ALBERT

Description: Aim. In the treatment of patients with chronic myelogenous leukemia (CML), neutropenia is a dose-limiting factor on the combination therapy of imatinib mesylate (STI571) and interferon-alpha. If STI571 combined with interferon-alpha effects on decreasing BCR-ABL-positive cells and granulocyte colony-stimulating factor (G-CSF) has an effect on increasing normal neutrophils, the combination therapy may improve current remission rates in CML. We evaluated in vitro combined effect of STI571, interferon-alpha and G-CSF on primary bone marrow cells from patients with CML in chronic phase (CP). Material and Methods. The primary bone marrow mononuclear cells (BMMNC) from patients with CML-CP were incubated in the medium containing STI571 (1 μM) and/or interferon-alpha (100 U/ml) with or without G-CSF (100 ng/ml). The viability of the cells was evaluated by trypan blue dye exclusion. Apoptosis was detected by the flow cytometry analysis with Annexin V (AV) and propidium iodide (PI) double staining. The colony formation assays of BMMNC were performed by methylcelulose. And then, BCR-ABL and normal cell colony rates were evaluated on14 days. Results. Treatment of BMMNC with STI571 revealed a decrease of cell number in dose-dependent and time-dependent manner. Incubation with STI571 and interferon-alpha for 24 and 48 hours decreased viable cell number by 58% and 39%, respectively. G-CSF did not stimulate the BCR-ABL-expressing cells proliferation in the combination therapy. Cytometry analysis with AV and PI showed about 70–75% of AV-positive cells in the combination therapy and monotherapy. There were no significant differences in apoptosis between STI571 plus G-CSF and interferon-alpha plus G-CSF. Colony formation of BMMNC from patients with CML-CP in combination of STI571 and interferon-alpha was more strongly suppressed than STI571alone, and G-CSF did not abrogate the suppressive effect of the combination therapy. From these observations, it is concluded that combination therapy of STI571 and interferon-alpha with G-CSF can induce apoptosis effectively in BCR-ABL-expressing cells. Conclusion. Obtained data provide an evidence for the effective and safe combination therapy of STI571 and interferon-alpha with G-CSF against CML-CP patients. Although further studies are needed to clarify whether combination therapy really increase normal cells or not, the combined therapy with G-CSF appears not to stimulate leukemic cell proliferation.

Description: Introduction: Blood donor recruitment and retention in the younger generation is an important concern in several countries with an aging population. In Japan, which is going to have an aging population, the number of blood donors has decreased by 15% over the past decade. As such, drastic measures must be taken to maintain the necessary blood stocks. The promotion of blood donation in high schools has been attempted in many countries. This strategy is particularly attractive because successful recruitment of young donors will ensure long term supplies of blood are maintained. To enhance the effectiveness of this approach it is important to communicate the need for blood donation by high school students and conduct appropriate surveys. Although there have been several reports on the promotion of blood donation by young people, these have not generally analyzed their psychology, personal environment and the views of large numbers of high school students. Materials and Methods: The study was accepted with each high school staff meeting and IRB in our university (#25-159). Inquiry anonymous surveys were designed for high school students, who answered by their own volition. The survey included 50 questions as follows; gender; age; build; previous blood donation by the individual as well as family members and friends; lifestyle; diet; views concerning blood demand in society; location of blood donation centers; knowledge of blood donation methods; blood recovery after donation; reasons for declining to give blood; ideas for an effective campaign to recruit blood donors; previous education on blood donation in their school etc. Individual views concerning an effective campaign to recruit new donors, events, characters and media were given in a free style. This work was supported by the Japanese Ministry of Health, Labor and Welfare (H25-medicine-general-022). Results:We obtained answers from 94% of students surveyed (16,333). The first and second studies were done in 2013 and 2014, and included 8,456 and 7,877 students, respectively. In the first study, the male/female ratio was 0.88. 1% and 26% of student body weights were

Description: Leukemic cells and tumor cells can be escaped from allogeneic recognition by usual cytotoxic T cells because of the low expression level of HLA class I molecules. It has recently been shown that inhibitory natural killer cell receptors (NKRs) on not only NK cells but also on T cells negatively regulate NK cell and T cell functions through their binding to MHC class I molecules. The C-type lectin superfamily inhibitory NKR (CD94/NKG2A) heterodimer recognizes an HLA-E that preferably bound to a peptide derived from the signal sequences of most HLA class I. Therefore, CD94 can monitor the global status of HLA class I on the tumor and leukemic cells and induce cytolytic attack without inhibitory signal against HLA class I decreased target cells. In this study, we expanded CD94-expressing T cells from four different sources of blood mononuclear cells (BMCs) and then investigated their cytolytic characteristics against patients’ primary leukemic cells in order to develop a potential strategy of cell therapy for hematological malignancy. We could get more than 100 fold expansion of CD94-expressing CD8 T cells from normal donor PBMC, apheresed PBMC without G-CSF mobilization from normal donor, apheresed PBMC with G-CSF mobilization from patients after chemotherapy and cord blood after 7 days culture with immobilized anti-CD3 monoclonal antibody (1μg/mL) and IL-15 (5 ng/mL). Cytolytic activities of purified CD94-expressing cells using magnetic cell sorting (MACS) (CD94 〉 90%) detected by 4 hours 51 Cr release assay against HLA class I intermediate primary leukemic cells (AML M0, M2, M4, CML CP, BC, MDS overt) (50 〈 mean fluorescence intensity (MFI) 〈 150) were 35.6 ± 12.8 % (n=21). However, CTL activities against HLA class I high primary leukemic cells (ATL, ALL, LBL)(MFI〉150) were lower than 10 % (6.5 ± 4.2, n=5). Also, CTL activities against HLA class I very high PHA autoblasts and alloblasts (MFI〉200) were lower than 5 % (4.0 ± 3.6, n=11). Although the cytolytic activity of CD94-expressing cells roughly depends on the expression of HLA class I molecules in inverse proportion, adhesion molecules and also activating molecules such as NKG2D on effector cells might be important for the regulation of the killing activity. In fact, anti-NKG2D mAb (50 μg/mL) suppressed the cytolytic activity of CD94-expressing cells against patients’ primary leukemic cells (% reduction of cytolytic activity, 22.5± 5.9, n=13). Furthermore, anti-LFA-1 mAb (20 μg/mL) suppressed the cytolytic activity of CD94-expressing cells much more effectively than did anti-NKG2D mAb(% reduction of cytolytic activity, 74.2±15.5, n=13, p

Description: CD94 is one of the C-type lectin family members, forms a heterodimer with NKG2 gene family, and CD94 /NKG2A are inhibitory receptors. Not only NK cells but a subset of T cells express CD94/NKG2A, and previously we revealed the proportion of CD94/NKG2A expressing CD8 T cells were higher in patients with chronic graft versus host disease (GVHD), and CD94 expressing T cells have suppressive effects on mixed lymphocyte culture(MLC). We focus on CD94 positive T cell during T cell reconstitution after allogeneic hematopietic stem cell transplantation (allo-HSCT). T cell receptor excision circles (TREC) are suggested to be a useful marker of recent thymic output. In this study, we attempt to study TREC-containing CD8 T cell subset expressing CD94, and to examine the relation of TREC DNA level in CD94 expressing CD8 T cell and GVHD. We analyzed peripheral blood mononuclear cells (PBMCs) isolated from 24 patients (82 samples) undergone allo-HSCT including 15 patients with bone marrow transplantation and 9 patients with non-myeloablative stem cell transplantation. Informed consent was obtained from all patients. CD4 positive T cells were separated from PBMCs by magnetic cell sorting, and CD4 negative cell population was divided into CD94 positive CD8 T cells and CD94 negative CD8 T cells by fluorescence activated cell sorter. Genomic DNA was extracted from these separated T cell subsets. TREC DNA copy numbers per 105 isolated T cells (TREC level) were quantified by real time PCR. We investigated TREC levels in clinical status with pre-allo-HSCT, no episodes of GVHD or before manifestation of GVHD (No GVHD), chronic GVHD on disease (C-GVHD), and no symptoms and remission status of GVHD after immunosuppressive therapy (R-GVHD). Statical analyses were carried out by Mann-Whitney U test. There were no significant differences in TREC level of sorted CD4 positive T cells in C-GVHD compared with No GVHD (p=0.75) and R-GVHD (p=0.61), and also CD94 negative CD8 T cells in C-GVHD compared with No GVHD (p=0.79) and R-GVHD (p=0.20). On the other hand, TREC level of CD94 positive CD8 T cells in C-GVHD decreased in comparison with No GVHD (p=0.015) and R-GVHD (p=0.0019). The reduction of TREC level is thought to be induced not only by low thymic output but also by dilution of TREC concentration due to peripheral T cell expansion without duplications of TREC. These results may suggest that CD94 positive T cells play a role in modulation of GVHD, and proliferate during chronic GVHD with dilution of TREC in CD94 positive CD8 T cells. It is suggested that TREC level of CD94 expressing CD8 T cells may be useful markers of chronic GVHD.

Description: Buckgrand. Graft-versus host disease (GVHD) is a major complication after hematopoietic stem cell transplantation (HSCT). Macrophage migration inhibitory factor (MIF) plays a pivotal role in systemic as well as local inflammatory and immune responses. Recent reports that MIF expression is up-regulated in the allo-immune reaction during renal transplantation. Otherwise, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein belong to the TNF family. The level of TRAIL expression in T cells as well as NK cells can be markedly up-regulated after cell activations. In this study we report that kinetics of serum level of MIF and TRAIL in GVHD patients before and after HSCT. Patients and Methods. Date randomly obtained from 16 patients (10 males and 6 females) who underwent allo-SCT for treatment of hematological malignancies at Hokkaido University Hospital during the period May 2001 to January 2005. All patients were informed consent of peripheral blood smpling. Eight patients were received conventional transplantation and the others were reduced intensity stem cell transplantation (RIST). Seven patients have HLA identical sibling donor, but the others were received unrelated donor. Twelve of the 16 patients was achieved acute GVHD (aGVHD), gradeIto IIin 8 patients. Twelve patients survived day 100 after allo-SCT, 9 of those 12 patients developed chronic GVHD(cGVHD). Serum MIF and TRAIL concentration were measured at various time points using enzyme-linked immunosorbent assays (ELISAs). Results. Serum MIF concentration analysis by ELISA showed that only patients who developed aGVHD significantly increased (two folds) before and after allo-SCT (avelage, from 7.34 ng/ml before allo-SCT to 14.7 ng/ml after allo-SCT, p=0.018). However, we could not detect any correlation of MIF levels and aGVHD severity, donor sources. On the other hand, serum TRAIL concentration analysis by ELISA showed that patients who developed aGVHD were not associated (avelage, from 458.6 pg/ml before allo-SCT to 484.12 pg/ml after allo-SCT, p=0.632). We could not detect association aGVHD severity, donor sources. However, peak titer in aGVHD patients tends to decrease in unrelated transplantation (related 580.86pg/ml, unrelated 415.59pg/ml, p=0.22). Interestingly, we showed that average serum TRAIL concentration before allo-SCT associated with aGVHD and cGVHD. Serum TRAIL concentration with aGVHD patients (n=12, 458.85pg/ml) was tended to increase than without aGVHD (n=4, 330.45pg/ml, P=0.063) and with cGVHD patients (n=9, 535.21pg/ml) was significantly increase without cGVHD patients (n=3, 282.0 ng/ml, P=0.007). Discussion. The present study demonstrated the kinetics of MIF and TRAIL. Systemic up-regulation of MIF expression is associated with the occurrence of aGVHD. This data suggested that MIF after allo-SCT might play a pathogenetic role in aGVHD. On the other hand, we suggested that high level of TRAIL before allo-SCT associated acute and cGVHD. Maybe we might be to estimate acute and cGVHD in examining TRAIL level before allo-SCT. In conclusion our data are the first to establish an association TRAIL and GVHD in allogeneic stem cell transplantation.