EBウイルス関連疾患におけるウイルス感染細胞の多様性

金沢大学大学院医学系研究科血管病態制御

ヒト新生児CD4抗原性T細胞(CD4細胞)の機能的分化誘導におけるCD45抗原意識

取得学位 : 博士（医学）, 学位授与番号 : 医博乙第1233号,学位授与年月日：平成5年6月1日,学位授与年：199

CD244抗原発現とCD8^+細胞傷害性T細胞の機能的分化

(1)新生児CD8+T細胞はCD244陰性であるのに対し,成人CD8+T細胞は約40-50%がCD244陽性であり,加齢に伴いCD244陽性比率は増加した.CD28,CD62L発現を3重解析するとCD244陰性細胞は全てCD28陽性CD62L陽性であるのに対し,CD244陽性細胞はCD28,CD62L陽性,陰性の亜群が存在した.伝染性単核球症(IM)や麻疹のCD8+T細胞ではCD244+細胞の増加が顕かで,特にCD28,CD62L陽性CD244陽性CD8+T細胞の増加が著明であった.(2)CD244陽性,陰性CD8+T細胞亜群における表面抗原,細胞内傷害性蛋白を解析した.CD244-細胞はどの表面分子発現からもナイーブ細胞として表面形質を示し,パーフォリン,グランザイムB, TIA-1発現も認めなかった.一方,CD244陽性細胞は,メモリー細胞表面形質を呈した.(3)Naive T細胞ではTCRレパートワは多様性に富むがmemory-T細胞では限られたT細胞レパートワであることが予想され,TCR CDR3 spectrum解析をCD244陽性、陰性各細胞亜群において解析した.CD244陰性細胞は全てのTCRVβでガウス分布を呈したが,CD244陽性分画は多くのTCRVβでskewedパターンを示した.また,CD28陽性細胞より陰性細胞で,CD62L陽性細胞より陰性細胞で複雑指数は低下していた。(4)CD244陽性CD8+T細胞への生体内でのCD244陰性細胞から陽性細胞への転換をin vitroにてCD244-CD8+T細胞を刺激培養して確認した.CD244発現はCD3刺激にて培養早期から増強した.一方,CD28,CD62L-分画への転換は2-3週間かかり緩除に進行した.(5)機能的分化における細胞分裂の程度を定量化するため、各細胞分画におけるTREC含有量を測定したところ、CD244陰性細胞は最も多くTREC量が多く、CD28CD62L陽性CD244陽性細胞、CD28/CD62L陰性CD244陽性細胞分画の順にTREC含有量は減少した。(1) About 40-50% adult CD8+ T cell were CD244 positive and CD244 positive fraction increased with aging whereas almost of newborn infant CD8+ T cell was CD 244 negative. CD28/CD62L positive, or negative two subgroup were present in CD244 positive cells when they analyzed three-color flow cytometry, whereas all CD244 negative CD8+ T cells were CD28 positive CD62L positive. Increase of CD244+ cells have been found in patients with infectious mononucleosis (IM) and measles., compared it with a normal subject, and in particular, and increase of CD28,CD62L positive CD244 positive CD8+ T cell were prominent.(2) We analyzed the difference of expression of surface antigen and intracellular cytotoxic granular proteins in CD244-positive, and CD244-negative CD8+ T cell subgroup. CD244-negative cells showed surface phenotype almost same as naive cell from all surface molecule expression and did not show perform, granzyme B, TIA-1 expression either. On the other hand, CD244-positive cells showed me mory cell surface phenotype.(3) Naive T cell have TCR repertoire with full of diversity, but T cell repertoire diversity was expected to be limited in memory-T cells, and TCR CDR spectrum were analyzed in each CD244-positive, CD244-negative cell subgroup. CD244 negative cells presented Gaussian distribution in all TCRVβ, but CD244-positive demarcation showed a skewed pattern in many TCRVβ. In addition, complexity index of CDR3 spectra in CD28-negative cell or CD62L negative cell decreased more than that in CD28 positive cells, or CD62L positive cell.(4) CD244-negative CD8+ T cell separated from normal adult CD8+T cells were stimulated and incubated, in vitro. We confirmed conversion from CD244-negative to CD244-positive phenotypte in CD8+ T cells. CD244 expression were increased with CD3 stimulation at early stage of culture. On the other hand, switch to CD28,CD62L- phenotype took two or three weeks and progressed in slowly.(5) We measured TREC content in each cell fractionation to quantify degree of mitosis in functional differentiation phase.CD 244 negative cells showed abundant TREC content, and the TREC content decreased in order of CD28CD62L positive CD244-positive cells, CD28/CD62L negative CD244-positive cell populations.研究課題/領域番号:16591013, 研究期間(年度):2004-2005出典：「CD244抗原発現とCD8^+細胞傷害性T細胞の機能的分化」研究成果報告書　課題番号16591013 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Similar Data Retrieval from Enormous Datasets on ELF/VLF Wave Spectrum Observed by Akebono

金沢大学総合メディア基盤センターAs the total amount of data measured by scientific spacecraft is drastically increasing, it is necessary for researchers to develop new computation methods for efficient analysis of these enormous datasets. In the present study, we propose a new algorithm for similar data retrieval. We first discuss key descriptors that represent characteristics of the VLF/ELF waves observed by the Akebono spacecraft. Second, an algorithm for similar data retrieval is introduced. Finally, we demonstrate that the developed algorithm works well for the retrieval of the VLF spectrum with a small amount of CPU load

Fas・Fasリガンド遺伝子異常の分子生物学的解析と遺伝子治療の基礎的検討

CD95(Fas)抗源は血液細胞の生理的アポトーシス細胞死を誘導するシグナルを伝達する分子として同定されたが,lprおよびgldマウスにおける自己免疫様リンパ球増殖症候群がFas,Fasリガンド(FasL)遺伝子異常に起因することが判明したことより,末梢における自己反応性リンパ球や過剰に産生され不用になった成熟リンパ球の排除におけるFas//Fasリガンドシステムによるアポトーシスの重要性が示唆されている.肝脾腫,リンパ節腫大,自己免疫性汎血球減少,高γglobulin血症,TCRαβ陽性CD4陰性CD8陰性T細胞の増加等lpr,gldマウス類似の臨床症状を示す自己免疫リンパ球増殖症候群の3家系4症例においてFas誘導性アポトーシス異常とFas遺伝子変異を解析した.末梢血より単核球及び顆粒球を分離し,PHA活性化T細胞,およびEB virus形質転換B細胞(LCL),顆粒球における抗Fas抗体にて誘導されるアポトーシスを比較検討し,LCLより抽出したRNAをRT-PCRにてFas cDNAを4分画するように設定したプライマーを用い増幅し,そのPCR産物を,Terminator Cycle Sequenxekitを用い塩基配列を検索した.抗Fas抗体を用いた患者由来細胞におけるFas抗原の発現は兄妹例(症例1,2)および症例4では正常であったが,症例3において全く欠如していた.抗Fas抗体により誘導されるアポトーシスは4例とも全く欠如し,患者由来活性化T細胞において抗CD3抗体にて誘導される細胞死も減弱していた.Fas遺伝子異常を解析すると,症例1,2ではintron7のsplicing donorの点突然変異によるexon7-8間に4bpの挿入が認められ,症例3ではexon4欠失のホモ接合体であり,症例4ではdeath domain内1014C-T置換に伴うnonsennse mutationのを認めた.症例1,2,4ではdeath domainを欠く異常Fas抗原によるdominant effectと考えられた.異常遺伝子は症例1,2では母親由来,症例3ではいとこ同士の両親由来であることが判明した.本邦初のFas抗原異常による自己免疫リンパ球増殖症候群を解析しFas抗原の免疫担当細胞のアポトーシスを介し免疫系の恒常性維持に重要な役割を果たすことが再認識された.Fas-mediated apoptosis and mutation of Fas and Fas ligand (FasL) gene were evaluated in four cases from three families with autoimmune lymphoproliferative syndrome. They showed clinical manifestations of hepatosplenomegaly, Iymphoadenopathy, autoimmune pancytopenia, hypergamma-globulinemia,. Apoptosis induced by anti-Fas antibody was determined PHA-activated T cells, EBV-transformed B cells and freshly isolated granulocytes from patients. Fas and Fas-ligand mRNA from patients\u27 cells was amplified by RT-PCR with primers specific for Fas and Fas-ligand gene and mutations of Fas gene was detected from PCR products. All cells from four cases showed resistance to Fas-induced apoptosis and the surface expression of Fas-receptor was completely diminished on cells from case 3. CD3-indeced activation cell death was also decreased in patients\u27 T cells. Three a different Fas gene mutations were detected. The heterozygous point mutations of intron 7 were detected in case 1 and 2 who are familial cases, resulting in skipping of exon 7 and premature termination. These mutation resulted in production of truncated Fas protein which lacks death domain. Case 3 showed homozygous mutation of intron 3 with lack of exon 4, In case 4, point mutation with exon 9 encoding death domain was detected. All four cases showed an increase of TCRαβ+CD4-CD8- T cells in circulation, which is a specific characteristic in mouse with Fas and FasL mutations.. Familial analysis showed that mutation of Fas gene were inherited from mother in Case 1 and 2 and from both parents in case 3. This is first report of Fas gene mutations in Japan. These results suggest that Fas-mediated apoptosis play pivotal role in maintenance of immune function.研究課題/領域番号:10670710, 研究期間(年度):1998-1999出典：「Fas・Fasリガンド遺伝子異常の分子生物学的解析と遺伝子治療の基礎的検討」研究成果報告書　課題番号10670710 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Fas誘導性アポトーシス細胞死における活性酸素の意義

活性酸素のアポトーシスにおける重要性を活性酸素の産生欠如が主病因である慢性肉芽腫症(CGD)患児由来細胞を用い検討し,Fas誘導性アポトーシスにおける活性酸素の意義を検討した。正常ヒト由来細胞は抗Fas抗体の添加によりアポトーシス誘導を受けるのに対し、CGD由来細胞は抗Fas抗体の点かによりアポトーシス誘導を受けるのに対し、CGD由来細胞では僅かであった。カタラーゼを添加することにより産生されるH_2O_2を分解すると濃度依存性に抗Fas抗体のアポトーシス増強作用は抑制され,一方、CGD由来細胞をH_2O_2存在下で培養するとFas誘導性アポトーシスの増強が認められた.FasあるいはFasリガンドの遺伝子変異による自己免疫疾患モデル1prgldマウスと類似した肝脾腫リンパ節腫大、汎血球減少,溶血性貧血,高γグロブリン血症等の臨床像を呈するヒト自己免疫性リンパ球増殖症候群の兄妹例においてFas誘導製アポトーシスを解析したところ,患児由来細胞においては全くFas誘導性アポトーシスが認められなかった.Western blot解析にて異常Fas抗原分子が同定され,遺伝子解析の結果,Fas intron7におけるT→C置換により患児由来のFas抗原はdeathdomainを含む細胞内部分を欠如すること,さらにこの遺伝子異常は母親由来であることが判明した.患児及び母親の末消血においてTCRαβ+CD4-CD8-T細胞の増加が認められた.Fas抗原を介したアポトーシスの生体内での免疫学的恒常性における重要性が示唆された.1) The involvement of reactive oxygen intermediates in Fas-mediated apoptosis was investigated using cells from patients with chronic granulomatous disease (CGD) that have no ability of reactive oxygen intermediates production. Although the treatments with anti-Fas antibody induced apoptosis of cells from normal subjects, cells from CGD were resistant to anti-Fas antibody. Catalase significantly inhibited Fas-mediated apoptosis of normal cells, on the other hands hydrogen peroxides induced apoptosis of CGD cells.2) Fas-mediated apoptosis was estimated in two siblings with autoimmune lymphoproliferative syndrome characterized with hepatosplenomegaly, lymphadenopathy, pancytopenia, hyper-gammagloblinemia, which is similar to clinicalmanifestations of lpr or gld mouse that have mutations of Fas or Fas ligand gene. Cells from two patients showed reduced Fas-induced apoptosis and smaller abnormal Fas protein was detected by western blot analysis. The T to C point mutation of intron 7 in patients Fas gene was detected and Fas antigen of patients lacks intracellular portion including death domain by this mutation TCRalphabeta+CD4-CD6- T cells were increased in blood I patients and analysis of family showed that mother has a same Fas mutation. These results indicated significance of Fas-mediated apoptosis in immunological homeostasis n vivo.研究課題/領域番号:08670862, 研究期間(年度):1996-1997出典：「Fas誘導性アポトーシス細胞死における活性酸素の意義」研究成果報告書　課題番号08670862(KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Differential expression of CD45RO (UCHL1) and its functional relevance in two subpopulations of circulating TCR-γ/δ+ lymphocytes

金沢大学大学院医学系研究科血管病態制御学We examined the developmental profile of TCR-γ/δ+ cells with respect to CD45RO expression. Although total TCR-γ/δ+ cells were negligible in the neonatal blood and increased with advancing age, most blood TCR-γ/δ+ cells markedly expressed CD45RO without a distinction of age, probably reflecting a different CD45RO expression of two subsets defined by BB3 and δTCS1 mAbs. The vast majority of BB3+ cells expressed CD45RO, whereas expression of CD45RO was virtually absent in the δTCS1+ population. Functional studies revealed that, while both TCR-γ/δ+ cell subsets showed CD3-mediated activation, only BB3+ (or TiγA+) cells, but not δTCS1+ cells, appeared to proliferate in response to PPD in PPD-reactive individuals. The results suggested that the CD45RO+ (BB3+ or TiγA+) subset among blood TCR-γ/δ+ cells may be mainly involved in the memory or primed component of the immune system responding to some foreign antigens

Extracorporeal membrane oxygenation following pediatric cardiac surgery: development and outcomes from a single-center experience

　Extracorporeal membrane oxygenation (ECMO) has emerged as an effective mechanical support following cardiac surgery with respiratory and cardiac failure. However, there are no clear indications for ECMO use after pediatric cardiac surgery. We retrospectively reviewed medical records of 76 pediatric patients [mean age, 10.8 months (0-86); mean weight, 5.16 kg (1.16-16.5)] with congenital heart disease who received ECMO following cardiac surgery between January 1997 and October 2010. Forty-five patients were treated with an aggressive ECMO approach (aggressive ECMO group, April 2005-October 2010) and 31 with a delayed ECMO approach (delayed ECMO group, January 1997-March 2005). Demographics, diagnosis, operative variables, ECMO indication, and duration of survivors and non-survivors were compared. Thirty-four patients (75.5%) were successfully weaned from ECMO in the aggressive ECMO group and 26 (57.7%) were discharged. Conversely, eight patients (25.8%) were successfully weaned from ECMO in the delayed ECMO group and two (6.5%) were discharged. Forty-five patients with shunted single ventricle physiology (aggressive: 29 patients, delayed: 16 patients) received ECMO, but only 15 (33.3%) survived and were discharged. The survival rate of the aggressive ECMO group was significantly better when compared with the delayed ECMO group (p<0.01). Also, ECMO duration was significantly shorter among the aggressive ECMO group survivors (96.5 ± 62.9 h, p<0.01). Thus, the aggressive ECMO approach is a superior strategy compared to the delayed ECMO approach in pediatric cardiac patients. The aggressive ECMO approach improved our outcomes of neonatal and pediatric ECMO

Role of interleukin 6 for differential responsiveness of naive and memory CD4+ T cells in CD2-mediated activation

金沢大学大学院医学系研究科血管病態制御学The present study was undertaken to elucidate different requirements for CD2-mediated activation of naive (CD45RO-) and memory (CD45RO+) CD4+ T cells. A mitogenic combination of anti-CD2 (anti-T112 and anti-T113) mAbs could effectively induce the proliferation of memory CD4+ T cells even in the absence of monocytes. In marked contrast, naive CD4+ T cells did not disclose any proliferative responses to antiCD2 mAbs, when monocytes were absent in culture. This differential responsiveness of naive and memory CD4+ T cells appeared to be related largely to a difference in IL-6 - producing ability between both populations. IL-6 among monocyte-derived cytokines could correct unresponsiveness of naive CD4+ T cells to anti-CD2 stimulation. Unlike naive CD4+ T cells, memory CD4+ T cells produced IL-6 by themselves, with its mRNA being expressed on anti-CD2 stimulation. Anti-IL-6R mAb significantly inhibited proliferation of memory CD4+ T cells seen in the anti-CD2-stimulated cultures without monocytes, indicating the involvement of their own production of IL-6 in CD2-mediated activation. The results suggest an essential role of IL-6 for triggering of CD4+ T cells via the CD2 molecule

A prospective randomized trial comparing the clinical effectiveness and biocompatibility of heparin-coated circuits and PMEA-coated circuits in pediatric cardiopulmonary bypass

OBJECT:　 We compared the clinical effectiveness and biocompatibility of poly-2-methoxyethyl acrylate (PMEA)-coated and heparin-coated cardiopulmonary bypass (CPB) circuits in a prospective pediatric trial.　 METHODS:　 Infants randomly received heparin-coated (n=7) or PMEA-coated (n=7) circuits in elective pediatric cardiac surgery with CPB for ventricular septum defects. Clinical and hematologic variables, respiratory indices and hemodynamic changes were analyzed perioperatively.　 RESULTS:　 Demographic and clinical variables were similar in both groups. Leukocyte counts were significantly lower 5 minutes after CPB in the PMEA group than the heparin group. Hemodynamic data showed that PMEA caused hypotension within 5 minutes of CPB. The respiratory index was significantly higher immediately after CPB and 1 hour after transfer to the intensive care unit (ICU) in the PMEA group, as were levels of C-reactive protein 24 hours after transfer to the ICU.　 CONCLUSION:　 Our study shows that PMEA-coated circuits, unlike heparin-coated circuits, cause transient leukopenia during pediatric CPB and, perhaps, systemic inflammatory respiratory syndrome after pediatric CPB