Production of Antibodies against Multipass Membrane Proteins Expressed in Human Tumor Cells Using Dendritic Cell Immunization

Antibody mediated therapeutic strategies against human malignant tumors have been widely authorized and clinically applied to cancer patients. In order to develop methods to generate antibodies reactive to the extracellular domains of multipass plasma membrane proteins specifically expressed in malignant tumors, we examined the use of dendritic cells (DCs) for immunization. DCs were transduced with genes encoding the human six transmembrane epithelial antigen of prostate 1 (STEAP1), STEAP4, and seven transmembrane prostate specific G-protein coupled receptor (PSGR). Mice were immunized with these DCs and followed by repeated booster immunization with plasmids expressing each protein. The immunized mice produced significant amounts of antibodies against these proteins. Our results suggest that DC immunization is an effective method to produce antibodies reactive to extracellular regions of plasma membrane proteins with multiple-transmembrane domains, and may be useful to develop antibody mediated antitumor therapies

コウムイン ホイクシ オ メザス ガクセイ エノ キャリア シエン ノ アリカタ ト コンゴ ノ カダイ

保育者を目指す学生の進路として、自治体の公立保育士や公立幼稚園教諭を目指す学生が増加傾向にある。公立保育士を目指す学生の採用試験対策への取り組みの支援、キャリア支援は、ここ数年を通して対応した教員、キャリア支援室職員にとっても一定の成果を見ることができた。このことは、教員間の連携とキャリア支援室との連携があったことの成果ではないかと考えられる。本稿では、３年間の公立保育士を目指す学生への支援体制について具体的に述べ、教員が関わった指導のデータ解析などを踏まえ、取り組みや支援を振り返り、今後の課題について検討する

Effect of aspirin treatment on serum levels of lipoprotein (a) : analysis from the apolipoprotein (a) isoforms

We have found that aspirin lowers elevated serum lipoprotein(a) [Lp(a)] levels via reduction of the transcriptional activity of apolipoprotein(a) [apo(a)] gene with suppression of apo(a) mRNA expression. In the present study, we evaluated the effect of aspirin treatment on serum Lp(a) level and analyzed its relation to type of apo(a) isoform. Serum levels of Lp(a) were measured by turbidimetric immunoassay before and after the oral administration of aspirin therapy (81 mg/day) in 57 patients with coronary artery disease or cerebral infarction. Apo(a) isoforms were determined by immunoblotting method. In patients with high serum Lp(a) levels (more than 30 mg/dl), aspirin reduced serum Lp(a) levels to approximately 80 % of the baseline after one month. Their levels sustained significantly low even after six months. The effect of aspirin in reducing elevated serum Lp(a) levels were stronger in patients with smaller-sized type or double-band type of apo(a) isoforms. The transcriptional efficiency of apo(a) gene is thought to be increased in patients with these apo(a) isoforms. Therefore, these findings suggest that aspirin reduces apo(a) gene transcription preferentialy in patients with high transcriptional efficiency of this gene

Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

Introduction: Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer.Methods: Whole genomes of influenza A and B viruses were amplified by multiplex RT-PCR from 13 clinical specimens collected in Tokyo, Japan. Barcode tags for multiplex MinION sequencing were added with each multiplex RT-PCR amplicon by nested PCR with custom barcoded primers. All barcoded amplicons were mixed and multiplex sequencing using the MinION sequencer with 1D2 sequencing kit. In addition, multiplex RT-PCR amplicons generated from each clinical specimen were sequenced using the Illumina MiSeq platform to validate the performance of MinION sequencer. The accuracy, recall, and precision rates of MinION sequencing were calculated by comparing the results of variant calling in the Illumina MiSeq platform and MinION sequencer.Results: Whole genomes of influenza A and B viruses were successfully amplified by multiplex RT-PCR from 13 clinical samples. We identified 6 samples as influenza type A virus H3N2 subtype and 7 as influenza B virus Yamagata lineage using the Illumina MiSeq platform. The overall accuracy, recall, and precision rates of the MinION sequencer were, respectively 99.95%, 89.41%, and 97.88% from 1D reads and 99.97%, 93.28%, and 99.86% from 1D2 reads.Conclusion: We developed a novel WGS method for influenza A and B viruses. It is necessary to improve read accuracy and analytical tools in order to better utilize the MinION sequencer for real-time monitoring of genetic rearrangements and for evaluation of newly emerging viruses

Drug retention rates and relevant risk factors for drug discontinuation due to adverse events in rheumatoid arthritis patients receiving anticytokine therapy with different target molecules

Objective: To compare reasons for discontinuation and drug retention rates per reason among anticytokine therapies, infliximab, etanercept and tocilizumab, and the risk of discontinuation of biological agents due to adverse events (AE) in patients with rheumatoid arthritis (RA). Method: This prospective cohort study included Japanese RA patients who started infliximab (n=412, 636.0 patientyears (PY)), etanercept (n=442, 765.3 PY), or tocilizumab (n=168, 206.5 PY) as the first biological therapy after their enrolment in the Registry of Japanese Rheumatoid Arthritis Patients for Long-term Safety (REAL) database. Drug retention rates were calculated using the Kaplan-Meier method. To compare risks of drug discontinuation due to AE for patients treated with these biological agents, the Cox proportional hazard model was applied. Results: The authors found significant differences among the three therapeutic groups in demography, clinical status, comorbidities and usage of concomitant drugs. Development of AE was the most frequent reason for discontinuation of biological agents in the etanercept and tocilizumab groups, and the second most frequent reason in the infliximab group. Discontinuation due to good control was observed most frequently in the infliximab group. Compared with etanercept, the use of infliximab (HR 1.69; 95% CI 1.14 to 2.51) and tocilizumab (HR 1.98; 95% CI 1.04 to 3.76) was significantly associated with a higher risk of discontinuation of biological agents due to AE. Conclusions: Reasons for discontinuation are significantly different among biological agents. The use of infliximab and tocilizumab was significantly associated with treatment discontinuation due to AE compared with etanercept

STEAP4 regulates focal adhesion kinase activation and CpG motifs within STEAP4 promoter region are frequently methylated in DU145, human androgen-independent prostate cancer cells

The possible roles of STEAP4 in cancer progression have not been reported. In this study, we report that STEAP4 expression is able to inhibit anchorage-independent cell growth. We also demonstrate that STEAP4 associates with focal adhesion kinase (FAK) and regulate the activity of FAK through Y397 phosphorylation. Furthermore, we show that CpG sequences in STEAP4 promoter region were frequently methylated in DU145, androgen-independent prostate cancer cells. Demethylation treatment induced STEAP4 expression in DU145, suggesting the possibility that STEAP4 expression in cancer cells is in part epigenetically regulated. Collectively, these data demonstrate a novel function of STEAP4 and that STEAP4 may play an important role in tumor malignancy

組織標的性を持つ遺伝子導入ベクターとしての次世代バキュロウイルスの開発

金沢大学医薬保健研究域薬学系本研究では出芽型組換えバキュロウイルスベクター(BV)に注目した。BVの大きなメリットとして、BV粒子のエンベロープに目的の膜タンパク質を発現させることが容易に可能であり、標的化遺伝子導入ベクターとしての応用が期待される。標的化分子の検索には、マラリア原虫には標的化に適した分子が多く存在することに着目した。肝細胞に高い親和性をもつCSPやTRAPを発現させたBVがヒト肝癌細胞(HepG2など)やヒト初代肝細胞PXB細胞において高い遺伝子導入効率を示すことを明らかにした。また補体制御因子の融合体を発現させることで、高い補体抵抗性をもつBVの作製に成功した。In this research, budded recombinant baculovirus vectors (BVs) have been developed because BVs have many advantages for tissue targeting gene delivery vectors.It is easily possible to display exogenous proteins on the surface of BV virion. CSP and TRAP, malaria sporozoite surface proteins have highly selective affinity to hepatocytes. BVs expressing CSP or TRAP molecule on the virion surface have been constructed and they were shown to greatly increase transduction efficacy to human hepatoma cell line (HepG2 et al.) and human primary hepatocytes (PXB cells). Furthermore, to overcome the vulnerability to serum complement system, fusion molecules of complement regulatory proteins were displayed to BV virion surfaces. These BVs were shown to greatly increase resistance against complement attack.研究課題/領域番号:15K07926, 研究期間(年度):2015-04-01 - 2018-03-3

差異的樹状細胞免疫法による前立腺癌幹細胞特異的モノクローナル抗体の網羅的作製

HAを発現する制御性樹状細胞をマウスに免疫するとHAに対する抗体産生は成熟活性型樹状細胞を用いた場合と比較すると強く抑制された。しかし、OVAタンパク質をその後免疫すると、OVAに対する抗体産生は抑制されなかった。これらのことから差異的樹状細胞免疫法の基盤が確立された。培養細胞から抽出された膜タンパク質は樹状細胞に貪食されることが確認された。貪食樹状細胞を免疫することで細胞表面タンパク質に対する抗体産生が誘導された。制御性樹状細胞を用いた場合は抗体産生が有意に抑制された。The regulatory dendritic cells expressing HA antigen were immunized into mice. The anti-HA antibody was less induced compared with normal dendritic cells. However, OVA protein immunization after HA immunization demonstrated anti-OVA antibody was similarly induced, suggesting differential dendritic cell immunization protocol worked properly. Dendritic cells were shown to phagocyte the membrane proteins extracted from cultured cells. These dendritic cells were immunized into mice. Anti-cell surface proteins of culture cells were produced, but it was less in the case of regulatory dendritic cells.研究課題/領域番号:21791532, 研究期間(年度):2009-201

マラリア原虫感染時における樹状細胞の発生分化プログラムに関する研究

金沢大学医薬保健研究域薬学系Plasmodium berghei ANKA(PbA)感染における脾臓内のcDCならびにpDCの細胞数の変動とそのメカニズムについて解析し、cDC, pDCとも細胞数が感染前と比較して有意に減少することが明らかになった。type Iならびにtype II interferonsがマラリア感染後のcDCの活性化、それに伴う細胞死に重要な役割を果たすことを明らかにした。\nマラリア感染を経験することで徐々にマラリア感染に抵抗性を持つようになることが知られているが、その詳細なメカニズムはいまだ不明である。PbA感染後ピリメタミンで治療したマウスでは脾臓cDCが有意に増加数することを明らかにした。The changes and mechanisms of number of splenic cDC and pDC after Plasmodium berghei ANKA (PbA) infection were analyzed. The number of cDC and pDC was significantly reduced after PbA infection. Further experiments demonstrate that type I and type II interferons were critically involved in activation and its-induced cell death of cDCs after PbA infection.It is well known that individuals with repeated malaria infection have acquired immunity against parasites, but its mechanisms remain unclear. To investigate the changes of DC after malaria infection-cure treatment, mice infected with PbA were cured using pyrimethamine. The analysis about DC shows that splenic cDC significantly increased in cured mice.研究課題/領域番号:24590507, 研究期間(年度):2012-04-01 - 2015-03-3