Controlling the kinetics of interferon transgene expression for improved gene therapy.

Interferon (IFN) gene based therapy has been studied for the treatment of many diseases such as viral infections, cancer and allergic diseases. Non-viral vectors, like plasmid DNA, are promising ways for delivering IFN genes, because of their low immunogenicity and toxicity compared with viral vectors. Potent therapeutic effects of IFN gene transfer will depend on the level and duration of transgene expression after in vivo administration. Therefore, controlling the kinetics of transgene expression of IFNs is a rational approach for improved gene therapy. The design and optimization of plasmid vectors, as well as their route/method of administration, is the key to obtaining high and persistent transgene expression. In this review, we aim to present experimental evidence about the relationships among the properties of plasmid vectors expressing IFNs, the kinetics of transgene expression, and therapeutic effects as well as safety issues

DNA density-dependent uptake of DNA origami-based two-or three-dimensional nanostructures by immune cells

DNA nanostructures are expected to be applied for targeted drug delivery to immune cells. However, the structural properties of DNA nanostructures required for the delivery have not fully been elucidated. In this study, we focused on the DNA density that can be important for the their recognition and uptake by immune cells. To examine this, DNA nanostructures with almost identical molecular weights and structural flexibility, but with different shapes and DNA densities, were designed using DNA origami technology. We compared the following five types of DNA nanostructures, all of which consisted of ten DNA helices using an identical circular, single-stranded scaffold and staples. Rec180 had a rectangular-shaped, almost flat structure. Rec90, Rec50 and Rec0 were bent forms of Rec180 at the center by 90, 50 or 0 degrees, respectively. Rec50/50 has two bends of 50 degrees each so that the both ends stick together to form a triangular prism shape. The fluctuation, or flexibility, of these DNA nanostructures under solution conditions was estimated using CanDo software. The DNA density estimated from the average distance between any two of the ten DNA helices in the DNA nanostructures was different among them; Rec50, Rec0 and Rec50/50 had a higher density than Rec180 and Rec90. Agarose gel electrophoresis and atomic force microscopy showed that all of the nanostructures were prepared with high yield. Flow cytometry analysis revealed that the uptake of DNA nanostructures by murine macrophage-like RAW264.7 cells was higher for those with higher DNA density than those with low density. There was a positive correlation between the density and cellular uptake. These results indicate that DNA nanostructures with high DNA density are suitable for delivery to immune cells

Blood concentrations of small extracellular vesicles are determined by a balance between abundant secretion and rapid clearance

Small extracellular vesicles (sEVs) are important mediators of cell–cell communication with respect to diverse physiological processes. To further understand their physiological roles, understanding blood sEV homoeostasis in a quantitative manner is desired. In this study, we propose novel kinetic approaches to estimate the secretion and clearance of mouse plasma–derived sEVs (MP-sEVs) based on the hypothesis that blood sEV concentrations are determined by a balance between the secretion and clearance of sEVs. Using our specific and sensitive sEV labelling technology, we succeeded in analysing MP-sEV clearance from the blood after intravenous administration into mice. This revealed the rapid disappearance of MP-sEVs with a half-life of approximately 7 min. Moreover, the plasma sEV secretion rate, which is presently impossible to directly evaluate, was calculated as 18 μg/min in mice based on pharmacokinetic (PK) analysis. Next, macrophage-depleted mice were prepared as a model of disrupted sEV homoeostasis with retarded sEV clearance. MP-sEV concentrations were increased in macrophage-depleted mice, which probably reflected a shift in the balance of secretion and clearance. Moreover, the increased MP-sEV concentration in macrophage-depleted mice was successfully simulated using calculated clearance rate constant, secretion rate constant and volume of distribution, suggesting the validity of our PK approaches. These results demonstrate that blood sEV concentration homoeostasis can be explained by the dynamics of rapid secretion/clearance

Hemobilia Derived from Cystic Artery Pseudoaneurysm

Cystic artery pseudoaneurysm (CAP) is a rare disease, with small number of previous reports related to CAP. Besides, it is frequently prone to critical condition due to arterial bleeding. Here, we presented a case of ruptured CAP with acute calculus cholecystitis and its subsequent successful management with temporary endoscopic biliary drainage for obstructive jaundice and embolization for the culprit artery without cholecystectomy. Since CAP is at high risk of bleeding, intravascular treatment, which is only one currently available therapeutic option, is urgently required in the clinical sites

Down-regulation of GATA1-dependent erythrocyte-related genes in the spleens of mice exposed to a space travel

Secondary lymphoid organs are critical for regulating acquired immune responses. The aim of this study was to characterize the impact of spaceflight on secondary lymphoid organs at the molecular level. We analysed the spleens and lymph nodes from mice flown aboard the International Space Station (ISS) in orbit for 35 days, as part of a Japan Aerospace Exploration Agency mission. During flight, half of the mice were exposed to 1 g by centrifuging in the ISS, to provide information regarding the effect of microgravity and 1 g exposure during spaceflight. Whole-transcript cDNA sequencing (RNA-Seq) analysis of the spleen suggested that erythrocyte-related genes regulated by the transcription factor GATA1 were significantly down-regulated in ISS-flown vs. ground control mice. GATA1 and Tal1 (regulators of erythropoiesis) mRNA expression was consistently reduced by approximately half. These reductions were not completely alleviated by 1 g exposure in the ISS, suggesting that the combined effect of space environments aside from microgravity could down-regulate gene expression in the spleen. Additionally, plasma immunoglobulin concentrations were slightly altered in ISS-flown mice. Overall, our data suggest that spaceflight might disturb the homeostatic gene expression of the spleen through a combination of microgravity and other environmental changes

メンタルヘルス不調者へのセルフマネジメントプログラムの効果に関する研究

本研究の目的は、心療内科に通院するメンタルへルス不調者に、セルフマネジメントプログラムを用いた介入を行い、セルフマネジメント力を高める効果があるかを実証的に検証することである。方法は、「治療のみの変化」「介入による変化」「介入後の持続効果」を比較した群間比較研究である。対象は、うつ・不安障害などで心療内科に通院している患者10 名で、1 回2 時間、3 回からなるプログラムに参加し、その前後と1ヶ月後に質問紙調査を実施した。調査内容は、自己効力感、自己抑制型行動特性、問題解決型行動特性、抑うつ度とした。プログラム内容は、メンタルヘルス不調の心理教育、自己理解、イメージセラピー、エンカウンターグループで構成した。調査の結果、介入前後の比較において、自己抑制型行動特性、問題解決型行動特性に有意差は認められなかった。自己効力感（p＜0.5）、抑うつ（p＜0.1〉で有意な改善が認められた。また、自己効力感の下位因子であるネガティブ因子に有意傾向が認められた（p＜1）。これらの効果は介入1ヵ月後まで維持されていた。さらに、治療のみの期間における尺度の変化は認められなかったことから、介入前後の変化は介入による効果の可能性が高い。セルフマネジメントプログラム実施により、抑うつ気分の改善と自己効力認知の改善が認められ、セルフマネジメントに向けた効果が確認された。自己抑制型行動特性と問題解決型行動特性に変化は認められず、行動変容に至る期間を加味した長期的なフォローアップが必要である。一部の参加者の記述から気分と認知にセルフマネジメントに向けた肯定的な変化があり、統計的分析結果が参加者の主観からも裏付けられた。The purpose of this study is to empirically indicate whether intervention of patientswith mental health disorders, who regularly see doctors, with a self-management program is effective in improving their self-management capabilities.The method used was intergroup research comparing “change only by treatment”,“change by intervention”and“continuing effects after intervention”.Subjects were10patients who regularly saw psychosomatic doctors with the symptoms of depression,anxiety disorder, etc. The program was comprised of three2-hour sessions, and a questionnaire survey was taken before and after those sessions plus one month later.Survey contents included a feeling of self-efficacy,self-restraint behavioral characteristics, problem solving behavioral characteristics,and degree of depression.The program was comprised of psychoeducation of mental health disorders,self-understanding,image therapy, and encounter groups.The results did not show any significance in self-restraint behavioral characteristics or problem solving behavioral characteristics when comparing the before and after intervention, but significant improvement was confirmed on self-efficacy（p<0.5）and depression（p<0.1）.Additionally, a negativefactor, which is the hypostatic factor of self-efficacy, showed a tendency of marginal significance（p<1）.These effects remained1month after intervention. Since the scale didn’t change in the period of only receiving doctor’s treatment, it is highly possible that the intervention was responsible for the effective changes.Improvement in depression and recognition of self-efficacy from the intervention was recognized and the researchers confirmed its effect toward patients’self-management. Some of the written answers from participants stated they had positive changes in their mood and cognition,which confirmed the results of the statistical analysis from the participants’subjective viewpoint

Saturation of transgene protein synthesis from mRNA in cells producing a large number of transgene mRNA.

Experimental results have suggested that transgene expression can be saturated when large amounts of plasmid vectors are delivered into cells. To investigate this saturation kinetic behavior, cells were transfected with monitoring and competing plasmids using cationic liposomes. Even although an identical amount of a monitoring plasmid expressing firefly luciferase (FL) was used for transfection, transgene expression from the plasmid was greatly affected by the level of transgene expression from competing plasmids expressing renilla luciferase (RL). Similar results were obtained by exchanging the monitoring and competing plasmids. The competing plasmid-dependent reduction in transgene expression from the monitoring plasmid was also observed in mouse liver after hydrodynamic injection of plasmids. On the other hand, the mRNA and protein expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous gene, in the liver hardly changed even when transgene expression process is saturated. The expression of FL from a monitoring plasmid was significantly restored by siRNA-mediated degradation of RL mRNA that was expressed from a competing plasmid. These results suggest that the efficiency of protein synthesis from plasmid vectors is reduced when a large amount of mRNA is transcribed with no significant changes in endogenous gene expression