Autoimmunity against melanoma differentiation-associated gene 5 induces interstitial lung disease mimicking dermatomyositis in mice.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis (DM) is characterized by amyopathic DM with interstitial lung disease (ILD). Patients with anti-MDA5 antibody-associated ILD frequently develop rapidly progression and present high mortality rate in the acute phase. Here, we established a murine model of ILD mediated by autoimmunity against MDA5. Mice immunized with recombinant murine MDA5 whole protein, accompanied with complete Freund's adjuvant once a week for four times, developed MDA5-reactive T cells and anti-MDA5 antibodies. After acute lung injury induced by intranasal administration of polyinosinic-polycytidylic acid [poly (I:C)] mimicking viral infection, the MDA5-immunized mice developed fibrotic ILD representing prolonged respiratory inflammation accompanied by fibrotic changes 2 wk after poly (I:C)-administration, while the control mice had quickly and completely recovered from the respiratory inflammation. Treatment with anti-CD4 depleting antibody, but not anti-CD8 depleting antibody, suppressed the severity of MDA5-induced fibrotic ILD. Upregulation of interleukin (IL)-6 mRNA, which was temporarily observed in poly (I:C)-treated mice, was prolonged in MDA5-immunized mice. Treatment with anti-IL-6 receptor antibody ameliorated the MDA5-induced fibrotic ILD. These results suggested that autoimmunity against MDA5 exacerbates toll-like receptor 3-mediated acute lung injury, and prolongs inflammation resulting in the development of fibrotic ILD. IL-6 may play a key role initiating ILD in this model.

Eribulin mesylate exerts antitumor effects via CD103.

Eribulin mesylate (ERB) is a synthetic analog of halichondrin B, inhibiting tumor cell growth by disrupting microtubule function. Recently, anticancer drugs have been shown to not only act directly on tumor cells but also to exert antitumor effects by modifying the tumor environment. Although ERB has also been speculated to modify the tumor microenvironment including the immune response to tumors, the precise mechanism remains unclear. In our study, ERB suppressed the tumor growth of MC38 colon cancer in wildtype mice, whereas ERB failed to inhibit the tumor growth in Rag1-deficient mice which lack both B and T cells. Moreover, depletion of either CD4+ or CD8+ T cells abrogated the antitumor effect of ERB, indicating that both CD4+ and CD8+ T cells play an important role in ERB-induced antitumor effects. Furthermore, ERB treatment increased the number of tumor infiltrating lymphocytes (TILs) as well as the expression of activation markers (CD38 and CD69), immune checkpoint molecules (LAG3, TIGIT and Tim3) and cytotoxic molecules (granzyme B and perforin) in TILs. ERB upregulated E-cadherin expression in MC38. CD103 is a ligand of E-cadherin and induces T-cell activation. ERB increased the proportion of CD103+ cells in both CD4+ and CD8+ TILs. The ERB-induced antitumor effect with the increased TIL number and the increased expression of activation markers, inhibitory checkpoint molecules and cytotoxic molecules in TILs was abrogated in CD103-deficient mice. Collectively, these results suggest that ERB exerts antitumor effects by upregulation of E-cadherin expression in tumor cells and subsequent activation of CD103+ TILs.

Immature Platelet Fraction and Its Kinetics in Neonates.

Thrombocytopenia is a common abnormality encountered in the neonatal period, and immature platelet fraction (IPF) may be an informative indicator of thrombopoiesis; however, data on IPF in neonates are scarce. To define reference intervals (RIs) and factors affecting IPF in neonates, we measured the IPF of 533 consecutive neonates. With a multiple regression analysis of 330 newborns with normal platelet counts at birth, premature delivery, neonatal asphyxia, intrauterine infection, chromosomal abnormalities, and respiratory disorders were identified as independent factors for IPF%. The RIs of IPF% and absolute IPF value in neonates were determined to be 1.3% to 5.7% and 3.2 to 14.5×10 9 /L, respectively. On day 14 after birth, IPF% increased to twice the value at birth and thereafter returned to the previous value on day 28. Reticulocyte counts, in contrast, were the lowest at day 14. IPF% was increased in 16 thrombocytopenic patients with various clinical conditions, especially those with immune-mediated thrombocytopenia. IPF in neonates may be evaluated essentially based on the same RIs as in adults, although some precautions must be taken when evaluating IPF in neonates in the first 2 weeks of life. IPF may be useful for evaluating thrombopoiesis and thrombocytopenia in neonates.

Glutathione Depletion and MicroRNA Dysregulation in Multiple System Atrophy: A Review.

Multiple system atrophy (MSA) is a rare neurodegenerative disease characterized by parkinsonism, cerebellar impairment, and autonomic failure. Although the causes of MSA onset and progression remain uncertain, its pathogenesis may involve oxidative stress via the generation of excess reactive oxygen species and/or destruction of the antioxidant system. One of the most powerful antioxidants is glutathione, which plays essential roles as an antioxidant enzyme cofactor, cysteine-storage molecule, major redox buffer, and neuromodulator, in addition to being a key antioxidant in the central nervous system. Glutathione levels are known to be reduced in neurodegenerative diseases. In addition, genes regulating redox states have been shown to be post-transcriptionally modified by microRNA (miRNA), one of the most important types of non-coding RNA. miRNAs have been reported to be dysregulated in several diseases, including MSA. In this review, we focused on the relation between glutathione deficiency, miRNA dysregulation and oxidative stress and their close relation with MSA pathology.

Investigation of patient factors associated with the number of transfusions required during chemotherapy for high-risk neuroblastoma.

BACKGROUND: Blood transfusion is an important supportive care for high-risk neuroblastoma. When the number of transfusions increases, transfusion-associated adverse reactions may be more problematic. However, the factors determining the degree of myelosuppression and the number of transfusions during chemotherapy for high-risk neuroblastoma remain unclear. MATERIALS AND METHODS: We investigated patient factors determining the number of required transfusions in 15 high-risk neuroblastoma patients who received five courses of chemotherapy. Clinical data, cytokine profile and colony-forming assay with bone marrow samples at diagnosis were analysed. RESULTS: The required number of transfusions of both platelets and erythrocytes decreased once in the second course and then increased as the course progressed. The variability among cases increased as the chemotherapy course progressed. In cases of low peripheral blood platelet count and lower fibrinogen level at diagnosis, the number of platelet transfusions was higher during chemotherapy. In contrast, there was a negative correlation between the forming ability of granulocyte-macrophage or erythroid colonies and the number of erythrocyte transfusions in the latter period. CONCLUSION: In the early stages of chemotherapy, bone marrow infiltration in neuroblastoma and/or coagulopathy complication may cause thrombocytopenia and requirement of platelet transfusion; conversely, in the later stages, the number of erythrocyte transfusions may be defined by the patient's inherent hematopoietic ability. These factors may be useful in predicting the required number of transfusions.

Anti-nuclear matrix protein 2 antibody-positive inflammatory myopathies represent extensive myositis without dermatomyositis-specific rash.

OBJECTIVES: Myositis-specific autoantibodies (MSAs) define distinct clinical subsets of idiopathic inflammatory myopathies (IIMs). The anti-nuclear matrix protein 2 (NXP2) antibody, a MSA detected in juvenile/adult IIMs, has been reported to be associated with a high risk of subcutaneous calcinosis, subcutaneous oedema and internal malignancies. The study aimed to clarify the clinical features of anti-NXP2 antibody-positive IIMs in detail. METHODS: This was a multicentre retrospective observational study on 76 anti-NXP2 antibody-positive patients. The antibody was detected via a serological assay using immunoprecipitation and western blotting. The patients were selected from 162 consecutive Japanese patients with IIMs. RESULTS: The cohort of anti-NXP2 antibody-positive IIMs included 29 juvenile patients and 47 adult patients. Twenty-seven (35.5%) patients presented with polymyositis phenotype without dermatomyositis-specific skin manifestations (heliotrope rash or Gottron sign/papules); this was more common in the adults than children (48.9% vs 15.8%, P < 0.01). Nine (11.8%) patients had subcutaneous calcinosis, and 20 (26.3%) patients had subcutaneous oedema. In addition, the proportion of patients with muscle weakness extending to the distal limbs was high (36 patients [47.4%]) in this cohort. Adult patients had a higher prevalence of malignancy than the general population (age-standardized incidence ratio of malignancies: 22.4). CONCLUSION: Anti-NXP2 antibody-positive IIMs, which include dermatomyositis sine dermatitis, are characterized by atypical skin manifestations and extensive muscular involvement.

Differential Involvement of Programmed Cell Death Ligands in Skin Immune Responses.

PD-1 is an immunoregulatory receptor that can bind PD-L1 or PD-L2 expressed on stimulated antigen-presenting cells. In this study, isolated antigen-presenting cells (macrophages and dendritic cells) were cultured with IFN-γ, IL-4, or IL-17A, and the expression of PD-L1 and PD-L2 was compared by flow cytometry. Strong upregulation of PD-L1 expression was observed on IFN-γ stimulation of both antigen-presenting cells as well as in response to IL-17A stimulation of macrophages compared with the expression in unstimulated controls. In contrast, only stimulation with IL-4 could upregulate PD-L2 expression on both antigen-presenting cells. Therefore, experiments were performed in murine models, including DNFB-induced contact hypersensitivity, calcipotriol-induced atopic dermatitis-like skin inflammation, and imiquimod-induced psoriasis-like dermatitis models, to trigger IFN-γ‒mediated T helper type (Th)1-, IL-4‒mediated Th2-, and IL-17A‒mediated Th17-type responses, respectively. In both Th1- and Th17-type immunity models, changes in ear thickness were more severe in Pd-l1‒deficient mice than in wild-type or Pd-l2‒deficient mice. In the Th2-type immunity model, changes in thickness in Pd-l2‒deficient mice were more severe than that in wild-type or Pd-l1‒deficient mice. Collectively, PD-L1 has predominant roles in Th1 and Th17 type immunity, whereas PD-L2 is involved in Th2-type immunity.

Combination Treatment of Topical Imiquimod Plus Anti-PD-1 Antibody Exerts Significantly Potent Antitumor Effect.

The exact mechanisms of the imiquimod (IMQ)-induced antitumor effect have not been fully understood. Although both topical IMQ treatment and anti-PD-1 antibody may be used for primary skin lesions or skin metastases of various cancers, the efficacy of each monotherapy for these lesions is insufficient. Using a murine tumor model and human samples, we aimed to elucidate the detailed mechanisms of the IMQ-induced antitumor effect and analyzed the antitumor effect of combination therapy of topical IMQ plus anti-PD-1 antibody. Topical IMQ significantly suppressed the tumor growth of MC38 in wildtype mice. IMQ upregulated interferon γ (IFN-γ) expression in CD8+ T cells in both the lymph nodes and the tumor, and the antitumor effect was abolished in both Rag1-deficient mice and IFN-γ-deficient mice, indicating that IFN-γ produced by CD8+ T cells play a crucial role in the IMQ-induced antitumor effect. IMQ also upregulated PD-1 expression in T cells as well as PD-L1/PD-L2 expression in myeloid cells, suggesting that IMQ induces not only T-cell activation but also T-cell exhaustion by enhanced PD-1 inhibitory signaling. Combination therapy of topical IMQ plus anti-PD-1 antibody exerted a significantly potent antitumor effect when compared with each single therapy, indicating that the combination therapy is a promising therapy for the skin lesions of various cancers.

Sequential Therapy of Inotuzumab Ozogamicin and Blinatumomab as a Bridge-to Hematopoietic Stem Cell Transplantation in a Pediatric Patient With Primary Refractory Acute Lymphoblastic Leukemia: A Case Report.

For relapsed/refractory (r/r) acute lymphocytic leukemia (ALL), there is a clinical question on how to combine blinatumomab and inotuzumab ozogamicin (InO), which are newly emerging immunotherapeutic agents, with conventional treatment. We report the case of an 11-year-old boy with B-cell ALL, who had a failed primary treatment and achieved molecular complete remission treated with a sequence therapy of InO and blinatumomab. Later, hematopoietic stem cell transplantation could be performed without major complications. Our case may suggest that the sequence therapy of InO and blinatumomab as a bridge-to hematopoietic stem cell transplantation could be effective in the treatment of pediatric r/r ALL.

Invasive and in situ lesions of squamous cell carcinoma are independent factors for postoperative surgical-site infection after outpatient skin tumors surgery: A retrospective study of 512 patients.

Surgical-site infection (SSI) is one of the major postoperative complications in surgery, which can cause significant morbidity. However, factors associated with SSI in dermatological surgery are not well understood. Here, we retrospectively investigated 512 patients who underwent outpatient surgery for skin tumors at the University of Tsukuba Hospital to analyze factors associated with postoperative SSI. The overall incidence of SSI was 28 (5.5%). Univariate logistic regression analysis revealed that SSI was significantly associated with invasive squamous cell carcinoma (iSCC), Bowen's disease (BD), actinic keratosis (AK), longer diameter of defects, presence of ulcer, reconstruction with full-thickness skin graft and local skin flaps, medical history of diabetes mellitus, and use of immunosuppressive agents. However, in the multivariate analysis only iSCC, BD, and AK retained significance. The frequencies of SSI in iSCC, BD, and AK were 22% (13/58 patients), 15.6% (5/32), and 25% (2/8), respectively; however, the frequency of other non-SCC tumors was only 1.9% (8/414). χ2 -Tests revealed that the frequency of SSI in iSCC, BD, and AK were all significantly higher than in non-SCC tumors, with the frequencies being more than eight times higher. These results suggest that invasive and in situ lesions of SCC are independent risk factors of SSI development after outpatient skin surgery.

IFN-γ-Stimulated Apoptotic Keratinocytes Promote Sclerodermatous Changes in Chronic Graft-Versus-Host Disease.

Patients with graft-versus-host disease (GVHD) develop characteristic mucocutaneous phenomena consisting of erosive erythema with histopathological findings including interface dermatitis and keratinocyte (KC) death, resulting in widespread sclerodermatous changes. We found that KCs exhibit marked production of TGFß1 in skin lesions of chronic GVHD but not in those of acute GVHD. To further investigate the roles of KCs, the main targets of donor T cells, in sclerodermatous changes followed by interface dermatitis, we established a murine model of chronic GVHD-like sclerodermatous changes followed by acute GVHD-like mucocutaneous injury in genetically modified mice transferred with KC-specific CD8 T cells. Although transfer of granzyme B-deficient CD8 T cells did not result in either mucocutaneous injury or sclerodermatous changes in recipients, IFN-γ-deficient CD8 T-cell recipients developed severe acute mucocutaneous injury but milder sclerodermatous changes than wild-type CD8 T-cell recipients. Moreover, IFN-γ-deficient CD8 T-cell recipients had a lower expression of TGFß1 in the epidermis than the control. Murine primary KCs undergoing FasL-induced apoptosis and incubated with IFN-γ produced TGFß1, the production of which was inhibited by a pan-caspase inhibitor. Our results indicate that IFN-γ promotes TGFß1 production by apoptotic KCs, which mediates the development of widespread sclerodermatous changes in KC-targeting GVHD.

Langerhans Cells Suppress CD8+ T Cells In Situ during Mucocutaneous Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (aGVHD) induced by allogenic hematopoietic stem cell transplantation is an immunological disorder in which donor lymphocytes attack recipient organs. It has been proven that recipient nonhematopoietic tissue cells, such as keratinocytes, are sufficient as immunological targets for allogenic donor T cells, whereas Langerhans cells (LCs) are potent professional hematopoietic antigen-presenting cells existing in the target epidermis and eliminated during the early phase of mucocutaneous aGVHD. Moreover, LCs have been reported to negatively regulate various types of immune responses. Here, we present data showing that initial depletion of recipient LCs exacerbates mucocutaneous lesions in a murine model of allogenic bone marrow transplantation-induced aGVHD. Furthermore, another murine model of mucocutaneous aGVHD induced in mice with keratinocytes genetically expressing chicken ovalbumin by transfer of ovalbumin-specific CD8+ OT-I cells also showed that LC-depleted recipient mice develop aggravated mucocutaneous disease owing to decreased apoptosis of skin-infiltrating OT-I cells. Moreover, coexisting LCs directly induce apoptosis and inhibit the proliferation of OT-I cells in vitro partially via B7 family proteins. Collectively, our results indicate that LCs negatively regulate mucocutaneous aGVHD-like lesions in situ by inhibiting the number of infiltrating CD8+ T cells.

Activation of CD8 T cells accelerates anti-PD-1 antibody-induced psoriasis-like dermatitis through IL-6.

Use of immune checkpoint inhibitors that target programmed cell death-1 (PD-1) can lead to various autoimmune-related adverse events (irAEs) including psoriasis-like dermatitis. Our observations on human samples indicated enhanced epidermal infiltration of CD8 T cells, and the pathogenesis of which appears to be dependent on IL-6 in the PD-1 signal blockade-induced psoriasis-like dermatitis. By using a murine model of imiquimod-induced psoriasis-like dermatitis, we further demonstrated that PD-1 deficiency accelerates skin inflammation with activated cytotoxic CD8 T cells into the epidermis, which engage in pathogenic cross-talk with keratinocytes resulting in production of IL-6. Moreover, genetically modified mice lacking PD-1 expression only on CD8 T cells developed accelerated dermatitis, moreover, blockade of IL-6 signaling by anti-IL-6 receptor antibody could ameliorate the dermatitis. Collectively, PD-1 signal blockade-induced psoriasis-like dermatitis is mediated by PD-1 signaling on CD8 T cells, and furthermore, IL-6 is likely to be a therapeutic target for the dermatitis.

PCDH17 functions as a common tumor suppressor gene in acute leukemia and its transcriptional downregulation is mediated primarily by aberrant histone acetylation, not DNA methylation.

We recently reported that methylation of PCDH17 gene is found in 30% of children with B-cell precursor acute lymphoblastic leukemia (ALL), and is significantly correlated to event-free or overall survival. We here evaluated PCDH17 mRNA expression in pediatric acute myeloid leukemia (AML) and ALL. PCDH17 mRNA expression levels in children with ALL/AML were lower than those in healthy counterparts. We next elucidated the mechanism underlying down-regulation of PCDH17 mRNA, using myeloid and lymphoid leukemic cell lines. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) resulted in restoration of PCDH17 mRNA expression and growth inhibition in K562, HL60, REH, and RCH-ACV cell lines. Upregulation of PCDH17 mRNA expression resulted from histone H3 acetylation. Knockdown of the PCDH17 gene, caused by transduction of PCDH17-targeted shRNA, significantly enhanced the proliferation of KU812 cells. Meanwhile, overexpression of PCDH17 via retroviral-particle transfection substantially inhibited the growth of Kasumi1 cells. The fold-increase in PCDH17 mRNA expression mediated by 5-azacytidine, an inhibitor of DNA methyltransferase, was fundamentally lower than that produced by TSA. In conclusion, our results suggest that PCDH17 gene functions as a common tumor suppressor gene in leukemic cells, and that histone deacetylase inhibitors re-express PCDH17 mRNA to a greater extent than demethylation reagents.

Analysis of the clinical characteristics of pediatric patients who experience ifosfamide-induced encephalopathy.

BACKGROUND: Several kinds of pediatric hematological and/or malignant diseases are treated with chemotherapy regimens including ifosfamide (IFO). IFO-induced encephalopathy (IIE) is one of the serious side effects, but there is not enough evidence regarding the clinical features of IIE in children. PROCEDURE: We performed a retrospective study on pediatric patients treated with chemotherapy regimens, including IFO, at a single center. We recorded the clinical characteristics of all patients; we compared the clinical characteristics between patients who developed IIE and those who did not. RESULTS: In total, 88 patients received a chemotherapy regimen including IFO. IIE developed in seven patients (8.0%). The median age of patients at the time of IIE development was 4.3 (range 1.4-6.5) years in the younger population. Six of seven patients with IIE improved with supportive therapy only; however, one patient died due to heart failure. Overall survival was not different between the two groups. Multivariable analysis revealed that the co-administration of cisplatin (CDDP) or carboplatin (CBDCA) was a significant risk factor associated with IIE. Although there was no significant difference in laboratory data between the groups before chemotherapy, patients who developed IIE showed exacerbation in several laboratory tests, including those for renal and liver functions. CONCLUSIONS: Renal dysfunction caused by the combination of nephrotoxic agents (IFO and CDDP/CBDCA) seems to be important for the development of pediatric IIE. It was thought to be difficult to predict IIE onset based on laboratory data before the initiation of chemotherapy regimens; however, careful observation of laboratory data during IFO chemotherapy regimens may help predict IIE onset and facilitate early treatment.