Search

BackgroundBecause of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive.MethodsWe conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide.ResultsIn vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels.ConclusionsThe trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS.Trial registration numberNCT02366546.

Introduction Quantitative measurement of anti-A/-B antibody titers is important during ABO-incompatible living kidney transplantation (ABOi-LKT). Methods We conducted a multi-institutional study to measure the antibody titers using the automated column agglutination technique (auto-CAT) and tube test (TT) method in ABOi-LKT recipients. Statistical analysis was performed to evaluate the two methods. Results We examined 111 samples from 35 ABOi-LKT recipients at four institutions. The correlation coefficient of the two methods was >0.9; the concordance rate and clinically acceptable concordance rate for the IgG titers were 60.4% and 88.3%, respectively. Perioperative status did not influence the statistical significance. Parallel changes were observed in the IgG antibody titers measured using the auto-CAT or TT technique by desensitizing therapy in time-course monitoring. Conclusion Auto-CAT is comparable with the TT technique and is feasible for IgG anti-A/B antibody titration in ABOi-LKT recipients. This article is protected by copyright. All rights reserved.

Background The basophil activation test (BAT), performed with patient blood samples and supernatants from transfused blood, was developed to elucidate the mechanistic relationship between transfusion and the resultant allergic transfusion reactions (ATRs). This test cannot be performed on myelosuppressed patients and neonates because of the absence of basophils. Therefore, we devised the passive immune basophil activation test (pi-BAT) using patients' plasma and residual transfused blood as sources of immunoglobulin E and allergen, respectively, and the basophils of healthy volunteers served as a source of the responder cells. The sensitivity and specificity of the pi-BAT, however, remained largely unknown. Study design and methods In this study, the pi-BAT was performed on 31 patients with nonhemolytic transfusion reactions including nine non-ATR and 22 ATR (12 mild and 10 moderate-to-severe) cases to examine its sensitivity and specificity. Results Nine of the 10 cases with moderate-to-severe ATR tested positive, whereas all the non-ATR cases negative, strongly indicating immunoglobulin E and allergens are involved in the pathogenesis underlying the blood transfusion-triggered adverse effects. Conclusion Thus, we propose that pi-BAT can be used to detect moderate-to-severe ATRs and their underlying mechanisms.

Macrophages play an indispensable role in both innate and acquired immunity, while the persistence of activated macrophages can sometimes be harmful to the host, resulting in multi-organ damage. Macrophages develop from monocytes in the circulation. However, little is known about the organ affinity of macrophages in the normal state. Using in vivo imaging with XenoLight DiR®, we observed that macrophages showed strong affinity for the liver, spleen and lung, and weak affinity for the gut and bone marrow, but little or no affinity for the kidney and skin. We also found that administered macrophages were still alive 168 hours after injection. On the other hand, treatment with clodronate liposomes, which are readily taken up by macrophages via phagocytosis, strongly reduced the number of macrophages in the liver, spleen and lung.

Background Despite recent progress in blood systems, transfusion errors can occur at any time from the moment of collection through to the transfusion of blood and blood products. This study investigated the actual statuses of blood transfusion errors at institutions of all sizes in Aichi prefecture. Materials and methods We investigated 104 institutions that perform 98 % of the blood transfusions in Aichi prefecture, and investigated the errors (incidents/accidents) that occurred at these facilities over 6 months (April to September, 2017). Incident/accident data were collected from responses to questionnaires sent to each institution; these were classified according to the categories and risk levels. Results Ninety-seven of the 104 institutions (93.3 %) responded to the questionnaire; a total of 688 incidents/accidents were reported. Most (682 cases; 99.2 %), were classified as risk level 2; however, 6 were level 3 and over, which included problems with autologous transfusion and inventory control. Approximately one-half of the incidents/accidents (394 cases; 57.3 %), were related to verification and the actual administration of blood products at the bedside; more than half of these incidents/accidents occurred at large-volume institutions. Meanwhile, a high frequency of incidents/accidents related to transfusion examination and labeling of blood products was observed at small- or medium-sized institutions. The reasons for most of these errors were simple mistakes and carelessness by the medical staff. Conclusions Our results emphasize the importance of education, operational training, and compliance instruction for all members of the medical staff despite advances in electronic devices meant to streamline transfusion procedures.

Plasma removal by washing platelet concentrates (PCs) is effective in preventing adverse reactions to PC transfusions. The Japanese Red Cross Society (JRCS) started releasing washed PCs (WPCs) as a commercially approved blood product in September 2016. This retrospective multicenter study investigated the change in the number of transfused WPCs and the impact on the incidence of adverse reactions to PCs before and after the release. The numbers and types of transfused PCs and the adverse reactions to the PCs for a year before the start of the WPC release and for a year after the release were reported by 27 medical institutes in Japan. Transfusion information for approximately 8% of the amount of PCs supplied in Japan was analyzed during the study period. After the start of WPC release by the JRCS, the number of transfused WPCs doubled. The rate of adverse reactions to PCs decreased significantly (p = 0.0223), from 4.30% before the release to 4.05% after the release. The rates of adverse reactions to unwashed and WPCs were 4.13% and 0.84%, respectively. Allergic adverse reactions were significantly decreased after the release (3.60% before versus 3.37% after). No severe allergic reactions to WPCs were reported. The release of WPCs by the JRCS significantly reduced transfusion-related adverse reactions to PCs in Japan.

BACKGROUND The plasma fraction of blood components has an essential role in the etiology of allergic transfusion reactions (ATRs). The difference of incidences of ATRs between fresh-frozen plasma (FFP) and platelet concentrates (PCs), in which plasma is the main component, is not clearly understood. This study compares the frequency of ATRs to FFP versus PCs on both first and subsequent (nonfirst) transfusions and considers the factors influencing the risk of ATRs. STUDY DESIGN AND METHODS Five hospitals agreed to systematically collect and share 2 years of data (January 2010 through December 2011). This was a retrospective observational analysis of data including the number of transfusion episodes and ATRs for FFP and PCs on first-transfusion patients (without transfusion history) and previously transfused patients. RESULTS The incidence of ATRs to PCs (2.51%) was significantly higher than to FFP (1.68%) on subsequent transfusions (p

Background Adoptive transfer of TCR-redirected T cells has been reported to exhibit efficacy in some patients with melanoma and sarcoma. However, cytokine release syndrome (CRS) or its relations to tumor response has not been well documented. This study aimed to evaluate clinical responses in association with the cell kinetics and CRSs after transfer of high-affinity NY-ESO-1 TCR-gene transduced T cells in cancer patients. Methods We developed a novel-type affinity-enhanced NY-ESO-1-specific TCR and an originally-developed retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1/TCR sequence was mutated for high affinity with replacements of G50A and A51E in CDR2 region. This is a first-in-human clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It was designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3 + 3 cohort design. Cyclophosphamide (1,500mg/m2) were administered prior to the TCR-T cell transfer as pre-conditioning. Results Nine patients were treated with the TCR-T cells that expanded in peripheral blood with a dose-dependent manner, associated with rapid proliferation within 5 days after infusion. Three patients receiving 5x109 cells developed early-onset CRSs, with elevated levels of serum IL-6, IFN-γ. The CRSs on day1 or 2 were well managed with tocilizumab treatment. Three synovial sarcoma patients exhibited tumor shrinkage and partial responses, and they all had high-expression of NY-ESO-1 in the tumor samples, namely, 75% or more. Exploratory analysis revealed that multiple chemotactic cytokines including CCL2 and CCL7, and IL-3 increased in the serum from the patients with CRS. The proportions of effector-memory phenotype T cells in the infused cell-product were significantly associated with CRS development. Conclusions The affinity-enhanced NY-ESO-1/TCR-T cell transfer exhibited early-onset CRS in association with in vivo cell proliferation and sequential tumor responses in the patients with high-NY-ESO-1-expressing synovial sarcoma. Clinical trial identification NCT02366546. Legal entity responsible for the study The authors. Funding Japan Agency for Medical Research and Development. Disclosure All authors have declared no conflicts of interest.

2530 Background: Adoptive transfer of TCR-redirected T cells has been reported to exhibit efficacy in some of melanoma and sarcoma patients. However, there have not been well known about cytokine release syndrome (CRS) or its relations to tumor responses. This study evaluates clinical responses in association with the cell kinetics and CRSs after transfer of high-affinity NY-ESO-1 TCR-gene transduced T cells in NY-ESO-1-expressiong cancer patients (NCT02366546). Methods: We developed a novel-type affinity-enhanced NY-ESO-1-specific TCR and an originally-developed retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1/TCR sequence is mutated for high affinity with replacements of G50A and A51E in CDR2 region. This is a first-in-man clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It was designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3+3 cohort design. Cyclophosphamide (1,500mg/m2) were administered prior to the TCR-T cell transfer as pre-conditioning. Results: 9 patients were treated with the NY-ESO-1/TCR-T cell transfer. The TCR-T cells expanded in peripheral blood with a dose-dependent manner, associated with rapid proliferation within 5 days after the cell transfer. 3 patients receiving 5x109 cells developed early-onset CRSs, with elevations of serum IL-6, IFN-γ. The CRSs developed on day1 or 2 after the cell transfer. They were well managed with tocilizumab treatment. 3 synovial sarcoma patients exhibited tumor shrinkages of partial responses, and they all had high-expression of NY-ESO-1 in the tumor samples, namely, 75% or more. Exploratory analysis revealed that multiple chemotactic cytokines including CCL2 and CCL7, and IL-3 increased in the serum from the patients with CRS. The proportions of effector-memory phenotype T cells in the infused cell-product were significantly associated with CRS development. Conclusions: The affinity-enhanced NY-ESO-1/TCR-T cell transfer exhibited early-onset CRS in association with in vivo cell proliferation and sequential tumor responses in the patients with high-NY-ESO-1-expressing synovial sarcoma. Clinical trial information: NCT02366546.

Background A surveillance system for transfusion-related adverse reactions and infectious diseases in Japan was started at a national level in 1993, but current reporting of events in recipients is performed on a voluntary basis. A reporting system which can collect information on all transfusion-related events in recipients is required in Japan. Methods We have developed an online reporting system for transfusion-related events and performed a pilot study in 12 hospitals from 2007 to 2010. Results The overall incidence of adverse events per transfusion bag was 1.47%. Platelet concentrates gave rise to statistically more adverse events (4.16%) than red blood cells (0.66%) and fresh-frozen plasma (0.93%). In addition, we found that the incidence of adverse events varied between hospitals according to their size and patient characteristics. Conclusion This online reporting system is useful for collection and analysis of actual adverse events in recipients of blood transfusions and may contribute to enhancement of the existing surveillance system for recipients in Japan.

Stromal cells and osteoblasts play major roles in forming and modulating the bone marrow (BM) hematopoietic microenvironment. We have reported that FGF2 compromises stromal cell support of normal hematopoiesis. Here, we examined the effects of FGF2 on the leukemia microenvironment. In vitro, FGF2 significantly decreased the number of stromal-dependent and stromal-independent G0-leukemia cells in the stromal layers. Accordingly, CML cells placed on FGF2-treated stromal layers were more sensitive to imatinib. Conversely, FGF2 increased the proliferation of osteoblasts via FGFR1 IIIc, but its effects on osteoblast support of leukemia cell growth were limited. We next treated a human leukemia mouse model with Ara-C with/without systemic FGF2 administration. BM sections from FGF2-treated mice had thickened bone trabeculae and increased numbers of leukemia cells compared to controls. Leukemia cell density was increased, especially in the endosteal region in FGF2/Ara-C -treated mice compared to mice treated with Ara-C only. Interestingly, FGF2 did not promote leukemia cell survival in Ara-C treated spleen. Microarray analysis showed that FGF2 did not alter expression of many genes linked to hematopoiesis in osteoblasts, but modulated regulatory networks involved in angiogenesis and osteoblastic differentiation. These observations suggest that FGF2 promotes leukemia cell growth in the BM by modulating osteoblast functions.

Livedoid vasculopathy (LV) is a chronic disease with recurrent reticularis and ulcers, mainly affecting the feet and lower legs. The pathogenesis of LV has not been yet thoroughly understood, but thrombosis is thought to play a major role because fibrin deposition within both the wall and lumen of affected vessels is pathologically detected. A 68-year-old woman first presented to our hospital in 2004 with a 6-year history of a reticular rash and ulceration on the lower legs. Screening tests for vasculitis and collagen disease were mostly normal, leading to diagnosis of LV. After failed treatment with steroid and aspirin, she was started on warfarin, to which she had a favorable response. However, she had to be admitted to the hospital because complication of swelling and infection in her left lower leg in 2004 + 10. Contrast-enhanced computed tomography showed thrombosis in the left popliteal artery. Screening tests for thrombotic tendency revealed that protein S activity was low (27%) although total protein S antigen was within normal range (73%). Analysis of protein S-alpha gene revealed 155 Lys>Glu mutation in exon VI, which was reported in 1994 and named as protein S Tokushima. Thus, we conclude that protein S deficiency could contribute to LV.

背景: 近年，輸血による細菌感染症の報告は増加傾向にある．しかし，輸血との因果関係を直接証明した症例はほとんどない．今回，濃厚血小板（PC）の細菌汚染により発症した敗血症の一例を経験したので報告する． 症例: 70歳男性．再生不良性貧血にて愛知医科大学病院外来通院中．血小板10,000/μl，ヘモグロビン7.9g/dlのためPC輸血，引き続き赤血球（RBC）輸血を受けた．RBC輸血開始後15分より，発熱，軽度の呼吸困難が出現し，感染症疑いにて入院となった．入院後無菌室にて抗生物質等の抗菌剤の投与を受け，全身状態は比較的安定していたが，翌朝死亡が確認された．病理解剖の承諾は得られなかった． 原因検索のため，発熱時に患者血液検査及び血液培養を行った．さらに，PCバッグ，セグメントチューブおよびRBCバッグ内の残余血液を用いて血液培養を施行した．患者白血球は著明に減少しており（300/μl），初期の血液培養にて黄色ブドウ球菌が検出されたことより，敗血症と診断された．一方，PCバッグ，セグメントチューブ双方から黄色ブドウ球菌が検出されたが，RBCバッグから菌は検出されなかった．これら3種（患者血液，PCバッグおよびセグメントチューブ）の菌株に対する薬剤感受性試験より，全てmethicillin-sensitive Staphylococcus aureus（MSSA）と判定され，さらに感受性パターン，コアグラーゼ試験，遺伝子検査により，全て同一と判定されたことより，PCに混入したMSSAにより発症した敗血症であることが明らかとなった． 結語: 輸血による細菌感染を疑った場合，患者のみならず，該当製剤バック内，さらにはセグメント内の血液を用いて細菌培養することが重要と考えられた．また，MSSAなどの皮膚常在菌は採血の際に血液製剤に混入する可能性があるため，特に常温保存のPCについては感染の危険性を認識する必要があると考えられた．

Background Since the introduction of adoptive immunotherapy (AIT) using autologous lymphokine-activated killer (LAK) cells for treating advanced cancer, many clinical trials were performed. However, their efficacy was much lower than expected. Thereafter, AIT with more specific targeting against tumour antigens, including tumour-infiltrating lymphocytes and cytotoxic T lymphocytes (CTL), was developed, and a better clinical outcome is anticipated. Recently, we have started CTL therapy as ‘an advanced therapy’, which needs approval from the government and a special facility known as the Cell Processing Room (CPR). Materials and Methods Most ordinary treatments are subject to the medical insurance system in Japan where 70% of costs are covered by the official health insurance and the rest is at patients’ expense. However, some sophisticated therapies including CTLs are only available through either clinical research or an advanced therapy, which needs official approval, and whose expense can be charged to patients in addition to the ordinary expense. With regard to cellular therapy, the Japanese government recently set regulation, including two main points: one is that cells should be used inside the hospital, indicating the responsibility of medical doctors for cellular therapy according to the Medical Practitioner Law; the other is that hospitals performing cellular therapy must establish a CPR to ensure the safety of cells processed. Mononuclear cells (MNCs) are separated from 40 ml of peripheral blood by a density gradient. They are exposed to irradiated autologous tumour cells, at a ratio of tumour cells : MNC = 1 : 100, with recombinant interleukin-2 (50 U/ml) for 4–5 days. Then, MNCs are stimulated by anti-CD3 monoclonal antibody for a similar time and subjected to culture for growth for 10 days, in total 17–19 days. Before harvesting, CTL is screened for contamination and specificity against tumour cells. Usually, about 1 × 109 cells are obtained and administered to patients intravenously. CTL is administered every 2 weeks, totalling four doses (one course). Two days before administration, a patient receives cyclophosphamide (Endoxan, 200 mg/body) to suppress regulatory T cell activity. Subjects of CTL therapy are patients (i) aged below 70 years old, with cancer; (ii) with no serious complications regarding major organs and who are durable for this treatment; (iii) with tumours or tumour markers that can objectively be evaluated; (iv) whose tumour cells are available as tumour antigen; (v) whose tumour cells express class I human leucocyte antigens (HLA); and (vi) who give informed consent. Results We treated 22 patients with various cancers, including melanomas and sarcomas, most of which had been refractory to current chemotherapy. From these, eight patients showed no progress of disease for at least 2 months. Among them, two cases revealed a convincing efficacy of CTL therapy, one showed relapse-free survival for 5 years and the other for 1 year. Conclusions Although in Japan circumstances to execute cellular therapy have become stricter, cellular therapy, including CTL, is considered feasible. Especially if it is taken into consideration that most of our cases are refractory to current chemotherapy and that two cases showed a remarkable response, it is a promising therapy for patients with advanced cancer.

【はじめに】2004年9月の「輸血療法の実施に関する指針」の一部改正, および翌年3月の「血液製剤等に係る遡及調査ガイドライン」によって輸血前後に測定するウィルスマーカーが具体的に示され, 輸血前後の検体保管が求められた. この様な背景の下, 輸血感染症に対する一連の対策が医療現場で採られているかどうか, 日本輸血細胞治療学会東海支部会領域の医療機関に対しアンケート調査を実施した.【対象・方法】2005年11月, 東海4県の232医療施設を対象に, (1) 施設情報, (2) 輸血部門管理体制, (3) 輸血前後の感染症検査, (4) 輸血前後の検体保管, (5) 説明と同意の内容, (6) 輸血製剤による細菌感染症への対応について調査した.【結果】回答施設数139, 回答率59.9%であった. 輸血前後の感染症検査を指針通りに実施している施設は前後共に2割程度と少なく, 実施施設の実際の輸血前患者に対する実施率も半数以下であった. 指針通り検査を実施しない理由として, 輸血前は「検査にお金がかかる」「事後に保管検体で対応する」, 輸血後は「輸血後の検査時期の把握が困難」が最も多かった. 輸血前検体は, 赤血球輸血に関しては84.9%の施設が保管していたが, 全ての輸血で保管している施設は40.3%であった. また, 輸血後検体について何らかの保管を行っている施設は20.8%しか認められなかった. 2004年9月改定の「輸血療法の実施に関する指針 (改定版)」で示された説明と同意を得るべき項目の内, 輸血感染症対策に関する項目についての記載または記載予定施設を77%認めたが, それ以外の項目についての記載または記載予定施設は5割以下であった. 細菌感染症対策として, 使用済みバッグの保管が望まれているが, ほとんどの施設が実施上不可能など否定的な回答であった.【結論】2004年に設けられた生物由来製品感染症等被害救済制度により輸血後肝炎など感染性輸血副作用を発症した患者が救済出来るようになった. しかしながら, 適応を受けるには輸血による感染である必要がある. それを証明するためには指針通りの輸血前後の感染症検査が重要である. しかし, 東海地区では指針通りの検査実施施設は少なく, 全ての輸血に対し輸血前検体を保管している施設も半数以下であった. この様な現状を考えると, 指針通りに感染症検査を実施するよう促すことは勿論であるが, 輸血後肝炎などの輸血感染症発症例に対しても事後の対応が可能なように, 全ての輸血患者の輸血前検体を保管する体制を構築することが急務と思われる.

【背景】静注用免疫グロブリン (IVIG) に関しては未だ明確な使用基準がなく, 適正使用を推進するには使用指針の作成が急務である. 我々は指針作成を最終的な目標とし, 先ずIVIGについて全国の中・大規模病院における疾患別の使用実態を調査, 検討した.【方法】全国の400床以上の732病院を対象に, 平成15年7月から9月までの3ヵ月間におけるIVIG使用状況をアンケート形式で調査した. 調査内容は疾患別の使用患者数および使用量である.【成績】本調査の有効回答率は36.6% (268/732病院) であった. 総使用患者数 (8,570例) 中の小児群は1,955例で自己免疫疾患が38%, 術後感染症を含めた感染症が33%であり, 成人群は6,615例で自己免疫疾患が10%, 感染症が73%と両群間に有意差が認められた. 総使用量 (171,018g) では, 小児群 (28,684g), 成人群 (142,334g) での自己免疫疾患の占める割合は各々60%, 35%であり, 感染症は各々15%, 45%であった. 感染症以外の患者数をみると, 小児群では川崎病が44.5%と高頻度であり, 成人群では特定の疾患に多く使用される傾向は認められなかった. 感染症に関しては両群とも細菌感染症に多く使用されていた. また, 重症感染症の指標として設定したCRP値の境界値 (小児群: 10mg/dl, 成人群: 15mg/dl) 以上での使用例は小児群では28%と低率であり, 成人群でも51%と約半数に過ぎなかった.【結論】本調査から自己免疫疾患ならびに感染症が今後のIVIG需要量を規定する主な疾患であると判断された. 特に, 感染症のうち, 重症と考え難く, 不適正と考えられる症例にもかなり使用されている実態が明らかになった.

Adoptive cell transfers of receptor gene-engineered T cells include chimeric antigen receptor-gene transduced T (CAR-T) cell therapy and TCR-gene transduced T (TCR-T) cell therapy. In CD19-CAR-T cell therapy, high incidence of cytokine release syndrome (CRS) is associated with in vivo CAR-T cell proliferation and its clinical efficacy. In human TCR-T cell therapies, there have not been well known about CRS and its association with in vivo T cell kinetics or tumor responses. We have been developing a novel-type affinity-enhanced NY-ESO-1-specific TCR, and an original retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1 TCR is mutated for high affinity with replacements of G50A and A51E in CDR2 region, which is restricted with HLA-A*02:01 and A*02:06. We extensively examined potential cross-reactivities to different antigen-peptides in preclinical studies, and the high-affinity NY-ESO-1 TCR did not recognize analogous peptides. The new generation retroviral TCR-vector provides enhanced expression of transduced tumor-specific TCRs and an inhibition effect of formations of self-reactive TCRs. This is a first-in-man clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It is designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3+3 cohort design. Cyclophosphamide with/without fludarabine were administered prior to the TCR-T cell transfer as pre-conditioning. Six patients were treated with the NY-ESO-1 TCR-T cell transfer, and evaluated for the safety and in vivo cell kinetics. The TCR-T cells appeared in peripheral blood with a dose-dependent manner, associated with in vivo proliferation in an early phase. In three patients given 5x108 cells, no toxicities were seen. Two patients receiving 5x109 cells developed early-phase CRS (G2), with elevations of serum IL-6 and IFN-gamma. They were managed the treatment of anti-IL-6 receptor monoclonal antibody, tocilizumab. In a patient who developed CRS, an event of lung injury (G3) occurred, which was associated with marked infiltration of the NY-ESO-1 TCR-T cells. It was successfully treated with steroid. Two synovial sarcoma patients exhibited tumor responses of PRs. In one patient, progression-free survival lasted more than 8 months. In summary, the affinity-enhanced NY-ESO-1 TCR-T cell transfer exhibited CRSs in association with in vivo cell proliferation and sequential tumor responses. Disclosures No relevant conflicts of interest to declare.

The plasma fraction of blood components has an essential role in the etiology of allergic transfusion reactions (ATRs). The difference of incidences of ATRs between fresh-frozen plasma (FFP) and platelet concentrates (PCs), in which plasma is the main component, is not clearly understood. This study compares the frequency of ATRs to FFP versus PCs on both first and subsequent (nonfirst) transfusions and considers the factors influencing the risk of ATRs.Five hospitals agreed to systematically collect and share 2 years of data (January 2010 through December 2011). This was a retrospective observational analysis of data including the number of transfusion episodes and ATRs for FFP and PCs on first-transfusion patients (without transfusion history) and previously transfused patients.The incidence of ATRs to PCs (2.51%) was significantly higher than to FFP (1.68%) on subsequent transfusions (p0.001). On the other hand, there were no significant differences in the incidences of ATRs to FFP (2.67%) and PCs (2.82%) on first transfusions. This discrepancy was most pronounced among males: FFP versus PCs on first transfusions, 2.02% versus 2.60% (p = 0.30); and on subsequent transfusions, 1.58% versus 2.46% (p = 0.0007). Among females, FFP versus PCs on first transfusions was 3.59% versus 3.13% (p = 0.61) and on subsequent transfusions was 1.87% versus 2.61% (p = 0.029).Repeated exposure rather than the total volume of transfused components may influence the incidence of ATRs.

Conventional cancer treatments are surgery, radiotherapy, and chemotherapy, but treatment efficiency is insufficient and cancer recurrence is common. Immunotherapy has been added as an important cancer treatment component, but no reports on its efficacy in oral and maxillofacial cancers exist. We evaluated the clinical efficacy of adoptive immunotherapy using ex vivo-activated cytotoxic T lymphocytes (CTL) in the treatment of 7 patients with advanced oral and maxillofacial cancers with stage IV disease at diagnosis. The mean follow-up period was 26.2 months. Phenotype of the lymphocyte assay revealed that the percentage of CD4(+) T cells decreased and that of CD8(+) T cells increased among infused lymphocytes compared to that in unstimulated peripheral blood mononuclear cells (PBMCs), and infused lymphocytes produced a significantly higher level of IFN-γ than PBMCs or tumor cells alone. In a representative patient who refused surgery tumor regression was confirmed after CTL infusion. Computed tomography clearly indicated a significant reduction in tumor size followed by the complete disappearance of the tumor. Histological examination showed that the cancers in patients receiving CTL therapy were heavily infiltrated with lymphocytes. The other 2 patients who received CTL therapy as adjuvant therapy showed neither recurrent disease nor new disease lesions. The 1-year survival rates showing response and those with progressive disease were 100 and 25%, respectively. Moreover, no significant adverse reactions were reported during the study period. CTL therapy remains in the early stages of treatment options, but it has potential as a valuable treatment and improvement of quality of life for patients with otherwise incurable cancers.

Background and Objectives This study compares the frequency of adverse transfusion reactions (ATRs) after first transfusions with the frequency of ATRs for subsequent (non-first) transfusions. Materials and Methods Five hospitals agreed to systematically collect and share 2 years of data. This was a retrospective observational analysis of data including the number of transfusion episodes and ATRs for red blood cells (RBCs), fresh frozen plasma (FFP) and platelet concentrates (PCs) given to first-time transfusion recipients and to those previously transfused. Results First transfusion ATRs to RBCs, FFP and PCs were 1·08%, 2·84% and 3·34%, respectively. These are higher than ATR incidences to RBCs (0·69%), FFP (1·91%) and PCs (2·75%) on subsequent transfusions. Specifically, first transfusion incidences of febrile non-haemolytic transfusion reactions (FNHTRs) to RBCs (0·43%) and allergic reactions to FFP (2·51%) were higher than on subsequent transfusions (RBCs: 0·23%, FFP: 1·65%). Conclusion There are risks of ATRs on the first transfusion as well as transfusions of patients with transfusion history.

Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

Objective In the homing process of hematopoietic progenitor cells (HPCs) to bone marrow, several adhesion molecules play important roles. However, HPCs are subjected to dramatic alteration of freeze-thawing that could affect these molecules. In this study, we investigated the effect of cryopreservation on the expression of adhesion molecules on HPCs. Methods The expression of different adhesion molecules on CD34 + cells was examined by flow cytometry using an immunofluorescence technique. Changes in expression before and after cryopreservation, and that of L-selectin on addition of dimethylsulfoxide (DMSO) or with serum incubation, were investigated. The relationship between expression and function was also determined using a transmigration assay system. Results L-selectin expression on CD34 + cells derived from mobilized peripheral blood (MPB), bone marrow (BM), and umbilical cord blood (CB) was significantly decreased from 37% to 23%, 48% to 11%, and 67% to 19%, respectively, by the freeze-thawing process, while the expression of other adhesion molecules was not appreciably changed. Within 30 minutes of incubation with DMSO, L-selectin expression on CD34 + cells significantly decreased from 65% to 22%. Furthermore, this was completely prevented by the matrix metalloproteinase (MMP) inhibitor, KB-R8301, indicating that the loss of L-selectin was due to shedding mediated by an MMP. To determine if L-selectin expression can be upregulated after cryopreservation, thawed samples were cultured overnight with serum. Values were observed to return to or rise higher than those of the fresh samples, this being particularly rapid and pronounced when the CD34 + cells were cocultured with the human BM stromal cell line, HS-27A. Therefore, this adhesion molecule could possibly be restored in vivo after transplantation in a way similar to the in vitro case. Conclusion Despite considerable damage to HPCs during cryopreservation, changes in these cells are reversible, in line with successful restoration of hematopoiesis after transplantation.

H. W. Reesink, S. Panzer, C. A. Gonzalez, N. Lena, P. Muntaabski, S. Gimbatti, E. Wood, M. Lambermont, V. Deneys, D. Sondag, T. Alport, D. Towns, D. Devine, P. Turek, M.-K. Auvinen, T. Koski, C. K. Lin, C. K. Lee, W. C. Tsoi, E. Lawlor, G. Grazzini, V. Piccinini, L. Catalano, S. Pupella, H. Kato, S. Takamoto, H. Okazaki, I. Hamaguchi, J. C. Wiersum-Osselton, A. J. W. van Tilborgh, P. Y. Zijlker-Jansen, K. M. Mangundap, M. R. Schipperus, D. Dinesh, P. Flanagan, O. Flesland, C. T. Steinsvag, A. Espinosa, M. Letowska, A. Rosiek, J. Antoniewicz-Papis, E. Lachert, M. B. C. Koh, R. Alcantara, M. Corral Alonso & E. Muniz-Diaz

The human retinoblastoma gene (RB) product, which is involved in the control of cell cycle and tumor suppression, is constitutively expressed as a nuclear phosphoprotein in normal human cells. We examined leukemic cells from 22 patients with blast crisis of chronic myelogenous leukemia (CML) for alterations of the RB expression. Western blotting and flow cytometry with anti-RB-protein antibodies showed that all of five cases with megakaryoblastic crisis lacked the expression of the RB-encoded protein, whereas none of 17 cases with the other phenotypes such as myeloblastic or lymphoblastic crisis showed any abnormality. These findings suggest that megakaryoblastic transformation of CML might be lineage-specifically associated with loss of the RB protein.

Careful assessment of risks and benefits has to precede each decision on allogenic red blood cell (RBC) transfusion. Physicians work to establish more appropriate transfusions of blood components according to the guidelines issued by the Ministry of Health, Welfare, and Labor in Japan. For many years the so-called "10/ 30 rule" was used as a hemoglobin/hematocrit transfusion trigger. However, this rule does not take into account the individual anemia tolerance of a patient nor its individual compensatory mechanisms. RBC transfusions should not be dictated by a single hemoglobin transfusion trigger, but instead should be on the patient's risk of developing complications of inadequate oxygenation. Therefore, transfusion decisions should be primarily based on an individual patient's need for global and regional oxygen supply as indicated by signs of inadequate global and regional oxygenation. However, a hemoglobin transfusion trigger may be useful if matched with some other makers of inadequate tissue perfusion. Therefore, RBC transfusion is recommended under the following circumstances: for hemoglobin levels6 g x dl(-1) and for physiologic signs of inadequate oxygenation.

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood, bone marrow, and umbilical cord blood (CB) and determined to be in heterogeneous populations; however, specific variations in their characteristics remain to be clarified. In this study, we observed that mononuclear cells (MNCs) of CB change in morphology to differentiate into mature endothelial cells (EC) after 6 weeks of culture. In early days of culture along with the differentiation, two distinct populations of EPCs were detected, defined by two-dimensional dot plots (forward scatter vs side scatter) with flow cytometry, namely, relatively small cells (S-EPCs) and relatively large cells (L-EPCs). S-EPCs were found to express CD34 but not CD14, while the converse was the case for L-EPCs. When CD34(+)/CD14(-) cells and CD34(-)/CD14(+) cells were isolated from original MNCs of CB and cultured independently, S-EPCs and L-EPCs were derived from CD34(+)/CD14(-) and from CD34(-)/CD14(+) cells, respectively. Furthermore, when the two EPCs at day 7 were separated by cell sorter and recultured, there was no crossover in terms of CD34 and CD14 expression. While expression of VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR-2) on L-EPCs was significantly greater than on S-EPCs, levels of CD31 were lower. In addition, L-EPCs exhibited greater proliferative ability on stimulation with VEGF. Although these two EPCs expressed different phenotypes, including growth factor receptors, and had different proliferative ability, they both eventually differentiated into mature ECs after more than 3 weeks of culture.

We carried out a survey on platelet transfusions performed in hospitals certified by the Japanese Society of Hematology. The average values of the pretransfusion platelet count (trigger value) for the day on which the transfusion was ordered, and the values on each day in the interval between the order and actual transfusion, were compared with the Guidelines. The average trigger value in aplastic anemia and myelodysplastic syndrome patients (A group) (1.41 x 10(4)/microl) for the same day on which the transfusion was ordered was higher than the Guideline, whereas those patients with hematological disorders undergoing chemotherapy (B group) and hematopoietic stem cell transplantation (C group), namely 2.08 x 10(4)/microl and 2.1 x 10(4)/microl, respectively, were acceptable values when compared with the Guidelines. On the other hand, in all groups, the transfusion trigger values at one or two days after ordering were higher than the Guidelines, being 2.56 x 10(4)/microl in the A group, 3.15 x 10(4)/microl in the B group, and 2.59 x 10(4)/microl in the C group. We have tried to formulate platelet count criteria for ordering a transfusion based on one day before the actual transfusion, because these platelet counts on ordering were relatively high. The criteria are 1.0-1.5 x 10(4)/microl in group A, 3.0 x 10(4)/microI in group B, and 3.0 x 10(4)/microl in group C. In order to perform platelet transfusion according to the Guidelines, the platelet count on ordering should be decreased as we proposed.

Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzyme-based methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. L-asparaginase (L-asp) therapy causes depletion of plasma asparagine followed by the loss of intracellular asparagine. Due to the lack of a rapid up-regulation of asparagine synthetase (ASNS) protein content in ALL cells, they are preferentially killed by L-asp. Elevated expression of ASNS within the leukemia cells causes decreased sensitivity to L-asp. The proof of ASNS deficiency in leukemia cells is considered to predictive for effectiveness of L-asp even in acute myeloid leukemia (AML) other than ALL patients. The establishment of quantitative estimation of ASNS protein content would be useful for the L-asp treatment in leukemia therapy. Objective: Our aim was to set up a flow cytometry system to check ASNS deficiency in leukemia cells and to investigate the sensitivity to L-Asp and the ASNS expression in AML leukemia cells. Methods: AML (KG-1, HL-60, U937) and ALL (MOLT-4, RS4;11) and CML (K562) cell lines were grown in RPMI1640 medium with 10% FCS. Primary leukemic cells from the peripheral blood or bone marrow of 20 AML patients were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was evaluated by cytosolic flow cytometry with Z5808 McAb (Hybridoma 31: 325-332.2012) and expressed as a ΔMFI(Difference of Mean Fluorescence Intensity(MFI) between by Z5808 and isotypic control) or MFI ratio(MFI by Z5808/MFI by isotypic control). When a sufficient amount of leukemic cells was available, sensitivity to L-asp (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of E. coliL-asp with the cells and by measuring the cell viability with a counting kit (WST1 viability assay) at day 3. Results: Determination of IC50 for the HL-60 (⊿MFI 48 ± 8.01, MFI ratio 1.77 ± 0.03) and U937 (⊿MFI 16.7 ± 0.47, MFI ratio 1.19 ± 0.02) demonstrated that these cells were equally sensitive to L-asp than the ALL cell line MOLT-4 in vitro (0.37 and 0.02IU/mL versus 0.15 IU/mL, respectively). K562 and KG-1 (⊿MFI 135.7 ± 5.66, MFI ratio 2.48 ± 0.09) cells with the highest ASNS expression exhibited resistance to L-asp (>10 IU/ml). Both of ASNS Expression by ⊿MFI and MFI ratio was inversely correlated with L-asp sensitivity judging from cell line studies. Judging from cell line study, the threshold for ASNS protein expression effective for L-asp treatment was considered to be 10 IU/mL) Remaining 5 patients were a moderate sensitivity (IC50 < 0.5 IU/mL). ASNS Expression in ALL was almost near zero. ASNS expression in fresh AML was low in all cases except for one M2 and one M4 cases. Indeed, at high ASNS expression, these cases were resistant in vitro to L-asp. The patients with blasts sensitive to L-asp had mainly M1, M5 or M7 AML. A good inverse correlation between ASNS expression and sensitivity to L-asp was observed also in primary leukemic cells from AML patients. Conclusions: We demonstrated here that some of AML cell lines with low ASNS expression are more sensitive to L-asp than leukemia cells with high ASNS expression. Also, fresh AML cells with low ASNS expression are more sensitive to L-asp than with high ASNS expression. 11/13AML or two AMLL cases were supposed to be effective for L-asp. Plasma asparagine depletion by L-asp in selected patients having low ASNS may be a promising therapeutic approach even for AML. This work was supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (C) KAKENHI Grant Number 24590713. Disclosures No relevant conflicts of interest to declare.

Midkine (MK) was originally cloned as a product of a retinoic acid-responsive gene. The rationale for studying MK expression is based on previous reports showing that it transforms 3T3 cells, and that it acts as an autocrine growth factor in Wilm's tumors, and that its overexpression has been associated with worse outcome in bladder carcinoma. Besides bladder carcinoma, its expression was reported in various solid tumors. We investigated the expression of MK protein and/or MK gene in biopsied specimens from 40 patients with primary malignant lymphoma, 21 with Hodgkin's disease (HD) and 19 with non-Hodgkin's lymphoma (NHL). Reed-Sternberg (R-S) cells were stained positive in 10 of 16 HD cases evaluated by immunohistochemical method, whereas 18 of 19 NHL cases did not stain, and one B-cell NHL stained weakly positive. Immunostaining analysis was extended to established cell lines and to normal lymphocytes with or without lectin stimulation or with EB virus transformation. Among hematopoietic cells examined, erythro- or megakaryoblastic leukemia cell lines (K562, MEG-01 and UT7) were positive, while normal lymphocytes (except the EB virus-transformed one) and most myeloid and lymphoid cell lines (except Raji cells) were negative. On the contrary, solid tumor cell lines showed high and strongly positive staining including cell lines derived from of lung gastric, colon, and a pancreatic cancer. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), which is suitable for the detection of weakly expressed mRNA, the relative ratio of MK mRNA to beta-actin mRNA of samples was measured and compared in cases where RNA was available. The mean values of relative ratio (MK/beta-actin) of HD were almost twice as those of NHL samples, peripheral blood T cells, and spleen B cells. Our findings showed that MK is expressed in Reed-Sternberg cells of HD, and that MK might play a role in the pathogenesis of HD.

Das vollstandige Manuskript dieser Zuschrift erscheint in: Angew. Chem. Suppl. 1983, 480. DOI: 10.1002/ange.198304800