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Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such "hu-NK mice" rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine., Competing Interests: Declaration of competing interest H.X., S.N., A.H., K.E., and N.S. have applied for patents related to this manuscript. K.E. is a founder of Megakaryon and a member of its scientific advisory board without salary. N.S. serves as an advisory for Megakaryon. S. Kaneko is a founder, shareholder, and director at Thyas Co., Ltd., and received research funding from Takeda Pharmaceutical Co., Ltd., Thyas Co., Ltd., Astellas Co., Ltd., KOTAI biotechnologies Co., Ltd., and Terumo Co., Ltd. This work was supported in part by grants from Megakaryon and Otsuka Pharmaceuticals. These interests were reviewed and are managed by Kyoto University in accordance with its conflict-of-interest policies., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by frameshift mutations in the DMD gene. DMD involves cardiac muscle, and the presence of ventricular arrhythmias or congestive failure is critical for prognosis. Several novel therapeutic approaches are being evaluated in ongoing clinical trials. Among them, exon-skipping therapy to correct frameshift mutations with antisense oligonucleotides is promising; however, their therapeutic efficacies on cardiac muscle in vivo remain unknown. In this study, we established induced-pluripotent stem cells (iPSCs) from T cells from a DMD patient carrying a DMD-exon 46-55 deletion, differentiated the iPSCs into cardiomyocytes, and treated them with phosphorodiamidate morpholino oligomers. The efficiency of exon-45 skipping increased in a dose-dependent manner and enabled restoration of the DMD gene product, dystrophin. Further, Ca 2+ -imaging analysis showed a decreased number of arrhythmic cells and improved transient Ca 2+ signaling after exon skipping. Thus, exon-45 skipping may be effective for cardiac involvement in DMD patients harboring the DMD-exon 46-55 deletion., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Fibrosis is an abnormal response to organ injury, characterized by accumulation of activated fibroblasts at the sites of injury. Fibroblasts arise from several sources, including resident fibroblasts and circulating fibrocytes that infiltrate organ tissue. Recently, epithelial-mesenchymal transition (EMT) has been recognized as a source of mesenchymal cells. EMT is induced by various growth factors, such as transforming growth factor (TGF)-β1, and enhanced by inflammatory cytokines. Recently the tumor necrosis factor superfamily member LIGHT has been implicated in the pathogenesis of inflammatory disease and airway remodeling in severe asthma. We hypothesized that LIGHT might contribute to the pathogenesis of airway fibrosis via enhancement of EMT. Therefore, we investigated LIGHT's ability to induce EMT. A549 cells were stimulated with LIGHT, TGF-β1 or both for 48h. To estimate EMT, we evaluated the expression of epithelial and mesenchymal markers using immunocytochemistry, Western blotting and quantitative RT-PCR. Signaling pathways for EMT were characterized by Western analysis to detect phosphorylation of Erk1/2 and smad2. LIGHT enhanced TGF-β1-induced EMT both morphologically, by suppressing E-cadherin and enhancing vimentin, and functionally, by enhancing cell contractility. Additionally, LIGHT induced EMT without TGF-β1. Evaluation of the mechanism showed that LIGHT did not induce TGF-β1 production or affect the smad-snai1 pathway. Inhibition of Erk1/2 phosphorylation reduced LIGHT-induced EMT, indicating the Erk1/2 pathway to be a key pathway in LIGHT-induced EMT. In summary, LIGHT enhanced TGF-β1-induced EMT but also induced EMT via the Erk1/2 pathway by itself, without TGF-β1 signaling. LIGHT may contribute to the pathogenesis of airway fibrosis through enhancement of EMT., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is one of the most common cancers worldwide and the third most common cause of cancer-related death. Imaging studies including ultrasound and computed tomography are recommended for early detection of HCC, but they are operator dependent, costly and involve radiation. Therefore, there is a need for simple and sensitive serum markers for the early detection of hepatocellular carcinoma (HCC). In our recent proteomic studies, a number of proteins overexpressed in HCC tissues were identified. We thought if the serum autoantibodies to these overexpressed proteins were detectable in HCC patients. Of these proteins, we focused on Ku86, a nuclear protein involved in multiple biological processes and aimed to assess the diagnostic value of serum anti-Ku86 in the early detection of HCC. Serum samples were obtained prior to treatment from 58 consecutive patients with early or relatively early hepatitis C virus (HCV)-related HCC and 137 patients with HCV-related liver cirrhosis without evidence of HCC. Enzyme immunoassays were used to measure serum levels of autoantibodies. Serum levels of anti-Ku86 antibodies were significantly elevated in HCC patients compared to those in liver cirrhosis patients (0.41±0.28 vs. 0.18±0.08Abs at 450nm, P<0001). Setting the cut-off level to give 90% specificity, anti-Ku86 was positive in 60.7% of stage I solitary tumor <2cm in diameter, whereas the sensitivities of alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist II (PIVKA-II) were 17.8% and 21.4%, respectively. The results of ROC analyses indicated the better performance of anti-Ku86 for early detection of HCC. Serum anti-Ku86 levels decreased after surgical resection of the tumors in the 12 HCC cases tested, Elevation of anti-Ku86 in solid tumors other than liver was minimal. Serum anti-Ku86 is a potential biomarker for early detection of HCV-related HCC. Further studies in a larger number of HCC patients with various etiologies are needed to further evaluate the diagnostic and pathophysiological roles of elevation of serum anti-Ku86 in early HCC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Lnk, SH2-B, and APS form a conserved adaptor protein family. All of those proteins are expressed in mast cells and their possible functions in signaling through c-Kit or FcRI have been speculated. To investigate roles of Lnk, SH2-B or APS in mast cells, we established IL-3-dependent mast cells from Ink-/-, SH2-B-/-, and APS -/- mice. IL-3-dependent growth of those cells was comparable. Proliferation or adhesion mediated by c-Kit as well as degranulation induced by cross-linking FcRI were normal in the absence of Lnk or SH2-B. In contrast, APS-deficient mast cells showed augmented degranulation after cross-linking FcRI compared to wild-type cells, while c-Kit-mediated proliferation and adhesion were kept unaffected. APS-deficient mast cells showed reduced actin assembly at steady state, although their various intracellular responses induced by cross-linking FcRI were indistinguishable compared to wild-type cells. Our results suggest potential roles of APS in controlling actin cytoskeleton and magnitude of degranulation in mast cells.

Murine leukemia virus (MLV) produces the unspliced RNA and the singly spliced RNA at a proper ratio at a time. To identify cis-elements involved in the production of the unspliced RNA, we examined the level of unspliced RNA in a series of mutants derived from a prototype Moloney MLV mutant gag-encoding G3.6. Our present data indicated that nt 1560-1906 region in the CA-encoding region in gag was the negative cis-element and nt 5119-5355 region, which was immediately upstream of the splice acceptor site, was the positive cis-element for expression of the unspliced RNA. It was found that the former element made expression of the unspliced RNA dependent upon the latter. These two elements were functional as discrete elements and their activities were relatively position-independent.

Erythromycin (EM), and related 14-member macrolide antibiotics, has attracted attention for its effectiveness in airway diseases including diffuse panbronchiolitis and sinobronchial syndrome. However, its molecular mechanisms remain unknown. We evaluated the effects of EM on activation of several transcription factors, including nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) in human bronchial epithelial cell line BET-1A, which are known to regulate the expression of many proinflammatory cytokines and chemokines such as interleukin-8 (IL-8). BET-1A cells were cultured with hormonally defined Ham's F12 medium, and were stimulated by phorbol myristate acetate (PMA). EM suppressed mRNA expression as well as the release of IL-8 at therapeutic and noncytotoxic concentrations (% inhibition of IL-8 protein release: 42.2 +/- 5.5%, at 10(-6) M). Furthermore, electrophoretic mobility shift assays revealed that EM inhibited the activations of NF-kappaB and AP-1 induced by PMA in BET-1A cells. These data indicate that EM has inhibitory effects not only on the mRNA expression and release of IL-8, but also on the activation of transcription factors NF-kappaB and AP-1. Our findings support the concept that the recruitment of neutrophils in airway diseases may be regulated by NF-kappaB and AP-1., (Copyright 2000 Academic Press.)

It has been emphasized that epithelial injury is closely correlated with airway hyperresponsiveness, which is one of the important pathophysiological characteristics of bronchial asthma. Growth of epithelial cells is important in the mucosal repair processes and is believed to be regulated by growth factors produced by inflammatory and immune effector cells as well as epithelial cells themselves. We studied the role of T cell-derived lymphokines IFN gamma and IL-4 on the proliferation of human bronchial epithelial cell line BEAS-2B. IFN gamma, but not IL-4, showed a dose-dependent growth inhibitory activity in vitro. Its activity was via its specific receptors on the cells, was augmented by TNF alpha, and was independent of the activity of endogenous TGF beta and nitric oxide. These results suggested that Th-1 T cells-derived lymphokine IFN gamma might be involved in the repair processes after mucosal injury found in bronchial asthma.

Eosinophils are believed to be one of the important sources of cytokines at the site of allergic inflammation. A novel antiallergic agent epinastine showed a dose- and time-dependent suppressive effect on IL-8, one of the chemokines for eosinophils, released from eosinophils isolated from atopic diseases. The time-dependent accumulation of IL-8 inhibited by cycloheximide and the evaluation of the IL-8 mRNA levels by reverse transcriptase-polymerase chain reaction suggested that its action occurred in the posttranscriptional processes. It was suggested that epinastine might prevent the autocrine cycle for recruitment of human eosinophils by inhibiting IL-8 release from these cells.

Airway epithelial cells have a potential to participate in local immune and inflammatory responses via releasing biologically active compounds. We studied the expression and release of interleukin 6 (IL-6), a multifunctional cytokine possibly involved in tissue immune responses. Primary culture of normal human bronchial epithelial cells and its transformed cell line BEAS-2B released significant amount of biologically and immunologically intact IL-6 into media. A protein synthesis inhibitor cycloheximide abolished the IL-6 release, suggesting a de novo synthesis. Northern blot analysis demonstrated the expression of the specific IL-6 mRNA. Human bronchial epithelial cells can produce IL-6 and contribute to the local activity of IL-6, suggesting that these cells may play a role in the regulation of airway immune responses.

We previously identified a 20KDa membrane glycoprotein 1F5 antigen which inhibits the assembly of homologous complement membrane attack complexes and we designate it as HRF20 standing for 20KDa homologous restriction factor. The amino acid sequence deduced from its coding base sequence resembles that of T cell activating protein, most conspicuously in cysteine residues, 10 out of 11 of which occupy identical positions in an overall sequence homology of 24.8%. Furthermore, proliferation of human T cells was stimulated by monoclonal antibody to HRF20.