Comparative Study of Metastasis Suppression Effects of Extracellular Vesicles Derived from Anaplastic Cell Lines, Nanog-Overexpressing Melanoma, and Induced Pluripotent Stem Cells.

Previous studies have demonstrated that extracellular vesicles (EVs) derived from an anaplastic mouse melanoma cell line made using Nanog overexpression of F10 (Nanog+F10) suppressed the metastasis of Nanog+F10. Here, an induced pluripotent stem (iPS) cell line was focused as a more anaplastic cell line, potentially producing EVs with higher metastasis-suppressive effects. The EVs were introduced into the tail vein nine times before introducing Nanog+F10 cells. Two weeks later, the liver and lung were resected and metastatic colonies were quantified. The involvement of macrophages (invasion inhibiting ability, phagocytic activity) and cytotoxic T cells (cytotoxicity) was evaluated using J774.1 and CTLL-2 cell lines. iPS EVs showed similar level effects to Nanog+F10 EVs in every item relevant to metastasis suppression. Differential expression analysis of miRNAs in EVs and functional network database analysis revealed that dominant regulatory miRNAs were predicted. The candidate hub genes most highly associated with the metastasis suppression mechanism were predicted as six genes, including Trp53 and Hif1a, for Nanog+F10 EVs and ten genes, including Ins1 and Kitl, for iPS EVs. Regarding the mechanism, Nanog+F10 EVs and iPS EVs were very different. This suggests synergistic effect when used together as metastasis preventive vaccine.

Connexin 45 is a novel suppressor of melanoma metastasis.

The expression spectra of connexin (Cx) isoforms were investigated in three mouse melanoma cell lines: B16-F1 (F1), B16-F10 (F10), and B16-BL6 (BL6). Metastatic potential intensity was higher in the order of F1, F10, and BL6. A remarkable behavior of Cx45 was found among 20 isoforms. The expression level of Cx45 was highest in F1 and lowest in BL6. It was inductively predicted that Cx45 might be a novel suppressor of metastasis. A Cx45-overexpressing BL6 cell line (Cx45 +BL6) was developed and its properties were compared with those of a wild-type cell line of BL6 (W-BL6). Compared to W-BL6, Cx45 +BL6 showed reduced wound healing, Transwell® permeability, and matrix metalloproteinase 9 expression, suggesting the suppression of cellular migration and invasion. The expression of E-cadherin and integrin ß1 in Cx45 +BL6 was also lower than in W-BL6, suggesting reduced cell adhesion. The decrease in cell adhesion was supported by the cell washing-out assay. In contrast, no difference between W-BL6 and Cx45 +BL6 was observed in cell proliferation, suggesting no effect on cell-cycle regulating factors. Finally, an in vivo assay revealed a significant decrease in the number of metastatic colonies of Cx45 +BL6 (176 ± 25/lung) in comparison with those of W-BL6 (252 ± 23/lung) in a mouse model. In conclusion, Cx45 is a novel suppressor of melanoma metastasis. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00563-x.

Successful baricitinib treatment of refractory anti-synthetase syndrome associated with interstitial lung disease.

A 47-year-old Japanese man was admitted with dyspnoea on exertion (DOE), skin rash and myalgia. Clinical findings of Gottron's sign and mechanic's hands were observed, with increased serum levels of Krebs von den Lungen-6, surfactant protein-D, creatine kinase, and anti-EJ on laboratory tests. In both lungs, chest computed tomography revealed diffuse reticular opacities and lower lobe predominance. The patient was diagnosed with anti-synthetase syndrome (ASS) and associated interstitial lung disease. Despite repeated administration of high-dose intravenous corticosteroids, cyclophosphamide and immunoglobulin, his skin rash, myalgia, and DOE followed a relapsing and remitting course. He was then given rituximab therapy. This was initially successful, but disease activity increased approximately 12 months after starting rituximab therapy. Finally, in addition to prednisolone and cyclosporine A, we administered baricitinib. There has been no relapse of the disease in the 12 months since he began baricitinib treatment.

Moderate hyperglycemia suppresses melanoma metastasis to liver.

The metastasis of various cancers is promoted by hyperglycemia. In contrast, melanoma and colorectal cancer seemed to be exceptional. We confirmed that the metastasis of melanoma B16-F10 could be suppressed by hyperglycemia. It was attractive from the prognostic point of view of the prevention of metastasis, though the problem of the risk of diabetes remained. Then, the effect of moderate hyperglycemic condition was investigated using a pre-diabetic model mouse (GKKO mouse). The metastasis of B16-F10 cells to liver was focused and the number and volume of metastatic colonies in liver were analyzed. The medians of the number of metastatic colonies in GKKO mice were 0.57-fold (P=0.06) compared to control mice. Analysis of macrophage markers revealed upregulation of CD86, a tumor-suppressive M1-type marker, and downregulation of CD206, a tumor-promotive M2-type marker. A tendency of upregulation of Cxcl10, a pro-inflammatory cytokine was also observed. Regarding cellular activities of B16-F10, migration activity and invasion activity were reduced by moderate hyperglycemia. In conclusion, metastasis of B16-F10 cells to liver could be suppressed by moderate hyperglycemia without the risk of diabetes. This information should contribute to dietary planning during prognosis.

Real-world clinical outcomes of treatment with molnupiravir for patients with mild-to-moderate coronavirus disease 2019 during the Omicron variant pandemic.

It is unclear whether molnupiravir has a beneficial effect on vaccinated patients infected with the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We here evaluated the efficacy of molnupiravir in patients with mild-to-moderate coronavirus disease 2019 (COVID-19) during the Omicron variant surge in Fukushima Prefecture, Japan. We enrolled patients with mild-to-moderate COVID-19 who were admitted to hospitals between January and April, 2022. Clinical deterioration after admission was compared between molnupiravir users (n = 230) and non-users (n = 690) after 1:3 propensity score matching. Additionally, we performed forward stepwise multivariate logistic regression analysis to evaluate the association between clinical deterioration after admission and molnupiravir treatment in the 1:3 propensity score-matched subjects. The characteristics of participants in both groups were balanced as indicated by covariates with a standardized mean difference of < 0.1. Regarding comorbidities, there was no imbalance between the two groups, except for the presence of hypertension, dyslipidemia, diabetes mellitus, and cardiac disease. The clinical deterioration rate was significantly lower in the molnupiravir users compared to the non-users (3.90% vs 8.40%; P = 0.034). Multivariate logistic regression analysis demonstrated that receiving molnupiravir was a factor for preventing deterioration (odds ratio 0.448; 95% confidence interval 0.206-0.973; P = 0.042), independent of other covariates. This real-world study demonstrates that molnupiravir contributes to the prevention of deterioration in COVID-19 patients after hospitalization during the Omicron variant phase.

Novel Roles of Nanog in Cancer Cells and Their Extracellular Vesicles.

The use of extracellular vesicle (EV)-based vaccines is a strategically promising way to prevent cancer metastasis. The effective roles of immune cell-derived EVs have been well understood in the literature. In the present paper, we focus on cancer cell-derived EVs to enforce, more thoroughly, the use of EV-based vaccines against unexpected malignant cells that might appear in poor prognostic patients. As a model of such a cancer cell with high malignancy, Nanog-overexpressing melanoma cell lines were developed. As expected, Nanog overexpression enhanced the metastatic potential of melanomas. Against our expectations, a fantastic finding was obtained that determined that EVs derived from Nanog-overexpressing melanomas exhibited a metastasis-suppressive effect. This is considered to be a novel role for Nanog in regulating the property of cancer cell-derived EVs. Stimulated by this result, the review of Nanog's roles in various cancer cells and their EVs has been updated once again. Although there was no other case presenting a similar contribution by Nanog, only one case suggested that NANOG and SOX might be better prognosis markers in head and neck squamous cell carcinomas. This review clarifies the varieties of Nanog-dependent phenomena and the relevant signaling factors. The information summarized in this study is, thus, suggestive enough to generate novel ideas for the construction of an EV-based versatile vaccine platform against cancer metastasis.

Coronavirus disease 2019 vaccination-induced acute exacerbation in idiopathic pulmonary fibrosis.

We report a rare case of acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) after coronavirus disease 2019 (COVID-19) vaccination. Clinicians should be aware of this COVID-19 vaccination-induced AE in IPF.

Clinical effect of early administration of tocilizumab following the initiation of corticosteroid therapy for patients with COVID-19.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first broke out in Wuhan in December 2019, and has since caused a global pandemic. The efficacy of several drugs has been evaluated, and it is now evident that tocilizumab has a beneficial effect, especially combined with corticosteroids, in patients with Coronavirus Disease 2019 (COVID-19). However, the optimal timing of tocilizumab administration has not yet been established. The goal of the present study was to determine the optimal timing of tocilizumab administration after starting corticosteroid therapy in patients with COVID-19. METHODS: We retrospectively analyzed the clinical characteristics of patients who were hospitalized for COVID-19 and treated with tocilizumab and corticosteroids in our hospital. The patients were divided into concurrent and sequential groups. The concurrent group received tocilizumab ≤24 h after corticosteroids, and the sequential group received tocilizumab >24 h after corticosteroid administration. RESULTS: The baseline clinical characteristics of tocilizumab administration were similar between the two groups. White blood cell counts were significantly lower and C-reactive protein levels were significantly higher in the concurrent group than the sequential group. In the concurrent group, tocilizumab administration led to a significant decrease in maximum body temperature. In addition, there were significantly more oxygen-free days in the concurrent group than in the sequential group. However, survival rate was not significantly different between the concurrent and the sequential groups. CONCLUSIONS: In the combination therapy with tocilizumab and corticosteroids, early administration of tocilizumab after starting corticosteroid treatment is effective when treating COVID-19.

Real-world clinical outcomes of treatment with casirivimab-imdevimab among patients with mild-to-moderate coronavirus disease 2019 during the Delta variant pandemic.

Background: Mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may reduce the efficacy of neutralizing monoclonal antibody therapy against coronavirus disease 2019 (COVID-19). We here evaluated the efficacy of casirivimab-imdevimab in patients with mild-to-moderate COVID-19 during the Delta variant surge in Fukushima Prefecture, Japan. Methods: We enrolled 949 patients with mild-to-moderate COVID-19 who were admitted to hospital between July 24, 2021 and September 30, 2021. Clinical deterioration after admission was compared between casirivimab-imdevimab users (n = 314) and non-users (n = 635). Results: The casirivimab-imdevimab users were older (P < 0.0001), had higher body temperature (≥ 38°C) (P < 0.0001) and greater rates of history of cigarette smoking (P = 0.0068), hypertension (P = 0.0004), obesity (P < 0.0001), and dyslipidemia (P < 0.0001) than the non-users. Multivariate logistic regression analysis demonstrated that receiving casirivimab-imdevimab was an independent factor for preventing deterioration (odds ratio 0.448; 95% confidence interval 0.263-0.763; P = 0.0023). Furthermore, in 222 patients who were selected from each group after matching on the propensity score, deterioration was significantly lower among those receiving casirivimab-imdevimab compared to those not receiving casirivimab-imdevimab (7.66% vs 14.0%; p = 0.021). Conclusion: This real-world study demonstrates that casirivimab-imdevimab contributes to the prevention of deterioration in COVID-19 patients after hospitalization during a Delta variant surge.

Development of dermatomyositis after anti-transcriptional intermediary factor 1-γ antibody seroconversion during treatment for small cell lung cancer.

BACKGROUND: Inflammatory myositis, such as dermatomyositis, is sometimes complicated by cancer and is recognized as cancer-associated myositis. Although some autoimmune antibodies are considered to be involved in the development of myositis in cancer patients, the precise mechanism has not been clarified. The findings of the present case shed light on the mechanism by which anti-transcriptional intermediary factor 1 (TIF1)-γ Ab was produced and the pathogenesis of cancer-associated myositis. CASE PRESENTATION: We describe a case of dermatomyositis that developed in a 67-year-old man who had been diagnosed with small cell lung cancer of clinical T4N3M0 stage IIIB/limited disease during treatment. He received systemic chemotherapy and radiation therapy, and dermatomyositis developed along with a significant decrease in tumor size. TIF1-γ Ab, which is one of the myositis-specific antibodies, was found to be seroconverted. In addition, immunohistochemical analysis showed that cancer cells were positive for the TIF1-γ antigen. CONCLUSION: The findings of the present case suggest that transcriptional intermediary factor 1-γ, which is released from tumor cells, induces the production of TIF1-γ Ab, leading to the development of dermatomyositis.

Immunoglobulin G4-positive interstitial pneumonia associated with pleuroparenchymal fibroelastosis.

A 79-year-old former smoking Japanese man was admitted to our hospital with a 2-year history of dry cough and dyspnoea on exertion. High-resolution computed tomography of the chest revealed reticulation and perilobular opacity with bronchial wall thickening and ground-glass opacities (GGOs) in both lungs, in addition to subpleural dense consolidation (pleuroparenchymal fibroelastosis-like lesion; PPFE-like lesion) predominantly in the bilateral upper lobes. Serum immunoglobulin G4 (IgG4) was elevated (348 mg/dl). Lung biopsy specimens obtained by video-assisted surgery revealed a mixture of usual interstitial pneumonia (IP) and non-specific IP pattern admixed with PPFE. In addition, immunohistochemical staining of IgG4 showed numerous IgG4-positive plasma cells. Consequently, he was diagnosed with IgG4-positive IP associated with PPFE. We initiated a combination therapy with prednisolone and cyclosporine as a calcineurin inhibitor. During prednisolone tapering, his clinical conditions and GGOs improved gradually over 12 months. However, reticular opacities and PPFE-like lesions remained unchanged, and pulmonary function test findings slightly deteriorated.

Anti-Metastatic Function of Extracellular Vesicles Derived from Nanog-Overexpressing Melanoma.

A metastatic melanoma cell line B16-F10 (F10) was modified to a more undifferentiated state by Nanog overexpression. The produced cell line Nanog+F10 showed a higher metastatic potential than F10. Instead of whole cells, the extracellular vesicles (EVs) therefrom were investigated about their possible role as an autovaccine against metastasis. EVs from Nanog+F10 cells (Nanog+F10-EVs) could suppress the metastasis, contrasting the EVs from less metastatic F10 cells (F10-EVs) enhanced metastasis. The involvement of TGF-ß1 in the role of Nanog+F10-EVs was analyzed, as TGF-ß1 was a secretory cytokine being affected most intensively by Nanog overexpression. It was suggested to be crucial that the TGF-ß1 concentration in Nanog+F10-EVs should be as low as 1.6 pg/µg for its metastasis-suppressive role. In response to Nanog+F10-EVs, immunoreaction was observed in liver, indicating the specific decrease in the number of tumor-promotive CD163-positive macrophages. These indicate a possibility of Nanog+F10-EVs as a novel autovaccine candidate against melanoma metastasis.

Usefulness of diurnal variation of fractional exhaled nitric oxide for predicting early therapeutic response to asthma treatment.

OBJECTIVE: Fractional exhaled nitric oxide (FeNO) is considered to be an adjunct for asthma management, although its usefulness remains controversial. Therefore, it may be necessary for new approaches to use FeNO for asthma management. We evaluated whether diurnal variations of FeNO can predict response to asthma treatment. METHODS: This pilot study consisted of 22 uncontrolled asthmatics and 16 healthy subjects. FeNO and peak expiratory flow (PEF) were measured by themselves twice daily at home for three weeks (asthmatics) or two weeks (healthy subjects), and daily mean and diurnal variations of FeNO and PEF levels were calculated. In uncontrolled asthmatics, treatment was intensified a week after study entry, and then control status was reevaluated after three to four weeks. Asthmatics were then divided into two groups; good or poor responders. RESULTS: Diurnal variations of FeNO levels, as well as daily mean FeNO and PEF levels, in uncontrolled asthmatics before intensive treatment were significantly higher than those in healthy subjects, regardless of treatment response (p < 0.01). Furthermore, in the good responders, diurnal variations of FeNO levels were significantly decreased in the 1st week (p < 0.05) of intensive treatment, whereas the daily mean FeNO levels significantly dropped in the 2nd week (p < 0.05). In the poor responders, no such changes were observed in FeNO levels. In terms of PEF, only the daily mean levels were significantly elevated after the initiation of intensive treatment, regardless of treatment response. CONCLUSIONS: Diurnal variations of FeNO may contribute to predicting early therapeutic response to asthma treatment.

Refinement of decision tree to assess the consequences of increased serum ALP in dogs: Additional analysis on toxicity studies of pesticides evaluated recently in Japan.

Recently we provided a new interpretation that increased serum ALP in dogs is not adverse if no hepatotoxic finding coexists in the analysis of toxicity studies of over 200 pesticides evaluated in Japan (Yokoyama et al., 2019). We also proposed a decision tree to evaluate the adversity of the increased ALP. The present analysis was conducted to validate the reliability of this interpretation with 129 pesticides more recently evaluated. Before applying, the decision tree was revised to be consistent in all steps. The pesticides showed similar characterization of increased ALP to the previous analysis in that the increase was more frequent than in rats and that liver hypertrophy and hepatotoxicity commonly coexisted with an increase in ALP in dogs. When short- and long-term studies of 58 pesticides inducing ALP activity in dogs were applied to the revised tree, the increased ALP in 8 pesticides was judged not adverse in either study. The revision of the tree did not affect the NOAEL judgment of these pesticides; however, the revised routes contributed to the judgment more robustly. This study showed the reliability of our interpretation and applicability of the decision tree to evaluate the adversity of increased ALP in dogs.

Effect of Nanog overexpression on the metastatic potential of a mouse melanoma cell line B16-BL6.

Nanog, a marker and regulator of the undifferentiated state in embryonic stem cells were anticipated to be an effective enhancer of cancer metastasis. We have developed a Nanog overexpressing mouse melanoma cell line B16-BL6 (BL6). BL6 was well recognized as a cell line with a high metastatic potential. In vitro tests revealed the enhancement of cell proliferation, wound healing activity, and matrix metalloproteinase 9 (MMP9) activity. Nanog-induced up- or down-regulated genes were comprehensively analyzed by transcriptome sequencing using Nanog+BL6 and wild-type BL6. Principally, up-regulated genes were involved in vesicle-aided glucose transport and oxidative phosphorylation, while down-regulated genes were associated with immunosuppression and apoptosis. A marked finding was that TGF-ß1 was down-regulated, because TGF-ß1 has been well discussed about its suppressive/progressive dual role in cancer. In vivo test showed that the number and volume of metastatic colonies of BL6 to lung were as high as 115 colonies/lung and 5.6 mm3/lung. Under this condition, Nanog overexpression caused a progressive effect (150 colonies/lung, p = 0.25; 9.2 mm3/lung, p = 0.13) rather than a suppressive effect on the metastasis. In this study, the effectiveness of Nanog overexpression in enhancing the metastatic potential of melanoma cell lines has been demonstrated for the first time.

Fluorescent Labeling of Connexin with As Complex and X-Y Coordinate Registration of Target Single Cells Based on a Triangle Standard Chip for the Image Analysis of Gap Junctional Communication.

Gap junction (GJ) research has entered a new stage focusing the concerted dynamic behavior of multiple isoforms of connexin (Cx) in the cell membrane, cytosolic vesicles, and space between them. To proceed with this research, imaging technologies are important. Here we describe two novel protocols for this purpose. At first, the adoption of a small motif of Cys-Cys-X-X-Cys-Cys as a visualization tag is described. An As complex, FlAsH, can bind to this tetra-Cys (TC) tag to form a fluorescent conjugate. Its introduction into the C-terminal of Cx43 is demonstrated. Next, a novel triangle chip for the accurate x-y registration is described. Target single cells of HeLa marked with a fluorescent dye can be easily recognized by electron microscopy based on this chip.

Effects of injured and dead cells of Escherichia coli on the colony-forming rate of live cells.

Osmotic stress-induced injured cells of Escherichia coli were prepared by sorting live cells onto tryptic soy agar (TSA) containing 10-50% sucrose. The time course of colony-forming rate (CFR%) was analyzed. A time delay in colony formation indicated a sublethal effect. The final CFR level at 24 h indicated the relative number of culturable cells irrespective of injury. A value of (100-CFR)% at 24 h indicated a lethal effect. When cells were grown on TSA containing 10% sucrose, the time delay was 4 h and the lethal effect was 4%. However, dead cells inhibited the growth of live cells. Physical contact with insoluble matter derived from dead cells or dead cells themselves might have caused growth inhibition. These findings highlight a novel perspective on colony count methods in practical situations, such as when sampling foods containing a high concentration of sucrose.

Monomorphic Epitheliotropic Intestinal T-cell Lymphoma Involving the Lung and Brain: A Rare Case Study.

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a primary intestinal T-cell lymphoma and other organ involvement is very rare. A rare case of MEITL involving the lung and brain is herein reported. The patient developed panperitonitis with a small intestinal perforation, and emergency surgery was performed. The pathological findings from the surgical specimens demonstrated atypical lymphoid cells which were positive for CD3, CD8, and CD56. Moreover, the pathological findings of lung specimens taken by bronchoscopy were consistent with those of the small intestine. It is therefore important to include the possibility of MEITL in the differential diagnosis of cancer patients.

Clinical significance of thyroid hormone and antibodies in patients with idiopathic interstitial pneumonia.

BACKGROUND: Hypothyroidism was recently reported to be common and to predict mortality in patients with idiopathic pulmonary fibrosis (IPF). In addition, a high prevalence of hypothyroidism was shown in patients with idiopathic pleuroparenchymal fibroelastosis. However, in idiopathic interstitial pneumonia (IIP), a clinical significance of thyroid function has not been clarified in detail. The goal of this study was to investigate the clinical significance of thyroid function and the presence of thyroid antibodies in IIP. METHODS: We have reviewed IIP patients, and analyzed the positivity of thyroid antibodies at first. Next, the relationship of clinical characteristics with thyroid function and the positivity of thyroid antibodies was analyzed. Lastly, the positivity of thyroid antibodies and other autoantibodies was evaluated. RESULTS: In IIP patients, thyroglobulin and thyroid peroxidase antibodies were positive in 17 and 16%, respectively, and 22% of patients had either or both antibodies. Subclinical and/or overt hypothyroidism was confirmed in 7% of IIP patients. The free thyrotropin level had a significant positive correlation with vital capacity and a significant negative correlation with the C-reactive protein and surfactant protein-A levels, and erythrocyte sedimentation ratio (ESR). In addition, autoantibodies suggestive of connective tissue diseases (CTDs) were positive in more than two thirds of IIP patients with the thyroid antibody, and the positive rate of antinuclear and proteinase-3 anti-neutrophil cytoplasmic antibodies was significantly higher in IIP patients with thyroid antibodies than those without the antibodies. CONCLUSIONS: Although thyroid dysfunction is not frequent, thyroid hormones and thyroid antibodies are possibly involved in the pathogenesis of IIP and their evaluation may be clinically useful to identify the clinical phenotype of IIP with autoimmune features.