Transgenic mice aberrantly expressing pyruvate dehydrogenase complex E2 component on biliary epithelial cells do not show primary biliary cirrhosis

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第1805号, 学位授与年月日 : 平成18年9月28日, 学位授与大学 : 金沢大学, 主査教授 : 中沼 安二, 副査教授 : 太田 哲生, 大井 章

Efficient and Directive Generation of Two Distinct Endoderm Lineages from Human ESCs and iPSCs by Differentiation Stage-Specific SOX17 Transduction

The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively

北陸地区におけるスクリーニング上部消化管内視鏡検査での咽頭観察の現状

[背景・目的] 現在, スクリーニング上部消化管内視鏡検査における咽頭領域の観察が十分に浸透しているとはいい難い状況である. 北陸地区における上部消化管内視鏡での咽頭観察の現状について調査した. [方法] 日本消化器内視鏡学会専門医114名にアンケートを送付し, 回答のあった73名を対象とし調査した. [結果] 咽頭観察を全例に行っている医師は79.5%, スクリーニングに画像強調観察(image-enhanced endoscopy:IEE)を用いた(I群)のは61.6%であった. 観察時間はI群が白色光(W群)と比べ有意に長く(p<0.001), 1年以内の癌の発見率はI群がW群と比べ有意に高かった(p=0.007). 問題点として, 観察の困難さ, 苦痛増強の可能性などの意見が多かった. [結論] スクリーニングにおける咽頭観察において, 癌の発見にはIEEにて時間をかけて観察することが重要である可能性が示唆された. 今後, さらなる咽頭観察の啓蒙活動が重要と考えられる.出版者照会後に全文公

Management of biliary stricture in patients with IgG4-related sclerosing cholangitis.

BACKGROUND:We aimed to determine the optimal approach with endoscopic biliary drainage (EBD) and corticosteroid (CS) for the treatment of IgG4-related sclerosing cholangitis (ISC). METHODS:To evaluate the safety of EBD for treatment of biliary stricture caused by ISC, we assessed the risk of stent dislodgement and sought to determine the most appropriate time for stent removal. We also assessed the safety of treatment with CS alone for patients with obstructive jaundice, and the rate of and risk factors for biliary tract complications. RESULTS:Sixty-nine patients with ISC treated with CS were enrolled. Twenty-eight patients (40.6%) were treated with EBD for biliary stricture before CS initiation. Intentional stent removal was performed in thirteen (46.4%) after confirming CS-induced improvement. Eleven of thirteen patients (84.6%) underwent stent removal within 1 month after CS initiation and all their stent removals were safely carried out without early (within two weeks) recurrence of obstructive jaundice. Ten of twenty-eight patients (35.7%) experienced spontaneous stent dislodgement after CS initiation, and seven (70%) of them developed stent dislodgement two weeks to two months after CS initiation. Among forty-one patients treated with CS alone without EBD, 10 patients had obstructive jaundice at the time of CS initiation and all of them achieved clinical improvement without biliary tract infection. During the follow-up, three patients (4.3%), all of whom had undergone EBD, developed bile-duct stones, while none of those treated with CS alone developed bile-duct stones (p = 0.032). Long-term biliary stenting was a risk factor for bile-duct stones. CONCLUSIONS:Biliary stent removal should be carried out within 2 weeks after CS initiation if biliary stricture improves to prevent stent dislodgement. Obstructive jaundice can be treated safely with CS alone in patients without infection. Clinicians should be aware of the possibility of bile-duct stones in patients treated with EBD

HHEX Promotes Hepatic-Lineage Specification through the Negative Regulation of Eomesodermin

<div><p>Human embryonic stem cells (hESCs) could provide a major window into human developmental biology, because the differentiation methods from hESCs mimic human embryogenesis. We previously reported that the overexpression of hematopoietically expressed homeobox (HHEX) in the hESC-derived definitive endoderm (DE) cells markedly promotes hepatic specification. However, it remains unclear how HHEX functions in this process. To reveal the molecular mechanisms of hepatic specification by HHEX, we tried to identify the genes directly targeted by HHEX. We found that HHEX knockdown considerably enhanced the expression level of eomesodermin (EOMES). In addition, HHEX bound to the HHEX response element located in the first intron of EOMES. Loss-of-function assays of EOMES showed that the gene expression levels of hepatoblast markers were significantly upregulated, suggesting that EOMES has a negative role in hepatic specification from the DE cells. Furthermore, EOMES exerts its effects downstream of HHEX in hepatic specification from the DE cells. In conclusion, the present results suggest that HHEX promotes hepatic specification by repressing EOMES expression.</p></div

Prognosis of type 1 autoimmune pancreatitis after corticosteroid therapy-induced remission in terms of relapse and diabetes mellitus.

Relapse and diabetes mellitus (DM) are major problems for the prognosis of autoimmune pancreatitis (AIP). We examined the prognosis of type 1 AIP after corticosteroid therapy (CST)-induced remission in terms of relapse and DM.The study enrolled 82 patients diagnosed with type 1 AIP who achieved remission with CST. We retrospectively evaluated the relapse rate in terms of the administration period of CST, clinical factors associated with relapse, and the temporal change in glucose tolerance.During follow-up, 32 patients (39.0%) experienced relapse. There was no significant clinical factor that could predict relapse before beginning CST. AIP patients who ceased CST within 2 or 3 years experienced significantly earlier relapse than those who had the continuance of CST (p = 0.050 or p = 0.020). Of the 37 DM patients, 15 patients (40.5%) had pre-existing DM, 17 (45.9%) showed new-onset DM, and 5 (13.5%) developed CST-induced DM. Patients with new-onset DM were significantly more likely to show improvement (p = 0.008) than those with pre-existing DM.It was difficult to predict relapse of AIP based on clinical parameters before beginning CST. Relapse was likely to occur within 3 years after the beginning of CST and maintenance of CST for at least 3 years reduced the risk of relapse. The early initiation of CST for AIP with impaired glucose tolerance is desirable because pre-existing DM is refractory to CST

The expression levels of DE markers in the si-HHEX-transfected cells were upregulated in hepatoblast differentiation from DE cells.

<p>(<b>A</b>) hESCs (H9) were differentiated into DE cells according to the protocol described in the <i>Materials and Methods</i> section. The DE cells were transfected with 50 nM si-control or si-HHEX on day 4, and cultured in the medium containing 20 ng/ml BMP4 and 20 ng/ml FGF4 until day 9. On day 9, the gene expression levels of hepatoblast markers (<i>AFP</i>, <i>EpCAM</i>, <i>TTR</i>, <i>HNF4α</i>, and <i>PROX1</i>) in si-control- or si-HHEX-transfected cells were examined by real-time RT-PCR. The gene expression levels in the si-control-transfected cells were taken as 1.0. (<b>B</b>) On day 9, the percentage of AFP-positive cells was measured by using FACS analysis to examine the hepatoblast differentiation efficiency. (<b>C</b>) The gene expression levels of DE (<i>EOMES</i>, <i>FOXA2</i>, <i>GATA4</i>, <i>GATA6</i>, <i>GSC</i>, and <i>SOX17</i>), pancreatic (<i>PDX1</i>, <i>NKX2.2</i>, and <i>NKX6.1</i>), intestinal (<i>CDX2</i> and <i>KLF5</i>), and pluripotent markers (<i>NANOG</i> and <i>OCT3/4</i>) in the si-control- or si-HHEX-transfected cells were examined by real-time RT-PCR. The gene expression levels in the si-control-transfected cells were taken as 1.0. (<b>D</b>) On day 9, the percentage of cells positive for the DE markers (CXCR4 and EOMES) was examined by using FACS analysis. All data are represented as means ± SD (<i>n = </i>3). *<i>p</i><0.05, **<i>p</i><0.01.</p

Hepatoblast differentiation was promoted by knockdown of EOMES in the presence of BMP4.

<p>(<b>A</b>) The hESCs (H9) were differentiated into the DE cells according to the protocol described in the <i>Materials and Methods</i> section. The hESC-derived DE cells were transfected with 50 nM si-control or si-EOMES on day 4, and then cultured with the medium containing BMP4 or FGF4. The percentage of AFP-positive cells was examined by FACS analysis on day 9. (<b>B</b>) The gene expression levels of hepatoblast markers (<i>AFP</i>, <i>EpCAM</i>, <i>TTR</i>, <i>HNF4α</i>, and <i>PROX1</i>) were measured by real-time RT-PCR on day 9. The gene expression levels in si-control-transfected cells were taken as 1.0. (<b>C</b>) The si-control- or si-EOMES-transfected cells were subjected to immunostaining with anti-AFP (green) antibodies. Nuclei were counterstained with DAPI (blue). The bar represents 50 μm. All data are represented as means ± SD (<i>n = </i>3). *<i>p</i><0.05, **<i>p</i><0.01.</p

HHEX suppresses EOMES expression by binding to the HRE located in the first intron of EOMES.

<p>(<b>A</b>) An overview of the EOMES mRNA precursor and the location of the putative HRE are presented. The HRE is located in the first intron of EOMES. (<b>B</b>) Luciferase reporter assays were performed to examine the regulation of EOMES expression by HHEX. HeLa cells were cotransfected with both firefly luciferase reporter plasmids (pControl-Luc, p5’ EOM-Luc, or p5’ EOM-mut-Luc) and effecter plasmids (control plasmids (pHMEF5) or HHEX expression plasmids (pHMEF5-HHEX)). The details of the luciferase reporter assays are described in the <i>Materials and Methods</i> section. The luciferase activities in the pControl-Luc- and pHMEF5-cotransfected cells were taken as 1.0. All data are represented as means ± SD (<i>n = </i>3). <i>*</i>, <i>p</i><0.05.</p