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非Down症児における急性巨核芽球性白血病 (AMKL) の発症は稀であり, 予後不良である. 症例は1歳4ヶ月の女児. 繰り返す発熱を主訴に他院にて治療されていたが, 末梢血中に芽球が出現したため, 香川大学医学部附属病院小児科に紹介入院となった. 血液検査の結果, 著明な白血球増多と貧血を呈しており, 芽球を2.0%認めたことから, 白血病の可能性を考慮し, 骨髄検査が行われた. 骨髄は高度過形成で芽球様細胞が78.8%認められた. 芽球様細胞は大小不同があり, 細胞質は好塩基性が強く, 辺縁はときにbleb様突起などの不整があり, ミエロペルオキシダーゼ (MPO) 染色は陰性であった. フローサイトメトリー (FCM) による細胞表面抗原はCD33, CD56, CD61, CD117陽性で, CD9, CD34, CD36, CD41, CD110などは陰性を示し, 細胞質内抗原ではTdT, CD3, CD79a, MPOがいずれも陰性であった. FCMの結果からAMKLの可能性を疑い, 細胞質内のCD41とCD61を追加で染色したところ共に陽性を示した. さらに染色体検査の結果が正常女性核型であったことから, 上記の所見と合わせて小児非Down症AMKL (non-DS-AMKL) と診断された. AMKLの巨核芽球は血小板関連抗原 (CD41, CD61, CD42b) のうち一つ以上が陽性を示すとされる. 本症例は形態的にAMKLの特徴を一部有し, 細胞表面のCD61が陽性でAMKLの条件を満たしたが, 非Down症児であることから, 芽球に付着した血小板による偽陽性の可能性を否定できず診断に苦慮した. しかし, 特異性が高いとされる細胞質内CD41, CD61の発現を証明できたことが確定診断をする上で根拠の一つとなり, FCMによる細胞内染色の重要性を再認識した症例であった. Acute megakaryoblastic leukemia (AMKL) in children without Down syndrome is rare and has a poor prognosis. A 16-month-old female, who was treated at another hospital with repeated fever, was referred to the Department of Pediatrics, Kagawa University Hospital, because of the appearance of blast cells in peripheral blood. A blood test showed marked leukocytosis and anemia, with 2.0% blast cells. Bone marrow was markedly hypercellular and 78.8% blast cells were observed. Blast cells were uneven in size, the cytoplasm was strongly basophilic, and the margins were sometimes irregular with bleb-like processes, and myeloperoxidase (MPO) staining was negative. Cell surface antigens by flow cytometry (FCM) were positive for CD33, CD56, CD61, and CD117, and negative for CD9, CD34, CD36, CD41, and CD110. The cytoplasmic antigens were negative for TdT, CD3, CD79a, and MPO. We suspected AMKL from FCM results, and when cytoplasmic CD41 and CD61 were additionally stained, both were positive. Furthermore, because the result of the chromosomal test showed a normal female karyotype, together with the above findings, a diagnosis of pediatric AMKL without Down syndrome (non-DS-AMKL) was made. AMKL megakaryoblasts are said to be positive for one or more of the platelet-related antigens (CD41, CD61, CD42b). This case had some morphological characteristics of AMKL, and the cells were positive for sCD61 which satisfied the conditions for AMKL. But because she was a non-Down syndrome child, the possibility of false positives due to platelets attached to blast cells could not be ruled out, making diagnosis difficult. However, the expression of cyCD41 and cyCD61, which are considered to be highly specific, was one of the criteria for making a definitive diagnosis. This case reaffirms the importance of intracellular staining by FCM.

多発性骨髄腫 （multiple myeloma : MM） に対する抗ヒト CD38 抗体であるdaratumumab（DARA） 投与中の患者検体では，間接抗グロブリン試験 （indirect antiglobulin test : IAT） が偽陽性となる可能性がある。今回，dithiothreitol （DTT） を使用しない，DARA の干渉を回避する新たな検出法を開発することを目的として検討を行った。 DARA と赤血球の結合を評価するために，各濃度のDARA 存在下で CD38 の発現を flow cytometry で測定したところ，CD38 の mean fluorescence intensity （MFI） は DARA 添加により低下した。さらに，DARA と His-tag 結合 recombinant human CD38 protein（ H-rhCD38）を反応させ，磁気ビーズを用いて上清から DARA の除去を検討した結果，DARA により阻害されていたAPC- 抗CD38 抗体の赤血球への結合が，DARA の除去により回復された。 これらの結果から，DARA 投与中の MM 患者の血清に H-rhCD38 を添加し，磁気ビーズで除去することで，DARA の IAT への干渉を回避できる可能性が示唆された。

Colchicine gel treatment is one of the major methods used to induce polyploid strains in plants. In this study, we examined several conditions of colchicine gel treatment in the diploid and tetraploid of Arabidopsis thaliana (ecotype: Columbia) to establish the most efficient method of tetraploid and octaploid induction, and compared the induction efficiency with that of one-drop colchicine solution treatment, which was previously reported. The shoot apex of diploid and tetraploid seedlings was treated with colchicine gel (0.5% or 1.0% colchicine dissolved in 1.0% agarose) for several days. Thus, 0.5% colchicine gel treatment of the diploid for two days induced tetraploid (7.5% of treated seedlings) most efficiently under all examined conditions, suggesting that one-drop colchicine solution treatment of the diploid is more effi-cient than colchicine gel treatment. 0.5% colchicine gel treatment of the tetraploid for two days induced ctaploid (5.0% of treated seedlings) most efficiently, which is similar to the one-drop colchicine solution treatment of the diploid. The survival rate of colchicine gel-treated seed-lings was much lower than that of one-drop colchicine-solution-treated seedlings. The survival rate difference resulted in a lower efficiency of polyploid induction by colchicine gel treatment. These results suggest that one-drop colchicine solution treatment of the tetraploid can most ef-ficiently induce octaploid., Departmental Bulletin Paper, テクニカルノート

我々は，当院で使用しているsysmex 社の多項目自動血球分析装置XN-9000に搭載されたhsA モードの基本性能を検討するにあたり，体腔液検体を対象としてBF モード・フローサイトメトリー法・目視法を用いて比較検討した｡比較的良好な相関が得られたが，マクロファージや腫瘍細胞など大型の細胞が出現する一部症例については，BF モード同様相関性が低くなった｡一方，低値細胞数症例においてhsA モードでのみHF 領域の細胞を検出できた症例を認めたことからBF モードと比較してより高感度に異常細胞を検出できる可能性が示唆された｡

[要旨]損傷菌の菌数測定には培養法が用いられることが多いが、細胞計測法のFlow cytometry (FCM) では、生菌、死菌、および損傷菌の迅速評価が可能である。本研究では、Listeria monocytogenesの代替株であるL.innocua (毒素陰性株)の薬剤損傷菌数測定について、培養法のThin agar layer (TAL)法とFCM法の比較 を行った。有効塩素謹度0～6ppmの強酸性電解水で処理したとき、TAL法では1ppmおよび2ppmの強酸性電解水処理において、選択培地よりもTAL培地で0.6log菌数が多く検出され、それぞれ59%および67%の損傷薗が生残したことが示唆された。 一方FCM法では、生菌を染色する膜透過型DNA染色剤 (thiazole orange:TO) と、死菌を染色する膜非透過型DNA染色剤 (propidium iodide:PI) での二重染色の結果、TOとPIの両方に染色された損傷菌の割合は、有効塩素濃度 1～3ppmでそれぞれ59%～78%となった。薬剤損傷L. innocuaの回復においては、TAL法では2時間後には損傷菌が未検出となったが、FCM法では4時間後まで損傷菌が生残していた。この結果より回復期の薬剤損傷菌の検出においては、二つの方法で違いがあることが示された。[Abstract]Culture-based methods are often used for the enumeration of sublethally injured bacteria,while flow cytometry(FCM)can provide rapid quantitation of live,dead,and injured bacteria. In this study,the culutur-based thin agar layer(TAL)method was compared with a FCM assay to enumerate chlorine-injured Listeria.innocua,a surrogate for L.monocytogenes.Bacteria were injured by mixing them with diluted electrolyed water containing 0 to 6 ppm available chlorine. In the TAL method,counts of L.innocua exposed to 1 to 2 ppm available chlorine were 0.6log higher on the TAL medium than on the selective medium than on the selective medium,indicating that 59 to 67% of the viable cells were injured by a chlorine level of 1 to 2 ppm.In comparison,the available chlorine level of 1 to 3 ppm caused sublethal injury ranging from 59 to 78% as determined by the FCM assay based on the quantitation of staned cells with both permeant DNA dye (thiazole orange:TO)and impermeant DNA dye (propidium iodide:PI),relative to live and dead cells that were stained only with TO and PI,respectively.With regard to recovery of chlorine-injured L.innocua after treatment with electrolyzed water,injured cells were inactivated after two hours when enumerated with the TAL method,whereas injured cells remained injured for four hours after the treatment as determined by the FCM assay.This result indicated a difference in the detection of chlorine-injured L.innocua between the two methods during the recovery period of injuredv cells.

Thrombosis is one of the most important health problems among developed countries. Roles of Platelet-leukocyte interaction in thrombosis has been gradually recognized. Increase in platelet-leukocyte aggregate has been reported among patients with acute myocardial infarction and cerebral infarction. Increase in platelet-leukocyte aggregate has also been reported among patients with myeloproliferative neoplasms, for whom thrombosis is the major clinical problem. Platelet-leukocyte aggregate may be useful as a marker of thrombosis risk. We measured platelet-leukocyte aggregate and platelet activation among healthy volunteers using flow cytometry. Percentage of platelet-monocyte aggregate was higher than that of platelet-granulocyte aggregate, and showed larger individual variation. Percentage of activated platelet showed better correlation with percentage of platelet-monocyte aggregate than with platelet-granulocyte aggregate. Percentage of platelet-leukocyte aggregate changed widely during storage periods.

急性骨髄性白血病（acute myeloblastic leukemia:以下AML）の微小残存病変（minimal residual disease：以下MRD）の検出は，骨髄移植後の再発を予測する重要な検査である。AMLの細胞は，様々な抗原発現異常を呈することが知られている。今回，AMLの患者にみられたCD34+CD15+CD7+細胞を指標として，flow cytometryを用いて骨髄移植後のMRDを検査し，MRDの陽性化が続いた後，骨髄再発を来した例を経験したので報告する。

薬物の精密投与を達成するための有効な手段として、微粒子担体を用いたDDS（ドラッグデリバリーシステム）製剤に対する期待が高まっている。薬物送達を目指した微粒子製剤を設計する場合、微粒子担体の粒子径、表面電荷及び表面特性などの粒子物性は、その物理化学的及び生体内安定性はもとより、その薬物送達機能を向上させる上で重要な因子である。微粒子担体としてリポソームは特に、これらの物性を制御しやすい担体として注目されている。従来、リポソームのようなサブミクロン粒子の物性を評価する手法としては、粒子径やゼータ電位測定、走査型及び透過型顕微鏡を用いた形状観察による評価などに限定されており、高機能な微粒子製剤を開発するためには、新しい視点での物性評価が強く求められている。本研究では、サブミクロン粒子の新規物性評価法の確立を目的とし、原子間力顕微鏡を用いて、リポソームの硬さについて評価を行った。また粒子の表面物性が生体組織との相互作用に与える影響に関してin vitro及びin vivoにて評価し、リポソームの経肺投与型製剤としての可能性についても検討した。, The Drug Delivery System (DDS) raises expectations as an effective method of achieving precise administration of active pharmaceutical ingredients. Concerning formulating fine-particle products for drug delivery, physicochemical properties of particles, such as particle size, surface charge or surface condition are important factors for increasing the function of drug delivery. Liposomes as drug carriers are quite useful for controlling these properties. Methods for evaluating physicochemical properties of submicron-sized particles are limited to the measurement of particle size or zeta potential and observation of morphology with a scanning or transmission electron microscope. Therefore, a novel evaluation method is needed to develop a functional product of fine particles. The rigidity of liposomes is evaluated with an atomic force microscope for the purpose of establishing a novel evaluation method. The effects of surface properties on bio-interaction are also evaluated in vitro or in vivo then the possibility of polymer-coated liposomes as the dosage form for pulmonary administration is investigated.

The ABO blood group system is the most important blood group system among the 29 International Society of Blood Transfusion（ISBT）-recognized systems, consisting of four antigens （A, B, O, and AB）. These antigens are known as oligosaccharide antigens, and widely expressed on the membrane of red cell and tissue cells as well as, in the saliva and body fluid. The ABO blood group system was discovered by the Austrian scientist Karl Landsteiner, who found three different blood types（A, B, and O）in 1900 from serological differences in blood. In 1902, DeCasterllo and Stürli discovered the fourth type, AB. The phenomenon of these serological differences was called the Landsteiner Law, and is still used to detect ABO blood group antigens in clinical laboratories. In 1924, Felix Bernstein predicted that the mechanism of inheritance involved three alleles at the ABO locus from extensive family studies. Furthermore, the structure and biochemical characteristics of the ABO antigens were elucidated by many investigators. In recent years, the ABO blood group at chromosome locus 9 has been determined, the gene cloned and the structures determined by Yamamoto et al . This has made it possible to genetically analyses of ABO blood group antigens using molecular biology techniques. Clinically, blood group antigens are important. In particular, the ABO blood group antigens are one of the most important issues in transfusion medicine, evaluation of the adaptability of donor blood cells with bone marrow transplantations, and survival confirmation of hemocytes. This article reviews the serology, biochemistry, biosynthesis and genetic characteristics of ABO antigens, and the analysis of ABO blood group using flow cytometry and polymerase chain reaction（PCR）amplification of specific alleles（PASA）-method and their application for clinical studies.

【背景】カルシニューリン阻害薬(calcineurin inhibitor:CNI)であるCyclosporin A(CsA)とTacrolimus(FK506)は,臓器移植に対する拒絶予防や同種造血幹細胞移植(allo-HSCT)後のgraft-versus-host disease(GVHD)に対する予防・治療における薬剤として重要な役割を果たしており,主にT細胞のサイトカイン産生を阻害することでその免疫抑制効果が発揮される.近年,特に臓器移植の分野においてはCNIの薬物体内動態や薬効指標の研究が進んできているが,allo-HSCTの分野においては,CNIの使用が標準化しているものの,その投与方法には施設問差があり,最適な投与方法が確立されていない.今回我々は,CsA,FK506それぞれの薬剤のallo-HSCTにおける至適投与法を考案する上で重要なサイトカイン産生抑制効果の特性を明らかにするために,フローサイトメトリーを用いて細胞内サイトカイン陽性細胞を検出し,in vitroにおけるCNIのサイトカイン産生抑制効果について検討した.【方法】健常人末梢血単核球(MNC)を分離し,Phorbor12-Myristate13-Acetate,ionomycin,brefeldinA存在下に37℃,4時間共培養後,MNCの表面抗原(CD4,CD8)を染色し,固定・膜透過処理を行った後,細胞内サイトカイン(IFN-γ,IL-2,IL-4)の染色を行い,フローサイトメトリーを用いて,CD4・CD8リンパ球分画による細胞内サイトカイン陽性T細胞の割合を測定・解析した.この方法を用いてCNI暴露とサイトカイン陽性T細胞の割合,CNI暴露解除後のサイトカイン陽性T細胞の回復,またCD3/CD28beads刺激により誘導した活性化T細胞を用いて,CNIが活性化T細胞へ及ぼす影響について検討した.【結果】CNI暴露後のサイトカイン陽性T細胞の非暴露T細胞との相対的割合は,CsAでは1-100ng/mlの濃度で濃度依存的に減少するが,FK506では1-10ng/mlとCsAに比し,より低濃度かつ狭い濃度域で急激に減少した.またCsAの活性化T細胞におけるサイトカイン抑制効果は,restingT細胞における効果よりも減弱されることが確認された.一方FK506では,活性化T細胞とrestingT細胞とで抑制効果に差を認めなかった。さらに,CsA暴露解除後のIL-2産生は,暴露濃度が低い場合には回復が有意であるが,高濃度のCsAやFK506では少なくとも4時間後までは抑制効果が持続することがin vitroで確認された.【結語】今回の得られた免疫抑制の特性から,allo-HSCT臨床におけるGVHD予防におけるCNI使用法を考察すると,CsAを用いる際は,一時的に高濃度(ピーク)を作る投与方法がCsAの特性を生かした投与法ではないかと考えられ,一方FK506は,毒性との兼ね合いから24時間持続点滴による投与法は許容される投与法であることが示唆された.

We used flow cytometry to estimate the polyploidy level of 304 carnation cultivars preserved at the National Institute of Floricultural Science, and counted chromosomes in root tips to verify the actual level. The results validated the use of flow cytometry to estimate polyploidy in carnation cultivars. It estimated that 297 carnation cultivars were diploid. Three cultivars ('Wiko', 'Scarlet Bell', and 'Spiral Vivid Red') were triploid and 'Saleya' was tetraploid. Another 3 cultivars ('Pink Roland', 'Youkihi', and 'Sonnet Sailor') might be tetraploid. We measured the stomatal length of 12 cultivars to test whether it is a convenient way to distinguish polyploidy. We could distinguish between diploid and polyploid, but not between triploid and tetraploid.

application/pdf, Rapid determination of ploidy levels in cyclamen by using flow cytometry of DAPI‐stained nuclei was examined. A strong positive linear correlation between DAPI fluorescent intensities of nuclei extracted from the leaf blades of cyclamen plants and the ploidy levels in diploid, triploid and tetraploid plants was confirmed. Thus, the flow cyto‐metry of DAPI‐stained nuclei in cyclamen was proven to be an effective and easy tool to determine the ploidy levels within a short time. DAPI fluorescent intensities of nuclei extracted from the pollen grains of diploid plants having and without giant pollen grains were also investigated. Three peaks of 1C, 2C and 4C were observed in the plants having giant pollen grains, whereas only 1C and 2C peaks were observed in the plants without giant pollen. The results suggested that the giant pollen grains were unreduced 2n pollen grains., フローサイトメトリーを用いた、シクラメンの簡易かつ迅速な倍数性検定法の確立を試みた結果、各倍数体レベルとDAPIにより染色された細胞内核DNAの蛍光強度との間に高い相関が認められ、フローサイトメトリーがシクラメンの簡易倍数性検定に有効な手段であることが示された。また、フローサイトメトリーを用いて、巨大花粉を分離しないまたは分離するシクラメン二倍体個体の花粉分析した結果、前者では1Cと2Cのピークが認められたのに対して、後者では1Cと2Cに加えて4Cのピークが認められ、巨大花粉は非還元性の2n花粉であることが強く示唆された。

The combination therapy of carcinurin inhibitor (CNI), and inhibitor of nucleic acid production, has been widely utilized in clinical kidney transplantation. Mycophenolate mofetil (MMF) has a strong immunosuppressive effect as compared with azathioprine. As a result, over immunosuppression is often experienced under the combination therapy of MMF and CNI. It is very difficult to adjust each immunosuppressive drug without knowing how much the immunosuppression and how much each drug affects the immunosuppression. The method of monitoring the immunosuppression has not yet become available for transplant patients under the combination therapy. We investigated the usefulness of flow cytometry of the lymphocyte stimulation test under the immunosuppressive drug in vitro. The peripheral blood from four healthy volunteers was separated by density gradient centrifugation. After lymphocytes were dyed with 5(and 6)-carboxy fluorescein diacetate succinimidyl ester(CFSE), cells were cultured with Staphylococcus enterotoxin B (SEB) for 4 days under the Mycophenolic acid (MPA) and/or Cyclosporine (CsA) in various concentrations. After cultured, Division Index (DI) was measured by FACS, and Stimulation Index (SI) was calculated using DI. From a series of multiple concentrations of MPA or CsA, SI 25, 50 and 75 of MPA groups were 54, 18 and 6 ng/ml, and SI 25, 50 and 75 of CsA groups were 250, 100 and 30 ng/ml. We defined the medium concentration of each drug as MPA 30 ng/ml and CsA 100 ng/ml, then 1/5 of these results as low concentration and concentration 5 times as high (setting up MPA: 6, 30 and 150 ng/ml, and CsA: 20, 100 and 500 ng/ml). The mean S.I. of MPA group was 71.27±11.55 at 6 ng/ml, 23.63±13.44 at 30 ng/ml and 4.87±1.33 at 150 ng/ml. The mean S.I. of CsA group were 78.02±9.05 at 20 ng/ml, 44.14±4.89 at 100 ng/ml and 16.51±5.17 at 500 ng/ml. At the combination of low doses of CsA and MPA, S.I. was 61.03±8.03. The addition of a medium dose of MPA to the low dose of CsA decreased S.I. to 12.23±0.75, which is almost the same result of the high dose of CsA group. These results have us conclude that the dose of CsA could be decreased when the medium dose of MMF is combined with CsA treatment. In addition, patterns of the histogram in Flow cytometry found that there were differences between CsA and MPA, leading to a better understanding as to which drug contributed to the over- immunosuppression.

Dendritic cells are strong antigen-presenting cells. Recently, antitumor immunity therapy using dendritic cells (DC therapy) has received attention. The clinical study of DC therapy for malignant diseases has been undertaken, and both cases of being effective responded and tumor-specific CTL-induced have been reported. However, no specific procedure for DC therapy has yet been established, so further detailed investigation is necessary. To perform a clinical study of such cell therapy, the technique for detecting and determining the cells to be used is critical. In this study, we devised a technique of deetecting peripheral myeloid dendritic cells by three-color flow cytometry. Gates were set for HLA-DR^+/CD33^+/CD14^- CD16^- CD34^- cells to determine one hundred thousands collected cells, leading to the clear identification of a cell population as being myeloid dendritic cells. The surface characteristics of this cell population were determined to be negative for CD3, CD19 and CD56, positive for CD2 and CD4, weakly positive for CD5, positive for CD11a and CD11c, negative to weakly positive for CD11b, negative for CD1a, CD80 and CD83, and positive for CD86. Such characteristics agree well with those previous- ly reported for peripheral myeloid dendritic cells. Thus, peripheral myeloid dendritic cells were judged to have been identified by this technique. The proportion of myeloid dendritic cells in normal subjects was 0.74±0.17%, with absolute numbers of 17.54±5.80/μl. In patients with chronic CML, incipient or recurrent malignant lymphoma, and acute leukemia in remission phase, the proportion of myeloid dendritic cells was reduced, suggesting a decrease in immunologic surveillance function. In patients with malignant lymphoma, following autologous peripheral blood stem cell transplantation, the proportion of myeloid dendritic cells was increased, representing the clinical effect of immunotherapy by autologous peripheral blood stem cell transplantation. This technique can readily deter- mine exact numbers of peripheral myeloid dendritic cells and will contribute to the future study of dendritic cells and the development of DC therapy.

レセプター型チロシンキナーゼであるc-kitとそのリガンドであるstem cell factor (SCF)は初期造血において重要な役割を果たしていることが知られている。c-kitとSCFの共発現がautocrine機序による腫瘍増殖に関与することがある種の腫瘍細胞において想定されているが, 白血病細胞での報告は未だない。急性白血病(AML)の細胞を対象に, c-kitとSCFによるautocrine機序を介した細胞増殖について検討した。AML28症例の白血病細胞を材料にc-kitとSCFのsoecific primerを用いたRT-PCR法によりmRNAの発現を検討し, flow cytometry (FCM)によりc-kitおよびSCFの細胞表面への発現を検討した。全例でc-kit mRNAの発現を認め, 28症例中2例ではc-kitとSCFの共発現をmRNAレベルと細胞表面蛋白レベルで確認した。共発現例の白血病細胞は低濃度のfetal calf serum (FCS)添加で増殖し, SCF中和抗体の添加で増殖が阻害された。また共発現を示した症例の培養細胞上清中にSCFが検出された。以上の結果より, AMLの症例中にはc-kitとSCFが共発現する例があり, auto-crine機序を介して白血病細胞の増殖に関与している可能性が示唆された。, Stem cell factor (SCF) is a pluripotent hematopoietic growth factor which is suggested to play an important role in early stage of constitutive hematopoiesis as the ligand of the c-kit proto-oncogene product. It is reported that some solid tumor cells such as small cell lung cancer, coexpress both c-kit and SCF, and the receptor/ligand system may have an autocrine function in such tumor cell growth. But little is known about the coexpression in hematological malignancies. We examined a panel of 28 acute myeloid leukemia (AMD specimens for coexpression of c-kit and SCF, using reverse transcription-polymelase chain reaction (RT-PCR) method for mRNA expression, and flow cytometry (FCM) for cell surface protein expression, c-kit mRNA was expressed in all cases tested, and in two cases, coexpression of c-kit/SCF was observed at mRNA and cell surface protein levels. These cells grew in spite of a low concentration of fetal calf serum, and the growth was blocked by addition of anti-SCF antibody. Enzyme-linked immunosorbent assay (ELISA) revealed elevation of soluble SCF levels in the supernatant of such cell culture. Thus, these data suggest that c-kit/SCF system may participate in autocrine stimulation of some AML cell growth.

In cells undergoing apoptosis, the characteristic "ladder" pattern of DNA fragments is observed on agarose gel electrophoresis. Such a pattern of DNA degradation generally serves as a criterion for the apoptosis. However, on agarose gel electrophoresis it is difficult to accurately quantify DNA fragmentation. Therefore, quantification of DNA fragmentation is determined by flow cytometry, DNA fragmentation assay etc. In this paper we improved the methods for the analysis of apoptosis by flow cytometry. Asa result, we can obtain much information (quantification of apoptosis and cell cycle analysis etc.) from a single sample (1～3×10 6 cells) with the improved methods. The sample is treated with phosphate-citrate buffer. These methods are very simple andconvenient.

The c-kit proto-oncogene encode a receptor tyrosine kinase that is considered to play an important role in hematopoiesis, especially at early stage. The expression of proto-oncogene c-kit was detected in human cell lines derived from leukemic cells of myeloid and megakaryocytic lineages and decresed depending on cell differentiation. The amount for c-Kit receptor protein on cells was analyzed by flow cytometry, and correlated with the level of its gene expression estimated by RT-PCR. The expression on the cells of patients with immature phenotype of acute myelogeneous leukemia (FAB M1 and M2) was high and was not detected on cells of lymphoid malignancies. Thus, the detection of c-kit gene or protein on leukemic cells was useful for determinination of the stage of differentiation and the lineages of cells and for diagnosis of leukemia, estimate of its prognosis, and the selection for treatment., c-kit遺伝子は初期造血に重要な役割を果たしているレセプター型チロシンキナーゼをコードしている。このc-kit遺伝子の発現は骨髄細胞性, 巨核芽球性白血病細胞から樹立した細胞株で検出され, それらの細胞の分化とともに減少した。c-kitレセプター蛋白の発現はFlow cytometryを用いた解析で, 遺伝子の発現と相関する結果が得られた。急性の造血器悪性疾患40例の細胞からRT-PCRを用いてその発現を検討したところ, 未分化なタイプの白血病(FABのM1, M2)に強く検出され, リンパ球系の細胞からは検出されなかった。このように細胞におけるc-kit遺伝子や, その蛋白の発現を検討することは, その細胞の分化段階, 系統を決定するうえで有用であり, 白血病の診断, 予後の決定, 治療法の選択等に重要な情報となることを推測させる。

We studied lymphocyte subsets, PHA-induced blastogenic rates of lymphocytes and Le^Y expression rates of them in 32 cases including 17 preoperative patients of gastric cancer, 8 healthy adults and 7 terminal patients of gastric cancer. In 17 patients who had underwent gastrectomy the relationship between histologic findings of cancer and lymphocyte in blood was examined. The PHA-induced blastogenic rates of lymphocytes were reduced in both operated and terminal stage groups. As regard to the change of lymphocyte subsets, the number of CD4^ lymphocytes were normal range with no relation about the depths of invastion of gastric cancer. However, CD8^ lymphocytes increased with the depth of invasion from mucosa to subserosa and CD19^ lymphocytes decreased with the depth of invasion from mucosa to muscularis propria. These changes in lymphocyte subsets were thought to be the results of depressed immune potency from early stages of gastric cancer. The Le^Y expression rate of lymphocytes did not depend on the depth of invasion of cancer cells in the operate group, but was significantly higher in the cancer-bearing state of gastric cancer and terminal stage patient. In the many cases of higher Le^Y expression rates of lymphocytes, blastogenic rates of them were low. Based on these results, measuring the rate of Le^Y expression on lymphocytes may be clinicaly usefull.

Thromboembolism is frequently observed in patients with prosthetic heart valve and is believed to be a major cause of post-operative morbidity and mortality. The aim of this study is to elucidate the role of activated platelets in the patients with prosthetic heart valve. Forty patients (23 female and 17 male) were operated on between 1967 and 1994 for prosthetic heart valve replacement in our university. Mean age was 55 years (range 2～68). All patients received anti-coagulant therapy. In 40 patients with prosthetic heart valve and 20 normal subjects, activated platelets were detected by flow-cytometric measurement of the proteins CD62P and CD63, which are expressed on the platelet surface membrane during platelet activation. Among healthy volunteers, a small percentage of platelets were activated (CD62P: 3.14±0.82%, CD63: 3.53±1.29%). In contrast, the patients group showed increased percentage of activated platelets. (CD62P: 4.08±2.25%, p=N.S., CD63: 4.82±2.70%, p

We observed adiamycin (ADR) uptake and cellular transmembrane potential [amount of intracellular fluorescence of 3,3'- (Di-n-hexyl)- 2,2'- oxacarbocyanine iodide (NK-2280)] in ADR-resistant cells established from Ehrlich ascites tumor cells (EATC) and wild type EATC. In ADR-resistant cells, ADR uptake and the cellular transmembrane potential decreased as the degree of resistance increased. 4,4'- diisothiocyanatostilbene- 2,2'- disulfonic acid (DIDS) induced markedly decreases of ADR uptake and the cellular transmembrane potential. A good correlation was observed between ADR uptake and transmembrane potential in cultured cells.

対象は他臓器転移を有する腎癌患者6名である.IFN-γ1, 000万/JRU/m2を1日1回点滴静注により5日間連続投与した後, 9日間休薬した.これを2回繰り返した後, 同量隔日3回投与し9日休薬し, さらに同量を3回隔日投与した.IFN-γ投与前後で, 末梢血のリンパ球数, 単球数に変動はなかった.CD 4陽性細胞は減少し, 逆にCD 8陽性細胞は有意に増加した.CD 16細胞も有意に増加し, Leu HLA-DRの値も有意に増加した.NK活性は増加の傾向, 非誘導LAK活性は有意の増加を示した.血清ネオプテリン値も有意に増加した.臨床的にIFN投与により画像診断的に転移巣の縮小はなかった.1例は投与10ヵ月後, 1例は3ヵ月後, 2例は1ヵ月後に死亡した, Immunologic and anti-tumor effects of interferon-gamma were studied in six patients with advanced renal cell carcinoma. A daily dose of 10 x 10(6) JRU/m2 of interferon-gamma (IFN-gamma) was given consecutively for five days and the treatment reiterated nine days later. Then the same dose was given every two days for three times and this regimen was also reiterated nine days later. Peripheral blood lymphocytes were analyzed before and one, three, five and eight weeks after the initial administration of IFN-gamma. Although the total number of lymphocytes and monocytes were not changed, CD8 (23.4 to 31.2%, p < 0.05), CD16 (12.4 to 19.3%, p < 0.01) and LeuHLA-DR (29.0 to 39.5%, p < 0.01) positive lymphocytes were significantly increased after the therapy. Non-induced LAK activity (0.6 to 10.8%, p < 0.05) and serum neopterin (3.5 to 10.8 nmol/L, p < 0.01) were also increased. These immunological parameters drastically changed during the consecutive administration of IFN-gamma, and tended to return to the previous value after this period. However, tumor regression was not clinically obtained. Our findings indicate that interferon-gamma stimulated the anti-tumor activity of the host, and administration of IFN-gamma is thought to be of immunological significance even in the advanced high stage cases.