シングルセルRNAシーケンシングによる1,25-ジヒドロキシビタミンD3反応性Fgf23発現骨細胞の同定

Fibroblast growth factor 23 (FGF23), a hormone, mainly produced by osteocytes, regulates phosphate and vitamin D metabolism. By contrast, 1,25-dihydroxyvitamin D3, the active form of vitamin D, has been shown to enhance FGF23 production. While it is likely that osteocytes are heterogenous in terms of gene expression profiles, specific subpopulations of Fgf23-expressing osteocytes have not been identified. Single-cell RNA sequencing (scRNA-seq) technology can characterize the transcriptome of an individual cell. Recently, scRNA-seq has been used for bone tissue analysis. However, owing to technical difficulties associated with isolation of osteocytes, studies using scRNA-seq analysis to characterize FGF23-producing osteocytes are lacking. In this study, we characterized osteocytes secreting FGF23 from murine femurs in response to calcitriol (1,25-dihydroxyvitamin D3) using scRNA-seq. We first detected Dmp1, Mepe, and Phex expression in murine osteocytes by in situ hybridization and used these as marker genes of osteocytes. After decalcification, enzyme digestion, and removal of CD45+ cells, femoral bone cells were subjected to scRNA-seq. We identified cell clusters containing osteocytes using marker gene expression. While Fgf23 expression was observed in some osteocytes isolated from femurs of calcitriol-injected mice, no Fgf23 expression was detected in untreated mice. In addition, the expression of several genes which are known to be changed after 1,25-dihydroxyvitamin D3 treatment such as Ccnd2, Fn1, Igfbp7, Pdgfa, and Timp1 was also affected by calcitriol treatment in Fgf23-expressing osteocytes, but not in those lacking Fgf23 expression, even after calcitriol administration. Furthermore, box-and-whisker plots indicated that Fgf23 expression was observed in osteocytes with higher expression levels of the Fam20c, Dmp1, and Phex genes, whose inactivating mutations have been shown to cause FGF23-related hypophosphatemic diseases. These results indicate that osteocytes are heterogeneous with respect to their responsiveness to 1,25-dihydroxyvitamin D3, and sensitivity to 1,25-dihydroxyvitamin D3 is one of the characteristics of osteocytes with Fgf23 expression. It is likely that there is a subpopulation of osteocytes expressing several genes, including Fgf23, involved in phosphate metabolism

Amphiregulin and Epiregulin mRNA expression in primary colorectal cancer and corresponding liver metastases

<p>Abstract</p> <p>Background</p> <p>Amphiregulin (AREG) and Epiregulin (EREG), ligands of EGFR, are reported to be predictive biomarkers of colorectal cancer patients treated with Cetuximab, an anti-EGFR antibody. The purpose of this study is to determine the correlation of AREG and EREG expression between primary colorectal cancer and corresponding liver metastases.</p> <p>Methods</p> <p>One hundred twenty colorectal cancer patients with liver metastases (100 with synchronous metastases, 20 with metachronous) were evaluated. No patients had ever received anti-EGFR antibody agents. AREG and EREG mRNA expression from both the primary tumor and liver metastases were measured using real-time RT-PCR. KRAS codon 12, 13 mutation status was analyzed by direct sequencing.</p> <p>Results</p> <p>Modest, but significant, correlations were observed between primary tumor and corresponding liver metastases in both AREG mRNA expression (Rs = 0.54, p < 0.0001) and EREG mRNA expression (Rs = 0.58, p < 0.0001). AREG and EREG mRNA expression was strongly correlated in both the primary tumor (Rs = 0.81, p < 0.0001) and the liver metastases (Rs = 0.87, p < 0.0001). No significant survival difference was observed between low and high AREG or EREG patients when all 120 patients were analyzed. However, when divided by KRAS status, KRAS wild-type patients with low EREG mRNA levels in the primary site showed significantly better overall survival rates than those with high levels (p = 0.018). In multivariate analysis, low EREG expression was significantly associated with better overall survival (p = 0.006).</p> <p>Conclusions</p> <p>AREG and EREG expression showed a modest correlation between primary tumor and liver metastases. As EREG mRNA expression was associated with decreased survival, it is appeared to be a useful prognostic marker in KRAS wild-type patients who never received anti-EGFR therapy.</p

Studies of the efficacy and safety of methotrexate at dosages over 8 mg/week using the IORRA cohort database

The maximum dosage of methotrexate (MTX) for treatment of rheumatoid arthritis (RA) formally approved in Japan is 8 mg/week. We intended to examine the efficacy and safety of MTX at dosages over 8 mg/week in Japanese rheumatoid arthritis patients using the large Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort database. Among 9,122 patients registered in the IORRA database from the October 2000 survey to the October 2007 survey, 5,201 patients who had been treated with MTX were selected. We attempted to overcome the drawbacks innate to nonrandomized studies by using longitudinal analyses and multifactorial logistic regression analyses. Cross-sectional analysis of data obtained from the October 2007 survey indicated that dosages of MTX higher than 8 mg/week were used in 27.5% of patients treated with MTX. Longitudinal analyses based on data from three consecutive phases showed that final Disease Activity Score-28 (DAS28) values were significantly lower [n = 260, mean difference 0.563, 95% confidence interval (CI) 0.438–0.688, P < 2.2 × 10−22, two-sided paired t test] than initial values when MTX was increased from 8 mg/week or lower to over 8 mg/week. In addition, longitudinal analyses based on data from two consecutive phases indicated decreases in DAS28 values of 0.26 ± 1.04 (n = 690, P = 6.78 × 10−11, two-sided paired t test) when MTX dosages were increased from 8 mg/week or lower to over 8 mg/week, compared with decreases of 0.07 ± 0.89 (n = 2,125, P = 0.000307) when the dosage was maintained at 8 mg/week. The decreases in DAS28 values were significantly larger in the former than the latter (P = 2.27 × 10−6, two-sided unpaired t test). Concerning safety of MTX at dosages over 8 mg/week, we performed logistic regression analysis in which the objective variable was the existence or nonexistence of self-reported side-effects and the explanatory variable was the MTX dosage in the former phase, with adjustments made for age, sex, body mass index (BMI), steroid administration, folic acid administration, concomitant pulmonary diseases, and renal dysfunction. The results indicated that MTX dosages over 8 mg/week did not have any association with either severe or severe + moderate side-effects. These data regarding both efficacy and safety of MTX at dosages over 8 mg/week in Japanese RA patients would provide the basis for use of the drug at dosages currently not formally approved by the Japanese government

Single-cell RNA sequencing identifies Fgf23-expressing osteocytes in response to 1,25-dihydroxyvitamin D3 treatment

Fibroblast growth factor 23 (FGF23), a hormone, mainly produced by osteocytes, regulates phosphate and vitamin D metabolism. By contrast, 1,25-dihydroxyvitamin D3, the active form of vitamin D, has been shown to enhance FGF23 production. While it is likely that osteocytes are heterogenous in terms of gene expression profiles, specific subpopulations of Fgf23-expressing osteocytes have not been identified. Single-cell RNA sequencing (scRNA-seq) technology can characterize the transcriptome of an individual cell. Recently, scRNA-seq has been used for bone tissue analysis. However, owing to technical difficulties associated with isolation of osteocytes, studies using scRNA-seq analysis to characterize FGF23-producing osteocytes are lacking. In this study, we characterized osteocytes secreting FGF23 from murine femurs in response to calcitriol (1,25-dihydroxyvitamin D3) using scRNA-seq. We first detected Dmp1, Mepe, and Phex expression in murine osteocytes by in situ hybridization and used these as marker genes of osteocytes. After decalcification, enzyme digestion, and removal of CD45+ cells, femoral bone cells were subjected to scRNA-seq. We identified cell clusters containing osteocytes using marker gene expression. While Fgf23 expression was observed in some osteocytes isolated from femurs of calcitriol-injected mice, no Fgf23 expression was detected in untreated mice. In addition, the expression of several genes which are known to be changed after 1,25-dihydroxyvitamin D3 treatment such as Ccnd2, Fn1, Igfbp7, Pdgfa, and Timp1 was also affected by calcitriol treatment in Fgf23-expressing osteocytes, but not in those lacking Fgf23 expression, even after calcitriol administration. Furthermore, box-and-whisker plots indicated that Fgf23 expression was observed in osteocytes with higher expression levels of the Fam20c, Dmp1, and Phex genes, whose inactivating mutations have been shown to cause FGF23-related hypophosphatemic diseases. These results indicate that osteocytes are heterogeneous with respect to their responsiveness to 1,25-dihydroxyvitamin D3, and sensitivity to 1,25-dihydroxyvitamin D3 is one of the characteristics of osteocytes with Fgf23 expression. It is likely that there is a subpopulation of osteocytes expressing several genes, including Fgf23, involved in phosphate metabolism

Rare Concurrence of Apical Hypertrophic Cardiomyopathy and Effusive Constrictive Pericarditis

A 78-year-old man with a history of pulmonary tuberculosis was referred for preoperative evaluation of cardiac function. Echocardiography and cardiac cine magnetic resonance imaging (MRI) indicated apical hypertrophic cardiomyopathy (HCM), a thickened visceral pericardium, and a large pericardial effusion. Cardiac late gadolinium-enhanced MRI revealed pericardial inflammation or fibrosis. Apical HCM with concurrent effusive constrictive pericarditis was diagnosed. Further studies are required to elucidate the pathophysiology of this condition