Tadashi Onoda

Elevated oxidative stress in erythrocytes due to a SOD1 deficiency causes anaemia and triggers autoantibody production

Reactive oxygen species are involved in the aging process and diseases. Despite the important role of Cu/Zn SOD (superoxide dismutase) encoded by SOD1, SOD1-/- mice appear to grow normally under conventional breeding conditions. In the present paper we report on a novel finding showing a distinct connection between oxidative stress in erythrocytes and the production of autoantibodies against erythrocytes in SOD1-/- mice. Evidence is presented to show that SOD1 is primarily required for maintaining erythrocyte lifespan by suppressing oxidative stress. A SOD1 deficiency led to an increased erythrocyte vulnerability by the oxidative modification of proteins and lipids, resulting in anaemia and compensatory activation of erythropoiesis. The continuous destruction of oxidized erythrocytes appears to induce the formation of autoantibodies against certain erythrocyte components, e.g. carbonic anhydrase II, and the immune complex is deposited in the glomeruli. The administration of an antioxidant, N-acetylcysteine, suppressed erythrocyte oxidation, ameliorated the anaemia, and inhibited the production of autoantibodies. These data imply that a high level of oxidative stress in erythrocytes increases the production of autoantibodies, possibly leading to an autoimmune response, and that the intake of antioxidants would prevent certain autoimmune responses by maintaining an appropriate redox balance in erythrocytes.

Cloning and characterization of an isoform of interleukin-21

Interleukin‐21 (IL‐21) has pleiotropic functions on the cells, which play roles in both innate and acquired immunity, such as T cells, B cells, natural killer (NK) cells and dendritic cells. In this study we identified a novel isoform of IL‐21, IL‐21iso in human and mouse. IL‐21iso might be an alternative splicing variant form and the C‐terminal region of predicted IL‐21iso amino acid sequences were different from original IL‐21 in both human and mouse. In spite of the differences in C‐terminal amino acid sequences, both human IL‐21 and IL‐21iso showed comparable proliferative effect on anti‐CD40 Ab‐activated primary B cells, anti‐CD3 Ab‐activated primary T cells and human NK cell line, NK0, and upregulated IFN‐γ production from NK0. Furthermore IL‐21 and IL‐21iso similarly activated STAT1 and STAT3. IL‐21iso mRNA was expressed in activated T cells as well as IL‐21 mRNA. However, cycloheximide treatment partially blocked the upregulation of IL‐21iso mRNA in activated T cells while little affected the IL‐21 mRNA expression suggesting that de novo protein synthesis is required for the full expression of IL‐21iso transcript. We also show that the secretion efficiency of hIL‐21iso is much lower than that of hIL‐21. These results may suggest there are some different regulatory mechanisms to produce IL‐21 or IL‐21iso in transcriptional and secretory steps.

Role of interleukin-21 isoform in dextran sulfate sodium (DSS)-induced colitis.

Interleukin-21 (IL-21) is overproduced in human intestines affected by inflammatory bowel disease (IBD) and in the gut of mice with DSS-induced colitis. IL-21-deficient mice are largely protected against DSS-induced colitis, indicating that IL-21 plays a key role in the development of IBD. We previously identified a novel IL-21 isoform named IL-21iso. In this study, we found that in addition to the conventional IL-21, IL-21iso mRNA was also expressed in the colon with DSS-induced colitis. To investigate whether IL-21iso plays a role in DSS-induced colitis, we established transgenic mice (mIL-21iso-Tg mice) that expressed mouse IL-21iso under the control of the lck proximal promoter. Although mIL-21iso-Tg mice did not have any gross physical abnormalities, their peripheral lymphocytes counts were higher than those in wild-type littermates. Notably, their CD8(+) T cell and CD4(+) effector memory T-cell populations were elevated. DSS-induced colitis was far more severe in the mIL-21iso-Tg mice than in wild-type mice, and was accompanied by a marked loss of body weight and by colon inflammation with increased cellular infiltration. In DSS-treated mice, colon tissues from mIL-21iso-Tg mice had significantly higher gene activation levels for cytokines such as IL-17A, TNF-α, IL-6, IL-10, and IL-4, and for transcription factors such as T-bet, GATA-3, RORγt, and Foxp3, than were found in wild-type mice. These results indicate that besides IL-21, IL-21iso may be another regulator of gut inflammation.

Regulation of interleukin-21 receptor expression and its signal transduction by WSB-2.

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain (gammac) receptor subunit. We identified novel IL-21R-binding molecule, WD-40 repeats containing SOCS-box-2, WSB-2. WSB-2 associated with the membrane-proximal intracytoplasmic region of IL-21R, including box1 and box2. Overexpression study of WSB-2 showed the reduction of IL-21R expression and IL-21-induced signal transduction. On the other hand, small interfering RNA for WSB-2 enhanced the expression level of IL-21R and IL-21-induced STAT3 activation, indicating that WSB-2 negatively controls the receptor expression. This report provides the first evidence that WSB-2 is a regulator of IL-21R expression and IL-21-induced signal transduction.

WSB-1, a novel IL-21 receptor binding molecule, enhances the maturation of IL-21 receptor

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain. We identified a novel IL-21R-binding molecule, WSB-1, which contains WD-40 repeats and a SOCS-box domain. WSB-1 associates with the middle part of intracytoplasmic region of IL-21R and enhances the maturation of IL-21R from N-linked glycosylated form to fully glycosylated mature form. Furthermore, WSB-1 moderates IL-21R degradation. Taken together, our present study suggests that WSB-1 has a role in the tuning of the maturation and degradation of IL-21R.

Identification of novel ALK rearrangement A2M–ALK in a neonate with fetal lung interstitial tumor

Fetal lung interstitial tumor (FLIT) is a recently reported type of congenital lung lesion comprising solid and cystic components. The pathological features include unique interstitial mesenchyme‐based cell proliferation, and differ from other neoplasms represented by pleuropulmonary blastoma or congenital peribronchial myofibroblastic tumor. FLIT is extremely rare and its gene expression profile has not yet been reported. We provide the first report of a novel chromosomal rearrangement resulting in α‐2‐macroglobulin (A2M) and anaplastic lymphoma kinase (ALK) gene fusion in a patient with FLIT. The tumor cells contained a t(2;12)(p23;p13) and were mesenchymal in origin (e.g., inflammatory myofibroblastic tumors), suggesting the involvement of ALK in this case of FLIT. Break apart fluorescence in situ hybridization demonstrated chromosomal rearrangement at ALK 2p23. Using 5′‐rapid amplification of cDNA ends, we further identified a novel transcript fusing exon 22 of A2M to exon 19 of ALK, which was confirmed by reverse‐transcription polymerase chain reaction. The corresponding chimeric gene was subsequently confirmed by sequencing, including the genomic break point between intron 22 and 18 of A2M and ALK, respectively. Discovery of A2M as a novel ALK fusion partner, together with the involvement of ALK, provides new insights into the pathogenesis of FLIT, and suggests the potential for new therapeutic strategies based on ALK inhibitors.

Apurinic/apyrimidinic endonuclease1/redox factor-1 (Ape1/Ref-1) is essential for IL-21-induced signal transduction through ERK1/2 pathway

IL-21 is a pleiotropic cytokine that regulates T-cell and B-cell differentiation, NK-cell activation, and dendritic cell functions. IL-21 activates the JAK-STAT, ERK, and PI3K pathways. We report here that Ape1/Ref-1 has an essential role in IL-21-induced cell growth signal transduction. Overexpression of Ape1/Ref-1 enhances IL-21-induced cell proliferation, but it is suppressed by overexpressing an N-terminal deletion mutant of Ape1/Ref-1 that lacks the redox domain. Furthermore, knockdown of the Ape1/Ref-1 mRNA dramatically compromises IL-21-induced ERK1/2 activation and cell proliferation with increasing cell death. These impaired activities are recovered by the re-expression of Ape1/Ref-1 in the knockdown cells. Our findings are the first demonstration that Ape1/Ref-1 is an indispensable molecule for the IL-21-mediated signal transduction through ERK1/2 activation.

Successful treatment of acute myeloid leukaemia in a patient with ataxia telangiectasia

Ataxia telangiectasia (AT) is a rare autosomal recessive multisystem disorder characterised by cerebellar degeneration, immunodeficiency and cancer predisposition. Around 10% of AT patients develop lymphoid malignancies, but the development of myeloid leukaemia with AT (AT‐AML) is extremely rare, and there have been no previous publications regarding suitable therapies. Here, we first describe a successful therapeutic experience in a patient with AT‐AML (FAB‐M1) who attained remission after induction therapy and maintained stable disease for a year. To minimise therapy‐induced toxicity, low‐dose induction was applied first, though this was obviously insufficient and the patient subsequently responded well to dose‐intensified short‐term chemotherapy. In this report, we suggest a curative therapeutic approach for AT‐AML, though the issue of how best to manage patients with cancer complicated by immunodeficiency remains undecided.

Eltrombopag with i.v. immunoglobulin for safe splenectomy in refractory immune thrombocytopenia

The first-line treatment for pediatric immune thrombocytopenia (ITP) consists of observation, i.v. immunoglobulin (IVIG), or corticosteroids. Although these procedures resolve the condition in most affected children, some are refractory to treatment or relapse. Recent second-line treatments, such as cyclosporin, rituximab or thrombopoietin receptor (TPO-R) agonist are promising, but some patients do not respond well to them. Splenectomy remains the only salvage intervention that may induce a curative effect in nearly two-thirds of such patients. We report a case of refractory ITP. The patient did not respond to firstand second-line treatment. Concomitant use of eltrombopag and IVIG resulted in transient increase of platelet count and allowed a safer execution of splenectomy that resulted in remission of ITP. This is the first successful treatment of pediatric refractory ITP using TPO-R agonist and IVIG. A 4-year-old girl saw the family physician with ecchymoses on the extremities and nasal bleeding. On laboratory analysis, platelets were 3 9 10/L, and platelet-associated IgG concentration was 140 ng/10 cells. She was admitted to a local hospital and was diagnosed with primary ITP. IVIG with oral prednisolone therapy increased the platelet count temporarily to 50 9 10/L, but it decreased to 1 9 10/L within 1 week (Fig. 1). The patient was then treated with methylprednisolone pulse, dexamethasone, and cyclosporine, with no improvement. After 4 months of this treatment, the patient was referred to the present hospital. Physical examination indicated many petechiae on the skin and oral mucosal bleeds. The patient had tampon gauzes in both nasal cavities. Hemoglobin fell to 7.5 g/dL. On bone marrow examination, megakaryocytes had increased (368 9 10/L) and nucleated cell count was normal (275.5 9 10/L). Bone marrow cell karyotype was normal. On peripheral blood cell smear there were only a few normal-sized platelets. The other laboratory results were as follows: thrombopoietin, 2.42 fmol/mL (normal, 1.97 0.28 fmol/mL); 13Curea breath test for Helicobacter pylori, negative; haptoglobin 1-1, 41 mg/dL (normal, 6.0–263.0 mg/dL); antinuclear antibody, negative; a disintegrin-like and metalloproteinase with thrombospondin type 1 motif 13 (ADAMTS-13) activity, 75.8% (normal, 70.0–120.0%); and anti-glycoprotein IIb/IIIa antibodysecreting B cells, 3.3/10 mononuclear cells (normal, 0.4 0.3/ 10 mononuclear cells). Concentrated platelet transfusion did not increase the platelet count. At 175 days after clinical onset, the patient was treated with 375 mg/m i.v. rituximab per week for 4 consecutive weeks, and this treatment temporarily increased the platelet count to 8 9 10/L. After informed consent from the patient’s guardians, TPO-R agonist eltrombopag was started at a dose of 12.5 mg/day (0.5 mg/kg/day) on day 258, and the dose was gradually increased to 37.5 mg/day (1.6 mg/kg/day). This led to a slight increase in platelet count from 2 9 10 to 6 9 10/L, and it was used for approximately 6 months. Ecchymoses and petechiae were present, and oral mucosal bleeds were still frequently observed. On a second bone marrow examination, including a trephine biopsy, nuclear cell count was 885 9 10/L; megakaryocyte count was 752 9 10/L; and there was a slight increase in the number of reticulin fibers without collagen fibers. Considering the synergy of the drugs, IVIG was added on days 445 and 446, which resulted in a temporary increase in the platelet count to 175 9 10/L. Based on this, we planned to perform a splenectomy shortly after the IVIG treatment. After vaccination against Haemophilus influenzae and pneumococcus, laparoscopic splenectomy was performed on day 595. After the splenectomy, eltrombopag was discontinued, and the patient maintained a platelet count >30 9 10/L with no bleeding and was discharged on day 611. One year after splenectomy, reticulin deposition was still observed on bone marrow examination. Megakaryocyte count was 208 9 10/L and blood cell dysplasia was not observed. Prednisolone was decreased gradually and finished 3 years after disease onset. At 4 years after onset, the platelet count was approximately 200 9 10/L. Data are scarce with regard to the use of TPO-R agonist combination therapy for refractory ITP. Recently, response to TPO-R agonist combination therapy has been reported in some cases of adult ITP. TPO-R agonists with IVIG may represent a possible therapeutic option for ITP refractory to second-line treatment to increase platelet count and reduce the perioperative risk of bleeding during splenectomy or other surgical interventions. Correspondence: Miyako Kanno, MD, Department of Pediatrics, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan. Email: mykanno@med.id.yamagata-u.ac.jp Received 16 January 2017; revised 28 September 2017; accepted 20 October 2017. doi: 10.1111/ped.13446

Giant thymolipoma in a young child initially diagnosed as cardiomegaly.

A 2-year-old girl was admitted to a local hospital because of sustained mild biphasic stridor. Chest X-ray appeared to show cardiomegaly, which was diagnosed as pseudocardiomegaly (Fig. 1a) because it was inconsistent with echocardiogram, which showed normal cardiac structure and function. Furosemide and milrinone were administered to reduce likely cardiac strain. Symptoms persisted and the patient was transferred to Yamagata University Hospital after detection of an unexpected giant mediastinal mass on computed tomography (CT). On admission, respiration was stable in room air, the lungs were almost clear to auscultation while sitting, and no cardiac murmur was noted. No surface lymph nodes were palpable. Laboratory investigations, including tumor markers and autoantibodies, were normal. Marrow smears showed normocellularity without malignant blasts or neoplastic infiltration. Contrast-enhanced magnetic resonance imaging (MRI) showed a lobulated anterior mediastinal mass with isointensity on T1and high signal intensity on T2and diffusion-weighted imaging consistent with the fat shown on contrast-enhanced CT (Fig. 1b–d), supporting a fat-saturated mass. There was no evidence of metastasis or adjacent tissue involvement. Subsequent pathological evaluation of an open biopsy specimen prompted complete resection. The tumor (140 × 100 × 70 mm, 354 g) was encapsulated but poorly demarcated from normal thymus, and consisted of normal thymic tissue and abundant adipose cells on pathology (Fig. 2). Karyotyping of tumor cells was as follows: 47,XX,-2,der(3)t(2;3)(p11.2;p25), add(5)(q13),der(6)t(5;6)(q11.2;q27),+2mar in one of 20 cells and 46,XX in the remaining cells. The tumor was conclusively diagnosed as a thymolipoma. At the time of writing the patient was well at 3 years after surgery, with no evidence of recurrence. Thymolipoma is a little-known, slowly progressive, rare anterior mediastinal neoplasm consisting of thymic and hyperplastic adipose tissues. It usually develops in adolescence and adulthood, being extremely rare in early childhood. In the present case, the tumor’s unique space-occupying nature resulted in the mimicking of enlarged cardiac silhouette chest on X-ray, leading to an initial diagnosis of pseudocardiomegaly, a diagnostic pitfall in young children with atypical mediastinal masses. Although MRI and CT can demonstrate the characteristic fat saturation of these tumors and resolve potential discrepancies between its asymptomatic, slow-growing nature and the radiological findings, detailed pathological confirmation is required. Total resection should be considered because of this tumor’s highly curative resectability and the requirement for surgical decompression. In the present case, given that the natural history and characteristics of these tumors, particularly in young children, are unknown, surgical removal was the optimal treatment option, but, because total resection potentially compromises future immunological competence, the immunological milieu should subsequently be monitored, especially in young children. Sustained respiratory symptoms or large thymus in young children are common, but further investigations should be considered when symptoms and radiological findings are ambiguous. Written informed consent was obtained from the patient’s parents in accordance with the Declaration of Helsinki, after approval by the institutional review board of Yamagata University Faculty of Medicine.