Ki-energy (Life-energy) Stimulates Osteoblastic Cells and Inhibits the Formation of Osteoclast-like Cells in Bone Cell Culture Models

Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese Ki expert, sent Ki-energy to the cells once for 5 or 10 min after they were seeded in culture dishes in the presence of 10% fetal bovine serum (FBS). They were incubated for 72 h and the cells were counted. The number in the dish with 10-min Ki-exposure was significantly greater than that in the control (P < 0.01 with n = 8). We performed a reverse transcription-polymerase chain reaction (RT–PCR) study using these cells, but the mRNA expressions did not change significantly. (ii) After cells were incubated for 72 h without Ki-exposure (in the presence of FBS), they were further cultured for 48 h (in the absence of FBS) to promote differentiation. At the beginning of the second culture stage, Ki was applied once for 10 min. After 48 h, RT–PCR was performed. The mRNA expressions which are related to bone mineralization, such as Runx2, α1(I) collagen, alkaline phosphatase and osteocalcin, increased significantly (P < 0.05 and n = 4 for all). For the bone resorption study, we used mouse marrow cultures, which can form osteoclast-like cells in the presence of (1–34) parathyroid hormone (PTH), and stimulate resorption. We exposed these cells to Ki-energy twice for the duration of 5 or 10 min on day 0 and day 4. On day 7, the cells were counted. The number of osteoclast-like cells in dishes with Ki exposure was significantly smaller than those in control dishes (P < 0.05 with n = 5). The difference between 5-min exposure and 10-min exposure was not statistically significant. All of our data suggest that the Ki-effect on osteoporosis should be further explored

子宮内膜症性嚢胞の悪性転化におけるHO-1発現マクロファージの特徴

Malignant transformation of endometriosis is a rare and still poorly understood event, but is associated with the distortion of the pro-oxidant and anti-oxidant balance. The aim of the present study was to quantify the numbers of macrophages polarized as M1 or M2 phenotypes and the expression of heme oxygenase (HO)-1 in tissue sections from patients with benign ovarian endometrioma (OE) and its malignant transformation (endometriosis-associated ovarian cancer, EAOC). We performed a retrospective study at the Department of Gynecology, Nara Medical University hospital from December 2012 to March 2015. This study included 53 patients with OE (n = 33) and EAOC (n = 20), and we evaluated polarized functional status of macrophages by immunohistochemical staining of CD68, CD11c, CD163 and HO-1. The number of the M1 phenotype (CD11c+, p = 0.001) and the M2 phenotype (CD163+, p = 0.009) was significantly lower in EAOC patients than in OE patients. Analyzing the correlations between the studied markers, the expression of CD68, CD11c, and CD163 proteins significantly correlated with each other (p < 0.001). The number of M2 phenotypes expressing HO-1 was significantly decreased in the EAOC group, compared with the OE group (P < 0.001), demonstrating sustained downregulation of an antioxidant marker, HO-1, in EAOC. In conclusion, reduced number of M2 macrophages expressing HO-1 may have an important role in promoting malignant transformation of OE.博士（医学）・乙第1434号・令和元年9月27日Copyright © 2018. Published by Elsevier GmbH

Long-Term Follow-Up after Surgical Management for Atypical Endometriosis: A Series of Nine Cases

Background and Objective: Atypical endometriosis is reported to possess a precancerous potential attributed to premalignant changes characterized by cytological atypia and architecture proliferation. Although the coexistence of atypical endometriosis and neoplasms has been reported, cases of atypical endometriosis transformation to carcinoma are rarely reported. The purpose of this case series was to evaluate the prognosis of atypical endometriosis. Subjects and Methods: Data from nine women who underwent surgical treatment including cystectomy and salpingo-oophorectomy with or without hysterectomy and diagnosed with atypical endometriosis was analyzed. Between January 2006 and January 2018, the clinical characteristics and prognosis of atypical endometriosis were evaluated. Results: During the follow-up period, eight of nine patients with atypical endometriosis did not develop malignant epithelial tumors, although one patient developed endometrioid carcinoma, grade 1, 48 months after her right laparoscopic cystectomy. The median overall survival period for all patients was 68 (range 13–131) months. Conclusion: When we encounter the cases of atypical endometriosis, it is necessary to consider the possibility of ovarian cancer and carefully follow those cases for long periods

子宮筋層の内外層に発生する子宮腺筋症おける、それぞれの組織学的特徴

OBJECTIVE: To estimate the phenotypic characterization of fibrotic process in adenomyosis occurring at the inner or the outer myometrium. METHODS: Eight cases of adenomyosis occurring at the inner myometrium (Subtype I) and 10 cases of adenomyosis occurring at the outer myometrium (Subtype II), and 10 normal counterparts were used in this study. A immunohistochemical study for smooth muscle cells (SMCs) was performed using cytoskeletal proteins, Type I and III collagen, TGF-β and its signaling molecules. RESULTS: An increased expression of Type I collagen was observed in the extracellular matrix of adenomyotic foci. In normal uteri, immunostaining of SMC differentiation marker proteins (Desmin, Smoothelin, Myosin heavy chain (MHC)) were absent or only found in low numbers at the inner myometrium, while all of these marker proteins were clearly stained at the outer myometrium. In both types of adenomyotic foci, Desmin, Smoothelin, and MHC commonly showed a negative staining at the adjacent area to the glands. A significant staining of Non-muscle myosin IIB, TGF-β, and phosphorylated TGF-β type I receptors were found only at the SMCs of Subtype II adenomyosis. The Smad3/2 ratio of Subtype II adenomyosis was significantly higher than that of Subtype I. CONCLUSIONS: The inner myometrium of normal uteri was composed of undifferentiated phenotypes of SMCs, while the outer myometrium was composed of terminally differentiated SMCs. Various fibrotic processes have been suggested in the development of uterine adenomyosis. Distinct expression patterns of fibrosis related proteins have been shown to be implicated with differences in the subtypes of adenomyosis.博士（医学）・甲第681号・平成30年3月15日Copyright: © 2017 Kishi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Vaporized Hydrogen Peroxide and Ozone Gas Synergistically Reduce Prion Infectivity on Stainless Steel Wire

Prions are infectious agents causing prion diseases, which include Creutzfeldt–Jakob disease (CJD) in humans. Several cases have been reported to be transmitted through medical instruments that were used for preclinical CJD patients, raising public health concerns on iatrogenic transmissions of the disease. Since preclinical CJD patients are currently difficult to identify, medical instruments need to be adequately sterilized so as not to transmit the disease. In this study, we investigated the sterilizing activity of two oxidizing agents, ozone gas and vaporized hydrogen peroxide, against prions fixed on stainless steel wires using a mouse bioassay. Mice intracerebrally implanted with prion-contaminated stainless steel wires treated with ozone gas or vaporized hydrogen peroxide developed prion disease later than those implanted with control prion-contaminated stainless steel wires, indicating that ozone gas and vaporized hydrogen peroxide could reduce prion infectivity on wires. Incubation times were further elongated in mice implanted with prion-contaminated stainless steel wires treated with ozone gas-mixed vaporized hydrogen peroxide, indicating that ozone gas mixed with vaporized hydrogen peroxide reduces prions on these wires more potently than ozone gas or vaporized hydrogen peroxide. These results suggest that ozone gas mixed with vaporized hydrogen peroxide might be more useful for prion sterilization than ozone gas or vaporized hydrogen peroxide alone

The group A Streptococcus interleukin-8 protease SpyCEP promotes bacterial intracellular survival by evasion of autophagy

Autophagy serves an innate immune function in defending the host against invading bacteria, including group A Streptococcus (GAS). Autophagy is regulated by numerous host proteins, including the endogenous negative regulator calpain, a cytosolic protease. Globally disseminated serotype M1T1 GAS strains associated with high invasive disease potential express numerous virulence factors and resist autophagic clearance. Upon in vitro infection of human epithelial cell lines with representative wild-type GAS M1T1 strain 5448 (M1.5448), we observed increased calpain activation linked to a specific GAS virulence factor, the IL-8 protease SpyCEP. Calpain activation inhibited autophagy and decreased capture of cytosolic GAS in autophagosomes. In contrast, the serotype M6 GAS strain JRS4 (M6.JRS4), which is highly susceptible to host autophagy-mediated killing, expresses low levels of SpyCEP and does not activate calpain. Overexpression of SpyCEP in M6.JRS4 stimulated calpain activation, inhibited autophagy and significantly decreased bacterial capture in autophagosomes. These paired loss- and gain-of-function studies reveal a novel role for the bacterial protease SpyCEP in enabling GAS M1 evasion of autophagy and host innate immune clearance

microRNA-345の過剰発現は、MUC1およびTJP2の発現を抑制することにより、膵管腺癌細胞株の浸潤能に影響を及ぼす

The majority of pancreatic carcinomas are pancreatic ductal adenocarcinomas (PDAC), and the presence of non-invasive pancreatic intraepithelial neoplasia or intraductal papillary mucinous neoplasm, as an associated lesion, is considered important. These microscopic hyperplastic or grossly papillomatous lesions exhibit varying degrees of morphological atypia and may develop into invasive carcinomas. In this study, we investigated whether mucin-1 (MUC1) is involved in the progression of pancreatic carcinoma and examined the mechanisms by which microRNAs regulate MUC1 expression in vitro. In PDAC cell lines, suppression of MUC1 expression reduced cell proliferation and invasion; PDAC cell lines transfected with an miR-345 precursor suppressed the expression of MUC1, and reduced cell proliferation and invasion. Tight junction protein 2 (TJP2), a putative target of miR-345, is regulated by MUC1. The suppression of TJP2 expression reduced cell proliferation by inducing apoptosis. These results suggest that MUC1 and TJP2, the putative target molecules of miR-345, are critical in maintaining the invasive potential of pancreatic carcinoma cells, and regulating their expression may prevent the progression of non-invasive pancreatic intraductal lesions to invasive carcinomas. This study provides new insights for the development of novel molecular targeted therapies for pancreatic carcinomas.博士（医学）・甲第866号・令和5年3月15

妊娠高血圧症候群におけるRAGEの関与

Preeclampsia/hypertensive disorders of pregnancy (PE/HDP) is a serious and potentially life-threatening disease. Recently, PE/HDP has been considered to cause adipose tissue inflammation, but the detailed mechanism remains unknown. We exposed human primary cultured adipocytes with serum from PE/HDP and healthy controls for 24 h, and analyzed mRNA expression of several adipokines, cytokines, and ligands of the receptor for advanced glycation endproducts (RAGE). We found that the mRNA levels of interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), high mobility group box 1 (HMGB1), and RAGE were significantly increased by the addition of PE/HDP serum. Among RAGE ligands, advanced glycation endproducts (AGE) and HMGB1 increased mRNA levels of IL-6 and CCL2 in SW872 human adipocytes and mouse 3T3-L1 cells. The introduction of small interfering RNA for RAGE (siRAGE) into SW872 cells abolished the AGE- and HMGB1-induced up-regulation of IL-6 and CCL2. In addition, lipopolysaccharide (LPS), a ligand of RAGE, increased the expression of IL-6 and CCL2 and siRAGE attenuated the LPS-induced expression of IL-6 and CCL2. These results strongly suggest that the elevated AGE, HMGB1, and LPS in pregnant women up-regulate the expression of IL-6 and CCL2 via the RAGE system, leading to systemic inflammation such as PE/HDP.博士（医学）・乙第1451号・令和2年3月16日© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

間欺的低酸素はヒト神経細胞においてGATA転写因子を介してPOMCとCARTのmRNAを増加させる

Sleep apnea syndrome (SAS) is characterized by intermittent hypoxia (IH) during sleep. SAS and obesity are strongly related to each other. Here, we investigated the effect of IH on the expression of major appetite regulatory genes in human neuronal cells. We exposed NB-1, SH-SY5Y, and SK-N-SH human neuronal cells to IH (64 cycles of 5 min hypoxia and 10 min normoxia), normoxia, or sustained hypoxia for 24 h and measured the mRNA levels of proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), galanin, galanin-like peptide, ghrelin, pyroglutamylated RFamide peptide, agouti-related peptide, neuropeptide Y, and melanocortin 4 receptor by real-time RT-PCR. IH significantly increased the mRNA levels of POMC and CART in all the neuronal cells. Deletion analysis revealed that the -705 to -686 promoter region of POMC and the -950 to -929 region of CART were essential for the IH-induced promoter activity. As possible GATA factor binding sequences were found in the two regions, we performed real-time RT-PCR to determine which GATA family members were expressed and found that GATA2 and GATA3 mRNAs were predominantly expressed. Therefore, we introduced siRNAs against GATA2 and GATA3 into NB-1 cells and found that GATA2 and GATA3 siRNAs abolished the IH-induced up-regulation of both POMC and CART mRNAs. These results indicate that IH stress up-regulates the mRNA levels of anorexigenic peptides, POMC and CART, in human neuronal cells via GATA2 and GATA3. IH can have an anorexigenic effect on SAS patients through the transcriptional activation of POMC and CART in the central nervous system.博士（医学）・甲第685号・平成30年6月27日© 2017 Elsevier Ltd. All rights reserved

乳腺腺様嚢胞癌においてサイトケラチン5/6の腺腔形成細胞の染色性は類似病変との鑑別に有用である

Adenoid cystic carcinoma (AdCC) of the breast is an uncommon but distinct neoplasm composed of a dual cell population polarized around true glandular (luminal) spaces and pseudolumina. The aim of this study was to clarify whether various immunohistochemical markers (CK7, EMA, CD117, p63, calponin, CD10, S100, CK5/6, CK14, vimentin, and type IV collagen) can distinguish between the two cell types in classical AdCC (n = 14) and in collagenous spherulosis (n = 5). The sensitivity and specificity of these 11 markers to distinguish luminal from abluminal cells were evaluated using a curve created by plotting the true-positive rate (sensitivity) against the false-positive rate (1 - specificity) at threshold settings of 0, 10, 50, and 70 %. The most sensitive and specific markers for luminal cells in AdCC were CK7 and EMA; those for abluminal cells were type IV collagen, p63, and vimentin. CD10 and S100 did not act as abluminal markers in AdCC. CK5/6, one of the basal/myoepithelial markers, was expressed more frequently in luminal than in abluminal cells of AdCC. Thus, CK5/6 immunostaining resulted in a reverse expression pattern, analogous to what we recently documented in clear cells in mammary adenomyoepithelioma. In conclusion, compared with myoepithelial/abluminal cells of normal breast or collagenous spherulosis, the neoplastic abluminal cells of classical AdCC are characterized by enhanced vimentin and attenuated CD10 and S100. Furthermore, the luminal cells of AdCC show a unique aberrant staining pattern for CK5/6 that may aid in the differential diagnosis.博士（医学）・乙第1389号・平成28年11月24日© Springer-Verlag Berlin Heidelberg 2016The final publication is available at Springer via http://dx.doi.org/10.1007/s00428-016-1963-