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We report current status of developing Soft X-ray Imager (SXI), the X-ray CCD camera onboard X-Ray Imaging and Spectroscopy Mission (XRISM). Four flight model (FM) CCDs have been selected considering several items including energy resolution at 5.9keV, CTI, dark current, etc. We have also completed calibration campaign for all the FM CCDs. Initial analyses show that the response function for monochromatic X-rays is basically the same as that of Hitomi CCDs. The focal plane including the single-stage Stirling cooler has been assembled. Production of key parts in SXI sensor body such as contamination blocking filter and onboard calibration source has been finished and they are waiting for assemble. The digitized signals of the CCD are corrected step by step before conversion to X-ray energy. We are preparing calibration database for the correction such as CTI, gain, and line redistribution function.

X-Ray Imaging and Spectroscopy Mission (XRISM) is the seventh Japanese X-ray astronomical satellite scheduled to be launched in the Japanese fiscal year 2022. XRISM has two mission instruments, “Resolve”, a soft X-ray spectrometer, and “Xtend”, a soft X-ray imager. The Former is an X-ray micro-calorimeter that has ∼ 5 eV of energy resolution with 3′ × 3 ′ of field of view. The Latter is an X-ray CCD camera with 38′ × 38′ of field of view. Both instruments are placed on the focal plane of X-ray telescopes, X-ray Mirror Assembly (XMA). Xtend CCDs are designed almost the same as those of Hitomi (ASTRO-H), whereas some improvements have been applied. In 2019, flight-model (FM) candidates of Xtend CCDs were fabricated by Hamamatsu Photonics K.K. We performed screening experiments to examine whether they met requirements or not, and then selected the best four chips as the FM. We then performed on-ground calibration on August 2019 and September 2019 for the FM chips to determine the gain correction parameters and to construct the detector response with several energies of monochromatic X-ray. In this paper, we report screening, selection, and on-ground calibration processes, especially focusing on the response verification.

We present experimental studies on the charge transfer inefficiency (CTI) of charge-coupled device (CCD) developed for the soft X-ray imaging telescope, Xtend, aboard the XRISM satellite. The CCD is equipped with a charge injection (CI) capability, in which sacrificial charge is periodically injected to fill the charge traps. By evaluating the re-emission of the trapped charge observed behind the CI rows, we find that there are at least three trap populations with different time constants. The traps with the shortest time constant, which is equivalent to a transfer time of approximately one pixel, are mainly responsible for the trailing charge of an X-ray event seen in the following pixel. A comparison of the trailing charge in two clocking modes reveals that the CTI depends not only on the transfer time but also on the area, namely the imaging or storage area. We construct a new CTI model with taking into account with both transfer-time and area dependence. This model reproduces the data obtained in both clocking modes consistently. We also examine apparent flux dependence of the CTI observed without the CI technique. The higher incident X-ray flux is, the lower the CTI value becomes. It is due to a sacrificial charge effect by another X-ray photon. This effect is found to be negligible when the CI technique is used., 8 pages, 8 figures, accepted for publication in NIMA, proceedings of the 12th International "Hiroshima" Symposium on the Development and Application of Semiconductor Tracking Detector (HSTD12)

We have been developing P-channel Charge-Coupled Devices (CCDs) for the upcoming X-ray Astronomy Satellite XRISM, planned to be launched in 2021. While the basic design of the CCD camera (Soft X-ray Imager: SXI) is almost the same as that of the lost Hitomi (ASTRO-H) observatory, we are planning to reduce the "light leakages" that is one of the largest problems recognized in Hitomi data. We adopted a double-layer optical blocking layer on the XRISM CCDs and also added an extra aluminum layer on the backside of them. We develop a newly designed test sample CCD and irradiate it with optical light to evaluate the optical blocking performance. As a result, light leakages are effectively reduced compared with that of the Hitomi CCDs. We thus conclude that the issue is solved by the new design and that the XRISM CCDs satisfy the mission requirement for the SXI., 6 pages, 4 figures, accepted for publication in NIMA, proceedings of the 12th International "Hiroshima" Symposium on the Development and Application of Semiconductor Tracking Detector (HSTD12)

X-ray Imaging Spectroscopy Mission (XRISM) is the next Japanese X-ray astronomical satellite to be launched in 2021 Japanese fiscal year. We are developing one of the XRISM instruments “Xtend”, which is an X-ray CCD camera combined with an X-ray telescope, and achieves the wide field of view of 3 8 ′ × 3 8 ′ in 0.4–13 keV. In 2019, twelve flight-model (FM) candidate CCD chips were fabricated by Hamamatsu Photonics K.K. We conducted screening experiments to examine whether the FM candidates met requirements for the Xtend CCDs, and selected the four FM chips from them. We constructed a screening system, with which we can examine various CCD performances by illuminating characteristic X-ray lines in a ∼ 0 . 5 –14 keV band or optical lights. With this system, all the twelve candidates were confirmed to satisfy the requirements. We then selected four chips with the best performance, in terms of e.g., their charge transfer inefficiencies, energy resolutions, soft X-ray sensitivities, and optical light leakages. In this paper, we report an overview of the screening system, and procedures and results of the screening process.

Entecavir (ETV) is a potent nucleoside analogue against hepatitis B virus (HBV), and emergence of drug resistance is rare in nucleoside-naive patients because development of ETV resistance (ETVr) requires at least three amino acid substitutions in HBV reverse transcriptase. We observed two cases of genotypic ETVr with viral rebound and biochemical breakthrough during ETV treatment of nucleoside-naive patients with chronic hepatitis B (CHB).Case 1: A 44-year-old HBeAg-positive man received ETV 0.1 mg/day for 52 weeks and 0.5 mg/day for 96 weeks consecutively. HBV DNA was 10.0 log(10) copies/ml at baseline, declined to a nadir of 3.1 at week 100, and rebounded to 4.5 at week 124 and 6.7 at week 148. Alanine aminotransferase (ALT) level increased to 112 IU/l at week 148. Switching to a lamivudine (LVD)/adefovir-dipivoxil combination was effective in decreasing HBV DNA. Case 2: A 47-year-old HBeAg-positive man received ETV 0.5 mg/day for 188 weeks. HBV DNA was 8.2 log(10) copies/ml at baseline, declined to a nadir of 2.9 at week 124, and then rebounded to 4.7 at week 148 and 6.4 at week 160. ALT level increased to 72 IU/l at week 172. The ETVr-related substitution (S202G), along with LVD-resistance-related substitutions (L180M and M204V), was detected by sequence analysis at week 124 in both case 1 and case 2.ETVr emerged in two Japanese nucleoside-naive CHB patients after prolonged therapy and incomplete suppression and in one patient after0.5 mg of dosing. ETV patients with detectable HBV DNA or breakthrough after extended therapy should be evaluated for compliance to therapy and potential emergence of resistance.

During progression to type 1 diabetes (T1D), the pancreatic islets of humans and of the widely studied mouse model of T1D, the nonobese diabetic (NOD) mouse, are infiltrated by cells of the immune system. Here we report that infiltrated mouse islets ("translucent islets") can be identified visually. We demonstrate the use of an efficient method for the isolation and culture of the islet-infiltrating cells of NOD mice, which results in a high percentage of CD8(+) T cells after seven days, with minimal manipulation. We show that islet-infiltrating cells exit the islets soon after they are placed in culture and can be used in flow cytometric experiments several hours later. Importantly, we demonstrate that the cultured cells are antigen-responsive and that specificities present at the beginning of the culture are generally still present on day seven. In addition, some reactivities are undetected without culture, supporting the validity of the seven-day expansion period. This technique greatly facilitates investigations of the CD8(+) T cell reactivities that play a pivotal role in the demise of pancreatic beta cells leading to the development of T1D.

OBJECTIVE—CD8+ T-cells specific for islet antigens are essential for the development of type 1 diabetes in the NOD mouse model of the disease. Such T-cells can also be detected in the blood of type 1 diabetic patients, suggesting their importance in the pathogenesis of the human disease as well. The development of peptide-based therapeutic reagents that target islet-reactive CD8+ T-cells will require the identification of disease-relevant epitopes.RESEARCH DESIGN AND METHODS—We used islet-infiltrating CD8+ T-cells from HLA-A*0201 transgenic NOD mice in an interferon-γ enzyme-linked immunospot assay to identify autoantigenic peptides targeted during the spontaneous development of disease. We concentrated on insulin (Ins), which is a key target of the autoimmune response in NOD mice and patients alike.RESULTS—We found that HLA-A*0201-restricted T-cells isolated from the islets of the transgenic mice were specific for Ins1 L3–11, Ins1 B5–14, and Ins1/2 A2–10. Insulin-reactive T-cells were present in the islets of mice as young as 5 weeks of age, suggesting an important function for these specificities early in the pathogenic process. Although there was individual variation in peptide reactivity, Ins1 B5–14 and Ins1/2 A2–10 were the immunodominant epitopes. Notably, in vivo cytotoxicity to cells bearing these peptides was observed, further confirming them as important targets of the pathogenic process.CONCLUSIONS—The human versions of B5–14 and A2–10, differing from the murine peptides by only a single residue, represent excellent candidates to explore as CD8+ T-cell targets in HLA-A*0201–positive type 1 diabetic patients.

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic β cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

The NOD mouse is a model for autoimmune type 1 diabetes in humans. CD8+ T cells are essential for the destruction of the insulin-producing pancreatic β cells characterizing this disease. AI4 is a pathogenic CD8+ T cell clone, isolated from the islets of a 5-wk-old female NOD mouse, which is capable of mediating overt diabetes in the absence of CD4+ T cell help. Recent studies using MHC-congenic NOD mice revealed marked promiscuity of the AI4 TCR, as the selection of this clonotype can be influenced by multiple MHC molecules, including some class II variants. The present work was designed, in part, to determine whether similar promiscuity also characterizes the effector function of mature AI4 CTL. Using splenocyte and bone marrow disease transfer models and in vitro islet-killing assays, we report that efficient recognition and destruction of β cells by AI4 requires the β cells to simultaneously express both H-2Db and H-2Kd class I MHC molecules. The ability of the AI4 TCR to interact with both H-2Db and H-2Kd was confirmed using recombinant peptide libraries. This approach also allowed us to define a mimotope peptide recognized by AI4 in an H-2Db-restricted manner. Using ELISPOT and mimotope/H-2Db tetramer analyses, we demonstrate for the first time that AI4 represents a readily detectable T cell population in the islet infiltrates of prediabetic NOD mice. Our identification of a ligand for AI4-like T cells will facilitate further characterization and manipulation of this pathogenic and promiscuous T cell population.

Ursodeoxycholic acid (UDCA) has been widely used in the treatment of patients with primary biliary cirrhosis (PBC). However, some patients are refractory to UDCA. The aim of this study is to clarify the additive improvement induced by bezafibrate in patients refractory to UDCA.This study was a prospective analysis of 37 consecutive PBC patients. All patients were treated first for 6 months with UDCA, and then with bezafibrate, if their alkaline phosphatase (ALP) levels did not decrease more than 40% or within the normal range after 6 months' treatment with UDCA. Clinical parameters were monitored for the subsequent 24 months.Twenty-two patients (59%) achieved improvement of ALP levels after the treatment with UDCA. Those patients (Group A) had significantly lower levels of ALP at diagnosis than those with abnormal ALP levels after 6 months' treatment with UDCA (Group B; P = 0.020). They continued UDCA monotherapy, and maintained normal ALP levels at subsequent follow ups. However, immunoglobulin M (IgM) levels remained abnormal in eight patients, whose IgM levels at the time of diagnosis were significantly higher than those whose IgM were normalized after 6 months' treatment with UDCA (P = 0.026). Those in Group B were treated additionally with bezafibrate, and 12 patients (80%) achieved normal ALP and IgM levels within 12 months of commencement of therapy.Higher ALP level at diagnosis is one of the predictors for UDCA failure. Combination treatment of bezafibrate in addition to UDCA may be an effective treatment for PBC patients refractory to UDCA.

Background and Aim: Ursodeoxycholic acid (UDCA) has been widely used in the treatment of patients with primary biliary cirrhosis (PBC). However, some patients are refractory to UDCA. The aim of this study is to clarify the additive improvement induced by bezafibrate in patients refractory to UDCA. Methods: This study was a prospective analysis of 37 consecutive PBC patients. All patients were treated first for 6 months with UDCA, and then with bezafibrate, if their alkaline phosphatase (ALP) levels did not decrease more than 40% or within the normal range after 6 months' treatment with UDCA. Clinical parameters were monitored for the subsequent 24 months. Result: Twenty-two patients (59%) achieved improvement of ALP levels after the treatment with UDCA. Those patients (Group A) had significantly lower levels of ALP at diagnosis than those with abnormal ALP levels after 6 months' treatment with UDCA (Group B; P = 0.020). They continued UDCA monotherapy, and maintained normal ALP levels at subsequent follow ups. However, immunoglobulin M (IgM) levels remained abnormal in eight patients, whose IgM levels at the time of diagnosis were significantly higher than those whose IgM were normalized after 6 months' treatment with UDCA (P = 0.026). Those in Group B were treated additionally with bezafibrate, and 12 patients (80%) achieved normal ALP and IgM levels within 12 months of commencement of therapy. Conclusion: Higher ALP level at diagnosis is one of the predictors for UDCA failure. Combination treatment of bezafibrate in addition to UDCA may be an effective treatment for PBC patients refractory to UDCA.

Aim: The average age of Japanese patients with drug-induced liver injury (DILI) is expected to rise as the population ages. The aim of this study was to evaluate the clinical characteristics of DILI in elderly Japanese subjects. Methods: A total of 142 hospitalized patients with DILI were divided into three groups by age (Group A

Activation of CD1d-restricted invariant NKT (iNKT) cells by α-galactosylceramide (αGalCer) significantly suppresses development of diabetes in NOD mice. The mechanisms of this protective effect are complex, involving both Th1 and Th2 cytokines and a network of regulatory cells including tolerogenic dendritic cells. In the current study, we evaluated a newly described synthetic αGalCer analog (C20:2) that elicits a Th2-biased cytokine response for its impact on disease progression and immunopathology in NOD mice. Treatment of NOD mice with αGalCer C20:2 significantly delayed and reduced the incidence of diabetes. This was associated with significant suppression of the late progression of insulitis, reduced infiltration of islets by autoreactive CD8+ T cells, and prevention of progressive disease-related changes in relative proportions of different subsets of dendritic cells in the draining pancreatic lymph nodes. Multiple favorable effects observed with αGalCer C20:2 were significantly more pronounced than those seen in direct comparisons with a closely related analog of αGalCer that stimulated a more mixed pattern of Th1 and Th2 cytokine secretion. Unlike a previously reported Th2-skewing murine iNKT cell agonist, the αGalCer C20:2 analog was strongly stimulatory for human iNKT cells and thus warrants further examination as a potential immunomodulatory agent for human disease.

Spontaneous autoimmune diabetes development in NOD mice requires both CD8+ and CD4+ T cells. Three pathogenic CD8+ T cell populations (represented by the G9C8, 8.3, and AI4 clones) have been described. Although the Ags for G9C8 and 8.3 are known to be insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein, respectively, only mimotope peptides had previously been identified for AI4. In this study, we used peptide/MHC tetramers to detect and quantify these three pathogenic populations among β cell-reactive T cells cultured from islets of individual NOD mice. Even within age-matched groups, each individual mouse exhibited a unique distribution of β cell-reactive CD8+ T cells, both in terms of the number of tetramer-staining populations and the relative proportion of each population in the islet infiltrate. Thus, the inflammatory process in each individual follows its own distinctive course. Screening of a combinatorial peptide library in positional scanning format led to the identification of a peptide derived from dystrophia myotonica kinase (DMK) that is recognized by AI4-like T cells. Importantly, the antigenic peptide is naturally processed and presented by DMK-transfected cells. DMK is a widely expressed protein that is nonetheless the target of a β cell-specific autoimmune response.

Cytotoxic T lymphocytes (CTL) specific for HLA-A2-binding MAGE-3 peptide (FLWGPRALV) were generated by repetitive stimulation of PBMC with the peptide in the presence of EBV-transformed B blasts and IL-2. Using these CTL, we investigated the expression of the HLA-A2-binding MAGE-3 peptide on lung cancer cell lines. Of 14 cell lines investigated, 1-87, PC-9, OU-LC-KI, 11-18 and LK87 were derived from HLA-A2 positive patients. But cytofluorometry analysis showed that 1-87, PC-9 and OU-LC-KI, but not 11-18 or LK87 expressed the HLA-A2 antigen. All five cell lines expressed MAGE-3 gene mRNA. Twelve of thirteen CTL lines from two HLA-A2 positive donors showed no cytotoxicity against any of the 14 lung cancer cell lines. CTL line TI-1 showed cytotoxicity against 1-87 but not against any of the other cell lines. Treatment of 1-87 with IFN-gamma greatly augmented the cytotoxicity of TI-1 and induced it in the other 12 CTL lines, confirming the expression of the peptide on 1-87. No cytotoxicity was induced by IFN-gamma treatment of PC-9 or OU-LC-KI. However, PC-9 and OU-LC-KI pulsed with the peptide were killed efficiently by all of the CTL lines, suggesting no expression of the peptide on those cells. A low level of cytotoxicity was induced on 11-18 but not LK87 by IFN-gamma treatment, although expression of the HLA-A2 antigen was not observed by cytofluorometry. These findings showed that expression of the HLA-A2-binding MAGE-3 peptide recognized by CTL was variable on lung cancer cell lines.