Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells.

Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.

Downregulation of miR-26 promotes invasion and metastasis via targeting interleukin-22 in cutaneous T-cell lymphoma.

It has been reported that certain microRNAs (miRNA) are associated with the pathogenesis of lymphoma. We have previously demonstrated that histone deacetylase inhibitors restore tumor-suppressive miRNAs, such as miR-16, miR-29, miR-150, and miR-26, in advanced cutaneous T-cell lymphoma (CTCL). Among these, the function of miR-26 remains unclear. In this study, we aimed to reveal the function of miR-26 in CTCL oncogenesis. First, we confirmed that the miR-26 family was markedly dysregulated in CTCL cell lines and primary samples. In vivo analysis using miR-26a-transduced CTCL cells injected into immunodeficient NOG mice demonstrated the significant prolonged survival of the mice, suggesting that the miRNA had a tumor-suppressive function. We performed gene expression assays and identified 12 candidate miR-26 targets, namely RGS13, FAM71F1, OAF, SNX21, CDH2, PTPLB, IL22, DNAJB5, CASZ1, CACNA1C, MYH10, and CNR1. Among these, IL22 was the most likely candidate target because the IL-22-STAT3-CCL20-CCR6 cascade is associated with tumor invasion and metastasis of advanced CTCL. In vitro analysis of IL22 and IL22RA knockdown and miR-26 transduction demonstrated inhibited CTCL cell migration. In particular, IL22 knockdown induced cell apoptosis. Finally, we conducted in vivo inoculation analysis of mice injected with shIL22-transfected CTCL cells, and found no tumor invasion or metastasis in the inoculated mice, although the control mice showed multiple tumor invasions and metastases. These results, along with our previous data, demonstrated that miR-26 is a tumor suppressor that is associated with tumor invasion and the metastasis of advanced CTCL by regulating the IL-22-STAT3-CCL20 cascade. Therefore, a IL-22-targeting therapy could be a novel therapeutic strategy for advanced CTCL.

Multiple myeloma with t(11;14)-associated immature phenotype has lower CD38 expression and higher BCL2 dependence.

CD38 expression on myeloma cells is a critical factor affecting the early response to the anti-CD38 antibody daratumumab. However, factors affecting CD38 expression in untreated multiple myeloma are not fully elucidated. In this study, we found that CD38 expression was significantly lower in myeloma patients with the translocation t(11;14)-associated immature plasma cell phenotype, and particularly in those expressing B-cell-associated genes such as PAX5 and CD79A. CD138, a representative marker of plasmacytic differentiation, was also significantly lower in these patients, suggesting that CD38 expression may be associated with the differentiation and maturation stages of myeloma cells. Furthermore, the BCL2/BCL2L1 ratio, a response marker of the BCL2 inhibitor venetoclax, was significantly higher in patients with the immature phenotype expressing B-cell-associated genes. The BCL2/BCL2L1 ratio and CD38 expression were significantly negatively correlated. We also confirmed that patients with translocation t(11;14) expressing B-cell-associated genes were indeed less sensitive to daratumumab-mediated direct cytotoxicity but highly sensitive to venetoclax treatment in ex vivo assays. Moreover, all-trans-retinoic acid, which enhances CD38 expression and induces cell differentiation in myeloma cells, reduced B-cell marker expression and the BCL2/BCL2L1 ratio in myeloma cell lines, leading to reduced efficacy of venetoclax. Venetoclax specifically induces cell death in myeloma with t(11;14), although why patients with translocation t(11;14) show BCL2 dependence is unclear. These results suggest that BCL2 dependence, as well as CD38 expression, are deeply associated with the differentiation and maturation stages of myeloma cells. This study highlights the importance of examining t(11;14) and considering cell maturity in myeloma treatment strategies.

Comprehensive analysis of microRNAs modulated by histone deacetylase inhibitors identifies microRNA-7-5p with anti-myeloma effect.

Basic research to expand treatment options for multiple myeloma is greatly needed due to the refractory nature of the disease. Histone deacetylase (HDAC) inhibitors, which are epigenetic regulators, are attractive but have limited applications. MicroRNAs (miRNAs), which are also epigenetic regulators, are important molecules that may lead to future therapeutic breakthroughs. In this study, we comprehensively searched for miRNAs that are altered by HDAC inhibitors in myeloma cells. We identified miR-7-5p (miR-7) as a miRNA downregulated by HDAC inhibitors. Transfection of myeloma cell lines with miR-7 suppressed cell proliferation, induced apoptosis, and enhanced the effects of the HDAC inhibitor panobinostat. Expression of miR-7 was downregulated by c-Myc inhibition, but upregulated by bortezomib. Comprehensive examination of miR-7 targets revealed four candidates: SLC6A9, LRRC59, EXOSC2, and PSME3. Among these, we focused on PSME3, an oncogene involved in proteasome capacity in myeloma cells. PSME3 knockdown increases myeloma cell death and panobinostat sensitivity. In conclusion, miR-7, which is downregulated by HDAC inhibitors, is a tumor suppressor that targets PSME3. This miR-7 downregulation may be involved in HDAC inhibitor resistance. In addition, combinations of anti-myeloma drugs that complement changes in miRNA expression should be considered.

Treatment Outcome in Head and Neck Cancer With Distant Metastasis at Initial Diagnosis.

OBJECTIVE: Recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) treatment has changed dramatically with the introduction of immune checkpoint inhibitors (ICIs). However, there are few reports of treatment outcomes on HNSCC with distant metastasis (M1) at initial diagnosis, and its treatment strategy has not been standardized. We aimed to analyze the treatment outcome and prognostic factors of patients with HNSCC with initial M1 disease. METHODS: In this multi-institutional retrospective study, 98 patients with HNSCC were initially diagnosed with M1 disease between 2007 and 2021. The patients were divided into the non-palliative (received any systemic chemotherapy, n = 60) and palliative (did not receive systemic chemotherapy, n = 38) groups. Overall survival (OS) was compared between the groups. In the non-palliative group, predictors of OS were explored based on patient characteristics and treatment details. RESULTS: The median OS in the non-palliative group was 15 months (95% confidence interval [CI], 10-20), which was significantly longer than that in the palliative group (3 months, 95% CI, 2-5) (p < 0.001). Multivariate analysis revealed that administration of locoregional radiation therapy (RT) (hazard ratio [HR] 0.407 [95% CI 0.197-0.844]; p = 0.016), ICIs (HR 0.216 [95% CI 0.088-0.532]; p < 0.001) and RT/surgery for distant metastasis (HR 0.373 [95% CI 0.150-0.932]; p = 0.034) were the independent prognostic factors of OS. CONCLUSION: An intensive treatment strategy combining systemic therapy using ICIs with RT/surgery for locoregional or distant metastasis may yield a survival benefit for patients with HNSCC with M1 disease. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:1679-1686, 2024.

Impairment of nuclear F-actin formation and its relevance to cellular phenotypes in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a mutation of lamin A, which contributes to nuclear architecture and the spatial organization of chromatin in the nucleus. The expression of a lamin A mutant, named progerin, leads to functional and structural disruption of nuclear organization. Since progerin lacks a part of the actin-binding site of lamin A, we hypothesized that nuclear actin dynamics and function are altered in HGPS cells. Nuclear F-actin is required for the organization of nuclear shape, transcriptional regulation, DNA damage repair, and activation of Wnt/ß-catenin signaling. Here we show that the expression of progerin decreases nuclear F-actin and impairs F-actin-regulated transcription. When nuclear F-actin levels are increased by overexpression of nuclear-targeted actin or by using jasplakinolide, a compound that stabilizes F-actin, the irregularity of nuclear shape and defects in gene expression can be reversed. These observations provide evidence for a novel relationship between nuclear actin and the etiology of HGPS.

[A case of Good syndrome accompanied by myasthenia gravis: immunological evaluations].

Good syndrome, characterized by both thymoma and hypogammaglobulinemia, is a rare immunodeficient disorder. We experienced a case of Good syndrome accompanied by myasthenia gravis (MG). A 58-year-old man was admitted to our hospital because of muscle weakness and fatigability. Based on the presence of anti-acetylcholine receptor (AChR) antibody and thymoma, he was diagnosed as having MG. Peripheral blood lymphocyte count was normal, but gammaglobulin levels were markedly decreased (IgG 283 mg/dl, IgA 17 mg/dl, IgM 1 mg/dl). Clinical remission of MG was achieved by thymectomy followed by high-dose corticosteroids. Despite monthly intravenous immunoglobulin supplementation, he suffered from repeated respiratory tract infections and candidiasis. Body CT revealed adrenal tumor and pancreatic cancer with liver metastasis, and he died of bacterial pneumonia. Immunological evaluations showed complete lack of CD19+ B cell in the peripheral blood and responses of peripheral blood mononuclear cells to mitogens. Peripheral blood T cells responded to a suboptimal concentration of a recombinant AChR fragment: this pattern of AChR-induced T cell response was typical of MG patients. We failed to detect IgG autoantibodies reactive with B cells in his serum. Patients with Good syndrome represent imbalance of immune responses, leading to both immunodeficiency and autoimmunity.

New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique.

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope (HVJ-E) and tissue maceration. METHODS: Cardiomyocytes (1.5 × 10(6)) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets (area: about 3.5 cm(2) including 2.1 × 10(6) cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or NaOH maceration: G1: HVJ-E(+), NaOH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaOH(+), Cardiomyocytes(+); G3: HVJ-E(+), NaOH(-), Cardiomyocytes(+); G4: HVJ-E(-), NaOH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin. RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas. CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaOH maceration.

The reaction mechanism for the high quantum yield of Cypridina (Vargula) bioluminescence supported by the chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-ones (Cypridina luciferin analogues).

To establish the reaction mechanism of the high-quantum-yield bioluminescence in Cypridina (Vargula), we investigated the chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-ones (1H) as Cypridina luciferin analogues in DMSO-1,1,3,3-tetramethylguanidine and in diglyme-acetate buffer. We found that the chemiluminescence of 1H with an electron-donating aryl group, such as a 4-(dimethylamino)phenyl, 3-indolyl or 3-(1-methyl)indolyl group, gave a high quantum yield (Phi(CL)) in diglyme-acetate buffer. This indicates that the reaction mechanism producing this high Phi(CL) involves the chemiexcitation of a neutral dioxetanone intermediate possessing an electron-donating aryl group to the singlet excited state of neutral acetamidopyrazine (the light emitter). In addition, we investigated the fluorescence of acetamidopyrazines and performed DFT calculations for neutral dioxetanones and the transition states (TS) of the dioxetanone's decomposition. The results made it clear that the electron-donating aryl group gives the TS and the singlet-excited acetamidopyrazine (S(1)) a strong intramolecular charge transfer (ICT) character, and their similar ICT character leads to the ICT TS --> S(1) route in the charge transfer-induced luminescence (CTIL) mechanism for efficient chemiexcitation. The reaction mechanism of the chemiluminescence of 1H can explain the highly efficient chemiexcitation of Cypridina bioluminescence.