👤 Yuji Oshima

Malignant phyllodes tumors (MPTs) are rare fibroepithelial breast tumors with no standard treatment for metastatic or recurrent cases. Comprehensive genomic profiling (CGP) has been conducted for MPT; however, its association with treatment remains unclear. A retrospective study was conducted on patients with advanced or recurrent MPTs treated with chemotherapy between 2013 and 2022 at two hospitals, analyzing clinical data, CGP, treatment outcomes, and survival. Five patients with metastatic MPTs who had received chemotherapy were identified. The median age was 55 years (range, 50-66), and all patients were female. As first-line treatment, four patients received doxorubicin plus ifosfamide (AI) combination therapy, while one received doxorubicin monotherapy. Among those treated with AI therapy, the best responses were partial response in three patients and stable disease in one. The median progression-free survival (PFS) for patients treated with AI therapy was 5.3 months. Of the five patients two proceeded to second-line therapy, and one patient received up to fourth-line treatment. Next-generation sequencing-based CGP testing was performed in four cases. One patient with an FGFR1-N546K-mutated MPT achieved a relatively long PFS of 6.8 months with pazopanib therapy, a multi-kinase inhibitor targeting FGFR1 among other kinases, as fourth-line therapy. AI therapy is useful for advanced or recurrent MPTs. The observed clinical benefit of pazopanib in a patient with FGFR1 N546K-mutated MPT suggests that FGFR1 kinase domain mutations may be a relevant factor in responsiveness of FGFR1-targeted therapy. Further data accumulation is warranted. Show less

Platelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications. The growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined. Multiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model. Our study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds. Show less

This study aimed to understand the mechanisms underlying the effects of maternal undernutrition (MUN) on liver growth and metabolism in Japanese Black fetal calves (8.5 months in utero) using an approach that integrates metabolomics and transcriptomics. Dams were fed 60% (low-nutrition; LN) or 120% (high-nutrition; HN) of their overall nutritional requirements during gestation. We found that MUN markedly decreased the body and liver weights of the fetuses; metabolomic analysis revealed that aspartate, glycerol, alanine, gluconate 6-phosphate, and ophthalmate levels were decreased, whereas UDP-glucose, UDP-glucuronate, octanoate, and 2-hydroxybutyrate levels were decreased in the LN fetal liver ( Show less

Some patients with inflammatory bowel disease (IBD) who were under mesalamine treatment develop adverse reactions called "mesalamine allergy," which includes high fever and worsening diarrhea. Currently, there is no method to predict mesalamine allergy. Pharmacogenomic approaches may help identify these patients. Here we analyzed the genetic background of mesalamine intolerance in the first genome-wide association study of Japanese patients with IBD. Two independent pharmacogenetic IBD cohorts were analyzed: the MENDEL (n = 1523; as a discovery set) and the Tohoku (n = 788; as a replication set) cohorts. Genome-wide association studies were performed in each population, followed by a meta-analysis. In addition, we constructed a polygenic risk score model and combined genetic and clinical factors to model mesalamine intolerance. In the combined cohort, mesalamine-induced fever and/or diarrhea was significantly more frequent in ulcerative colitis vs Crohn's disease. The genome-wide association studies and meta-analysis identified one significant association between rs144384547 (upstream of RGS17) and mesalamine-induced fever and diarrhea (P = 7.21e-09; odds ratio = 11.2). The estimated heritability of mesalamine allergy was 25.4%, suggesting a significant correlation with the genetic background. Furthermore, a polygenic risk score model was built to predict mesalamine allergy (P = 2.95e-2). The combined genetic/clinical prediction model yielded a higher area under the curve than did the polygenic risk score or clinical model alone (area under the curve, 0.89; sensitivity, 71.4%; specificity, 90.8%). Mesalamine allergy was more common in ulcerative colitis than in Crohn's disease. We identified a novel genetic association with and developed a combined clinical/genetic model for this adverse event. Show less

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-β. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-β. Surprisingly, addition of TGF-β induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-β significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs. Show less

Peritoneal fibrosis (PF) is a severe complication of peritoneal dialysis, but there are few effective therapies for it. Recent studies have revealed a new biological function of trehalose as an autophagy inducer. Thus far, there are few reports regarding the therapeutic effects of trehalose on fibrotic diseases. Therefore, we examined whether trehalose has anti-fibrotic effects on PF. PF was induced by intraperitoneal injection of chlorhexidine gluconate (CG). CG challenges induced the increase of peritoneal thickness, ColIα Show less

To evaluate the destruction complex of beta-catenin by the expression of the proteins beta-catetenin, adenomatous polyposis coli, GSK3β, axin and ubiquitin in colorectal carcinoma and colonic adenoma. Tissue samples from 64 patients with colorectal carcinoma and 53 patients with colonic adenoma were analyzed. Tissue microarray blocks and slides were prepared and subjected to immunohistochemistry with polyclonal antibodies in carcinoma, adjacent non-neoplastic mucosa, and adenoma tissues. The immunoreactivity was evaluated by the percentage of positive stained cells and by the intensity assessed through of the stained grade of proteins in the cytoplasm and nucleus of cells. In the statistical analysis, the Spearman correlation coefficient, Student's t, χ2, Mann-Whitney, and McNemar tests, and univariate logistic regression analysis were used. In colorectal carcinoma, the expressions of beta-catenin and adenomatous polyposis coli proteins were significantly higher than in colonic adenomas (p<0.001 and p<0.0001, respectively). The immunoreactivity of GSK3β, axin 1 and ubiquitin proteins was significantly higher (p=0.03, p=0.039 and p=0.03, respectively) in colorectal carcinoma than in the colonic adenoma and adjacent non-neoplastic mucosa. The immunohistochemistry staining of these proteins did not show significant differences with the clinical and pathological characteristics of colorectal cancer and colonic adenoma. These results suggest that, in adenomas, the lower expression of the beta-catenin, axin 1 and GSK3β proteins indicated that the destruction complex of beta-catenin was maintained, while in colorectal carcinoma, the increased expression of beta-catenin, GSK3β, axin 1, and ubiquitin proteins indicated that the destruction complex of beta-catenin was disrupted. Avaliar o complexo de destruição da betacatenina no carcinoma colorretal e no adenoma do colo pela expressão das proteínas betacatenina, adenomatous polyposis coli, GSK3β, axina e ubiquitina. Amostras de tecidos de 64 doentes com carcinoma colorretal e de 53 pacientes com adenoma do colo foram analisadas. Blocos de tecidos foram submetidos ao estudo imuno-histoquímico com anticorpos policlonais nos tecidos do carcinoma, mucosa não neoplásica adjacente e adenoma. A imunorreatividade foi avaliada pela porcentagem de positividade de células coradas e pela intensidade do grau de coloração das proteínas no citoplasma e no núcleo das células. Na análise estatística, foram utilizados o coeficiente de correlação de Spearman, os testes t de Student, χ2, Mann-Whitney e de McNemar, e a análise de regressão logística univariada. No carcinoma colorretal, as expressões da betacatenina e da adenomatous polyposis coli foram significativamente maiores do que em adenomas do colo (p<0,001 e p<0,0001, respectivamente). A imunorreatividade das proteínas GSK3β, axina 1 e ubiquitina foi significativamente maior (p=0,03, p=0,039 e p=0,03, respectivamente) no carcinoma colorretal do que no adenoma e na mucosa não neoplásica adjacente. A coloração imuno-histoquímica dessas proteínas não apresentou diferenças significantes em relação às características clinicopatológicas do câncer colorretal e do adenoma. Em adenomas, as menores expressões de betacatenina, axina 1 e GSK3β indicaram que o complexo de destruição da betacatenina estava conservado, enquanto que, no carcinoma colorretal, o aumento das expressões da betacatenina, GSK3β, 1 axina, e ubiquitina indicaram que o complexo de destruição de betacatenina estava alterado. Show less

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly. Previous sequencing studies of AMD susceptibility genes have revealed the association of rare coding variants in CFH, CFI, C3 and C9 in European population; however, the impact of rare or low-frequency coding variants on AMD susceptibility in other populations is largely unknown. To identify the role of low-frequency coding variants on exudative AMD susceptibility in a Japanese population, we analysed the association of coding variants of 34 AMD candidate genes in the two-stage design by a multiplex PCR-based target sequencing method. We used a total of 2,886 (1st: 827, 2nd: 2,059) exudative AMD cases including typical AMD, polypoidal choroidal vasculopathy, and retinal angiomatous proliferation and 9,337 (1st: 3,247 2nd: 6,090) controls. Gene-based analysis found a significant association of low-frequency variants (minor allele frequency (MAF) < 0.05) in CETP, C2 and CFB. The association of CETP remained after conditioned with all known genome-wide association study (GWAS) associated variants. In addition, when we included only disruptive variants, enrichment of rare variants (MAF < 0.01) was also observed after conditioned with all GWAS associated variants (P = 1.03 × 10−6, odds ratio (OR) = 2.48). Haplotype and conditional analysis of the C2-CFB-SKIV2L locus showed a low-frequency variant (R74H) in CFB would be individually associated with AMD susceptibility independent of the GWAS associated SNP. These findings highlight the importance of target sequencing to reveal the impact of rare or low-frequency coding variants on disease susceptibility in different ethnic populations. Show less

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor. Show less

Gprc5b, a retinoic acid-inducible orphan G protein-coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins possibly involved in non-canonical Wnt signaling. Many GPCR transcripts are alternatively spliced, which diversifies this class of proteins in their cell- and tissue-specific signaling, regulatory and/or pharmacological properties. We previously generated p97FE65 isoform-specific knockout mice that showed learning/memory deficits. In this study, we further characterized the 97FE65 null mice using cDNA microarray and RT-PCR analyses. We discovered a novel brain-specific C-terminal splice variant of Gprc5b, Gprc5b_v2, which was differentially expressed in p97FE65 wild type and null mouse brains. The null mice were generated in 129/Sv ES cells, and backcrossed to C57Bl/6J for ten generations. We found that expression of Gprc5b_v2 mRNA in the brains of p97FE65 null mice was dramatically down-regulated (more than 20 fold) compared to their wild type littermates. However, expression profiles of Gprc5b variants and SNP analysis surrounding the FE65 locus suggest that the down-regulation is unlikely due to the altered FE65 function, but rather is caused by gene retention from the 129/Sv ES cells. Consistently, in contrast to ubiquitously expressed Gprc5b_v1, Gprc5b_v2 was predominantly expressed in the brain tissues of C57Bl/6J mice. The alternative splicing of the 3' terminal exon also altered the protein coding sequences, giving rise to the characteristic C-termini. Levels of Gprc5b_v2 mRNA were increased during neuronal maturation, paralleling the expression of synaptic proteins. Overexpression of both Gprc5b variants stimulated neurite-like outgrowth in a neuroblastoma cell line. Our results suggest that Gprc5b-v2 may play a role during brain maturation and in matured brain, possibly through the regulation of neuronal morphology and protein-protein interaction. This study also highlights the fact that unexpected gene retention following repeated backcrosses can lead to important biological consequences. Show less

Vitreous samples collected in retinopathic surgeries have diverse properties, making proteomics analysis difficult. We report a cluster analysis to evade this difficulty. Vitreous and subretinal fluid samples were collected from 60 patients during surgical operation of non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy, and rhegmatogenous retinal detachment. For controls we collected vitreous fluid from patients of idiopathic macular hole, epiretinal, and from a healthy postmortem donor. Proteins from these samples were subjected to quantitative proteomics using two-dimensional gel electrophoresis. We selected 105 proteins robustly expressed among ca 400 protein spots and subjected them to permutation test. By using permutation test analysis we observed unique variations in the expression of some of these proteins in vitreoretinal diseases when compared to the control and to each other: 1) the levels of inflammation-associate proteins such as AAT, APOA4, ALB, and TF were significantly higher in all four types of vitreoretinal diseases, and 2) each vitreoretinal disease elevates a unique set of proteins which can be interpreted based on the pathology of retinopathy. Our protocol will be effective for the study of protein expression in other types of clinical samples of diverse property. Show less