Chlorhexidine possesses unique cytotoxic actions in rat thymic lymphocytes : Its relation with electrochemical property of membranes

Chlorhexidine (CHX) is an antibacterial agent used in various types of pharmaceutical products. Therefore, CHX is easily found around us. Owing to its positive charge, the electrochemical property of cell membranes was assumed to be a key point of cytotoxic action of CHX. Depolarization of membranes attenuated the cytotoxic action of CHX in rat thymic lymphocytes. CHX interfered with annexin V binding to membranes. Manipulations to induce exposure of phosphatidylserine on the outer membrane surface augmented the cytotoxic action of CHX, indicating that changes in the electrochemical property of membranes affected the cytotoxic action of CHX. Hence, CHX might kill cells physiologically undergoing apoptosis, resulting instead in necrotic cell death. However, the threshold CHX concentration in this in vitro study was slightly higher than blood CHX concentrations observed clinically. Therefore, these results may support the safety of CHX use although CHX possesses unique cytotoxic actions described in this study

Nonivamide, a natural analog of capsaicin, affects intracellular Ca2+ level in rat thymic lymphocytes

Effect of nonivamide, a natural analog of capsaicin, on intracellular Ca2+ level of rat thymocytes was examined using a flow-cytometric technique with appropriate fluorescent probes in order to further characterize the cytotoxicity because nonivamide can be used as an active intergradient of antifouling paints. Nonivamide at concentrations ranging from 30 μM to 300 μM significantly increased the intensity of Fluo-3 fluorescence. The potency of 100 μM nonivamide to increase the fluorescence was similar to that of 100 μM capsaicin. The increase in Fluo-3 fluorescence by 100 μM nonivamide was attenuated under an external Ca2+-free condition. Nonivamide at 100 μM also increased the intensity of Fluo-3 fluorescence in the continued presence of 100 μM capsaicin. It is suggested that nonivamide at high micromolar concentrations increases intracellular Ca2+ level via the activation of vanilloid receptors. Nonivamide concentrations (30 μM or more) that increase intracellular Ca2+ level in rat thymocytes are comparable to those in algal cells. However, it is something hard to argue the implications in environmental science because nonivamide doesn’t seem to be released into environment in such a high concentration, and because bioaccumulation of nonivamide has not been reported

Case report: Pathological complete response of pregnancy associated pulmonary enteric adenocarcinoma to chemoradiotherapy

Pulmonary enteric adenocarcinoma (PEAC) is a rare lung adenocarcinoma with morphological features similar to those of primary and metastatic colorectal adenocarcinoma. To date, only a few studies have reported the therapeutic effects of chemoradiotherapy on PEAC. This report describes the case of a 28-year-old woman with pregnancy-related PEAC who presented with left shoulder pain. A superior sulcus tumor was identified in the left thoracic cavity, and the biopsy indicated more than 50% intestinal differentiation components. Moreover, immunohistochemical staining revealed positive CDX2 and CK7 expression. Positron emission tomography-computed tomography, upper endoscopy, colonoscopy, and small intestinal capsule endoscopy revealed no gastrointestinal malignancies. The patient was diagnosed with locally advanced PEAC (clinical stage T4N0M0; stage IIIA). Therefore, the patient was treated with preoperative chemoradiotherapy and underwent gross total resection during surgery. Pathological evaluation of the specimen revealed no residual tumor, indicating that the chemoradiotherapy for PEAC was highly effective. One subsequent brain metastasis was also resected, and the patient has not experienced recurrence in 28 months since this resection and continues to be monitored regularly. This is the first pathologically confirmed report of the use of chemoradiotherapy (carboplatin [CBDCA] and paclitaxel [PTX]) for PEAC and its clinical efficacy. Unlike previous reports, the efficacy of this treatment is attributed to the use of PTX in preoperative chemotherapy and the p21− status of the patient, which may have increased sensitivity to chemoradiation therapy. Therefore, chemoradiotherapy (CBDCA + PTX) may be a viable treatment option for advanced intestinal lung adenocarcinoma

Zinc increases vulnerability of rat thymic lymphocytes to arachidonic acid under in vitro conditions

Previous studies on the cytotoxicity of arachidonic acid (ARA) elucidated the involvement of oxidative stress and Ca2+. In the present study, the Zn2+-related cytotoxicity of ARA was studied by a flow cytometric technique with appropriate fluorescent probes in rat thymocytes. Addition of 10 μM ZnCl2 enhanced the increase in cell lethality induced by 10 μM ARA. The removal of Zn2+ by Zn2+ chelators attenuated the ARA-induced increase in cell lethality. Thus, Zn2+ is suggested to be involved in ARA cytotoxicity. ARA at 3–10 μM elevated intracellular Zn2+ level. The Zn2+ chelators attenuated the ARA-induced increase in intracellular Zn2+ level while ARA significantly increased intracellular Zn2+ level in the presence of 3 μM ZnCl2, suggesting the involvement of external Zn2+. Zn2+ reportedly exerts cytotoxic action under oxidative stress induced by hydrogen peroxide, via an excessive increase in intracellular Zn2+ levels. Since ARA induces oxidative stress, the simultaneous administration of zinc and ARA may be harmful

Zinc-related actions of sublethal levels of benzalkonium chloride : Potentiation of benzalkonium cytotoxicity by zinc

Benzalkonium chloride (BZK) is a common preservative used in pharmaceutical and personal care products. ZnCl2 was recently reported to significantly potentiate the cytotoxicity of some biocidal compounds. In the present study, therefore, we compared the cytotoxic potency of BZK and then further studied the Zn2+-related actions of the most cytotoxic agent among BZK, using flow cytometric techniques with appropriate fluorescent probes in rat thymocytes. Cytotoxicity of benzylcetyldimethylammonium (BZK-C16) was more potent that those of benzyldodecyldimethylammonium and benzyldimethyltetradecylammonium. ZnCl2 (1–10 μM) significantly potentiated the cytotoxicity of BZK-C16 at a sublethal concentration (1 μM). The co-treatment of cells with 3 μM ZnCl2 and 1 μM BZK-C16 increased the population of both living cells with phosphatidylserine exposed on membrane surfaces and dead cells. BZK-C16 at 0.3–1.0 μM elevated intracellular Zn2+ levels by increasing Zn2+ influx, and augmented the cytotoxicity of 100 μM H2O2. Zn2+ is concluded to facilitate the toxicity of BZK. We suggest that the toxicity of BZK is determined after taking extracellular (plasma) and/or environmental Zn2+ levels into account

Effects of iguratimod on glucocorticoid-induced disorder of bone metabolism in vitro

Introduction: Glucocorticoids are widely used to treat various diseases including rheumatoid arthritis (RA); however, one of the most frequent and severe adverse effects is glucocorticoid-induced osteoporosis (GIOP). Iguratimod (IGU) is a novel conventional synthetic disease-modifying anti-rheumatic drug developed in Japan. The aim of this study is to investigate the effects of IGU on glucocorticoid-induced disorder of bone metabolism in vitro. Materials and methods: In osteoclastogenesis of mouse bone marrow-derived cells, tartrate-resistant acid phosphatase staining, resorption pit assay, western blotting, real-time polymerase chain reaction (PCR), and mRNA sequencing were performed. In osteoblastogenesis of MC3T3-E1 cells, alkaline phosphatase (ALP) staining and activity, alizarin red staining, and mRNA sequencing were performed, and real-time PCR and western blotting were conducted in MC3T3-E1 cells and murine osteocyte-like cell line MLO-Y4 cells. Results: IGU significantly suppressed a dexamethasone-induced increase in osteoclasts, differentiation, and bone resorption activity by inhibition of the receptor activator of the nuclear factor kappa-B (RANK)/tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)/nuclear factor kappa-B (NFκB)-p52 pathway. In MC3T3-E1 cells, IGU significantly upregulated dexamethasone-induced downregulation of ALP activity, bone mineralization, and osteoblast-related gene and protein expression. In MLO-Y4 cells, IGU significantly upregulated dexamethasone-induced downregulation of the gene expression of ALP and osteocalcin, and also downregulated receptor activator of NFκB ligand (RANKL)/osteoprotegerin gene expression ratio without dexamethasone. Conclusion: These results suggest that IGU may improve glucocorticoid-induced disorder of bone metabolism and may exhibit positive effects against GIOP associated with RA.This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1007/s00774-021-01206-5Miyama A., Ebina K., Hirao M., et al. Effects of iguratimod on glucocorticoid-induced disorder of bone metabolism in vitro. Journal of Bone and Mineral Metabolism 39, 639 (2021

A novel anti-TNF-α drug ozoralizumab rapidly distributes to inflamed joint tissues in a mouse model of collagen induced arthritis

In clinical studies, the next-generation anti-tumor necrosis factor-alpha (TNF-α) single domain antibody ozoralizumab showed high clinical efficacy shortly after the subcutaneous injection. To elucidate the mechanism underlying the rapid onset of the effects of ozoralizumab, we compared the biodistribution kinetics of ozoralizumab and adalimumab after subcutaneous injection in an animal model of arthritis. Alexa Fluor 680-labeled ozoralizumab and adalimumab were administered by subcutaneous injection once (2 mg/kg) at five weeks after induction of collagen-induced arthritis (CIA) in an animal arthritis model. The time-course of changes in the fluorescence intensities of the two compounds in the paws and serum were evaluated. The paws of the CIA mice were harvested at four and eight hours after the injection for fluorescence microscopy. Biofluorescence imaging revealed better distribution of ozoralizumab to the joint tissues than of adalimumab, as early as at four hours after the injection. Fluorescence microscopy revealed a greater fluorescence intensity of ozoralizumab in the joint tissues than that of adalimumab at eight hours after the injection. Ozoralizumab showed a significantly higher absorption rate constant as compared with adalimumab. These results indicate that ozoralizumab enters the systemic circulation more rapidly and is distributed to the target tissues earlier and at higher levels than conventional IgG antibodies. Our investigation provides new insight into the mechanism underlying the rapid onset of the effects of ozoralizumab in clinical practice.Oyama S., Ebina K., Etani Y., et al. A novel anti-TNF-α drug ozoralizumab rapidly distributes to inflamed joint tissues in a mouse model of collagen induced arthritis. Scientific Reports 12, 18102 (2022); https://doi.org/10.1038/s41598-022-23152-6

Basic fibroblast growth factor promotes meniscus regeneration through the cultivation of synovial mesenchymal stem cells via the CXCL6–CXCR2 pathway

Objective: To investigate the efficacy of basic fibroblast growth factor (bFGF) in promoting meniscus regeneration by cultivating synovial mesenchymal stem cells (SMSCs) and to validate the underlying mechanisms. Methods: Human SMSCs were collected from patients with osteoarthritis. Eight-week-old nude rats underwent hemi-meniscectomy, and SMSCs in pellet form, either with or without bFGF (1.0 × 106 cells per pellet), were implanted at the site of meniscus defects. Rats were divided into the control (no transplantation), FGF (−) (pellet without bFGF), and FGF (+) (pellet with bFGF) groups. Different examinations, including assessment of the regenerated meniscus area, histological scoring of the regenerated meniscus and cartilage, meniscus indentation test, and immunohistochemistry analysis, were performed at 4 and 8 weeks after surgery. Results: Transplanted SMSCs adhered to the regenerative meniscus. Compared with the control group, the FGF (+) group had larger regenerated meniscus areas, superior histological scores of the meniscus and cartilage, and better meniscus mechanical properties. RNA sequencing of SMSCs revealed that the gene expression of chemokines that bind to CXCR2 was upregulated by bFGF. Furthermore, conditioned medium derived from SMSCs cultivated with bFGF exhibited enhanced cell migration, proliferation, and chondrogenic differentiation, which were specifically inhibited by CXCR2 or CXCL6 inhibitors. Conclusion: SMSCs cultured with bFGF promoted the expression of CXCL6. This mechanism may enhance cell migration, proliferation, and chondrogenic differentiation, thereby resulting in superior meniscus regeneration and cartilage preservation.Goshima A., Etani Y., Hirao M., et al. Basic fibroblast growth factor promotes meniscus regeneration through the cultivation of synovial mesenchymal stem cells via the CXCL6–CXCR2 pathway. Osteoarthritis and Cartilage , (2023); https://doi.org/10.1016/j.joca.2023.07.010

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target