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Currently there is no effective treatment available for osteoarthritis (OA). We have recently developed Kyoto University Substances (KUSs), ATPase inhibitors specific for valosin-containing protein (VCP), as a novel class of medicine for cellular protection. KUSs suppressed intracellular ATP depletion, endoplasmic reticulum (ER) stress, and cell death. In this study, we investigated the effects of KUS121 on chondrocyte cell death. In cultured chondrocytes differentiated from ATDC5 cells, KUS121 suppressed the decline in ATP levels and apoptotic cell death under stress conditions induced by TNFα. KUS121 ameliorated TNFα-induced reduction of gene expression in chondrocytes, such as Sox9 and Col2α. KUS121 also suppressed ER stress and cell death in chondrocytes under tunicamycin load. Furthermore, intraperitoneal administration of KUS121 in vivo suppressed chondrocyte loss and proteoglycan reduction in knee joints of a monosodium iodoacetate-induced OA rat model. Moreover, intra-articular administration of KUS121 more prominently reduced the apoptosis of the affected chondrocytes. These results demonstrate that KUS121 protects chondrocytes from stress-induced cell death in vitro and in vivo, and indicate that KUS121 is a promising novel therapeutic agent to prevent the progression of OA.

In glaucoma, retinal ganglion cells degenerate progressively, leading to visual field loss and blindness. Presently, the only treatment strategy for glaucoma is lowering the intraocular pressure. However, there are cases in which patients develop progressive visual field loss even though their intraocular pressures are within normal ranges. Therefore, the development of novel therapeutic strategies is an urgent endeavor. Besides high intraocular pressure, several other factors have been proposed to be associated with glaucoma progression, e.g., myopia, blood flow impairment, and amyloid β accumulation. We have previously reported that hop flower extracts possess γ-secretase inhibitory activities and reduce amyloid β deposition in the brains of Alzheimer’s disease model mice. In the current study, we showed that administration of hop flower extracts to glutamate-aspartate transporter (GLAST) knockout mice, the glaucoma model mice, attenuated glaucomatous retinal ganglion cell degeneration. Preservation of retinal ganglion cells in hop flower extract-administered mice was confirmed using optical coherence tomography, confocal scanning laser ophthalmoscopy, and retinal flatmount and histological evaluations. Hop flower extracts are, therefore, deemed a possible candidate as a novel therapeutic agent to treat glaucoma.

Keratoconus is a common ocular disorder that causes progressive corneal thinning and is the leading indication for corneal transplantation. Central corneal thickness (CCT) is a highly heritable characteristic that is associated with keratoconus. In this two-stage genome-wide association study (GWAS) of CCT, we identified a locus for CCT, namely STON2 rs2371597 (P = 2.32 × 10−13), and confirmed a significant association between STON2 rs2371597 and keratoconus development (P = 0.041). Additionally, strong STON2 expression was observed in mouse corneal epithelial basal cells. We also identified SMAD3 rs12913547 as a susceptibility locus for keratoconus development using predictive analysis with IBM’s Watson question answering computer system (P = 0.001). Further GWAS analyses combined with Watson could effectively reveal detailed pathways underlying keratoconus development., Yoshikatsu Hosoda et al. study the genetic basis for central corneal thickness (CCT) that is associated with keratoconus. They identify two susceptibility loci, STON2 rs2371597 and SMAD3 rs12913547, using two-step genome-wide association study (GWAS) and predictive analysis with IBM’s Watson question answering computer system, respectively.

In glaucoma, retinal ganglion cells are damaged, leading to the progressive constriction of the visual field. We have previously shown that the valosin-containing protein (VCP) modulators, Kyoto University Substance (KUS)121 and KUS187, prevent the death of retinal ganglion cells in animal models of glaucoma, including the one generated by N-methyl-D-aspartate (NMDA)-induced neurotoxicity. KUSs appeared to avert endoplasmic reticulum (ER) stress by maintaining ATP levels, resulting in the protection of ganglion cells from cell death. To further elucidate the protective mechanisms of KUSs, we examined gene expression profiles in affected ganglion cells. We first injected KUS-treated mice with NMDA and then isolated the affected retinal ganglion cells using fluorescence-activated cell sorting. Gene expression in the cells was quantified using a next-generation sequencer. Resultantly, we found that KUS121 upregulated several genes involved in energy metabolism. In addition, we observed the upregulation of Zfp667, which has been reported to suppress apoptosis-related genes and prevent cell death. These results further support the suitability of KUS121 as a therapeutic drug in protecting retinal ganglion cells in ophthalmic disorders, such as glaucoma.

We have established a stepwise method to differentiate induced pluripotent stem cells (iPSCs) into retinal pigment epithelium (RPE) (iPSC-RPE), which enables efficient isolation and purification of patient-derived iPSC-RPE cells with high quality. Here, we describe in detail the process of differentiating iPSCs into iPSC-RPE.

We have previously shown that maintenance of ATP levels is a promising strategy for preventing neuronal cell death, and that branched chain amino acids (BCAAs) enhanced cellular ATP levels in cultured cells and antagonized cell death. BCAAs attenuated photoreceptor degeneration and retinal ganglion cell death in rodent models of retinal degeneration or glaucoma. This study aimed to elucidate the mechanisms through which BCAAs enhance ATP production.Intracellular ATP concentration was measured in HeLa cells under glycolysis and citric acid cycle inhibited conditions. Next, glucose uptake was quantified in HeLa cells and in 661W retinal photoreceptor-derived cells under glycolysis inhibition, endoplasmic reticulum stress, and glucose transporters (GLUTs) inhibited conditions, by measuring the fluorescence of fluorescently labeled deoxy-glucose analog using flow cytometry. Then, the intracellular behavior of GLUT1 and GLUT3 were observed in HeLa or 661W cells transfected with enhanced green fluorescent protein-GLUTs.BCAAs recovered intracellular ATP levels during glycolysis inhibition and during citric acid cycle inhibition. BCAAs significantly increased glucose uptake and recovered decreased glucose uptake induced by endoplasmic reticulum stress or glycolysis inhibition. However, BCAAs were unable to increase intracellular ATP levels or glucose uptake when GLUTs were inhibited. Fluorescence microscopy revealed that supplementation of BCAAs enhanced the translocation of GLUTs proteins to the plasma membrane over time.BCAAs increase ATP production by promoting glucose uptake through promotion of glucose transporters translocation to the plasma membrane. These results may help expand the clinical application of BCAAs in retinal neurodegenerative diseases, such as glaucoma and retinal degeneration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

We have established a stepwise method to differentiate induced pluripotent stem cells (iPSCs) into retinal pigment epithelium (RPE) (iPSC-RPE), which enables efficient isolation and purification of patient-derived iPSC-RPE cells with high quality. Here, we describe in detail the process of differentiating iPSCs into iPSC-RPE.

Post-traumatic osteoarthritis (PTOA) is a major cause which hinders patients from the recovery after intra-articular injuries or surgeries. Currently, no effective treatment is available. In this study, we showed that inhibition of the acute stage chondrocyte death is a promising strategy to mitigate the development of PTOA. Namely, we examined efficacies of Kyoto University Substance (KUS) 121, a valosin-containing protein modulator, for PTOA as well as its therapeutic mechanisms. In vivo, in a rat PTOA model by cyclic compressive loading, intra-articular treatments of KUS121 significantly improved the modified Mankin scores and reduced damaged-cartilage volumes, as compared to vehicle treatment. Moreover, KUS121 markedly reduced the numbers of TUNEL-, CHOP-, MMP-13-, and ADAMTS-5-positive chondrocytes in the damaged knees. In vitro, KUS121 rescued human articular chondrocytes from tunicamycin-induced cell death, in both monolayer culture and cartilage explants. It also significantly downregulated the protein or gene expression of ER stress markers, proinflammatory cytokines, and extracellular-matrix-degrading enzymes induced by tunicamycin or IL-1β. Collectively, these results demonstrated that KUS121 protected chondrocytes from cell death through the inhibition of excessive ER stress. Therefore, KUS121 would be a new, promising therapeutic agent with a protective effect on the progression of PTOA., 京大開発の薬剤「KUS121」の変形性膝関節症への効果を確認 --外傷性変形性関節症の治療薬として臨床応用へ--. 京都大学プレスリリース. 2020-12-17.

Introduction This study was designed to identify the minimal and necessary cell morphologies to be considered for high-precision diagnosis of high-grade urothelial carcinoma (HGUC) in a routine urinary cytology practice. Materials and methods We included 338 urine cytology specimens from 11 medical facilities in Japan. Six experts evaluated these Papanicolaou-stained specimens using their own diagnostic criteria to categorize them within an initial 4-tiered classification system. Of the 338 cases, 70 HGUC and 32 benign cases (with a complete consensus diagnosis of 6 experts) were included for the analysis. Two of the cytologists evaluated the specimens for 20 specific cellular features. The results were analyzed using a contingency table and by discriminant analysis. Results Of the original 338 cases, 165 were originally diagnosed as HGUC, but only 70 (42.4%) were scored as malignant by all participating cytologists; of the 101 benign cases, only 32 (31.7%) were classified as such in all examinations. These specimens were re-evaluated by 6 experts using a panel of 20 specific cellular features used to distinguish between HGUC and benign diseases; tests of significance and discriminant analyses identified 7 critical features that were most useful for cytological diagnosis. Statistical analysis revealed that a focus on these 7 features led to a diagnosis of HGUC with a probability of over 95%. Conclusions The accuracy of our presently used method to evaluate urinary cytology is not consistently high. This novel classification system, which focuses on 7 critical features, facilitates the high accurate diagnosis of HGUC in routine cytology practice.

Retinitis pigmentosa (RP) is an incurable retinal degenerative disease with an unknown mechanism of disease progression. Mer tyrosine kinase (MERTK), which encodes a receptor of the Tyro3/Axl/Mer family of tyrosine kinases, is one of the causal genes of RP. MERTK is reportedly expressed in the retinal pigment epithelium (RPE) and is essential for phagocytosis of the photoreceptor outer segment. Here, we established induced pluripotent stem cells (iPSC) from patients with RP having homozygous or compound heterozygous mutations in MERTK, and from healthy subjects; the RP patient- and healthy control-derived iPSCs were differentiated into RPE cells. Although cytoskeleton staining suggested that polarity may have been disturbed mildly, there were no apparent morphological differences between the diseased and normal RPE cells. The internalization of photoreceptor outer segments in diseased iPSC-RPE cells was significantly lower than that in normal iPSC-RPE cells. This in vitro disease model may be useful for elucidating the mechanisms of disease progression and screening treatments for the disease.

Retinal neuronal cell death underlies many incurable eye diseases such as retinitis pigmentosa (RP) and glaucoma, and causes adult blindness. We have shown that maintenance of ATP levels via inhibiting ATP consumption is a promising strategy for preventing neuronal cell death. Here, we show that branched chain amino acids (BCAAs) are able to increase ATP production by enhancing glycolysis. In cell culture, supplementation of the culture media with BCAAs, but not glucose alone, enhanced cellular ATP levels, which was canceled by a glycolysis inhibitor. Administration of BCAAs to RP mouse models, rd10 and rd12, significantly attenuated photoreceptor cell death morphologically and functionally, even when administration was started at later stages. Administration of BCAAs in a glaucoma mouse model also showed significant attenuation of retinal ganglion cell death. These results suggest that administration of BCAAs could contribute to a comprehensive therapeutic strategy for retinal neurodegenerative diseases such as RP and glaucoma.

Age-related macular degeneration (AMD) is a leading cause of blindness among elderly people. The appearance of drusen is a clinical manifestation and a harbinger of both exudative and atrophic AMD. Recently, antibody-based medicines have been used to treat the exudative type. However, they do not restore good vision in patients. Moreover, no effective treatment is available for atrophic AMD. We have created small chemicals (Kyoto University Substances; KUSs) that act as ATP regulators inside cells. In the present study, we examined the in vivo efficacy of KUS121 in C-C chemokine receptor type 2-deficient mice, a mouse model of AMD. Systemic administration of KUS121 prevented or reduced drusen-like lesions and endoplasmic reticulum stress, and then substantially mitigated chorioretinal pathologies with significant preservation of visual function. Additionally, we confirmed that long-term oral administration of KUS121 caused no systemic complications in drusen-affected monkeys. ATP regulation by KUSs may represent a novel strategy in the treatment of drusen and prevention of disease progression in AMD.

Significance Bietti’s crystalline dystrophy (BCD) is an autosomal recessive, progressive chorioretinal degenerative disease. Retinal pigment epithelium (RPE) cells are impaired in patients with BCD, but the underlying mechanisms of RPE cell damage have not yet been determined because cells from lesions cannot be readily acquired from patients with BCD. In the present study, we successfully generated a human in vitro model of BCD, BCD patient-specific iPSC-RPE cells, and demonstrated that the accumulation of free cholesterol caused RPE cell damage and subsequent cell death via the induction of lysosomal dysfunction and impairment of autophagy flux in BCD-affected cells. We believe these findings provide evidence of the possible therapeutic efficacy of reducing intracellular free cholesterol in BCD., Bietti’s crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.

Retinal neuronal cell death underlies many incurable eye diseases such as retinitis pigmentosa (RP) and glaucoma, and causes adult blindness. We have shown that maintenance of ATP levels via inhibiting ATP consumption is a promising strategy for preventing neuronal cell death. Here, we show that branched chain amino acids (BCAAs) are able to increase ATP production by enhancing glycolysis. In cell culture, supplementation of the culture media with BCAAs, but not glucose alone, enhanced cellular ATP levels, which was canceled by a glycolysis inhibitor. Administration of BCAAs to RP mouse models, rd10 and rd12, significantly attenuated photoreceptor cell death morphologically and functionally, even when administration was started at later stages. Administration of BCAAs in a glaucoma mouse model also showed significant attenuation of retinal ganglion cell death. These results suggest that administration of BCAAs could contribute to a comprehensive therapeutic strategy for retinal neurodegenerative diseases such as RP and glaucoma.

アポクリン腺癌はまれな皮膚付属器腫瘍の1つであり, 細胞学的にもその報告はきわめて少ない.今回われわれは, 腋窩原発のアポクリン腺癌と診断され, 両肺と縦隔リンパ節に転移した1例を経験したので報告する.症例は63歳男性.14年前, 右腋窩腫瘍摘出.組織診断はアポクリン腺癌であった.5年後, 両肺に転移.以来, 3度にわたり腫瘍摘出術が施行された.縦隔リンパ節転移の内容液細胞診では, 壊死性背景に軽度重積性を示す乳頭状の大型細胞集塊を多数認めた.細胞質は明るく豊富で顆粒状を呈していた.核は類円形～楕円形, 一部の核に搬壁を認め, 明瞭な核小体を1個有していた.細胞集塊内には粘液塊が多数みられ, 特に乳頭状突出部で顕著であった.組織学的には, 好酸性の細胞質を有する腫瘍細胞が主として乳頭状に増殖し, 部分的に管状あるいは充実性に増殖する部分が認められ, 多様な像を呈していた.乳頭状増殖を示す腫瘍細胞には断頭分泌像を認めた.細胞質内や管腔内にジアスターゼ抵抗性PAS反応陽性物質を認めた.免疫組織化学所見では, GCDFP-15, EMA, S-100蛋白陽性, CEA陰性であった.本腫瘍は長年にわたる前駆病巣の存在が指摘されており, この時期に早期発見, 治療することが望ましいと思われた.

腫瘍が多彩な組織像を呈し, 術前の尿細胞診検査においての組織型の判定が困難であった腎盂移行上皮癌の1例について報告する.症例は, 59歳男性. 肉眼的血尿を訴えて来院され臨床的には超音波検査, CT検査にて腎細胞癌が最も疑われたが, 細胞診的には, 移行上皮癌あるいは腺癌いずれとも診断し難い像であった. 術前の尿細胞診標本では, 壊死性背景に多数の異型細胞が孤在性あるいは集簇をなして出現していた. 胞体が, basophilicでhomogenousな細胞また泡沫状で粘液様空胞を有する細胞もみられた. 一方, 核は偏在あるいは中心性でクロマチンは粗網状から細顆粒状に増量し, 好酸性の明瞭な核小体をもつ細胞も出現していた. 摘出された腫瘍は, 肉眼的に腎盂から左腎上極に浸潤性に増殖し, 腎盂腔内への乳頭状病変はみられなかった. 組織学的には, 一部に高分化な移行上皮癌の像を認めるものの多くは, 肉腫様の部分, 扁平上皮化生を起こした部分よりなっていた. すなわち, 尿細胞診でみられた細胞は扁平上皮化生を起こした部分からのものと考えられた.

The determination of cobalt(II) with 3-mercapto-1, 5-diphenyl formazan (dithizone) by a reversed-phase partition high performance liquid chromatography is described. Co(HDz)3 was separated from a mixture of Ni(HDz)2, Zn(HDz)2, Pb(HDz)2 and Cd(HDz)2 on a stainless steel column packed with Shimadzu Zorbax ODS by the use of methanol-water as a mobile phase. Co(HDz)3was first eluted to give a sharp elution peak, and the other complexes were eluted with almost the same retention time. The recommended procedure for the determination of cobalt(II) is as follows: A 10 ml of the sample solution containing cobalt(II), other metal ions and 100 mg of disodium tartrate should be shaken for 20 min with 10 ml of 5.00 × 10-3 mol dm-3 dithizone in chloroform at pH 8.5. An excess of dithizone in the extract can be removed by washing the extract with an ammonia-water mixture. A 10μl of the extract can be injected into the column by means of a micro syringe. Cobalt(II) can be determined in the range of 5.2 to 30.0 ng with the peak area calibration curve obtained at 550 nm in the presence of 24.2 ng nickel(II), 18.8 ng zinc(II), 52.6 ng lead(II) and 5.2 ng cadmium(II) using 85%(v/v) methanol-water at the flow rate of 1.0 ml/min within a relative error of + 4%. The analysis time was about 25 min.