WT1-specific CD8 + cytotoxic T cells with the capacity for antigen-specific expansion accumulate in the bone marrow in MDS.

Wilms' tumor 1 (WT1) is a tumor-associated antigen and immunotherapy target in myelodysplastic syndrome (MDS). Further information is needed on the characteristics of WT1-specific CD8 + T cells to develop immunotherapeutic strategies for MDS. To clarify the frequency, distribution, and phenotype of WT1-specific CD8 + T cells, which occur innately in MDS patients, we analyzed paired peripheral blood (PB) and bone marrow (BM) samples from 39 patients with MDS or acute myeloid leukemia with myelodysplasia-related changes. The median frequency of WT1 tetramer-binding CD8 + T cells in the CD8 + T cell population was 0.11% in PB and 0.18% in BM. A further tetramer assay combined with mixed lymphocyte peptide culture (MLPC assay) was used to detect functional WT1-specific CD8 + T cells that could respond to the WT1 peptide. Functional WT1-specific CD8 + T cells were detected in BM in 61% of patients, which was significantly higher than in PB (23%, p = 0.001). The frequency of these cells estimated by the MLPC assay was tenfold higher in BM than in PB. The majority of WT1 tetramer-binding CD8 + T cells in BM had a unique phenotype with co-expression of CD39 and CXCR4. These findings will facilitate the development of novel immunotherapeutic strategies for MDS.

Rhnull phenotype caused by a novel RHAG mutation, c.945+1G>A, in the Japanese population.

BACKGROUND: The Rh complex contributes to cell membrane structural integrity of erythrocytes. Rhnull syndrome is characterized by the absence of the Rh antigen on the erythrocyte membrane, resulting in chronic hemolytic anemia. We recently came across 3 Rhnull phenotype probands within two families with the same novel RHAG mutation in the Japanese population. MATERIALS AND METHODS: Detailed Rh phenotyping by hemagglutination was performed using monoclonal and polyclonal anti-D, -C, -c, -E, and -e; monoclonal and polyclonal anti-Rh17 antibodies; and polyclonal anti-Rh29 antibodies. RHAG mRNA transcripts were analyzed by reverse transcription-polymerase chain reaction, and the mutation was verified by genomic sequencing. RESULTS: The genomic region spanning exon 6 contained a G > A transition in the invariant GT motif of the 5' donor splice-site of Intron 6 (c.945+1G>A). The Rhnull phenotype was caused by an autosomal recessive mutation in Probands 1 and 2, determined by family history. Regarding clinical features, the degree of hemolysis varied slightly between these individuals, with Proband 3 displaying acute hemolytic anemia with an infection. While no standard therapy has been established, the condition of the patient in this study improved with conservative treatment, including hydration and antibiotics. CONCLUSION: The mechanisms of hemolysis due to the Rhnull phenotype can vary, but our findings indicate that acute hemolytic crisis caused by the Rhnull syndrome could be associated with infection.

[Myelodysplastic syndrome with refractory hemorrhage due to reduced platelet aggregation activity].

A 75-year-old woman suffered a cat bite 10 months after myelodysplastic syndrome (MDS) diagnosis. She visited our hospital because the internal bleeding of the wound did not improve. Although the wound was treated, the bleeding did not stop. She was hospitalized for emergency medical treatment because the bleeding volume exceeded 200 ml. Although her platelet count was normal, the platelet function test showed a decrease in collagen and arachidonic acid aggregation. After platelet transfusion, her bleeding stopped. Patients with MDS may potentially have platelet dysfunction. In the case of bleeding without thrombocytopenia, a platelet function test should be performed and treatment intervention, such as platelet transfusion, should be considered.

Development of potent antagonists for formyl peptide receptor 1 based on Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH.

While stimulation of formyl peptide receptors (FPRs) on the surface of human neutrophils induces several immune responses, under conditions of continuous activation of the receptor by agonists such as formyl-Met-Leu-Phe-OH (fMLP), neutrophil-dependent tissue damage ensues. Thus, FPR antagonists could be anticipated as drugs for FPR-related disease. In this study, Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH (Boc-FlFlF), one of several FPR subtype selective antagonists, was chosen and the positions at the Phe residues were optimized. We found that substitution with unnatural amino acids resulted in an improvement of two orders of magnitude. The most potent antagonist indicated FPR subtype selectivity at 1 µM. In addition to finding a potent antagonist, the structure-activity trends observed in this study should be valuable in designing a new type of FPR subtype selective antagonist.

Anti-tumor activities of Au(I) complexed with bisphosphines in HL-60 cells.

We found that Au(I) complexed with 2,3-bis(tert-butyl(methyl)phosphino) quinoxaline (10) was a potent anti-tumor agent (half-maximal growth inhibitory concentration, GI50=0.87µM) with broad anti-tumor activity. In particular, the activity of complex 10 was high in tumor cell lines derived from the colon and ovary. Treatment with complex 10 resulted in the apoptosis of HL-60 cells. The ligand for the preparation of complex 10 is commercially available implying that complex 10 might be a good drug candidate for cancer therapy.

WT1 peptide vaccination in combination with imatinib therapy for a patient with CML in the chronic phase.

Although tyrosine kinase inhibitors is effective for dramatically reducing CML cells, it might be difficult to eradicate completely the CML stem cells. We aimed to clarify the safety and effects of WT1 peptide vaccination in combination with imatinib therapy for a CML patient. A 51 year-old male with CML in CP, who showed a resistance against imatinib therapy for 2.5 years, began to be treated with 9 mer modified-type WT1 peptides in combination with standard dose of imatinib. Although every 2-week-administration of WT1 peptides for 22 weeks did not show definite effects on the quantification of bcr-abl transcripts, by changing the administration from every 2 weeks to 4 weeks bcr-abl transcripts decreased remarkably. After 11 months of every 4-week-administration of the peptides and 12 months post cessation of the peptides bcr-abl transcripts achieved to the level below detection by RQ/RT-PCR (complete molecular response). WT1/MHC tetramer(+)CD8(+) CTLs, which appeared after the second administration of WT1 peptides and remained more than 15 in number among 10(6) CD8(+) T cells throughout the administration of WT1 peptides, are still present in the blood on 14th month post cessation of the peptides. An in vitro study as to the cytotoxicity of lymphocytes induced by mixed lymphocyte peptide culture demonstrated that cultured lymphocytes possessed cytotoxicity against WT1 expressing leukemia cells and the cytotoxicity was WT1-specific and MHC class I restricted. The present study showed that WT1 peptide vaccination in combination with TKI is feasible and effective in the therapy for imatinib-resistant CML.

A chronic myeloid leukemia patient with atypical karyotype and BCR-ABL e13a3 transcript caused by complex chromosome rearrangement.

Philadelphia (Ph) chromosome as a result of t (9; 22) (q34; q11) is observed in more than 90% of chromic myeloid leukemia (CML) patients. Cases in which the typical Ph chromosome is not visible at the karyotype level comprise 5-10% of CML patients. CML cases with fusion transcripts such as e13a3 in which ABL exon 3 rather than exon 2 has fused to BCR are very rare. Such reported cases with the e13a3 transcript show the Ph chromosome on karyotype analysis. We reported an atypical karyotype CML patient with the e13a3 BCR-ABL transcript caused by complex translocation. Fluorescence in situ hybridization (FISH) analysis of the metaphase led to a precise cytogenetical characterization. The patient showed favorable response to imatinib, and achieved major molecular responses.

[Effective combination chemotherapy with rituximab for acute lymphoblastic leukemia with bone relapse after bone marrow transplantation].

A 26-year-old woman with acute lymphoblastic leukemia (ALL) relapsed three times after HLA-matched related bone marrow transplantation. Initially, ALL relapsed in the central nervous system (CNS) 1 year after transplantation. Then, ALL relapsed as a single bone tumor involving the CNS and pelvis 4 years after transplantation. Finally, multiple bone tumors in the pelvis and lumbar bones were found as well as spread to the bone marrow 5 years after transplantation. Bone marrow aspiration also showed ALL relapse. Flow cytometry analyses detected CD20-positive cells in the bone tumor. Though the initial bone tumor was resistant to hyper CVAD, radiation was effective and this patient achieved complete remission. At that time, the total radiation dose had already reached the upper limit. After the third relapse, bone marrow achieved complete remission with the administration of pirarubicin, vincristine, prednisolone, and L-asparaginase (arranged DVP-L), though this combination chemotherapy itself was not effective in multiple bone tumors. Thereafter, arranged DVP-L plus rituximab was administered, which resulted in significant tumor reduction. Biweekly rituximab administration as maintenance therapy has completely prevented the regrowth of bone tumors. Rituximab for relapsed CD20-positive ALL patients after stem cell transplantation could be beneficial.

Imatinib mesilate inhibits neointimal hyperplasia via growth inhibition of vascular smooth muscle cells in a rat model of balloon injury.

Restenosis is a major problem in percutaneous catheter intervention (PCI) for coronary artery stenosis in patients with acute myocardial infarction. Coronary restenosis arises from intimal hyperplasia, i.e., hyperplasia of the vascular smooth muscle cells (SMCs) caused by endothelial cell (EC) damage due to PCI. Drug eluting stent (DES), a novel stent coated with a cell-growth inhibitor, such as rapamycin, has been utilized to block SMC proliferation, but DES also blocks EC repair and thus requires the administration of anti-platelets for a long time to prevent thrombus formation after PCI. Moreover, insufficient prevention of platelet aggregation sometimes induces restenosis after PCI. One of the signal transduction inhibitors, imatinib mesilate, blocks tyrosine kinase activity of platelet-derived growth factor receptor (PDGFR), and therefore it may block the development of neointima through growth inhibition of SMCs without the obstructive effect on EC-repair. We therefore studied the effects of imatinib on neointimal hyperplasia in a balloon injury model of rat carotid arteries. Rats were orally administered with imatinib for 14 days after balloon injury, and sacrificed to analyze the neointimal formation. Intimal hyperplasia was inhibited by imatinib in a dose-dependent manner. Therefore imatinib presumably obstructed the growth of SMCs via interception on growth-signaling of PDGFR. The administration of imatinib after coronary stenting or the use of an imatinib-eluting stent may further reduce the risk of restenosis in patients.

Serum erythropoietin level as a marker of limb ischemia.

Bone marrow implantation (BMI) has been utilized for the treatment of limb ischemia, however, serum markers have not yet been reported to express the degree of limb ischemia. We analyzed the serum levels of several cytokines including erythropoietin (EPO) in the treated legs and the contralateral ones in 11 patients with limb ischemia treated with BMI. The EPO level in the pre-treated legs in the 5 patients with arteriosclerosis obliterans revealed a good correlation with ankle-brachial pressure index. The EPO level, but not the levels of TNF-alpha, VEGF, and bFGF in the pre-treated legs was significantly higher than that in the contralateral legs in the 11 patients, and the EPO level decreased in 4 weeks after BMI. The serum EPO level may express the degree of limb ischemia presumably through the reactive production of EPO in ischemic tissue.

[Prognostic factors for critical limb ischemia after autologous bone marrow implantation].

OBJECTIVES: Autologous bone marrow implantation (BMI) is effective to treat critical limb ischemia, but the long-term prognosis is not clear. The outcome of BMI treatment for ischemic legs was investigated related to the clinical background of the patient, and short-term effects of BMI. The end event was defined as unexpected lower limb amputation. METHODS AND RESULTS: This study included 21 consecutive patients (mean age 60.0 +/- 13.6 years) with peripheral arterial disease who underwent BMI between December 2001 and March 2005. Twelve patients had arteriosclerosis obliterans (ASO), 5 had Buerger disease (thromboangiitis obliterans), 3 had thromboembolism, and 1 had hypereosinophilic syndrome. The patients with ASO had severe complications such as diabetes and hyperlipidemia. The total number of transplanted CD34-positive cells, ankle-brachial pressure index (ABI), and tissue oxygen pressure (TcO2) were lower in ASO patients than non-ASO patients. Significant risk factors for the event were diagnosis of ASO and low TcO2 (< 30 mmHg) according to the Kaplan-Meier survival curve and log rank test. All 6 patients who required limb amputation had ASO simultaneously with low TcO2 (6 of 9, 67%). In contrast, there was no correlation between the end event and short-term effect of BMI such as improvements in ABI and TcO2. CONCLUSIONS: Treatment with BMI could not save legs in some patients with ASO associated with severe leg ischemia.

Successful treatment of adult T-cell leukemia with unrelated cord blood transplantation.

This study reports the first well-documented case of adult T-cell leukemia (ATL) successfully treated with unrelated cord blood transplantation (UCBT). A 49-year-old woman was diagnosed with acute-type of ATL. Chemotherapy induced complete remission, but the human T-cell leukemia virus type 1 (HTLV-1) proviral load was detected in mononuclear cells of her peripheral blood. The patient received UCBT with a conditioning regimen consisting of total body irradiation, cytarabine, and cyclophosphamide. She remains in remission 30 months after UCBT and the HTLV-1 proviral load has fallen to undetectable levels. This result suggests that UCBT should be a therapeutic option for ATL patients who do not have suitable donors and those who urgently require treatment.