膵特異的RECK発現の不活化は、膵発癌、上皮間葉転換、転移を引き起こす

京都大学新制・課程博士博士(医学)甲第25194号医博第5080号京都大学大学院医学研究科医学専攻(主査)教授 藤田 恭之, 教授 小濱 和貴, 教授 川口 義弥学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Fluid dynamics in patients with nasal disease

Computational fluid dynamics （CFD） analysis is useful for quantitative assessment in patients with upper airway obstructions. We compared CFD analysis with rhinomanometry （RM） and acoustic rhinometry （AR）. Twenty patients with nasal and paranasal diseases who required computed tomography assessment underwent RM and AR. We measured the pressure and velocity at four parts of the upper airway using CFD analysis. Then we evaluated the correlation among CFD analysis, RM, and AR. CFD analysis detected obstruction sites in the nasal airway and pharynx in 14 and 2patients, respectively. High negative pressure accompanied the nasal obstruction, even behind the nasal cavity. Nasal airway pressure measured using CFD analysis strongly correlated with nasal resistance in RM （Spearman correlation coefficient＝0.853）. CFD analysis’s sensitivity and specificity to detect the obstruction were 84.6％ and 57.1％, respectively （compared to those of RM） and 83.3％ and 50.0％, respectively （compared to those of AR）. The CFD analysis’s ability to detect obstruction was comparable to that of RM and AR; therefore, it may help evaluate the upper airways in patients with nasal and paranasal diseases. We found impaired nasal ventilation also affected other parts of the upper airway. Further studies with a larger sample size are required to validate the use of CFD analysis for assessing the degree of upper airway ventilation disorders

Medial meniscus posterior root repair decreases posteromedial extrusion of the medial meniscus during knee flexion

Background Medial meniscus (MM) medial extrusion in the coronal plane does not always improve, even after repair. This study aimed to determine the extent of posteromedial extrusion of the MM during knee flexion before and after MM pullout repair using three-dimensional magnetic resonance imaging (MRI). Methods Data from 14 patients (mean age, 63.4 years; 86% female) who had undergone MM pullout repair at the current institution between August 2017 and October 2018 were retrospectively reviewed. The MRIs were performed pre-operatively and ≥ 3 months postoperatively. Three-dimensional MRIs of the tibial surface and MM were evaluated using Tsukada's measurement method before and after pullout repair. The expected center of MM posterior root attachment (point A), the point on the extruded edge of the MM farthest away from point A (point E), and the point of intersection of a line through the posteromedial corner of the medial tibial plateau and a line connecting points A and E (point I) were identified. Subsequently, the pre-operative and postoperative AE and IE distances were calculated and compared. Results Point E was laterally shifted by the pullout repair, whereas point I showed no significant change. The postoperative IE distance (6.7 mm) was significantly shorter than the pre-operative one (9.1 mm, P < 0.01). The postoperative AE distance (29.3 mm) was significantly shorter than the pre-operative one (31.5 mm, P < 0.01). Conclusions The AE and IE distances significantly decreased after MM posterior root repair, suggesting that transtibial pullout repair may be useful in reducing posteromedial extrusion of the MM

spERt Technology: A novel strategy to improve productivity through enhanced polyribosome assembly on the endoplasmic reticulum in CHO cells

In cell line development process, it is frequently observed that increased mRNA levels do not always correlate with protein expression levels in CHO cells. In line with this gap, the endoplasmic reticulum (ER) in CHO cells is much less proliferated as compared with that in terminally differentiated (i.e., professional) secretory cells, suggesting that there is still room to improve their specific productivity if translational efficiency on the ER can be up-regulated. Here we present a novel engineering approach (spERt Technology) to improve specific production rates by mimicking the ER translational apparatus of professional secretory cells. In spERt Technology, we exploit the unique factors that are required for translationally active polyribosome formation on the ER to directly enhance the translational efficiency (1, 2). A high antibody (Ab) producing clone generated by a novel screen using flow cytometry (3) was used as a model cell line. The factors were introduced into the high producer and a series of the spERt Technology - introduced cell lines were generated Among these cell lines, we selected one of the best clones (spERt-f9) having stable and high productivity. Polyribosome analysis of these cell lines revealed that enhanced assembly of the ER polyribosomes as expected (1). Consistent with the highly developed polyribosomes, the spERt-introduced cell lines produced higher levels of Ab than that of parental cells, and showed prominent increase of specific production rates. Further optimization of feeding process resulted in remarkable increase of productivity in spERt-f9 cells: Ab titers of 7.6 g/L and 9.5 g/L on day 14 and 17, respectively, were achieved in shake flask fed-batch cultures by using chemically defined media. Importantly, high cell viabilities were maintained in spERt-f9 cells throughout the culture periods. In addition, lower glucose consumption and reduced accumulation of ammonia were observed. Product quality in these cells were analyzed and compared with that in the parental cells. In conclusion, spERt Technology enables to improve productivity of high Ab producers, associated with reduced accumulation of waste metabolites and high cell viabilities

Computational fluid dynamics analysis in patients with nasal disease

Computational fluid dynamics （CFD） analysis is useful for quantitative assessment in patients with upper airway obstructions. We compared CFD analysis with rhinomanometry （RM） and acoustic rhinometry （AR）. Twenty patients with nasal and paranasal diseases who required computed tomography assessment underwent RM and AR. We measured the pressure and velocity at four parts of the upper airway using CFD analysis. Then we evaluated the correlation among CFD analysis, RM, and AR. CFD analysis detected obstruction sites in the nasal airway and pharynx in 14 and 2patients, respectively. High negative pressure accompanied the nasal obstruction, even behind the nasal cavity. Nasal airway pressure measured using CFD analysis strongly correlated with nasal resistance in RM （Spearman correlation coefficient＝0.853）. CFD analysis’s sensitivity and specificity to detect the obstruction were 84.6％ and 57.1％, respectively （compared to those of RM） and 83.3％ and 50.0％, respectively （compared to those of AR）. The CFD analysis’s ability to detect obstruction was comparable to that of RM and AR; therefore, it may help evaluate the upper airways in patients with nasal and paranasal diseases. We found impaired nasal ventilation also affected other parts of the upper airway. Further studies with a larger sample size are required to validate the use of CFD analysis for assessing the degree of upper airway ventilation disorders

Transtibial pullout repair of medial meniscus posterior root tear restores physiological rotation of the tibia in the knee-flexed position

BACKGROUND: Medial meniscus posterior root tear (MMPRT) results in joint overloading and degenerative changes in the knee. Favorable clinical outcomes have been reported after transtibial pullout repair of MMPRT. To date, however, in vivo tibial rotational changes before and after root repair remain poorly understood. The purpose of this study was to investigate postoperative changes in tibial rotation following MMPRT pullout repair. HYPOTHESIS: Pathological external rotation of the tibia in the knee-flexed position is caused by MMPRT and is reduced after transtibial pullout repair. PATIENTS AND METHODS: Fifteen patients who underwent MMPRT pullout repair and 7 healthy volunteers were included. Magnetic resonance imaging examinations were performed in the 10° and 90° knee-flexed positions. The angles between the surgical epicondylar axis and a line between the medial border of the patellar tendon and the apex of the medial tibial spine were measured. Baseline was defined as a line lying at a right angle to the other, and a value was positive and negative when the tibia rotated internally and externally, respectively. RESULTS: In the volunteer's normal knees, tibial internal rotation was +1.00°±3.27° at 10° flexion and +4.14°±3.46° at 90° flexion. In the MMPRT preoperative knees, tibial internal rotation was +1.07°±3.01° at 10° flexion and +1.27°±2.96° at 90° flexion. In the postoperative knees, tibial internal rotation was +1.60°±2.85° at 10° flexion and +4.33°±2.89° at 90° flexion. DISCUSSION: This study demonstrates discontinuity of the MM posterior root may induce a pathological external rotation of the tibia during knee flexion and that MMPRT pullout repair reduces the pathological external rotation of the tibia in the knee-flexed position

Epithelial EP4 plays an essential role in maintaining homeostasis in colon

Colonic epithelial cells comprise the mucosal barrier, and their dysfunction promotes microbial invasion from the gut lumen and induces the development of intestinal inflammation. The EP4 receptor is known to mediate the protective effect of prostaglandin (PG) E2 in the gastrointestinal tract; however, the exact role of epithelial EP4 in intestinal pathophysiology remains unknown. In the present study, we aimed to investigate the role of epithelial EP4 in maintaining colonic homeostasis by characterizing the intestinal epithelial cell-specific EP4 knockout (EP4 cKO) mice. Mice harboring the epithelial EP4 deletion showed significantly lower colonic crypt depth and lower numbers of secretory cell lineages, as well as impaired epithelial cells in the colon. Interestingly, EP4-deficient colon epithelia showed a higher number of apoptotic cells. Consistent with the defect in mucosal barrier function of colonic epithelia and secretory cell lineages, EP4 cKO colon stroma showed enhanced immune cell infiltration, which was accompanied by increased production of inflammatory cytokines. Furthermore, EP4-deficient colons were susceptible to dextran sulfate sodium (DSS)-induced colitis. Our study is the first to demonstrate that epithelial EP4 loss resulted in potential "inflammatory" status under physiological conditions. These findings provided insights into the crucial role of epithelial PGE2/EP4 axis in maintaining intestinal homeostasis

A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions

<p>Abstract</p> <p/> <p>Prostate cancer metastasizes to bone in the majority of patients with advanced disease leading to painfully debilitating fractures, spinal compression and rapid decline. In addition, prostate cancer bone metastases often become resistant to standard therapies including androgen deprivation, radiation and chemotherapy. There are currently few models to elucidate mechanisms of interaction between the bone microenvironment and prostate cancer. It is, thus, essential to develop new patient-derived, orthotopic models. Here we report the development and characterization of PCSD1 (Prostate Cancer San Diego 1), a novel patient-derived intra-femoral xenograft model of prostate bone metastatic cancer that recapitulates mixed osteolytic and osteoblastic lesions.</p> <p>Methods</p> <p>A femoral bone metastasis of prostate cancer was removed during hemiarthroplasty and transplanted into <it>Rag2^-/-;γ_c^-/-</it>mice either intra-femorally or sub-cutaneously. Xenograft tumors that developed were analyzed for prostate cancer biomarker expression using RT-PCR and immunohistochemistry. Osteoblastic, osteolytic and mixed lesion formation was measured using micro-computed tomography (microCT).</p> <p>Results</p> <p>PCSD1 cells isolated directly from the patient formed tumors in all mice that were transplanted intra-femorally or sub-cutaneously into <it>Rag2^-/-;γ_c^-/-</it>mice. Xenograft tumors expressed human prostate specific antigen (PSA) in RT-PCR and immunohistochemical analyses. PCSD1 tumors also expressed AR, NKX3.1, Keratins 8 and 18, and AMACR. Histologic and microCT analyses revealed that intra-femoral PCSD1 xenograft tumors formed mixed osteolytic and osteoblastic lesions. PCSD1 tumors have been serially passaged in mice as xenografts intra-femorally or sub-cutaneously as well as grown in culture.</p> <p>Conclusions</p> <p>PCSD1 xenografts tumors were characterized as advanced, luminal epithelial prostate cancer from a bone metastasis using RT-PCR and immunohistochemical biomarker analyses. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely resembled the bone lesions in the patient. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study metastatic disease in the bone and to develop novel therapies for inhibiting prostate cancer growth in the bone-niche.</p

JNK pathway plays a critical role for expansion of human colorectal cancer in the context of BRG1 suppression

Tumor stem cells (TSCs), capable of self-renewal and continuous production of progeny cells, could be potential therapeutic targets. We have recently reported that chromatin remodeling regulator Brg1 is required for maintenance of murine intestinal TSCs and stemness feature of human colorectal cancer (CRC) cells by inhibiting apoptosis. However, it is still unclear how BRG1 suppression changes the underlying intracellular mechanisms of human CRC cells. We found that Brg1 suppression resulted in upregulation of the JNK signaling pathway in human CRC cells and murine intestinal TSCs. Simultaneous suppression of BRG1 and the JNK pathway, either by pharmacological inhibition or silencing of c-JUN, resulted in even stronger inhibition of the expansion of human CRC cells compared to Brg1 suppression alone. Consistently, high c-JUN expression correlated with worse prognosis for survival in human CRC patients with low BRG1 expression. Therefore, the JNK pathway plays a critical role for expansion and stemness of human CRC cells in the context of BRG1 suppression, and thus a combined blockade of BRG1 and the JNK pathway could be a novel therapeutic approach against human CRC

Cytotoxic T Lymphocytes Regenerated from iPS Cells Have Therapeutic Efficacy in a Patient-Derived Xenograft Solid Tumor Model

Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/β genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors