Although glucocorticoid (GC) is widely used for treating hematopoietic malignancies including adult T-cell leukemia (ATL), the mechanism by which leukemic cells become resistant to GC in the clinical course remains unclear. Using a series of T-cell lines infected with human T lymphotropic virus type-I (HTLV-I), the causative virus of ATL, we have dissected the transformation from interleukin (IL)-2-dependent to -independent growth stage. The transformation associates the loss of thioredoxin-binding protein-2 (TBP-2), a tumor suppressor and regulator of lipid metabolism. Here we show that TBP-2 is responsible for GC-induced apoptosis in ATL cells. In the IL-2-dependent stage, dexamethasone induced TBP-2 expression and apoptosis, both of which were blocked by GC receptor (GR) antagonist RU486. Knockdown of TBP-2 consistently reduced the amount of GC-induced apoptosis. In IL-2-independent stage, however, expression of GR and TBP-2 was suppressed and GC failed to induce apoptosis. Forced expression of GR led the cells to mild sensitivity to GC, which was also accomplished by treatment with suberoylanilide hydroxamic acid, a TBP-2 inducer. A transfection experiment showed that TBP-2 expression induced apoptosis in IL-2-independent ATL cells. Thus, TBP-2 is likely to be one of the key molecules for GC-induced apoptosis and a potential target for treating the advanced stage of ATL.

Despite the outstanding results generally obtained with imatinib mesylate (IM) in the treatment of chronic myeloid leukemia (CML), some patients show a poor molecular response. To evaluate the relationship between steady-state trough plasma IM concentration (IM-C(min)) and clinical response in CML patients, we integrated data from six independent Japanese studies. Among 254 CML patients, the mean IM-C(min) was 1,010.5 ng/ml. Importantly, IM-C(min) was significantly higher in patients who achieved a major molecular response (MMR) than in those who did not (P = 0.002). Multivariate analysis showed that an MMR was associated with both age (odds ratio (OR) = 0.97 (0.958-0.995); P = 0.0153) and with IM-C(min) (OR = 1.0008 (1.0003-1.0015); P = 0.0044). Given that patients with IM-C(min) values >1,002 ng/ml had a higher probability of achieving an MMR in our large cohort (P = 0.0120), the data suggest that monitoring of IM levels in plasma may improve the efficacy of IM therapy for CML patients.

Continuous ambulatory peritoneal dialysis (CAPD) is an effective therapy for the treatment of patients with end-stage renal failure.1 Peritoneal fibrosis is one of the most common morphological changes observed in long-term CAPD patients and includes loss of mesothelial cells and severe accumulation of collagen fibres.2 The prevention of peritoneal fibrosis is necessary for maintenance of long-term CAPD. It has previously been reported that transforming growth factor-beta 1 …

Adult T-cell leukemia (ATL) is a human malignancy associated with human T-lymphotropic retrovirus type-1 and diagnosed based on clinicopathological findings and the presence of integrated human T-lymphotropic retrovirus type-1 provirus in the DNA of tumor cells.1 Despite recent progress in combination chemotherapy regimens, acute-type ATL usually has a poor prognosis.2 Allogeneic SCT is expected to be another therapeutic option for patients with ATL.3, 4 Recently, cord blood transplantation (CBT) has been used for adult patients with hematological malignancies as donor coordination is not necessary, and the incidence and severity of acute GVHD are lower than those for BMT.5 Inflammatory pseudotumor (IPT) is a rare tumor that can occur in various organs and tissues.6 Although its pathogenesis remains unclear, the terms inflammatory myofibroblastic tumor or inflammatory myofibrohistiocytic proliferation have been proposed as being more descriptive of IPT arising in the lung.7 IPT of different organs associated with hematopoietic SCT (HSCT), although rare, has also been reported.8 We describe a patient who developed a lung IPT after CBT. To the best of our knowledge, this report of lung IPT after CBT is also the first report of any IPT after CBT.

Cellular microvesicles (MVs) are released by various cell types such as circulating blood cells and cells from the vessel wall during cell activation by agonists and physical stress.1 MVs contribute to many pathophysiologic processes and are able to transfer various receptors. It is previously reported that MVs play a role in SCT.2, 3 In particular, Janowska-Wieczorek et al.2 reported that platelet-derived MVs bind to hematopoietic stem/progenitor cells and enhance their engraftment. Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine and a known chemotactic for lymphocytes and monocytes. SDF-1 is also the predominant chemokine that mobilizes stem/progenitor cells and has been shown to be upregulated in many damaged tissues as part of the injury response.4 In contrast, endothelial progenitor cells are a subtype of BM-derived progenitor cells expressing surface antigens of both hematopoietic stem cells and endothelial cells.5 Recently, Deregibus et al.6 found that endothelial progenitor cell-derived MVs were incorporated in endothelial cells by interaction with α4- and β1-integrins expressed on MV surfaces and suggested that endothelial progenitor cells may activate angiogenesis in endothelial cells by releasing MVs, which are able to trigger an angiogenic program. Some previous experimental studies have suggested that transplantation of stem and progenitor cells may have a beneficial effect on functional and structural recovery in several organs. However, there have been few investigations of the relationship between MVs and SDF-1 during PBSC harvest. We measured the levels of CD42b-negative/α4-integrin-positive MVs, SDF-1 and soluble CD40 ligand in plasma obtained from patients undergoing PBSC harvest. For mobilization of PBSC, malignant lymphoma patients (n=22) received priming chemotherapy and administration of rhG-CSF. For CD34 assay, a sample of each patient's PBSCs was analyzed by flow cytometry (Cytoron Absolute, Ortho Diagnostic Systems, Tokyo).7 All samples were washed to remove platelets and incubated with two kinds of monoclonal antibody (FITC-α4-integrin and PE-CD42b). MVs were defined as elements <1.5 μ in size with CD42b-negative and α4-integrin-positive.6 We also measured plasma levels of SDF-1 (R&D Systems, Mineapolis MN. USA) and soluble CD40 ligand (Chemicon International Inc., Temecula, CA, USA). We compared samples on days 0 and 5 after rhG-CSF treatment; numbers of CD34-positive cells peaked on day 5 (days 0 vs 5; 3.2±0.8 vs 77.9±8.4/μl, respectively, P<0.0001; Figure 1). MVs, SDF-1 and soluble CD40 ligand exhibited the same changes (days 0 vs 5: MVs, respectively, 219±35 vs 492±53/μl, P<0.01; SDF-1, 1.219±271 vs 1.789±430 pg/ml, P<0.05; soluble CD40 ligand, 1.8±0.4 vs 4.1±0.6 ng/ml, P<0.01; Figure 1). Janowska-Wieczorek et al.8 suggested that MVs actively released from cells may have an important role in cell-to-cell communication. The reports by Deregibus et al.6 and Ratajczak et al.9 have developed this view. In particular, the indication that MVs may deliver mRNA to target cells is very interesting, as MVs express surface molecules characteristic of the original cells. Although we observed that CD42b-negative/α4-integrin-positive MVs exhibited the same changes as SDF-1 and soluble CD40 ligand in company with an increase of CD34+ stem cells during PBSC harvest, the importance of our findings is presently unclear both for the mechanism of mobilization and for events post transplantation. However, SDF-1 has a major role in the pathology of tissue repair, and α4-integrin contributes to hematopoietic progenitor cell recruitment into BM following transplantation.10 Therefore, CD42b-negative/α4-integrin-positive MVs are possible to have an unique role in SCT. Further investigation is necessary to better elucidate this possibility.

The complete remission (CR) rate in acute leukemia (AL) has been improved due to progressive chemotherapy. However, relapse frequently occurs as minimal residual disease (MRD). To eradicate MRD, immunotherapy using autologous cytotoxic T-lymphocytes has been available. Leukemia cells (LC) of the patients were stored frozen before chemotherapy. Furthermore, after achieving CR, an EBV-transformed lymphoblastoid B-cell line (LCL) was established in each case. To induce Cytotoxic T-lymphocytes (CTL), co-culture system was carried out in two types. One is co-culture of peripheral blood mononuclear cells (PBMCs) and original leukemia cells (LC), the other is co-culture of PBMCs and the patient’s own LCL. After co-culture for 5 days, cytotoxic activity was examined by FACS with PKH-26 staining. Interestingly, cytotoxicity against not only autologous LC but the patient’s own LCL was observed in almost cases. This phenomenon is called cross-killing. Moreover, regulatory T-cells (Treg) were detected in the effecter phase determined with FACS (CD4+ CD25+) and FOXP3 mRNA by RT-PCR. Enhanced cytotoxicity was observed in inverse relation to a low proportion of Treg. These results suggested that more cytotoxicity may be induced by deletion of the Treg population. In conclusion, cell therapy as an immunotherapy should be useful tool to improve the prognosis, and if stocked LC is not available, CTL activity must be induced by co-culture with the patient’s own LCL.

Objective: Some patients who had carried out long-term continuous ambulatory peritoneal dialysis discontinued the treatment because of progressive peritoneal fibrosis. It has been previously reported that transforming growth factor-β1 (TGF-β1) is one of the factors that induces peritoneal fibrosis. Also, hepatocyte growth factor (HGF) plays a role in the prevention of fibrosis and in inhibiting TGF-β1 production. In this study, we examined the effects of HGF on peritoneal fibrosis by TGF-β1 induced by high concentrations of D-glucose. Design: We transfected a full-length human HGF cDNA in an expression vector into human peritoneal mesothelial cells (HPMCs) using the calcium phosphate method. Transfected HPMCs were cultured with high concentrations of D-glucose solution and co-cultured with fibroblasts using a transwell system. Cell proliferation was determined using the Tetra Color One method. TGF-β1 and HGF protein were measured by enzyme-linked immunosorbent assay. Results: In addition to recombinant HGF, the growth inhibition of HPMCs by high concentration D-glucose or TGF-β1 was significant. By transfecting HGF cDNA into HPMCs, growth inhibition by high concentration D-glucose was completely restored. Furthermore, the production of TGF-β1 was also significantly decreased. Conclusion: These results suggested that exogenous HGF could possibly prevent peritoneal fibrosis.

Human peritoneal mesothelial cells (HPMCs) play an important role in peritoneal functions. During long term peritoneal dialysis, it has been reported that HPMCs are damaged by high glucose solution via the signal of transforming growth factor (TGF)- ß1 produced by HPMCs. In this study, we focused on the effect of hepatocyte growth factor (HGF), known as an anti-fibrotic and anti-TGF-ß1 agent, on HPMCs damaged by high glucose solution. HPMCs were isolated from specimens of the omentum from nonuremic patients after informed consent had been obtained. After confirming adhesion for 6 hours, 100 μL of DMEM with 0.5%FCS were added at different concentrations (D-glucose; 6, 30mM) with or without HGF (10, 30, 100 ng/mL) for 48 hours. We examined the effects of a high concentration of glucose and then focused on following four critical points: 1) the production of HGF from HPMCs exposed to a high concentration of glucose, 2) the expression of c-Met on HPMCs, 3) the viability of those cells, and 4) matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase-2 (TIMP-2). The following significant changes are described herein: high glucose solution and TGF-ß1 i) decreased HGF production from HPMCs and ii) up-regulated expression of c-Met on HPMCs, and addition of HGF iii) restored viability of HPMCs damaged by glucose, iv) suppressed TGF-ß1 production by HGF, and v) induced up-regulation of MMP-2 and decreased TIMP-2 production by HPMCs. Levels of HGF decreased by high concentrations of glucose in the peritoneal cavity may induce the loss of HPMCs and thereby result in peritoneal fibrosis. These results suggest that HGF is an effective agent in the regeneration of peritoneal membrane damaged by high glucose solution.

CD34 + progenitors from MDS patients are unresponsive to SDF-1, despite high levels of SDF-1 in bone marrow plasma

The frequent involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in ATL. Epstein-Barr virus (EBV) infection has been observed in the clinical course of ATL, and ATL cells in all skin cases were strongly positive for EBV nuclear antigen (EBNA)-1 and latent membrane protein (LMP)-1 by immunohistochemical staining. Interestingly, ATL cells in all skin cases were positive for CD45R0 (UCHL1) and CD20 (L26), indicating that they have both T and B cells characterization. We suggested that HTLV-1 enhances the entry of EBV into endothelial cells, epithelial cells and EBV presents for an active role of HTLV-1 in the development of ATL. Here, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both B cell lines. On characterization of the cell lines, enhanced expression of intercellular adhesion molecule-1 or-3 (CD54, CD50), lymphocyte function-associated antigen-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) was observed compared to an EBV-infected B cell line, Raji. Moreover, EBV oncogenes such as EBV-encoded small RNA (EBER)-1, EBNA-1,-2 and LMP-2A were also detected. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donor's lymphocytes, Raji cells and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was observed in double transfectants (EBV and HTLV-1). Furthermore, we detected high production levels of interleukin-4 (IL-4) in those B-cell lines and transfectants. These results indicated co-expression of HTLV-1 and EBV may induce aggressive organ involvement by enhanced expression of adhesion molecules via IL-4 signaling. Furthermore, HTLV-1 could greatly enhance EBV infectivity and attribute to increased replication of EBV. EBV also could be a necessary cofactor for ATL development in immunodeficiency states. ATL patients who have been coinfected with EBV and HTLV-1 have a high risk of worse progress of disease. ATL patients have a poor treatment outcome, even with strong chemotherapy, especially who have severe skin involvements have a resistance for chemotherapy. Viewed from a different angle, our observations suggest that EBV infection may be used as one of bad prognostic factors and an identification of bad-risk patients who need to benefit from early high dose chemotherapy. Collectively, a reactivation of EBV infection and EBV oncogenes can provide an acceleration of organ involvement in ATL.These results can explain why EBV-associated ATL is more frequent and clinically more aggressive.

Adult T-cell leukemia (ATL) is a leukemia of mature peripheralT lymphocytes associated with human T-lymphotropic virustype I (HTLV-I). We previously reported that all-transretinoic acid (ATRA) inhibited cell growth and inducedapoptosis in HTLV-I (þ) T-cell lines as well as fresh ATL cellsobtained from ATL patients.

We previously reported that all-trans retinoic acid (ATRA) inhibits growth in HTLV-1-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed the NF-κB transcriptional activity as well as cell growth in HTLV-1-positive T-cell lines decreased significantly in these cells in the presence of ATRA. However, no significant growth inhibition was observed after treatment with IFN-γ, TNF-α, and TGF-β of growth inhibitory cytokines induced by ATRA. Thereafter, we observed that ATRA reduced HTLV-1 proviral DNA, gag and tax mRNA load using a real time quantitative polymerase chain reaction and soluble IL-2 receptor in the culture supernatant using ELISA in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited reverse transcriptase activity similar to azidothymidine in HTLV-1-positive T-cell lines. These results suggested that ATRA could inhibit reverse transcriptase activity in the growth inhibition of ATL cells. Additionally, an effect of ATRA on replication of the human immunodeficiency virus (HIV) was also observed. ATRA significantly reduced the HIV proviral DNA load of both a HIV-1-positive cell line and HIV-1-infected patients. These results indicated that ATRA may be useful for HIV treatment in a clinical setting.