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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

INTRODUCTION: Liquid-based cytology (LBC)-fixed samples can be used for preparing multiple specimens of the same quality and for immunocytochemistry (ICC); however, LBC fixing solutions affect immunoreactivity. Therefore, in this study, we examined the effect of LBC fixing solutions on immunoreactivity. METHODS: Samples were cell lines, and specimens were prepared from cell blocks of 10% neutral buffered formalin (NBF)-fixed samples and the four types of LBC-fixed samples: PreservCyt®, CytoRich™ Red, CytoRich™ Blue, and TACAS™ Ruby, which were post-fixed with NBF. ICC was performed using 24 different antibodies, and immunocytochemically stained specimens were analyzed for the percentage of positive cells. RESULTS: Immunoreactivity differed according to the type of antigen detected. For nuclear antigens, the highest percentage of positive cells of Ki-67, WT-1, ER, and p63 was observed in the NBF-fixed samples, and the highest percentage of positive cells of p53, TTF-1, and PgR was observed in the TACAS™ Ruby samples. For cytoplasmic antigens, the percentage of positive cells of CK5/6, Vimentin, and IMP3 in LBC-fixed samples was higher than or similar to that in NBF-fixed samples. The percentage of positive cells of CEA was significantly lower in CytoRich™ Red and CytoRich™ Blue samples than in the NBF-fixed sample (p < 0.01). Among the cell membrane antigens, the percentage of positive cells of Ber-EP4, CD10, and D2-40 was the highest in NBF-fixed samples and significantly lower in CytoRich™ Red and CytoRich™ Blue samples than that in NBF-fixed samples (p < 0.01). The NBF-fixed and LBC-fixed samples showed no significant differences in the percentage of positive cells of CA125 and EMA. DISCUSSION/CONCLUSION: ICC using LBC-fixed samples showed the same immunoreactivity as NBF-fixed samples when performed on cell block specimens post-fixed with NBF. The percentage of positive cells increased or decreased based on the type of fixing solution depending on the amount of antigen in the cells. Further, the detection rate of ICC with LBC-fixed samples varied according to the type of antibody and the amount of antigen in the cells. Therefore, we propose that ICC using LBC-fixed samples, including detection methods, should be carefully performed.

DOI： 10.1159/000526131

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Phosphatidylinositol 3-kinase (PI3K) signaling promotes the differentiation and proliferation of regulatory B (Breg) cells, and the lipid phosphatase phosphatase and tensin homolog deleted on chromosome 10 (PTEN) antagonizes the PI3K-Akt signaling pathway. We previously demonstrated that cardiac Akt activity is increased and that restraint stress exacerbates hypertension and both heart and adipose tissue (AT) inflammation in DS/obese rats, an animal model of metabolic syndrome (MetS). We here examined the effects of restraint stress and pharmacological inhibition of PTEN on heart and AT pathology in such rats. Nine-week-old animals were treated with the PTEN inhibitor bisperoxovanadium-pic [bpV(pic)] or vehicle in the absence or presence of restraint stress for 4 weeks. BpV(pic) treatment had no effect on body weight or fat mass but attenuated hypertension in DS/obese rats subjected to restraint stress. BpV(pic) ameliorated left ventricular (LV) inflammation, fibrosis, and diastolic dysfunction as well as AT inflammation in the stressed rats. Restraint stress reduced myocardial capillary density, and this effect was prevented by bpV(pic). In addition, bpV(pic) increased the proportions of Breg and B-1 cells as well as reduced those of CD8+ T and B-2 cells in AT of stressed rats. Our results indicate that inhibition of PTEN by bpV(pic) alleviated heart and AT inflammation in stressed rats with MetS. These positive effects of bpV(pic) are likely due, at least in part, to a reduction in blood pressure, an increase in myocardial capillary formation, and an altered distribution of immune cells in fat tissue that result from the activation of PI3K-Akt signaling.

DOI： 10.14814/phy2.15165

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INTRODUCTION: Liquid-based cytology (LBC) is increasingly used for nongynecologic applications. However, the cytological preparation of LBC specimens is influenced by the processing technique and the preservative used. In this study, the influence of the processing techniques and preservatives on cell morphology was examined mathematically and statistically. METHODS: Cytological specimens were prepared using the ThinPrep (TP), SurePath (SP), and AutoSmear methods, with 5 different preservative solutions. The cytoplasmic and nuclear areas of Papanicolaou-stained specimens were measured for all samples. RESULTS: The cytoplasmic and nuclear areas were smaller in cells prepared using the 2 LBC methods, compared to that prepared using the AutoSmear method, irrespective of the preservative used. The cytoplasmic and nuclear areas of cells prepared using the SP method were smaller than those of cells prepared using the TP method, irrespective of the preservative used. There were fewer differences among the cytoplasmic areas of cells prepared with different preservative solutions using the TP method; however, the cytoplasmic areas of cells prepared using the SP method changed with the preservative solution used. CONCLUSIONS: The most significant difference affecting the cytoplasmic and nuclear areas was the processing technique. The TP method increased the cytoplasmic and nuclear areas, while the methanol-based PreservCyt solution enabled the highest enlargement of the cell. LBC is a superior preparation technique for standardization of the specimens. Our results offer a better understanding of methods suitable for specimen preparation for developing precision AI-based diagnosis in cytology.

DOI： 10.1159/000519335

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INTRODUCTION: Deep learning is a subset of machine learning that has contributed to significant changes in feature extraction and image classification and is being actively researched and developed in the field of cytopathology. Liquid-based cytology (LBC) enables standardized cytological preparation and is also applied to artificial intelligence (AI) research, but cytological features differ depending on the LBC preservative solution types. In this study, the relationship between cell detection by AI and the type of preservative solution used was examined. METHODS: The specimens were prepared from five preservative solutions of LBC and stained using the Papanicolaou method. The YOLOv5 deep convolutional neural network algorithm was used to create a deep learning model for each specimen, and a BRCPT model from five specimens was also created. Each model was compared to the specimen types used for detection. RESULTS: Among the six models, a difference in the detection rate of approximately 25% was observed depending on the detected specimen, and within specimens, a difference in the detection rate of approximately 20% was observed depending on the model. The BRCPT model had little variation in the detection rate depending on the type of the detected specimen. CONCLUSIONS: The same cells were treated with different preservative solutions, the cytologic features were different, and AI clarified the difference in cytologic features depending on the type of solution. The type of preservative solution used for training and detection had an extreme influence on cell detection using AI. Although the accuracy of the deep learning model is important, it is necessary to understand that cell morphology differs depending on the type of preservative solution, which is a factor affecting the detection rate of AI.

DOI： 10.1159/000526098

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Language：English   Publisher：Japanese Pharmacological Society  

<p>Neonicotinoid insecticides and organophosphorus (OP) insecticides that act on the cholinergic nervous system of insects, are major insecticides used in agriculture throughout the world. The effects of neonicotinoid insecticides and OP insecticides cause overstimulation and desensitization in the nervous system by interacting with the nicotinic acetylcholine receptors (nAChRs) and inhibiting acetylcholine-esterase, respectively. The immunotoxicity of OP insecticides to human has been reported, including exacerbation of inflammation and asthma, and we have reported that diazinon, one of OP insecticides, modulated macrophage functions. Although neonicotinoid insecticides are thought to be relatively low toxic for human compared to OP insecticides due to the low affinity of mammalian nAChRs, less is known about the exact effects of neonicotinoid insecticides. </p><p>　In this study, we investigated the effects of neonicotinoid insecticides and OP insecticides on macrophage functions, such as cytokine production, the levels of cell-surface molecule expression, and phagocytosis in RAW264.7 cells. We also investigated the induction of autophagy by neonicotinoid insecticides and OP insecticides.</p>

DOI： 10.1254/jpssuppl.wcp2018.0_po3-13-24

CiNii Research

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Toxicology  

Diazinon is an organophosphorus (OP) insecticide and is widely used not only in agriculture but also homes and garden in Japan. Diazinon has been reported to increase TNF-α production in rat serum and brain, suggesting that it can modify the proinflammatory response. In this study, we investigated the effects of diazinon on macrophage functions, such as cytokine production, reactive oxygen species (ROS) generation, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) expressions, cell-surface molecule expressions, and phagocytosis in RAW264.7 cells. In RAW264.7 cells, diazinon induced the production of TNF-α and IL-6. Diazinon induced ROS generation and the expressions of COX-2, iNOS, and cell-surface molecules CD40, CD86, and MHC class II, but reduced phagocytic activity in RAW264.7 cells. ERK and p38, but not JNK and p65 were involved in diazinon-induced IL-6 expression in RAW264.7 cells. We also examined these proinflammatory responses in bone marrow-derived macrophages (BMDM) and bronchoalveolar lavage fluid (BALF) cells. These results suggested that diazinon can activate macrophages and enhance inflammatory responses.

DOI： 10.1016/j.tox.2017.01.014

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Language：Japanese  

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Language：English   Publisher：Pharmacology  

Respiratory viral infections that cause chronic airway and lung disease can result in the activation of the innate immune response. Alveolar macrophages (AMs), one of the first lines of defense in the lung, are abundantly located in alveoli and the respiratory tract. Flavonoids found in fruits and vegetables exhibit cytoprotective effects on various cell types. In this study, we investigated the effect of quercetin on activation of AMs that had been exposed to imiquimod, a ligand of Toll-like receptor (TLR) 7. In both a mouse AM cell line (AMJ2-C11 cells) and mouse bronchoalveolar fluid cells, we demonstrated that quercetin attenuated TLR7-induced the expression of TNF-α and IL-6. In AMJ2-C11 cells, quercetin also attenuated the TLR7-induced CD40 expression; attenuated the translocation of p65; induced translocation of Nrf2 from cytosol to nucleus; and induced heme oxygenase (HO)-1 expression. Notably, tin protoporphyrin IX (SnPP), an inhibitor of HO-1, also attenuated TLR7-induced transcription of the TNF-α and IL-6 genes, suggesting that the effect of quercetin is mediated by HO-1. These results suggest that dietary supplementation with quercetin may have efficacy in the treatment of respiratory viral infection.

DOI： 10.1159/000438993

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Cellular Biochemistry  

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme-free globin (Apo Hb), induced IDO expression in bone marrow-derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of IκBα. Hb-induced IDO expression was inhibited by inhibitors of PI3-kinase (PI3K), PKC and nuclear factor (NF)-κB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb-induced IDO expression was inhibited by anti-oxidant N-acetyl-L-cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3-amino-1,2,4-triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O2-, H2O2, and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF-κB through the PI3K-PKC-ROS and PI3K-Akt pathways is required for the Hb-induced IDO expression in BMDCs. © 2009 Wiley-Liss, Inc.

DOI： 10.1002/jcb.22308

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Language：English   Publisher：Immunology Letters  

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway in some dendritic cells (DC) such as plasmacytoid DC (pDC) regulates T-cell responses. It is unclear whether bone marrow-derived myeloid DC (BMDC) express functional IDO. The IDO expression was examined in CD11c+CD11b+ BMDC differentiated from mouse bone marrow cells using GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with the production of nitric oxide (NO) in BMDC in cultures for 24 h. In the enzyme assay using cellular extracts of BMDC, the IDO activity of BMDC stimulated with CpG was enhanced by the addition of a NO synthase (NOS) inhibitor, suggesting that IDO activity was suppressed by NO production. On the other hand, the concentration of Kyn in the culture supernatant of BMDC was not increased by stimulation with CpG. Exogenously added Kyn was taken up by BMDC independently of CpG stimulation and NO production, and the uptake of Kyn was inhibited by a transport system L-specific inhibitor or high concentrations of tryptophan. The uptake of tryptophan by BMDC was markedly lower than that of Kyn. In conclusion, IDO activity in BMDC is down-regulated by NO production, whereas BMDC strongly take up exogenous Kyn. © 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2007.11.016

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Event date： 2017.3

Language：Japanese   Presentation type：Poster presentation  

Venue：長崎ブリックホール、長崎新聞文化ホール　アストピア   Country：Japan  

Event date： 2017.3

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Event date： 2016.8 - 2016.9

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Venue： 神戸常磐大学   Country：Japan  

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Country：Australia  

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Country：Australia  

Event date： 2016.7

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Venue：近畿大学11月ホール   Country：Japan  

Event date： 2015.5

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Venue：グランドプリンスホテル新高輪国際館パミール   Country：Japan  

Event date： 2014.8

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Venue：大田区産業プラザ   Country：Japan  

Event date： 2014.8

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Venue：大田区産業プラザ   Country：Japan  

Event date： 2014.7

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Country：Japan  

Event date： 2014.7

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Event date： 2014.6

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Venue：福岡ソフトリサーチパーク（SRP）センタービル   Country：Japan  

Event date： 2013.8

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Venue：MiCo Milano Congressi   Country：Italy  

Event date： 2013.8

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Venue：MiCo Milano Congressi   Country：Italy  

Event date： 2013.8

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Venue：MiCo Milano Congressi   Country：Italy  

Event date： 2013.7

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Venue：神奈川   Country：Japan  

Event date： 2012.3

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Venue：北海道   Country：Japan  

Event date： 2008.12

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Venue：京都   Country：Japan  

Event date： 2008.12

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Venue：岡山   Country：Japan