB-431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), which is a potent and selective inhibitor of TGF-beta receptor I (TbetaRI), also known as activin receptor-like kinase 5 (ALK5). Treatment of B6-ILD mice with SB-431542 resulted in improvement of ILD, delay in mortality, reduction of the expression of interferon (IFN)-gamma and IL-6 in the lungs. The same treatment also decreased significantly the percentage of natural killer (NK) cells in the lungs (P < 0.05) and mRNA expression levels of certain chemokines such as CCL2, CCL3, CCL4, CCL5 and CXCL10 in B6-ILD. These findings were confirmed by IL-18 plus IL-2 treatment of Smad3-deficient (Smad3(-/-)) mice (P < 0.05). Our results showed that inhibition of TGF-beta signalling reduced the percentage of NK cells and the expression of certain chemokines in the lungs, resulting in improvement of ILD. The findings suggest that TGF-beta signalling may play an important role in the pathogenesis of IL-18 plus IL-2-induced ILD in mice.

Oral administration of heat-killed Lactobacillus brevis SBC8803 elevates the ratio of acyl/des-acyl ghrelin in blood and increases short-term food intake.

It is known that gastrointestinal microbiota, probiotics and heat-killed microbes can regulate intestinal immunity; however, their effect on the secretion of gastrointestinal hormones is unclear. The secretion of gastrointestinal hormones can be mediated by the elevation of intracellular Ca2+ concentration, suggesting that these hormones may act through common mechanisms. We have previously shown that heat-killed Lactobacillus brevis SBC8803 (hk-SBC8803) induced the secretion of serotonin and elevated intracellular Ca2+ concentration in serotonin-producing RIN-14B cells, suggesting that hk-SBC8803 could potentially cause the same effect on other gastrointestinal hormones, including hunger hormone ghrelin. Here, we tested this hypothesis by treating cultured cells and experimental animals with hk-SBC8803 and assessing ghrelin secretion, expression of ghrelin-related genes, and food intake. The results indicated that hk-SBC8803 treatment for 30 min significantly upregulated the secretion of acyl ghrelin (active form) (P=0.046) and mRNA expression of the Syt3 (synaptotagmin 3) gene related to ghrelin exocytosis (P<0.05) in primary mouse stomach cells. In addition, oral administration of 500 mg/kg hk-SBC8803 to rats tended to upregulate acyl ghrelin concentration (P=0.10) and significantly increased the ratio of acyl to des-acyl (inactive) ghrelin (P=0.027) in blood, which corresponded to a tendency of stimulating food intake (P=0.087) at 30 min post-treatment. However, when in order to minimise individual differences we normalised the data on food intake to those on one-day food intake prior to food deprivation, the resultant food intake ratio showed a significant increase (by 5% compared to control; P=0.032) at 30 min after hk-SBC8803 treatment, indicating that hk-SBC8803 administration stimulated rats to take more food during the first meal after fasting. These results suggest that hk-SBC8803 induces short-term ghrelin secretion and transiently increases appetite, which is an important effect for individuals with low energy intake.

Invariant NKT cells produce IL-17 through IL-23-dependent and -independent pathways with potential modulation of Th17 response in collagen-induced arthritis.

Invariant natural killer T (iNKT) cells play a protective role in the development of certain autoimmune diseases. However, their precise role in the pathogenesis of autoimmune arthritis remains unclear. In this study, we examined the possible contribution of iNKT cells in collagen-induced arthritis (CIA) by using iNKT cell-deficient mice (Jalpha281-/- mice). CIA in these mice was markedly suppressed and interleukin (IL)-17 production was reduced in a native type II collagen (CII)-specific T cell response. Draining lymph nodes of CII-immunized Jalpha281-/- mice contained a significantly low number of IL-17-producing T helper cells. To determine whether iNKT cells produce IL-17, we measured IL-17 by enzyme-linked immunosorbent assay in iNKT cells stimulated with the ligand, alpha-galactosylceramide (alpha-GalCer). Notably, splenocytes from Jalpha281-/- mice stimulated in this way were negative for IL-17, whereas those from C57BL/6 mice produced IL-17. Immunostaining for IL-17 in iNKT cells confirmed intracellular staining of the protein. RT-PCR analysis showed that iNKT cells expressed retinoid-related orphan receptor gammaT and IL-23 receptor. Moreover, cell sorting demonstrated that NK1.1- iNKT cells were the main producers of IL-17 compared with NK1.1+ iNKT cells. IL-17 production by iNKT cells was induced by IL-23-dependent and -independent pathways, since iNKT produced IL-17 when stimulated with either IL-23 or alpha-GalCer alone. Our findings indicate that iNKT cells are producers and activators of IL-17 via IL-23- dependent and -independent pathways, suggesting that they are key cells in the pathogenesis of CIA through IL-17.

The transition state in the folding-unfolding reaction of four species of three-disulfide variant of hen lysozyme: the role of each disulfide bridge.

The effects of lacking a specific disulfide bridge on the transition state in folding were examined in order to explore the folding-unfolding mechanism of lysozyme. Four species of three-disulfide variant of hen lysozyme (3SS-lysozyme) were prepared by replacing two Cys residues with Ala or Ser: C6S/C127A, C30A/C115A, C64A/C80A and C76A/C94A. The recombinant hen lysozyme was studied as the standard reference containing four authentic disulfide bridges and the extra N-terminal Met: the recombinant hen lysozyme containing the extra N-terminal. Folding rates were measured by monitoring the change in fluorescence intensity associated with tri-N-acetyl-d-glucosamine binding to the active site of refolded lysozyme. It was confirmed that the folding rate of the recombinant hen lysozyme containing the extra N-terminal was the same as that of wild-type lysozyme, and that the folding rate was little affected by the presence of tri-N-acetyl-d-glucosamine (triNAG). The folding rate of C64A/C80A was found to be the fastest and almost the same as that of the recombinant hen lysozyme containing the extra N-terminal, and that of C30A/C115A the second, and that of C6S/C127A the third. The folding rate of C76A/C94A was particularly slow. On the other hand, the unfolding rates which were measured in the presence of triNAG showed the dependence on the concentration of triNAG. The intrinsic unfolding rate in the absence of triNAG was determined by extrapolation. Also in the unfolding rate, C76A/C94A was markedly slower than the others. It was found from the analysis of binding constants of triNAG to C64A/C80A during the unfolding process that the active site of C64A/C80A partly unfolds already prior to the unfolding transition. On the basis of these kinetic data, we suggest that C64A/C80A folding transition can occur with leaving the loop region around SS3 (C64-C80) flexible, while cross-linking by SS4 (C76-C94) is important for the promotion of folding, because it is an indispensable constraint on the way towards the folding transition state.

Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments.

Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.

Efficient in vitro folding of the three-disulfide derivatives of hen lysozyme in the presence of glycerol.

Four derivatives of hen lysozyme, each lacking one native disulfide bond of the four in authentic lysozyme, were produced in Escherichia coli by expressing synthetic mutant genes. In the reoxidation reaction of the reduced derivatives purified from inclusion bodies, the addition of glycerol significantly enhanced the efficiency of folding and 'correct' disulfide bond formation. This enabled simple chromatographical purification of refolded materials. Purified 3SS-derivatives all showed lytic activities and secondary structures comparable to authentic lysozyme, which directly showed that none of the four native disulfide bonds is a prerequisite for 'correct' in vitro folding.

Embryonic and paralarval development of the central nervous system of the loliginid squid Sepioteuthis lessoniana.

The embryonic development of the central nervous system (CNS) in the oval squid Sepioteuthis lessoniana is described. It has three distinct phases: (1) The ganglionic accumulation phase: Ganglionic cell clusters develop by ingression, migration, and accumulation of neuroblasts. (2) The lobe differentiation phase: Ganglia differentiate into lobes. The phase is identified by the beginning of an axogenesis. During this phase, neuropils are first formed in the suboesophageal mass, then in the basal lobe system, and finally in the inferior frontal lobes and the superior frontal-vertical lobe systems. (3) The neuropil increment phase: After the shape of the lobes reached its typical form, neuropil growth occurs, specifically in the vertical lobe. The paralarval central nervous system (CNS) is characterized by neuronal gigantism of the giant fibers and some suboesophageal commissures and connectives. The neuropil formation in the CNS of S. lessoniana occurs somewhat earlier than in Octopus vulgaris, although the principal developmental plan is quite conservative among the other coleoids investigated. Some phylogenetic aspects are discussed based on the similarities in the morphologic organization of their brains.