マクロファージはクロストーク機構により子宮内膜細胞を酸化ストレスから保護する

This aim of this study was to investigate whether macrophages protect endometriotic cells from oxidative injury and to elucidate the underlying mechanisms of any protection. Endometriotic cells cultured with or without differentiated macrophages (dTHP-1 cells) were treated with hydrogen peroxide (H2O2) or methemoglobin, a major component of hemoglobin species in endometriotic cyst fluid. Co-culture experiments, microarray analysis, screening and validation of differentially expressed genes (DEGs), cell proliferation and viability assays, and experiments using a specific inhibitor were conducted to investigate the functional cross-talk between endometriotic cells and macrophages. Microarray analysis revealed that endometriotic cells co-cultured with dTHP-1 differentially express several genes compared with monoculture. Quantitative enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis identified TGF-β1 as a promising candidate gene expressed in endometriotic cells co-cultured with dTHP-1 cells. TGF-β1 stimulated the expression of heme oxygenase-1 (HO-1) in dTHP-1 cells. HO-1 expression was increased in dTHP-1 cells co-cultured with endometriotic cells compared with the dTHP-1 monoculture. Both H2O2 and methemoglobin upregulated the expression of the HO-1 protein in the dTHP-1 monoculture; moreover, co-culture with endometriotic cells further enhanced HO-1 production. The co-culture with dTHP-1 protected endometriotic cells against oxidative injury. Blockade of HO-1 abolished the protective effects of macrophages. In an oxidative stress environment, TGF-β1 produced by endometriotic cells may protect against oxidative injury through the upregulation of macrophage-derived HO-1. The cross-talk between endometriotic cells and macrophages may contribute to the progression and pathogenesis of endometriosis.博士（医学）・甲第849号・令和4年9月28日Copyright © 2022, Society for Reproductive Investigation.© 2022 Springer Nature Switzerland AG.The version of record of this article, first published in Reproductive Sciences, is available online at Publisher's website: https://doi.org/10.1007/s43032-022-00890-6.発行元が定める登録猶予期間終了の後、本文を登録予定（2023.08

HNF-1β過剰発現を呈する卵巣明細胞癌においてGSK-3βは新たなシグナル伝達経路を介在する

Deubiquitinase USP28 is a target gene of the transcription factor HNF1 homeobox β (HNF-1β), which promotes the survival of ovarian clear cell carcinoma (OCCC) cell lines. However, the pharmacological inhibition of HNF-1β can cause several adverse effects as it is abundantly expressed in numerous organ systems, including the kidney, liver, pancreas and digestive tract. Therefore, small interfering RNA (siRNA) screening was performed in the current study to identify other potential downstream targets of the HNF-1β-mediated pathway. The results revealed that glycogen synthase kinase-3β (GSK-3β) may be a potential downstream target affecting cell viability. To further clarify the effects of GSK-3β, two human OCCC cell lines, TOV-21G (HNF-1β overexpressing line) and ES2 (HNF-1β negative) were transfected with siRNA targeting GSK-3β or control vectors. Loss-of-function studies using RNAi-mediated gene silencing indicated that HNF-1β facilitated GSK-3β expression, resulting in the loss of phosphorylated nuclear factor-κB (p-NFκB) and the reduction of TOV-21G cell proliferation. The cell proliferation assay also revealed that GSK-3β inhibitors rescued the effects of HNF-1β silencing on cell viability in a dose-dependent manner. Furthermore, the GSK-3β inhibitor, AR-A014418, effectively inhibited tumor cell proliferation in a xenograft mouse model. In conclusion and to the best of our knowledge, the current study was the first to determine that GSK-3β is a target gene of HNF-1β. In addition, the results of the present study revealed the novel HNF-1β-GSK-3β-p-NFκB pathway, occurring in response to DNA damage. Targeting this pathway may therefore represent a putative, novel, anticancer strategy in patients with OCCC.博士（医学）・甲第785号・令和3年3月15日Copyright: © Kawaharaet al. This is an open access article distributed under theterms of CreativeCommons Attribution License(https://creativecommons.org/licenses/by-nc-nd/4.0/)

Sh3bp2 Gain-Of-Function Mutation Ameliorates Lupus Phenotypes in B6.MRL-Faslpr Mice

SH3 domain-binding protein 2 (SH3BP2) is an adaptor protein that is predominantly expressed in immune cells, and it regulates intracellular signaling. We had previously reported that a gain-of-function mutation in SH3BP2 exacerbates inflammation and bone loss in murine arthritis models. Here, we explored the involvement of SH3BP2 in a lupus model. Sh3bp2 gain-of-function (P416R knock-in; Sh3bp2KI/+) mice and lupus-prone B6.MRL-Faslpr mice were crossed to yield double-mutant (Sh3bp2KI/+Faslpr/lpr) mice. We monitored survival rates and proteinuria up to 48 weeks of age and assessed renal damage and serum anti-double-stranded DNA antibody levels. Additionally, we analyzed B and T cell subsets in lymphoid tissues by flow cytometry and determined the expression of apoptosis-related molecules in lymph nodes. Sh3bp2 gain-of-function mutation alleviated the poor survival rate, proteinuria, and glomerulosclerosis and significantly reduced serum anti-dsDNA antibody levels in Sh3bp2KI/+Faslpr/lpr mice. Additionally, B220+CD4-CD8- T cell population in lymph nodes was decreased in Sh3bp2KI/+Faslpr/lpr mice, which is possibly associated with the observed increase in cleaved caspase-3 and tumor necrosis factor levels. Sh3bp2 gain-of-function mutation ameliorated clinical and immunological phenotypes in lupus-prone mice. Our findings offer better insight into the unique immunopathological roles of SH3BP2 in autoimmune diseases

Determination of the genetic structure of remnant Morus boninensis Koidz. trees to establish a conservation program on the Bonin Islands, Japan

BACKGROUND: Morus boninensis, is an endemic plant of the Bonin (Ogasawara) Islands of Japan and is categorized as "critically endangered" in the Japanese red data book. However, little information is available about its ecological, evolutionary and genetic status, despite the urgent need for guidelines for the conservation of the species. Therefore, we adopted Moritz's MU concept, based on the species' current genetic structure, to define management units and to select mother tree candidates for seed orchards. RESULTS: Nearly all individuals of the species were genotyped on the basis of seven microsatellite markers. Genetic diversity levels in putative natural populations were higher than in putative man-made populations with the exception of those on Otouto-jima Island. This is because a limited number of maternal trees are likely to have been used for seed collection to establish the man-made populations. A model-based clustering analysis clearly distinguished individuals into nine clusters, with a large difference in genetic composition between the population on Otouto-jima Island, the putative natural populations and the putative man-made populations. The Otouto-jima population appeared to be genetically differentiated from the others; a finding that was also supported by pairwise F(ST )and R(ST )analysis. Although multiple clusters were detected in the putative man-made populations, the pattern of genetic diversity was monotonous in comparison to the natural populations. CONCLUSION: The genotyping by microsatellite markers revealed strong genetic structures. Typically, artificial propagation of this species has ignored the genetic structure, relying only on seeds from Otouto-jima for replanting on other islands, because of a problem with inter-specific hybridization on Chichi-jima and Haha-jima Islands. However, this study demonstrates that we should be taking into consideration the genetic structure of the species when designing a propagation program for the conservation of this species

母体血中における分娩形式による分娩前後の、羊水塞栓症のバイオマーカーとなりうるSCC 抗原の大きな変化

Amniotic fluid embolism (AFE) is a serious disease in which amniotic fluid components enter the maternal systemic circulation, causing cardiopulmonary collapse in pregnant women. It has been previously reported that amniotic fluid contains extremely high levels of squamous cell carcinoma antigen (SCCA), and that pregnant women who do not survive due to AFE have high blood SCCA levels. The aim of the present study was to determine the possible mechanisms through which SCCA in amniotic fluid enters the maternal blood, as well as the potential origin of SCCA. The prospective study included a cohort of 464 women (339 normal vaginal deliveries, 97 cesarean deliveries without labor, and 28 cesarean deliveries with labor). The dynamic changes in maternal serum SCCA levels were determined before and after delivery in relation to the mode of delivery, and SCCA levels were measured in the placenta, fetal skin, amniotic fluid cell components, amniotic fluid and neonatal urine. Serial serum samples collected at the time of admission, at 2 h postpartum, and on postpartum day 3 were quantitatively measured for SCCA by enzyme‑linked immunosorbent assay. Amniotic fluid and neonatal urine SCCA levels were also measured. The protein expression of SCCA in the placenta and fetal skin was assessed by immunohistochemistry. In vaginal deliveries, there was a significant increase in serum SCCA levels from admission to 2 h postpartum, and SCCA levels decreased on postpartum day 3. In cesarean deliveries, the SCCA levels at the time of admission and 2 h postpartum did not differ. The SCCA levels were significantly higher in women who underwent vaginal deliveries compared to those that underwent cesarean deliveries without labor (P=0.033). Immunohistochemical staining revealed no SCCA expression in the placenta and fetal skin. The SCCA levels in neonatal urine immediately after birth were as high as those in the amniotic fluid, suggesting that SCCA may originate from fetal urine. The present study thus suggests that amniotic fluid SCCA levels, which may be derived from fetal urine, can enter the maternal circulation during vaginal delivery. The onset of labor and full cervical dilatation are the main causes of entry of amniotic fluid components into the maternal circulation.博士（医学）・甲第796号・令和3年6月25日Copyright: © Nakano et al. This is an open access article distributed under the terms of Creative Commons Attribution License (https://creativecommons.org/licenses/by-nc-nd/4.0/)

卵巣子宮内膜症性嚢胞の嚢胞液鉄濃度が不妊に及ぼす影響について

The causes of infertility in women with endometriosis may range from anatomical distortions to various pathophysiological disturbances. The aim of the present study was to examine the effects of the cyst fluid (CF) concentration of iron on infertility in patients with ovarian endometrioma (OMA). Patients with histologically confirmed OMA were enrolled at the Department of Obstetrics and Gynecology, Nara Medical University Hospital between 2013 and 2019. The patients were divided into 2 groups, namely women experiencing current infertility (infertile group) and those without complaints of infertility (non‑infertile group). There were 2 types of patients in the infertile group: Patients who failed to achieve a clinical pregnancy following ≥12 months of regular unprotected sexual intercourse and those who had already been treated at fertility clinics. The CF concentration of iron was measured by the inductively coupled plasma‑optical emission spectrometry (ICP‑OES) method. The clinical data were analyzed retrospectively. A total of 77 patients were enrolled in the present study. Among these, 32 (41.6%) patients had infertility. When compared with the non‑infertile women, the infertile women were significantly younger (median age, 35 years; range, 24‑47 years; vs. median age, 40 years; range, 21‑53 years; respectively; P=0.003). The CF concentrations of iron (median, 324.8 mg/l; range, 71.3‑1046.3 mg/l; vs. median, 226.5 mg/l; range, 65.3‑737.5 mg/l; respectively; P=0.019) were significantly higher in the infertile group compared with the non‑infertile group. Multivariate logistic regression analysis indicated that age at diagnosis (≤38 years), the CF concentrations of iron (>326.6 mg/l) and the infertility index (iron/age ratio, >8.37) were independent risk factors for endometriosis‑related infertility. Multivariate analysis revealed that age (HR, 6.44; 95% CI, 2.06‑20.12) and iron (HR, 4.90; 95% CI, 1.48‑16.22) were independent factors for the identification of patients with OMA who presented with a complaint of infertility. In addition, the infertility index (iron/age ratio, >8.37; HR, 4.85; 95% CI, 1.01‑23.27) was an important predictor of infertility. ROC curve analysis also revealed that the areas under the ROC (AUC) for age, iron and infertility index were 0.699, 0.666 and 0.731, respectively. On the whole, the findings of the present study demonstrate that age at diagnosis and the CF concentration of iron may be potentially effective markers for predicting infertility in women with OMA.博士（医学）・乙第1500号・令和3年3月15日Copyright © Nagayasuet al. This is an open access article distributed under theterms of CreativeCommons Attribution License(https://creativecommons.org/licenses/by-nc-nd/4.0/)

卵巣子宮内膜症性嚢胞の癌化におけるCD44v9と8-OHdGの発現

Aim: Expression of CD44 variant isoforms (CD44v) promotes the synthesis of reduced glutathione and contributes to reactive oxygen species defense through up-regulation of the intracellular antioxidant. The aim of the study was to investigate the expression of CD44v9 and oxidative DNA damage marker, 8-OHdG, in benign ovarian endometrioma (OE) and OE harboring clear cell carcinomas (CCC). Methods: A retrospective study was performed at the Department of Gynecology, Nara Medical University hospital from January 2006 to December 2012. Patients with histologically confirmed benign OE (n = 27) and OE harboring areas of CCC (n = 8) were selected. Tissue samples were immunohistochemically analyzed for the presence of CD44v9 and 8-OHdG using avidin-biotin complex method. Results: CD44v9 was located on the cell membrane of endometriotic epithelial cells and expressed in 88.9% (24/27) of benign OE tissues. Only 25.0% (2/8) of benign endometriotic lesions adjacent to CCC was found to stain weakly for CD44v9. Percentage of CD44v9 positive cells was 68.5 ± 20.2% (mean ± standard deviation) of benign OE, 16.7 ± 16.5% of CCC endometriotic tissue (P < 0.001). Compared to benign OE, CCC endometriotic tissue showed a significant increase in the proportion of 8-OHdG expression (77.3 ± 22.5% vs 94.9 ± 3.0%, P = 0.049). A significant negative correlation was observed between CD44v9 status and 8-OHdG nuclear expression (r = -0.458, P = 0.006). Conclusion: Alterations in CD44v9 and 8-OHdG may be associated with malignant transformation of benign OE.博士（医学）・乙第1452号・令和2年3月16日© 2019 Japan Society of Obstetrics and Gynecology.This is the peer reviewed version of the following article: [https://obgyn.onlinelibrary.wiley.com/doi/full/10.1111/jog.14093], which has been published in final form at [https://doi.org/10.1111/jog.14093]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

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