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Cytokines are important intercellular communication tools for immunity. Many cytokines promote gene transcription and proliferation through the JAK/STAT (Janus kinase/signal transducers and activators of transcription) and the Ras/ERK (GDP/GTP-binding rat sarcoma protein/extracellular signal-regulated kinase) pathways, and these signaling pathways are tightly regulated. The SOCS (suppressor of cytokine signaling) family members are representative negative regulators of JAK/STAT-mediated cytokine signaling and regulate the differentiation and function of T cells, thus being involved in immune tolerance. Human genetic analysis has shown that SOCS family members are strongly associated with autoimmune diseases, allergy and tumorigenesis. SOCS family proteins also function as immune-checkpoint molecules that contribute to the unresponsiveness of T cells to cytokines.

Heart failure is a complex clinical syndrome characterized by insufficient cardiac function. Heart-resident and infiltrated macrophages have been shown to play important roles in the cardiac remodeling that occurs in response to cardiac pressure overload. However, the possible roles of T cells in this process, have not been well characterized. Here we show that T cell depletion conferred late-stage heart protection but induced cardioprotective hypertrophy at an early stage of heart failure caused by cardiac pressure overload. Single-cell RNA sequencing analysis revealed that CD8+T cell depletion induced cardioprotective hypertrophy characterized with the expression of mitochondrial genes and growth factor receptor genes. CD8+T cells regulated the conversion of both cardiac-resident macrophages and infiltrated macrophages into cardioprotective macrophages expressing growth factor genes such as Areg, Osm, and Igf1, which have been shown to be essential for the myocardial adaptive response after cardiac pressure overload. Our results demonstrate a dynamic interplay between cardiac CD8+T cells and macrophages that is necessary for adaptation to cardiac stress, highlighting the homeostatic functions of resident and infiltrated macrophages in the heart.

Inflammation and immune responses after tissue injury play pivotal roles in the pathology, resolution of inflammation, tissue recovery, fibrosis and remodeling. Regulatory T cells (Tregs) are the cells responsible for suppressing immune responses and can be activated in secondary lymphatic tissues, where they subsequently regulate effector T cell and dendritic cell activation. Recently, Tregs that reside in non-lymphoid tissues, called tissue Tregs, have been shown to exhibit tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. Unlike other tissue Tregs, the role of Tregs in the brain has not been well elucidated because the number of brain Tregs is very small under normal conditions. However, we found that Tregs accumulate in the brain at the chronic phase of ischemic brain injury and control astrogliosis through secretion of a cytokine, amphiregulin (Areg). Brain Tregs resemble other tissue Tregs in many ways but, unlike the other tissue Tregs, brain Tregs express neural-cell-specific genes such as the serotonin receptor (Htr7) and respond to serotonin. Administering serotonin or selective serotonin reuptake inhibitors (SSRIs) in an experimental mouse model of stroke increases the number of brain Tregs and ameliorates neurological symptoms. Knowledge of brain Tregs will contribute to the understanding of various types of neuroinflammation.

Ten-eleven translocation (TET) proteins regulate DNA methylation and gene expression by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although Tet2/Tet3 deficiency has been reported to lead to myeloid cell, B-cell and invariant natural killer T (iNKT) cell malignancy, the effect of TET on regulatory T cells (Tregs) has not been elucidated. We found that Tet2/Tet3 deficiency in Tregs led to lethal hyperproliferation of CD4+Foxp3+ T cells in the spleen and mesenteric lymph nodes after 5 months of age. Additionally, in aged Treg-specific Tet2/Tet3-deficient mice, serum IgG1, IgG3, IgM and IgE levels were markedly elevated. High IL-17 expression was observed in both Foxp3+ and Fopx3- CD4+ T cells, and adoptive transfer of Tet2/Tet3-deficient Tregs into lymphopenic mice inhibited Foxp3 expression and caused conversion into IL-17-producing cells. However, the conserved non-coding DNA sequence-2 (CNS2) region of the Foxp3 gene locus, which has been shown to be particularly important for stable Foxp3 expression, was only partly methylated. We identified novel TET-dependent demethylation sites in the Foxp3 upstream enhancer, which may contribute to stable Foxp3 expression. Together, these data indicate that Tet2 and Tet3 are involved in Treg stability and immune homeostasis in mice.

Cytokines are important intercellular communication tools for immunity. Most cytokines utilize the JAK-STAT and Ras-ERK pathways to promote gene transcription and proliferation; however, this signaling is tightly regulated. The suppressor of cytokine signaling (SOCS) family and SPRED family are a representative negative regulators of the JAK-STAT pathway and the Ras-ERK pathway, respectively. The SOCS family regulates the differentiation and function of CD4+ T cells, CD8+ T cells, and regulatory T cells, and is involved in immune tolerance, anergy, and exhaustion. SPRED family proteins have been shown to inactivate Ras by recruiting the Ras-GTPase neurofibromatosis type 1 (NF1) protein. Human genetic analysis has shown that SOCS family members are strongly associated with autoimmune diseases, allergies, and tumorigenesis, and SPRED1 is involved in NF1-like syndromes and tumors. We also identified the NR4a family of nuclear receptors as a key transcription factor for immune tolerance that suppresses cytokine expression and induces various immuno-regulatory molecules including SOCS1.

In addition to maintaining immune tolerance, FOXP3+ regulatory T (Treg) cells perform specialized functions in tissue homeostasis and remodelling1,2. However, the characteristics and functions of brain Treg cells are not well understood because there is a low number of Treg cells in the brain under normal conditions. Here we show that there is massive accumulation of Treg cells in the mouse brain after ischaemic stroke, and this potentiates neurological recovery during the chronic phase of ischaemic brain injury. Although brain Treg cells are similar to Treg cells in other tissues such as visceral adipose tissue and muscle3–5, they are apparently distinct and express unique genes related to the nervous system including Htr7, which encodes the serotonin receptor 5-HT7. The amplification of brain Treg cells is dependent on interleukin (IL)-2, IL-33, serotonin and T cell receptor recognition, and infiltration into the brain is driven by the chemokines CCL1 and CCL20. Brain Treg cells suppress neurotoxic astrogliosis by producing amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand. Stroke is a leading cause of neurological disability, and there are currently few effective recovery methods other than rehabilitation during the chronic phase. Our findings suggest that Treg cells and their products may provide therapeutic opportunities for neuronal protection against stroke and neuroinflammatory diseases. In a mouse model of ischaemic stroke, regulatory T cells infiltrate the injured brain in response to the chemokines CCL1 and CCL20 and suppress excessive astrogliosis via the production of amphiregulin.

Enhanced infiltration of regulatory T cells (Treg) into tumor tissue is detrimental to patients with cancer and is closely associated with poor prognosis as they create an immunosuppressive state that suppresses antitumor immune responses. Therefore, breaking Treg-mediated immune tolerance is important when considering cancer immunotherapy. Here, we show that the Nr4a nuclear receptors, key transcription factors maintaining Treg genetic programs, contribute to Treg-mediated suppression of antitumor immunity in the tumor microenvironment. Mice lacking Nr4a1 and Nr4a2 genes specifically in Tregs showed resistance to tumor growth in transplantation models without exhibiting any severe systemic autoimmunity. The chemotherapeutic agent camptothecin and a common cyclooxygenase-2 inhibitor were found to inhibit transcriptional activity and induction of Nr4a factors, and they synergistically exerted antitumor effects. Genetic inactivation or pharmacologic inhibition of Nr4a factors unleashed effector activities of CD8+ cytotoxic T cells and evoked potent antitumor immune responses. These findings demonstrate that inactivation of Nr4a in Tregs breaks immune tolerance toward cancer, and pharmacologic modulation of Nr4a activity may be a novel cancer treatment strategy targeting the immunosuppressive tumor microenvironment. Significance: This study reveals the role of Nr4a transcription factors in Treg-mediated tolerance to antitumor immunity, with possible therapeutic implications for developing effective anticancer therapies. Cancer Res; 78(11); 3027–40. ©2018 AACR.

Retroviral TET1 or hypoxia stabilize Foxp3 in iTregs generated in vitro, Since induced regulatory T cells (iTregs) can be produced in a large quantity in vitro, these cells are expected to be clinically useful to induce immunological tolerance in various immunological diseases. Foxp3 (Forkhead box P3) expression in iTregs is, however, unstable due to the lack of demethylation of the CpG island in the conserved non-coding sequence 2 (CNS2) of the Foxp3 locus. To facilitate the demethylation of CNS2, we over-expressed the catalytic domain (CD) of the ten-eleven translocation (TET) protein, which catalyzes the steps of the iterative demethylation of 5-methylcytosine. TET-CD over-expression in iTregs resulted in partial demethylation of CNS2 and stable Foxp3 expression. We also discovered that TET expression was enhanced under low oxygen (5%) culture conditions, which facilitated CNS2 DNA demethylation and stabilization of Foxp3 expression in a TET2- and TET3-dependent manner. In combination with vitamin C treatment, which has been reported to enhance TET catalytic activity, iTregs generated under low oxygen conditions retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model compared with untreated iTregs. Our data indicate that the induction and activation of TET enzymes in iTregs would be an effective method for Treg-mediated adoptive immunotherapy.

Regulatory T cells (Tregs) play essential roles in maintaining immunological self-tolerance and preventing autoimmunity. The adoptive transfer of antigen-specific Tregs has been expected to be a potent therapeutic method for autoimmune diseases, severe allergy, and rejection in organ transplantation. However, effective Treg therapy has not yet been established because of the difficulty in preparing a limited number of antigen-specific Tregs. Chimeric antigen receptor (CAR) T cells have been shown to be a powerful therapeutic method for treating B cell lymphomas, but application of CAR to Treg-mediated therapy has not yet been established. Here, we generated CD19-targeted CAR (CD19-CAR) Tregs from human PBMCs (hPBMCs) and optimized the fraction of the Treg source as CD4(+)CD25(+)CD127(lo)CD45RA(+)CD45RO(–). CD19-CAR Tregs could be expanded in vitro while maintaining Treg properties, including high expression of the latent form of TGF-β. CD19-CAR Tregs suppressed IgG antibody production and differentiation of B cells via a TGF-β–dependent mechanism. Unlike conventional CD19-CAR CD8(+) T cells, CD19-CAR Tregs suppressed antibody production in immunodeficient mice that were reconstituted with hPBMCs, reducing the risk of graft-versus-host disease. Therefore, the adoptive transfer of CD19-CAR Tregs may provide a novel therapeutic method for treating autoantibody-mediated autoimmune diseases.

Objectives Tocilizumab (TCZ) is a humanised anti‐interleukin (IL)‐6 receptor (IL‐6R) monoclonal antibody that is a promising agent to treat various autoimmune diseases. However, the mechanism of TCZ efficacy is unclear. This study aims to elucidate the relationship between Tregs and IL‐6R blockade in autoimmunity‐mediated renal disease based on a TCZ‐treated cohort of patients with anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV) and in an experimental model of crescentic glomerulonephritis (cGN). Methods We examined multiple serum levels of cytokines and chemokines and peripheral blood mononuclear cells in patients with AAV who received TCZ monotherapy and achieved drug‐free remission. Moreover, we investigated the mechanistic role of IL‐6R blockade in accelerated cGN model to analyse the local sites of inflammation. Results Serum chemokines CCL22 and CCL17, in addition to the CCR4+Foxp3+ Treg population, increased in patients who demonstrated drug‐free remission after the cessation of TCZ. In the cGN model, IL‐6R blockade ameliorated the disease, elevated CCL22/17 in CD206+CD11b+CD11c+ kidney M2‐like type macrophages, and increased the migration of Tregs into the kidney and regional lymph nodes. The local administration of CCL22 in the kidney facilitated Treg accumulation and reduced glomerular crescent formation. Conclusions This study revealed a new mechanism whereby effector Tregs migrate into the inflammatory kidney via the CCL22/17–CCR4 axis that is facilitated by M2‐like type macrophages that are induced by IL‐6R blockade., Our study has revealed a new remission mechanism whereby effector Tregs migrate into the crescentic glomerulonephritis via the CCL22/17–CCR4 axis that is facilitated by M2‐like type macrophages that are induced by IL‐6R blockade.

CD8+T cells are important in protective immunity against intracellular pathogens and tumors. In chronic infections or cancer, CD8+T cells are constantly exposed to antigens and inflammatory signals. Such excessive and constitutive signals lead to the deterioration of T cell function, called ‘exhaustion’. Exhausted T cells are characterized by low proliferation in response to antigen stimulation, progressive loss of effector function (cytokine production and killing function), expression of multiple inhibitory receptors such as PD-1, Tim3, and LAG3, and metabolic alterations from oxidative phosphorylation to glycolysis. These dysfunctions are associated with altered transcriptional programs and epigenetic regulations and recent studies suggested that NR4a and TOX transcription factors are deeply involved in exhaustion phenotypes. However, an increase the early memory T cells including stem cell memory T (TSCM) cells is critical for T cell persistence and efficient tumor killing especially for adoptive cancer immunotherapy such as CAR-T cell therapy. An increasing amount of evidence supports the therapeutic potential of targeting exhausted T cells and TSCM cells. We have begun to understand the molecular mechanisms of T cell exhaustion and early memory formation, and the clinical application of converting exhausted T cells to rejuvenated early memory T cells is the goal of our study.

Inflammation and immune responses after tissue injury play pivotal roles in the resolution of inflammation, tissue recovery, fibrosis, and remodeling. Regulatory T cells (Tregs) are responsible for immune tolerance and are usually activated in secondary lymphatic tissues. Activated Tregs subsequently regulate effector T cell and dendritic cell activation. For clinical applications such as the suppression of both autoimmune diseases and the rejection of transplanted organs, methods to generate stabilized antigen-specific Tregs are required. For this purpose, transcriptional and epigenetic regulation of Foxp3 expression has been investigated. In addition to conventional Tregs, there are some Tregs that reside in tissues and are called tissue Tregs. Tissue Tregs exhibit tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. Such tissue Tregs could also be useful for Treg-based cell therapy. We recently discovered brain Tregs that accumulate in the brain during the chronic phase of ischemic brain injury. Brain Tregs resemble other tissue Tregs, but are unique in expressing neural cell-specific genes such as the serotonin receptor (Htr7); consequently, brain Tregs respond to serotonin. Here, we describe our experiences in the use of Tregs to suppress graft-versus-host disease and to promote neural recovery after stroke.

T helper type 1 (Th1) cells form one of the most stable CD4 T-cell subsets, and direct conversion of fully differentiated Th1 to regulatory T (Treg) cells has been poorly investigated. Here, we established a culture method for inducing Foxp3 from Th1 cells of mice and humans. This is achieved simply by resting Th1 cells without T-cell receptor ligation before stimulation in the presence of transforming growth factor-beta (TGF-β). We named the resulting Th1-derived Foxp3+ cells Th1reg cells. Mouse Th1reg cells showed an inducible Treg-like phenotype and suppressive ability both in vitro and in vivo. Th1reg cells could also be induced from in vivo-developed mouse Th1 cells. Unexpectedly, the resting process enabled Foxp3 expression not through epigenetic changes at the locus, but through metabolic change resulting from reduced mammalian target of rapamycin complex 1 (mTORC1) activity. mTORC1 suppressed TGF-β-induced phosphorylation of Smad2/3 in Th1 cells, which was restored in rested cells. Our study warrants future research aiming at development of immunotherapy with Th1reg cells.

Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess immune responses in autoimmune diseases, allergy, rejection after organ transplantation and graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation. Although in vitro-expanded antigen-specific induced Tregs (iTregs) have been considered to be a promising therapeutic agent against such excessive immune reactions, the instability of iTregs after transfer is a fundamental problem in their clinical application. In this study, we searched for the optimal way to generate stable iTregs for the prevention of the murine GVHD model, in which conventional iTregs are reported to be inefficient. Allo-antigen-specific iTregs were generated by co-culturing naive T cells with allogenic dendritic cells in the presence of TGF-β and retinoic acid. By examining various agents and genes, we found that vitamin C stabilized Foxp3 expression most effectively in adoptively transferred iTregs under a GVHD environment. Vitamin C treatment caused active DNA demethylation specifically on the conserved non-coding sequence 2 (CNS2) enhancer of the Foxp3 gene locus in allo-antigen-specific iTregs and reduced iTreg conversion into pathogenic exFoxp3 cells. Vitamin C-treated iTregs suppressed GVHD symptoms more efficiently than untreated iTregs. Vitamin C also facilitated induction of a FOXP3high iTreg population from human naive T cells, which was very stable even in the presence of IL-6 in vitro. The treatment of vitamin C for iTreg promises innovative clinical application for adoptive Treg immunotherapy.

Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. Several Smad-independent pathways including the JNK and the RhoA-ROCK pathways have been implicated in the induction of RORγt, the master regulator of Th17, however, molecular mechanisms underlying Smad-independent pathway remain largely unknown. To identify novel pathways involved in Th17 differentiation, we screened 285 chemical inhibitors for known signaling pathways. Among them, we found that Kenpaullone, a GSK3-β and CDK inhibitor, efficiently suppressed TGF-β-mediated RORγt induction and enhanced Foxp3 induction in primary T cells. Another CDK inhibitor, Roscovitine, but not other GSK3-β inhibitors, suppressed Th17 differentiation and enhanced iTreg development. Kenpaullone and Roscovitine suppressed experimental autoimmune encephalomyelitis (EAE), a typical Th17-mediated autoimmune disease model. These two compounds enhanced STAT5 phosphorylation and restored IL-2 production in the presence of TGF-β. These data suggest that CDK inhibitors modulate TGF-β-signaling pathways, which restore TGF-β-mediated suppression of IL-2 production, thereby modifying the Th17/iTreg balance.

Post-ischemic inflammation is an essential step in the progression of ischemic stroke. However, it has not been sufficiently clarified how post-ischemic inflammation begins. Brain is a sterile organ, but Toll-like receptor (TLR) 2 and TLR4 have a pivotal role in the initiation of inflammation. Some endogenous danger associated molecular patterns (DAMPs) are released from injured brain cells. Among them, high mobility group box 1 (HMGB1) and peroxiredoxin (Prx) family proteins are key initiators of post-ischemic inflammation. HMGB1 induces blood brain barrier breakdown at the hyperacute phase; on the other hand, Prxs directly activate infiltrating macrophage through TLR2/4 and induce inflammatory cytokines production at the acute phase. Thus, there is a time lag as well as functional differences between HMGB1 and Prxs. Novel neuroprotective treatment could be developed through more detailed elucidation of the regulation of post-ischemic inflammation. Rec.12/10/2012, Acc.1/29/2013, pp150-155

Damage-associated molecular patterns (DAMPs) trigger sterile inflammation after tissue injury, but the mechanisms underlying the resolution of inflammation remain unclear. In this study, we demonstrate that common DAMPs, such as high-mobility-group box 1 (HMGB1), peroxiredoxins (PRXs), and S100A8 and S100A9, were internalized through the class A scavenger receptors MSR1 and MARCO in vitro. In ischemic murine brain, DAMP internalization was largely mediated by MSR1. An elevation of MSR1 levels in infiltrating myeloid cells observed 3 d after experimental stroke was dependent on the transcription factor Mafb. Combined deficiency for Msr1 and Marco, or for Mafb alone, in infiltrating myeloid cells caused impaired clearance of DAMPs, more severe inflammation, and exacerbated neuronal injury in a murine model of ischemic stroke. The retinoic acid receptor (RAR) agonist Am80 increased the expression of Mafb, thereby enhancing MSR1 expression. Am80 exhibited therapeutic efficacy when administered, even at 24 h after the onset of experimental stroke. Our findings uncover cellular mechanisms contributing to DAMP clearance in resolution of the sterile inflammation triggered by tissue injury.

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Inflammasomes are cytosolic protein complexes that stimulate the activation of caspase-1, which in turn induces the secretion of the inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. Recent studies have indicated that the inflammasome known as the NOD-like-receptor-family, pyrin domain-containing 3 (NLRP3) inflammasome recognizes several RNA viruses, including the influenza and encephalomyocarditis viruses, whereas the retinoic acid-inducible gene I (RIG-I) inflammasome may detect vesicular stomatitis virus. We demonstrate that measles virus (MV) infection induces caspase-1-dependent IL-1β secretion in the human macrophage-like cell line THP-1. Gene knockdown experiments indicated that IL-1β secretion in MV-infected THP-1 cells was mediated by the NLRP3 inflammasome but not the RIG-I inflammasome. MV produces the nonstructural V protein, which has been shown to antagonize host innate immune responses. The recombinant MV lacking the V protein induced more IL-1β than the parental virus. THP-1 cells stably expressing the V protein suppressed NLRP3 inflammasome-mediated IL-1β secretion. Furthermore, coimmunoprecipitation assays revealed that the V protein interacts with NLRP3 through its carboxyl-terminal domain. NLRP3 was located in cytoplasmic granular structures in THP-1 cells stably expressing the V protein, but upon inflammasome activation, NLRP3 was redistributed to the perinuclear region, where it colocalized with the V protein. These results indicate that the V protein of MV suppresses NLRP3 inflammasome-mediated IL-1β secretion by directly or indirectly interacting with NLRP3.

Inflammation and immune responses after stroke including ischemic cerebral infarction play pivotal roles in the pathology, resolution of inflammation, and neurological recovery. Regulatory T (Treg) cells are the cells responsible for immune tolerance, usually activated by secondary lymphatic tissues, which subsequently regulate effector T cell activation and dendritic cell activation. Recently, Tregs that are present in tissues, called tissue Tregs, have been shown to exhibit tissue-specific functions in addition to immune regulation, contributing to the maintenance of homeostasis and tissue repair. We found that Tregs accumulate in the brain at the chronic phase of ischemic brain injury and control astrogliosis throgh secretion of amphiregulin. Unlike other tissue Tregs, brain Tregs express a serotonin receptor (Htr7) that is characteristic of the nervous system, and are proliferated and activated by serotonin. Administering a serotonin or selective serotonin reuptake inhibitor (SSRI) in an experimental stroke mouse model increased the number of brain Tregs and improved neurological symptoms. Elucidation of the significance of brain Tregs should also contribute to the understanding of other types of neuroinflammation.

Inflammation is an essential step in the progression of ischemic stroke pathology. Various mechanisms of innate immunity are implicated in the acute sterile inflammation following ischemic brain injury. Ischemic insults induce the necrotic death of brain cells, which leads to the extracellular release of danger-associated molecular patterns (DAMPs). DAMPs are recognised by pattern recognition receptors (PRRs) in innate immune cells. IL-1β and various neurotoxic mediators are produced by the activation of PRRs. Caspase-1 activation and the formation of inflammasome complex are essential for the production of the active form of IL-1β in ischemic stroke. Recent evidence has supported the use of new therapeutic opportunities for ischemic stroke by targeting DAMPs and inflammasome. This review examines the evidence regarding the essential roles of innate immunity in the creation of novel treatments for ischemic stroke.

To clarify the diagnostic issues and treatment of patients with cryptogenic or symptomatic generalized epilepsies, not including West syndrome (WS), we investigated electroclinical change during the clinical course, and treatment effects in these patients. The selection criteria were minor generalized seizures as their main seizure type and diffuse epileptic discharges as their main EEG findings. Regarding EEG, we included EEGs that predominantly displayed multifocal spike-waves because of the inclusion of severe epilepsy with multiple independent spike foci (SE-MISF). We divided the subjects into two groups according to their main seizure types: Group A (54 patients) with brief tonic seizures and Group B (24 patients) with myoclonic seizures and/or atypical absences. The main epileptic syndromes were considered to be Lennox-Gastaut syndrome and SE-MISF in Group A, and epilepsy with myoclonic-astatic seizures in Group B. A history of WS was often seen in Group A, but it was exceptional in Group B. During the clinical course, seizure types did not basically change in Group A. EEG patterns were changeable in both groups. Although there was some overlap in electroclinical manifestations among epileptic syndromes, a transition between the two groups was not seen. High-dose valproate and ethosuximide were the most effective in Groups A and B, respectively. Long-term prognosis was significantly more favorable in Group B than in Group A.

Cytokines are key modulators of immunity. Most cytokines use the Janus kinase and signal transducers and activators of transcription (JAK-STAT) pathway to promote gene transcriptional regulation, but their signals must be attenuated by multiple mechanisms. These include the suppressors of cytokine signaling (SOCS) family of proteins, which represent a main negative regulation mechanism for the JAK-STAT pathway. Cytokine-inducible Src homology 2 (SH2)-containing protein (CIS), SOCS1, and SOCS3 proteins regulate cytokine signals that control the polarization of CD4(+) T cells and the maturation of CD8(+) T cells. SOCS proteins also regulate innate immune cells and are involved in tumorigenesis. This review summarizes recent progress on CIS, SOCS1, and SOCS3 in T cells and tumor immunity.

The Non-Clinical Evaluation Subcommittee of the Drug Evaluation Committee, Japan Pharmaceutical Manufacturers Association (JPMA) collected historical control data from developmental and reproductive toxicity studies, which were conducted by the member companies and associated contract laboratories between 1986 and 1993. The data include spontaneous incidences of fetal morphological alterations and other observations made at terminal cesarean sections in rats, rabbits and mice. In addition, mating and natural delivery data in rats are also provided. There were strain differences as well as inter-laboratory variations in the incidences of fetal visceral and skeletal alterations for both rats and rabbits. These inter-laboratory variations were attributed to differences in the selection of observation parameters, observation criteria and classification of the findings.

Objectives Matrix-assisted laser desorption time-of flight ionization (MALDI)-imaging MS (IMS) with MSMS analysis using on-tissue tryptic digests is a powerful tool for identification of disease-related proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections. We applied this novel IMS technique, not only to identify tryptic peptides of deposited amyloidogenic proteins but also to clarify topologies of these proteins in amyloidosis tissue sections. Methods Sequence determinations of tryptic peptides derived from amyloidogenic proteins were performed using MALDI-MSMS analysis directly from Congo red positive regions in tissue sections with/without procedure for retrieval of epitopes before on-tissue digestion. Results Tryptic peptides, m/z = 1073.5 and 1924.3 were identified with the sequences, from 48th to 56th and 1st to 19th positions of Ig lambda V-III region, respectively. Other peptides, m/z = 1365.5 and 1523.5 were with the sequences, from 22nd to 34th and 36th to 48th positions of TTR, respectively. Heat-map images of all four tryptic peptides were overlapped with Congo red positive regions. Immunohistochemistry of FFPE tissue sections was confirmed to only react with anti-λ chain antibody in a case of AL-type amyloidosis or anti-TTR antibody in two cases of TTR-type amyloidosis. Conclusion IMS with MSMS analysis using on-tissue tryptic digestion enables us not only to identify amyloidogenic molecule in a sliced tissue section but also to play a complementary role with the conventional pathological examination.

Summary: Purpose: A retrospective study was carried out on 53 cases with childhood epilepsy to evaluate the validity of the initial selection of antiepileptic drug (AED). Methods: We investigated the AEDs selected at the beginning of the treatment from the medical records of 53 untreated cases. A follow-up study was undertaken to evaluate the effects of the AEDs. In the second study, we investigated the AEDs of 10 cases with atypical benign partial epilepsy (ABPE), to clarify whether the initial AEDs selected for rolandic epilepsy were related to the appearance of ABPE. Results: The AEDs used at the initial stage consisted of carbamazepine (CBZ), valproic acid (VPA), phenobarbital (PB), and vitamin B6. The main AEDs were CBZ and VPA for localizationrelated epilepsy, and VPA for generalized epilepsy. The initial selection of AEDs in 41 (85.4%) of 48 cases treated with AEDs were considered to be correct from the results of follow-up. We could not specify any AEDs that related to the appearance of ABPE. Conclusions: The selection of AED in this series was considered to be most appropriate. We proposed a criterion to determine whether to begin the AED treatment immediately at the initial seizure. Ke yW ords: Childhood epilepsy—Antiepileptic drugs—Medication—Beginning of treatment—Initial seizure.

Signal transduction pathways elicited by cytokines and hormones have been shown to regulate distinct stages of development. Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of cytokine signaling mediated by the JAK-STAT signaling pathway. In particular, SOCS1 and SOCS3 are potent inhibitors of JAKs and can play pivotal roles in inflammation, as well as in the development and progression of cancers. Abnormal expression of SOCS1 and SOCS3 in cancer cells has been reported in human carcinoma associated with dysregulation of signals from cytokine receptors, Toll-like receptors (TLRs), and hormone receptors, resulting in malignancies. In this review, we focus on the role of SOCS1 and SOCS3 in cancer development. In addition, the potential of SOCS as a therapeutic target and diagnostic aid will be discussed.

The main purpose of this study was to examine the developmental process of personal and social orientedness in young adults in relation to changes in loneliness types. Two hundred and sixty-two male and 416 female college students completed the loneliness, personal and social orientedness, and self-esteem scales. They were classified into four loneliness types (by Ochiai, 1983), and Type A (Loneliness from conformity to others) and D (Loneliness from independence) were compared, in accordance with a previous finding that youths tend to change from the former to latter. Although students of the two types scores was high for Type A, but was not significant for Type D. Second, self-esteem was correlated with only personal orientedness for Type A, but with both scores of orientedness for Type D. It is argued from the results that in the development of personal and social orientedness in young adults, the stage where the self emerges from a fused state of self and other, evolves on the basis of self-other independence, to the stage where both personal and social orientedness become important after only personal orientedness has played a central role.

Nod-like receptors (NLRs) comprise a large family of intracellular pattern- recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed the inflammasomes. The NLR family, pyrin domain-containing 3 (NLRP3) is triggered by a diverse set of molecules and signals, and forms the NLRP3 inflammasome. Recent studies have indicated that both DNA and RNA viruses stimulate the NLRP3 inflammasome, leading to the secretion of interleukin 1 beta (IL-1β) and IL-18 following the activation of caspase-1. We previously demonstrated that the proton-selective ion channel M2 protein of influenza virus activates the NLRP3 inflammasome. However, the precise mechanism by which NLRP3 recognizes viral infections remains to be defined. Here, we demonstrate that encephalomyocarditis virus (EMCV), a positive strand RNA virus of the family Picornaviridae, activates the NLRP3 inflammasome in mouse dendritic cells and macrophages. Although transfection with RNA from EMCV virions or EMCV-infected cells induced robust expression of type I interferons in macrophages, it failed to stimulate secretion of IL-1β. Instead, the EMCV viroporin 2B was sufficient to cause inflammasome activation in lipopolysaccharide-primed macrophages. While cells untransfected or transfected with the gene encoding the EMCV non-structural protein 2A or 2C expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells transfected with the gene encoding the EMCV 2B or influenza virus M2 protein. 2B proteins of other picornaviruses, poliovirus and enterovirus 71, also caused the NLRP3 redistribution. Elevation of the intracellular Ca2+ level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca2+ did not reduce virus-induced IL-1β secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca2+ flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation., Author Summary The innate immune system, the first line of defense against invading pathogens, plays a key role not only in limiting microbe replications at early stages of infection, but also in initiating and orchestrating antigen-specific adaptive immune responses. The innate immune responses against viruses usually rely on recognition of viral nucleic acids by host pattern-recognition receptors such as Toll-like receptors and cytosolic helicases. In addition, recent studies have indicated that certain viruses activate the NLRP3 inflammasome, a multiprotein complex containing the intracellular pattern-recognition receptor NLRP3, which in turn induces secretion of proinflammatory cytokines. We have previously revealed the role of the NLRP3 inflammasome in innate recognition of influenza virus, in which the influenza virus proton-selective ion channel M2 protein, but not viral RNA, is required. Here, we demonstrate that another RNA virus, encephalomyocarditis virus (EMCV), also activates the NLRP3 inflammasome in a viral RNAindependent manner. Instead, the EMCV viroporin 2B, which is involved in Ca2+ flux from intracellular storages into the cytosol, activates the NLRP3 inflammasome. Our results highlight the importance of viroporins, virusencoded transmembrane pore-forming proteins, in recognition of virus infections by NLRP3.

The purpose of this study is to clarify the usefulness and problems of the newly proposed classification of epilepsies (International League Against Epilepsy: ILAE, 2001) in the epidemiological studies of epilepsy. We previously conducted an epidemiological study in Okayama Prefecture, Japan, in 1999, using the ILAE 1989 classification. Among 250,997 children under 13 years of age, 2220 epileptic patients were ascertained. In this study, we reclassified them according to the ILAE 2001 classification, focusing on axes 2 (seizure types) and 3 (syndromes). We were able to classify 1803 (95.0%) seizure types out of 1899 with detailed clinical information. In focal seizures, the most common were secondarily generalized seizures (88.6%), which generally do not represent a unique anatomic substrate. In generalized seizures, topic-clonic seizures (40.7%) and spasms (21.0%) were the most common. We identified only 269 (12.1%) patients with specific epilepsy syndromes out of the 2220. We classified 1761 patients without specific syndromic diagnoses only by axis 2, but the new concept of epileptic seizure types, representing a unique pathophysiologic mechanism and anatomic substrate, was not very meaningful in most cases, even in those with focal seizures.

Absence seizures are classified into typical and atypical absences according to clinical and EEG characteristics. Although missense mutations in the GABA(A) receptor gamma2 subunits (GABRG2) gene have recently been detected in two families with typical absence seizures, no study has been carried out to clarify the relationship between atypical absence and GABA(A) receptors. We performed mutation analysis of all the coding exons of GABA(A) receptor alpha1, beta2 and gamma2 subunit (GABRA1, GABRB2 and GABRG2) genes by direct sequencing to clarify whether there was common molecular biological mechanism underlying both typical and atypical absences. We recruited 52 unrelated Japanese patients, thirty-eight with typical absences and 14 with atypical absences. They consisted of 38 with childhood absence epilepsy, three with Lennox-Gastaut syndrome, two with epilepsy with myoclonic-astatic seizures and nine with epilepsy with continuous spike-waves during slow wave sleep. All of the subjects were idiopathic or cryptogenic cases without any organic brain lesions or underlying diseases. We detected five polymorphisms (T156C in GABRA1, C1194T in GABRB2, and C315T, T588C and C1230T in GABRG2), and they are silent mutations. In conclusion, mutations in GABRA1, GABRB2 and GABRG2 do not seem to be a major genetic cause of epilepsy with typical and atypical absences in Japanese subjects.

Objectives: Febrile seizures plus (FS+) are attracting attention for their corresponding genetic abnormalities, and are defined as febrile seizures (FS) continuing beyond 6 years of age (late FS) or those associated with afebrile seizures. We tried to elucidate their clinical and EEG characteristics as compared with those of children having only FS. Subjects and methods: We reviewed clinical records in a pediatric neurology clinic to identify 31 patients with FS+ (group FS+) and 51 with only FS (group FS). Their family history of seizures, clinical features and EEG findings were compared. Results: A family history of seizures was noted in 14 patients (45.2%) of group FS+ and in 24 (47.1%) of group FS. In group FS+, 19 patients had late FS, 11 had afebrile seizures, and the remaining one had both types of seizures. Two patients had seizures induced by TV/video-game as well, and another suffered from absences. Epileptic EEG abnormalities, which included diffuse spike-waves and focal spikes, were noted in 13 patients (41.9%) of group FS+ and 12 (23.5%) of group FS. Conclusions: The clinical and EEG characteristics of the children having FS+ were diverse, without significant differences from those with FS except for the seizures types.

Background: The lesions of gastric antral vascular ectasia take two endoscopic forms, diffuse red spots and red stripes. In addition, they are often associated with cirrhosis. The main aim of the present retrospective study was to determine whether differences in endoscopic appearance and presence or absence of cirrhosis have relationships to clinical features and course. Methods: Gastric antral vascular ectasia in 30 patients was classified into 2 endoscopic subtypes, punctate type (21 patients) and striped type (8 patients); only 1 patient could not be categorized to either type. The 30 patients were divided into groups based on the presence (25) or absence (5) of cirrhosis. Results: All patients with punctate-type vascular ectasias had cirrhosis, whereas only 38% of patients with the striped type had cirrhosis. All patients without cirrhosis had the striped pattern. For patients with the 2 endoscopic types as well as those with and without cirrhosis, the outcomes of endoscopic treatment were similar. Conclusions: The findings of the present study suggest that cirrhosis is strongly associated with the development of punctate-type vascular ectasias. The endoscopic appearance of vascular ectasias and the presence or absence of cirrhosis did not determine outcome of patients. (Gastrointest Endosc 2001;53:764-70.)

Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) (MIM 125370)1 is a rare autosomal dominant neurodegenerative disorder found mostly in Japanese people and caused by an expansion of the CAG repeat in the DRPLA gene on chromosome 12p.2 Childhood-onset patients generally have a phenotype of progressive myoclonus epilepsy (PME) with a large expansion of the CAG repeat.2 In the present study, we report a patient with DRPLA who showed epilepsy with electrical status epilepticus during slow sleep (ESES) or the extraordinary EEG abnormality of continuous spike-waves during slow wave sleep (CSWS) and epileptic negative myoclonus in childhood. Epilepsy with CSWS generally occurs in cryptogenic patients or symptomatic ones with nonprogressive brain pathology and is an age-related and self-limited disorder.3 Remarkably, his CSWS on EEG remitted spontaneously with age, as it does in other patients without progressive disorder, although the underlying degeneration of the brain continued relentlessly. This patient, who was born after an uneventful pregnancy, had a father with ataxia and dementia and a diagnosis of DRPLA in later life. The patient's early …