Pathogenicity of functionally activated PD-1+CD8+ cells and counterattacks by muscular PD-L1 through IFNγ in myositis.

Programmed-cell-death 1 (PD-1) expression is associated not only with T-cell activation but with exhaustion. Specifically, PD-1+ T cells present an exhausted phenotype in conditions of chronic antigen exposure, such as tumor microenvironments and chronic viral infection. However, the immune status regarding exhaustion of PD-1+CD8+ T cells in chronic autoimmune diseases including idiopathic inflammatory myopathies (IIMs) remains unclear. We aimed to clarify the role of PD-1+CD8+ T cells and PD-1 ligand (PD-L1) in IIMs. We showed that PD-1+ cells infiltrated into PD-L1-expressing muscles in patients with IIMs and immune checkpoint inhibitor-related myopathy. According to the peripheral blood immunophenotyping, the PD-1+CD8+ cell proportions were comparable between the active and inactive patients. Of note, PD-1+CD8+ cells in the active patients highly expressed cytolytic molecules, indicating their activation, while PD-1-CD8+ cells expressed low levels of cytolytic molecules in the active and inactive patients. A part of PD-1+CD8+ cells expressed the HMG-box transcription factor TOX highly and presented the exhausted phenotype in the active patients. Among PD-1+CD4+ T cells, PD-1highCXCR5-CD45RO+CD4+ peripheral helper T cells were increased in the active patients. PD-L1-deficient mice developed severer C-protein-induced myositis (CIM), a model of polymyositis, with abundant infiltration of PD-1+CD8+ cells expressing cytolytic molecules than wild-type mice, indicating pathogenicity of the PD-1+CD8+ cells and the protective role of PD-L1. The deficiency of IFNγ, a general PD-L1-inducer, impaired muscular PD-L1 expression and exacerbated CIM, indicating IFNγ-dependent muscular PD-L1 regulation. IFNγ-induced PD-L1 on myotubes was protective in an established muscle injury model. In conclusion, PD-1+CD8+ T cells rather than PD-1-CD8+ T cells were a pathogenic subset of IIMs. Muscular PD-L1 was regulated by IFNγ and exerted protective properties in IIMs.

Pathogenesis of Gastrointestinal Follicular Lymphomas: Consideration Based on Histopathology and Endoscopic Findings.

Gastrointestinal (GI) follicular lymphoma (FL) is the most frequently diagnosed extranodal FL; however, its pathogenesis is debatable. We investigated the distribution, endoscopic, and histopathologic findings of 366 GI FL samples obtained from 298 patients. FLs were most frequently observed in the small intestine (71%), including the duodenum (52%), but were also commonly found in the stomach (15%) and colon (12%). The proportion of granular lesions in the duodenum, terminal ileum, colon, and stomach was 74%, 39%, 24%, and 0%, respectively. Submucosal or ulcerated tumors were frequently observed in the stomach (48%) and colon (52%). Most GI FL showed grade 1 to 2 histology (89%) as well as CD10 + (93%) and BCL2 + (98%) positivity. There were no significant differences in the endoscopic or histologic findings between primary and secondary GI FLs. As known, the mucosa of the small intestine is thin and villous, while the mucosa of the stomach and colon is thicker and has a smooth surface. Granular lesions corresponding to very small FL were detected in the former but rarely in the latter. Nine (7%) patients with primary GI FL developed histologic transformation to diffuse large B-cell lymphoma (n=8) or high-grade B-cell lymphoma (n=1) 10 months to 14 years after the diagnosis of FL. Two patients died of lymphoma. In conclusion, the incidence and endoscopic findings differed, but the histopathology was similar in FLs in each site. These differences might be attributed to variations in each GI site's mucosal structure and the neoplastic follicles' size. Due to its characteristic structure, very small classic FLs might be detectable mainly in the small intestine.

Diagnostic clues of BCL2-negative, faint, or controversial follicular lymphomas: a study of 103 cases.

BCL2 positivity by immunohistochemistry is helpful for the diagnosis of follicular lymphoma (FL); however, a minority of FL cases are BCL2-negative, and the diagnosis is thus challenging. We retrospectively analyzed the incidence, morphology, immunophenotype, and genetic status of BCL21+ (weakly/focally positive by clone 124), BCL20 (negative), and BCL2controversial FLs compared with BCL22+ (strongly positive) FLs to clarify diagnostic clues. In 1068 FL cases, 103 (10%) with BCL21+ (37 cases, 4%), BCL20 (61 cases, 6%), or BCL2controversial (5 cases, 0.5%) were included in the final analysis. BCL21+ and BCL20 FLs tended to have limited stage disease, nodal disease, and grades 3A/3B histology and showed a higher complete response rate than BCL22+ FLs. Among 103 BCL20, BCL21+, or BCL2controversial FL cases, 34 (33%) had a diffuse area composed of CD20-positive small-to medium-sized lymphoid cells, a feature of low-grade B-cell lymphoma. Interfollicular dense CD20-positive cells and interfollicular clusters of CD10-positive cells were observed in 59% and 37% of cases, respectively. In remaining 13/40 cases (33%), BCL2 was converted to BCL22+ by other clones E17/SP66. CD23 and MUM1 were positive in 10/40 (25%) and 1/40 (3%) cases, respectively. IGH/BCL2 fusion and clonality were detected in 6/37 (16%) and 31/34 (91%) cases, respectively. In conclusion, morphological examination of the distribution of CD20-and/or CD10-positive cells and the presence of diffuse area could be used to diagnose FL in most cases. The majority of the remaining FL cases could be diagnosed using other BCL2 clones and clonality analyses.

Factors associated with impaired physical function in elderly rheumatoid arthritis patients who had achieved low disease activity.

OBJECTIVE: We aimed to investigate factors associated with impaired physical function (defined as HAQ Disability Index [HAQ-DI] >0.5) of old-old (aged 75-84) patients with rheumatoid arthritis (RA). METHODS: Data from 15,185 RA patients in the National Database of Rheumatic Disease in Japan were extracted from 2017 to 2018. We enrolled 3,708 patients aged 55-84 in simplified disease activity index (SDAI) ≤11 and Steinbrocker stage I/II. Factors associated with HAQ-DI >0.5 were analyzed by multivariable logistic regression. RESULTS: About half of the old-old patients received methotrexate, which was lower than middle-aged (55-64) and young-old patients (65-74). The proportion of glucocorticoids in the old-old patients was highest among the three groups, and biological disease-modifying anti-rheumatic drugs were similarly used. The prevalence of HAQ-DI >0.5 was significantly higher in old-old patients with low disease activity than in those with remission. The same was true in the middle-aged and young-old patients. Multivariable analysis showed age, higher SDAI, glucocorticoid use, and methotrexate non-use were significantly associated with HAQ-DI >0.5 in the old-old patients. CONCLUSIONS: SDAI remission was an ideal goal for old-old patients in terms of physical function. Glucocorticoids and a low proportion of methotrexate use may influence the physical function of old-old patients.

Linear imaging theory for differential phase contrast and other phase imaging modes in scanning transmission electron microscopy.

We propose a linear imaging theory for differential phase contrast under the weak-phase-weak-amplitude object approximation. Contrast transfer functions are defined for thin and thick weak objects, and they successfully describe several imaging characteristics of differential phase contrast. We discuss the defocus dependence of the contrast for several examples: atomic resolution, a p-n junction, a heterointerface, and grain boundaries. Understanding the imaging characteristics helps in adjusting aberrations in DPC STEM.

Development of organ-specific autoimmunity by dysregulated Aire expression.

Deficiency for AIRE/Aire in both humans and mice results in the development of organ-specific autoimmune disease. We tested whether augmented and/or dysregulated AIRE/Aire expression might be also prone to the breakdown of self-tolerance. To define the effect of augmented Aire expression on the development of autoimmunity, antigen-specific clonal deletion and production of clonotypic regulatory T cells (Tregs) in the thymus were examined using mice expressing two additional copies of Aire in a heterozygous state (3xAire-knockin mice: 3xAire-KI). We found that both clonal deletion of autoreactive T cells and production of clonotypic Tregs in the thymus from 3xAire-KI were impaired in a T-cell receptor-transgenic system. Furthermore, 3xAire-KI females showed higher scores of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein than wild-type littermates, suggesting that augmented Aire expression exacerbates organ-specific autoimmunity under disease-prone conditions. In humans, we found that one patient with amyopathic dermatomyositis showed CD3- CD19- cells expressing AIRE in the peripheral blood before the treatment but not during the remission phase treated with immunosuppressive drugs. Thus, not only loss of function of AIRE/Aire but also augmented and/or dysregulated expression of AIRE/Aire should be considered for the pathogenesis of organ-specific autoimmunity. We suggest that further analyses should be pursued to establish a novel link between organ-specific autoimmune disease and dysregulated AIRE expression in clinical settings.

CD3-and CD20-negative extramedullary non-Hodgkin leukemia/lymphoma: a histopathological study of 118 cases.

Histopathological diagnoses are challenging for rare CD3-and CD20-negative extramedullary leukemias/lymphomas. We report 118 cases of CD3- CD20-extramedullary leukemias/lymphomas (2.4% of 4977 cases). CD45 was positive in 68% of cases. Forty-nine (41%) cases were anaplastic large cell lymphomas. Thirty-five (30%) cases were large B-cell lymphomas/plasmablastic lymphomas positive for at least one of the following markers: CD79a, PAX5, CD19, CD138, and MUM1. Nine (8%) cases were peripheral T/NK-cell lymphomas, where at least CD43, CD45RO, or cytotoxic molecules were positive; 4, 3, and 2 cases were extranodal NK/T-cell lymphoma, nasal type, peripheral T-cell lymphoma-not otherwise specified, and adult T-cell leukemia/lymphoma, respectively. The remaining 25 (21%) cases included 11, 8, and 6 cases of myeloid sarcoma, blastic plasmacytoid dendritic cell neoplasm, and B- or NK-cell lymphoblastic leukemia/lymphoma, respectively. For large B-cell lymphoma/plasmablastic lymphoma diagnosis, MUM1 (92%) was the most sensitive marker, followed by CD79a (63%), PAX5 (52%), CD138 (42%), and CD19 (36%). EBER 1 and HHV8 were positive in 32% and 0% of the cases. For peripheral T/NK-cell lymphomas other than ALCL, CD45RO and CD43 were positive in nine cases; however, cytotoxic molecules (TIA1, 86%; granzyme B, 71%) were the most sensitive markers. In conclusion, most cases of the 118 (2.4%) CD3- CD20- extramedullary leukemia/lymphoma were represented by anaplastic large cell lymphomas (41%). The second most frequent group of neoplasia, large B-cell lymphoma/plasmablastic lymphoma (30%), characterized a special diagnostic challenge when B-cell markers were not expressed, requiring immunohistochemistry for multiple B-cell markers and molecular analysis in some cases.

Arterial and Venous Thrombosis Complicated in COVID-19: A Retrospective Single Center Analysis in Japan.

Background: Thrombosis is a characteristic complication in coronavirus disease 2019 (COVID-19). Since coagulopathy has been observed over the entire clinical course, thrombosis might be a clue to understanding the specific pathology in COVID-19. Currently, there is limited epidemiological data of COVID-19-associated thrombosis in the Japanese population and none regarding variant strains of SARS-CoV-2. Here, we elucidate the risk factors and the pattern of thrombosis in COVID-19 patients. Methods: The patients consecutively admitted to Tokyo Medical and Dental University Hospital with COVID-19 were retrospectively analyzed. SARS-CoV-2 variants of concern/interest (VOC/VOI) carrying the spike protein mutants E484K, N501Y, or L452R were identified by PCR-based analysis. All thrombotic events were diagnosed by clinical symptoms, ultrasonography, and/or radiological tests. Results: Among the 516 patients, 32 patients experienced 42 thromboembolic events. Advanced age, severe respiratory conditions, and several abnormal laboratory markers were associated with the development of thrombosis. While thrombotic events occurred in 13% of the patients with a severe respiratory condition, those events still occurred in 2.5% of the patients who did not require oxygen therapy. Elevated D-dimer and ferritin levels on admission were independent risk factors of thrombosis (adjusted odds ratio 9.39 and 3.11, 95% confidence interval 2.08-42.3, and 1.06-9.17, respectively). Of the thrombotic events, 22 were venous, whereas 20 were arterial. While patients with thrombosis received anticoagulation and antiinflammatory therapies with a higher proportion, the mortality rate, organ dysfunctions, and bleeding complications in these patients were higher than those without thrombosis. The incidence of thrombosis in COVID-19 became less frequent over time, such as during the replacement of the earlier strains of SARS-CoV-2 by VOC/VOI and during increased use of anticoagulatory therapeutics. Conclusion: This study elucidated that elevated D-dimer and ferritin levels are useful biomarkers of thrombosis in COVID-19 patients. The comparable incidence of arterial thrombosis with venous thrombosis and the development of thrombosis in less severe patients required further considerations for the management of Japanese patients with COVID-19. Further studies would be required to identify high-risk populations and establish appropriate interventions for thrombotic complications in COVID-19.

COVID-19 pneumonia complicated by bilateral pneumothorax: A case report.

BACKGROUND: Pneumothorax is a rare but life-threatening complication associated with pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). CASE PRESENTATION: Informed consent was obtained from the patient himself.A 50-year-old man presented with a 9-day history of fever, cough, and dyspnoea. He was diagnosed with coronavirus disease 2019 (COVID-19) pneumonia and was admitted to the Medical Hospital, Tokyo Medical and Dental University. Chest CT showed diffuse patchy ground-glass opacities (GGOs). His state of oxygenation deteriorated, and mechanical ventilation was initiated on day 4 after admission (12th day from onset). He improved gradually and was weaned from ventilation on day 15. Sudden onset of bilateral pneumothorax occurred on day 21 with severe respiratory failure, and chest CT revealed pneumatocele formation on both lower lobes. CONCLUSIONS: Pneumothorax is a notable complication in cases of severe COVID-19 pneumonia, especially in those who require positive-pressure ventilation.

Quantitative electric field mapping of a p-n junction by DPC STEM.

Local electromagnetic fields in a specimen is measured at high spatial resolutions using differential phase contrast (DPC) imaging in scanning transmission electron microscopy (STEM). According to previous studies, DPC signals can be quantified by measuring the center of mass of the diffraction pattern intensity and/or performing a deconvolution method based on a phase contrast transfer function (PCTF). However, when using a segmented detector, the field strength has been considerably underestimated for a very thick specimen. The main cause of the underestimation is assumed to be inelastic scattering, mainly bulk plasmon scattering. In this study, we develop a method to remove this inelastic scattering effect from segmented detector DPC signals by modifying the PCTF deconvolution method. Field quantification results using this new technique are compared with those using pixelated detector DPC and electron holography, and all results indicated good agreement within an error margin.

[Richter syndrome successfully treated with ibrutinib monotherapy: two case reports].

The Richter syndrome (RS) is defined as a histologically diagnosed diffuse large B-cell lymphoma (DLBCL) or Hodgkin lymphoma (HL) in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma. A standard treatment for RS has not yet been established. Most patients with RS are treated with combination chemotherapy regimens used for de novo DLBCL or HL. Recently, the Bruton's tyrosine kinase inhibitor, ibrutinib (IBR), has shown remarkable efficacy in CLL; however, limited evidence exists regarding its single agent efficacy in RS. We encountered two patients with RS in whom CLL transformed to DLBCL, confirmed by G-banding/spectral karyotyping analysis. Both patients achieved durable responses for 12 and 10 months, with IBR alone. Hemorrhagic cystitis due to adenovirus occurred in one patient at an initial dose of 420 mg/day, but a dose reduction to 280 mg/day made long-term continuation of IBR possible. Interestingly, retreatment with IBR alone achieved disease control again for 5.5 and 2 months, after these patients underwent salvage chemotherapies for aggressive relapse.

Impact of chromosomal abnormalities on the efficacy of lenalidomide plus dexamethasone treatment in patients with relapsed/refractory multiple myeloma.

Lenalidomide is an effective therapeutic agent for multiple myeloma (MM). However, its efficacy in the context of chromosomal abnormalities (CA) is poorly understood. We retrospectively analyzed 83 patients with relapsed/refractory (RR) MM, who received lenalidomide plus low-dose dexamethasone (Ld), in the context of CA. The median age and number of prior therapies were 69 and 2, respectively. Three, 11, 45, and 19 patients achieved complete response, very good partial response, partial response, and stable disease, respectively. Median progression-free survival (PFS) and overall survival (OS) were 11.1 and 38.8 months, respectively. Seventy-two patients were evaluated for frequently observed translocations; median PFS was 24.4 months in 20 patients with t(11;14), 13.0 months in 16 patients with t(4;14), and 3.7 months in seven patients with t(14;16). G-banded karyotype analysis detected 11 hypodiploid patients, who had shorter PFS and OS (2.5 and 6.2 months, respectively) compared to others (13.0 and 43.7 months, respectively). Hypodiploid patients showed poor clinical outcome, whereas patients with t(11;14) showed favorable outcome. In summary, the present study presents the clinical impact of chromosomal abnormalities on the outcome of Ld therapy, and contributes to understanding the appropriate choice of lenalidomide-based therapy to achieve effective treatment of RR MM.

Exophiala dermatitidis Fungemia Diagnosed Using Time-of-flight Mass Spectrometry during Chemotherapy for Malignant Lymphoma and Successful Treatment with Voriconazole.

Infectious diseases, including those caused by fungi, remain important issues in patients receiving malignant lymphoma chemotherapy. We herein report a rare case of Exophiala dermatitidis fungemia during chemotherapy in a 67-year-old woman admitted to our hospital. She had a fever that could not be resolved despite antifungal therapy. Yeast-like fungi were detected in blood culture samples, but biochemical identification was difficult. E. dermatitidis, a black mold, was identified using time-of-flight mass spectrometry. The patient finally improved after her treatment was switched to voriconazole. Fungal infection is difficult to diagnose and treat, but this novel approach can improve patients' outcomes.

Pre- and post-transplant ponatinib for a patient with acute megakaryoblastic blast phase chronic myeloid leukemia with T315I mutation who underwent allogeneic hematopoietic stem cell transplantation.

A 42-year-old female complaining of fever and night sweats was diagnosed with acute megakaryoblastic blast phase chronic myeloid leukemia (CML-BP). She had massive splenomegaly, left pleural effusion with leukemia infiltration, and moderate myelofibrosis. She received dasatinib monotherapy (140 mg/day) as for induction, after which her pleural effusion rapidly resolved and hematological remission was achieved. However, CML relapsed 4 months after starting dasatinib due to increased BCR-ABL fusion signals in the peripheral blood. The T315I mutation was also detected at the recurrence of CML. As a salvage treatment, ponatinib monotherapy (45 mg/day) was started immediately. After 5 months, BCR-ABL fusion signals decreased to 5%, and myelofibrosis improved from MF Grade 2 to 1; she then underwent allogeneic bone marrow transplantation from an unrelated donor. However, the graft failed, and cord blood transplantation (CBT) was performed. Ponatinib (15 mg/day) was continued after CBT as a maintenance treatment, with molecular complete response continuing for more than 1 year with no severe adverse events, including cardiovascular events. There is limited evidence regarding the optimal dose and schedule of ponatinib before and after allogeneic hematopoietic stem cell transplantation, especially in patients with CML-BP having T315I mutation; thus, well-designed clinical trials are warranted.

Accurate measurement of electric potentials in biased GaAs compound semiconductors by phase-shifting electron holography.

The innate electric potentials in biased p- and n-type GaAs compound semiconductors and the built-in potential were successfully measured with high accuracy and precision by applying in situ phase-shifting electron holography to a wedge-shaped GaAs specimen. A cryo-focused-ion-beam system was used to prepare the 35°-wedge-shaped specimen with smooth surfaces for a precise measurement. The specimen was biased in a transmission electron microscope, and holograms with high-contrast interference fringes were recorded for the phase-shifting method. A clear phase image around the p-n junction was reconstructed even in a thick region (thickness of ~700 nm) at a spatial resolution of 1 nm and precision of 0.01 rad. The innate electric potentials of the unbiased p- and n-type layers were measured to be 12.96 ± 0.17 V and 14.43 ± 0.19 V, respectively. The built-in potential was determined to be 1.48 ± 0.02 V. In addition, the in situ biasing measurement revealed that the measured electric-potential difference between the p and n regions changed by an amount equal to the voltage applied to the specimen, which indicates that all of the external voltage was applied to the p-n junction and that no voltage loss occurred at the other regions.

Current diagnosis and treatment of polymyositis and dermatomyositis.

Idiopathic inflammatory myopathies (IIMs) are heterogeneous disorders that affect the skeletal muscles. Polymyositis, dermatomyositis, and inclusion body myositis are major IIM subsets. Immune-mediated necrotizing myopathy became recognized as a potentially new IIM subset. Since the new classification criteria published by the International Myositis Classification Criteria Project have higher sensitivity and specificity for IIM classification and subclassification than the previous criteria, they should help precise diagnosis. It should be noted that several tests available in current clinical practice, such as electromyography, magnetic resonance imaging, and other myositis-specific autoantibodies than anti-Jo-1 antibodies, were not included in the new criteria. As for treatment, glucocorticoids are used empirically as the first-line treatment despite their various adverse effects. Concomitant treatment with steroid-sparing immunosuppressive agents, including methotrexate, azathioprine, calcineurin inhibitors, mycophenolate mofetil, and cyclophosphamide, reduces successfully initial glucocorticoid doses for the remission induction, the relapse risk during glucocorticoid tapering, and adverse effects of glucocorticoids. Treatment with biologics, including rituximab and abatacept, seems promising in some IIM patients. Multi-target treatment with glucocorticoids and several steroid-sparing immunosuppressive agents is effective in refractory IIM patients. Considering proven steroid-sparing efficacy and tolerability of multi-target treatment in patients with other autoimmune diseases, it should be a good therapeutic option for IIMs.

Photoluminescence color stability of green-emitting InP/ZnS core/shell quantum dots embedded in silica prepared via hydrophobic routes.

In this work, green-emitting InP/ZnS quantum dots (QDs) modified with 1-dodecanethiol were embedded into silica by two methods to improve their photostability while maintaining a high photoluminescence quantum yield (PLQY) and a color coordinate. A monolithic QD-silica composite prepared by a non-aqueous route with tetraethyl orthosilicate and lactic acid featured low transparency, a loss of the color purity of green, and a PLQY of 1.6%, which was considerably lower than that of the original QDs (67%). The decrease of the PLQY was attributed to QD aggregation in the sol-gel process and degradation of the QDs by the acid. The alternative method involved stirring a toluene dispersion of the QDs with tetramethyl orthosilicate (TMOS) for 20 h or 7 days. The PLQY of the TMOS-modified InP/ZnS QDs (20 h) was 62%, which was only slightly lower than that of the original QDs. The PLQY decreased to 52% when the duration of aging was prolonged to 7 days. This decrease was attributed to desorption of surface modifiers from the QD surface and oxidative degradation by oxygen dissolved in toluene. Herein, the color coordinate was maintained stably. Photostability was evaluated by continuous irradiation of the samples by a blue light emitting diode. The decrease of photoluminescence (PL) intensity was suppressed by the silica encapsulation. In particular, the PL intensity of the TMOS-modified InP/ZnS QD sample (7 d) maintained 99% of its initial intensity. Silica encapsulation of InP/ZnS QDs prevented contact of the QDs with oxygen in the air, resulting in improved photostability.

Precise method for measuring spatial coherence in TEM beams using Airy diffraction patterns.

We have developed a method to precisely measure spatial coherence in electron beams. The method does not require an electron biprism and can be implemented in existing analytical transmission electron microscopes equipped with a post-column energy filter. By fitting the Airy diffraction pattern of the selector aperture, various parameters such as geometric aberrations of the lens system and the point-spread function of the diffraction blurring are precisely determined. From the measurements of various beam diameters, components that are attributed to the partial spatial coherence are successfully separated from the point-spread functions. A linear relationship between the spatial coherence length and beam diameter is revealed, thus indicating that a wide range of coherence lengths can be determined by our proposed method as long as the coherence length remains >80% of the aperture diameter. A remarkable feature of this method is its ability to simultaneously determine diffraction blurring and lens aberrations. Possible applications of this method are also discussed.