Eiichi Azuma

A Pivotal Role of Rho GTPase in the Regulation of Morphology and Function of Dendritic Cells

Dendritic cell (DC) is the most potent activator of CD4+ T cells and has unique dendrites and veils. To explore the function of Rho in DC, exoenzyme C3 from Clostridium botulinum was used as a specific inhibitor of Rho. Treatment of DC with C3 (DC/C3) resulted in profound morphological changes by losing dendrites and emerging of shrunk membrane processes that were in parallel with marked reduction of polymerized actin in the marginal area. Inactivation of Rho-associated coiled coil-containing kinase (p160ROCK) by a specific ROCK inhibitor Y-27632 also led to disappearance of dendrites of DC with retaining large membrane expansions. In scanning electron microscopy, untreated DCs interacted with CD4+ T cells more efficiently than DC/C3. Conjugate formation assay showed that the number of DCs associated with CD4+ T cells was 2-fold higher in untreated DCs than that of DC/C3. Alloantigen-presenting capacity of DC/C3 was significantly suppressed in a dose-dependent manner. Because C3 treatment did not affect the surface expression of HLA, costimulatory, and adhesion molecules of DC, we examined cytokine production of DC and naive CD4+ T cells to further elucidate the inhibitory mechanism of MLR. Unexpectedly, DC/C3 increased IL-12 production after LPS stimulation. Naive CD4+ T cells cocultured with DC/C3 produced the increased percentage of IFN-γ-producing cells, whereas the percentage of IL-2-producing T cells was decreased. These results demonstrate that Rho GTPase in DC controls both characteristic shape and immunogenic capacity.

Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia

Purpose: During an ongoing immune response, cytokines produced by T helper types 1 (Th1) and 2 (Th2) together with T cytotoxic types 1 (Tc1) and 2 (Tc2) are critical to the effectiveness of that response. Dysregulated expansion of one or the other subset may contribute to the impaired function of the T-cell-mediated immune system in cancer patients. In the present study we have investigated whether such dysregulation might exist in children with acute lymphoblastic leukemia (ALL). Methods: We analyzed 61 blood samples from 45 children with B cell precursor ALL and 16 healthy children. Interleukin(IL)-2, IL-4, and interferon γ (IFNγ) production of their respective purified CD4+ and CD8+ T cells were assessed at the single-cell level by intracellular-cytokine-staining flow cytometry. Results: At the time of diagnosis, IL-2-producing cell populations in CD4+ and CD8+ T cells were reduced below the normal range in 31 of 44 (70.5%) and 23 of 38 (60.5%) cases respectively. Similarly, IFNγ-producing cell populations in CD4+ and CD8+ T cells decreased in 17 of 44 (38.6%) and 18 of 38 (47.4%) cases respectively. Conversely cell populations capable of IL-4 production in CD4+ and CD8+ T cell subsets were increased in 13 of 30 (43.3%) and 15 of 30 (50.0%) cases respectively. Therefore, the Th1-to-Th2 and Tc1-to-Tc2 ratios (1.6 ± 2.2 and 7.7 ± 6.7 respectively) were significantly lower in peripheral blood T cells of ALL patients (n = 30) than those (6.0 ± 2.9 and 20.1 ± 10.3 respectively) in 15 healthy controls (P < 0.0001). Although both CD45RA+/CD4+ and CD45RA+/CD8+ cells significantly increased in 43 ALL patients (P < 0.05), there existed no apparent correlation between CD45 isoform expression and cytokine (IL-2 and IFNγ) production. Interestingly, the ability to produce both IL-2 and IFNγ was recovered in 8 cases examined, after complete remission had been achieved. Conclusion: These observations suggest that, in both CD4+ and CD8+ T cells of ALL patients, there is a dysregulation in the functionality of Th1 (Tc1) and Th2 (Tc2) cells with a gross reduction of Th1 (Tc1) cell populations and an expansion in Th2 (Tc2).

BK virus-associated fatal renal failure following late-onset hemorrhagic cystitis in an unrelated bone marrow transplantation.

The human polyomavirus BK (BKV)-associated hemorrhagic cystitis (HC) has been a frequent and seldom life-threatening complication after bone marrow transplantation (BMT). The authors report a male with myelodysplastic syndrome, who developed BKV-associated late-onset HC 12 days after HLA-matched unrelated BMT. His urine contained epithelial cells with intranuclear inclusion bodies suggestive of BKV infection and was positive for BKV in polymerase chain reaction. He did not respond to any treatment for HC. In addition, he developed BKV-associated acute renal failure on day 26, followed by hepatic veno-occlusive disease on day 42. This is the first case in which BKV may be associated with fatal progressive renal failure.

Cellular immunosuppression in children with acute lymphoblastic leukemia: Effect of consolidation chemotherapy

SummaryThe present study was designed to evaluate the chemotherapy-induced cellular immunosuppression in 20 children with acute lymphoblastic leukemia (ALL) in remission and receiving maintenance chemotherapy. Peripheral blood was serially obtained from leukemic children during vincristine/cyclophosphamide/6-mercaptopurine/prednisone combined consolidation chemotherapy. The mean absolute number of peripheral blood lymphocytes as well as the mean absolute numbers of lymphocyte subsets (T cells, T cell subsets, B cells, and natural killer cells) from leukemic children before consolidation chemotherapy were all significantly lower than in control subjects; however, the percentages of lymphocyte subsets were similar in both groups. After consolidation chemotherapy, the percentages of CD4+ T lymphocytes and natural killer (NK) cells were significantly decreased and the percentages of monocytes and CD8+ T lymphocytes were significantly increased. Phytohemagglutinin- and 12-O-tetradecanoylphorbol-13-acetate-induced production of interleukin-2 (IL-2) and NK-cell-mediated cytotoxic activity by peripheral blood mononuclear cells (PBMC) were also substantially decreased in the post-therapy groups. NK activity correlated with the percentage of NK cells in PBMC. In contrast, OK432-induced production of tumor necrosis factor α (TNFα) and killer activity against NK-resistant target cells were significantly increased after therapy as compared with the pre-therapy and control groups. TNFα production correlated with the percentage of monocytes in PBMC. These results demonstrate that substantial quantitative and qualitative chemotherapy-induced abnormalities of the cellular immune system are present in the majority of patients treated with ALL. It is also suggested that the increased TNFα production by monocytes and the appearance of potent killing activity against NK-resistant targets might compensate for the defects of IL-2 production and NK activity during intensive consolidation chemotherapy.

Chemical sensitization and regulation of TRAIL-induced apoptosis in a panel of B-lymphocytic leukaemia cell lines.

Summary.  Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) effectively kills tumour cells but not normal cells. We investigated TRAIL sensitivity and the TRAIL‐induced apoptosis signalling pathway in a panel of B‐lymphocytic leukaemia cell lines. Depending upon TRAIL sensitivity, leukaemia cells could be divided into three groups: highly sensitive, moderately sensitive and resistant. TRAIL receptor‐2 (DR5) plays an important role in transducing apoptosis signals. DR5 was internalized into the cytoplasm where it recruited FAS‐associated death domain protein (FADD) under TRAIL stimulation in both sensitive and resistant cells. However, the active form of caspase‐8 was recruited to FADD and only sensitive cells showed increased caspase‐8 activity upon TRAIL stimulation. The caspase‐8 specific inhibitor, Z‐IETD, impaired caspase‐8 activation and completely abrogated TRAIL‐induced apoptosis. These results suggest that TRAIL resistance in B‐lymphocytic leukaemia cells is due to negative regulation at the level of caspase‐8 activation and that caspase‐8 activation is an indispensable process in TRAIL‐induced apoptosis. However, FADD‐like interleukin‐1 β‐converting enzyme inhibitory protein (c‐FLIPL) was similarly expressed and down‐regulated after TRAIL stimulation in both sensitive and resistant cells. Interestingly, in some cell lines, TRAIL sensitivity and caspase‐8 activity was enhanced or restored with the treatment of cycloheximide (CHX). In addition, X‐linked inhibitor of apoptosis (XIAP) levels decreased significantly and rapidly following treatment with CHX. Down‐regulation of XIAP may be responsible for enhancement or restoration of TRAIL sensitivity after CHX treatment in B‐lymphocytic leukaemia cells.

Fas/APO-1 (CD95)-mediated cytotoxicity is responsible for the apoptotic cell death of leukaemic cells induced by interleukin-2-activated T cells.

Apoptotic cell death is induced by the cross‐linking of Fas/APO‐1 receptor (CD95) in acute myelogenous leukaemia (AML) cells. Since CD95 ligand (CD95L) is expressed on interleukin‐2 (IL‐2)‐activated T cells, we investigated the involvement of CD95‐CD95L pathway in T cell‐mediated cytotoxicity against AML cells. Activated CD8+ T cells could efficiently kill a parental CD95‐sensitive AML cell line, MML‐1 and, to a lesser extent, a CD95‐resistant clone, MML‐1R. Neither MML‐1 nor MML‐1R cells were killed by activated CD4+ T cells. The blocking monoclonal antibody (MoAb) against CD95, ZB4, caused a significant inhibition of T‐cell‐mediated cytotoxicity against MML‐1 cells, but not against MML‐1R cells. Interestingly, MML‐1 cells underwent the classic nuclear morphologic changes and DNA fragmentation characteristic of apoptosis when cultured with activated T cells. Enumeration of apoptotic and necrotic nuclei showed that both apoptosis and necrosis were induced in MML‐1 cells, whereas necrosis was exclusively observed in MML‐1R cells. Apoptosis of MML‐1 cells was completely blocked in the presence of ZB4 MoAb. Similarly, blocking by ZB4 MoAb significantly inhibited T‐cell‐mediated lysis of fresh AML cells in a CD95‐sensitive group, but not in a CD95‐refractory group. In addition CD8+ T cells expressed CD95L mRNA more abundantly than CD4+ T cells upon activation by IL‐2. These findings suggest that T‐cell‐mediated cytotoxicity against AML cells requires participation of CD95‐CD95L pathway for cytotoxic signal transduction leading to target apoptosis.

Successful hematopoietic reconstitution by transplantation of umbilical cord blood cells in a transfusion-dependent child with Diamond-Blackfan anemia

A 4-year-old boy with Diamond–Blackfan anemia and a history of multiple transfusions underwent umbilical cord blood transplantation from his HLA-identical female sibling born by vaginal delivery at 38 weeks. The patient was prepared with busulfan, cyclophosphamide and antilymphocyte globulin. Methotrexate and cyclosporin A were given for the prophylaxis of GVHD. Regimen-related toxicity was not observed and successful engraftment occurred, including the erythroid series. No evidence of acute or chronic GVHD has been observed for 14 months after transplantation. This is the first case of successful umbilical cord blood transplantation to a patient with Diamond–Blackfan anemia.

Transient renal tubular acidosis in a neonate following transplacental acetazolamide

Renal tubular acidosis (RTA) was observed in a preterm boy shortly after birth. His mother had glaucoma and had been treated during pregnancy with oral acetazolamide, a carbonic anhydrase inhibitor. When RTA developed, acetazolamide was detected in his serum demonstrating transplacental acetazolamide passage.

mRNA expression of Fas receptor (CD95)-associated proteins (Fas-associated phosphatase-1/FAP-1, Fas-associating protein with death domain/FADD, and receptor-interacting protein/RIP) in human leukaemia/lymphoma cell lines.

mRNA expression of Fas (CD95)‐associated proteins [Fas‐associating protein with death domain (FADD), receptor‐interacting protein (RIP), and Fas‐associated phosphatase‐1 (FAP‐1)] has been investigated in 26 Fas‐positive human leukaemia/lymphoma cell lines. Reverse transcriptase‐polymerase chain reaction analysis revealed that FADD and RIP mRNA were invariably expressed in both Fas‐sensitive and Fas‐insensitive cell lines. However, FAP‐1 mRNA was detected in only 11 of 26 cell lines. Interestingly 7/14 cell lines in the Fas‐sensitive group were positive for FAP‐1 mRNA expression. 8/12 cell lines in the Fas‐refractory group did not express FAP‐1 mRNA, but half of these cell lines were susceptible to tumour necrosis factor α‐induced growth inhibition. These findings suggest that the presence or absence of FAP‐1 mRNA expression did not always correlate with relative sensitivity of Fas‐mediated growth inhibition. Furthermore, it is assumed that leukaemia/lymphoma cells could possess structural or functional defects of Fas or Fas‐associated proteins resulting in the failure to trigger apoptotic cell death.

Resistance to TRAIL-induced apoptosis caused by constitutional phosphorylation of Akt and PTEN in acute lymphoblastic leukemia cells.

OBJECTIVE Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, which induces apoptosis in cancer cells but not in normal cells. Akt/protein kinase B, when phosphorylated to its active form, promotes cell survival and blocks apoptosis. The aim of this study was to investigate the role of Akt pathway in acquired TRAIL resistance of acute lymphoblastic leukemia cells. MATERIALS AND METHODS MB-IT and NALM-24 cells that developed resistance to TRAIL, i.e., TRAIL-resistant cells (MB-IT R and NALM-24 R) were established from TRAIL-sensitive acute lymphoblastic leukemia cell lines (MB-IT S and NALM-24 S), respectively, through application of TRAIL and repetitive limiting dilution. Apoptosis was measured by flow cytometry using propidium iodide/Annexin-V fluorescein isothiocyanate staining. TRAIL receptor cell surface expression of MB-IT and NALM-24 were analyzed by flow cytometry. Protein levels were analyzed by Western blot analysis. RESULTS The obtained resistant cell lines presented the same pattern of receptor expression as sensitive parent cells, and the internalization of DR5 after TRAIL treatment was similar. Caspase-8/3, FLIP, BID, XIAP were cleaved/downregulated in sensitive cells after treatment with TRAIL, but not in the resistant cells. We also observed that phosphoinositide-3-kinase (PI3K)/Akt pathway was constitutively active in resistant clones, and was not downregulated upon TRAIL treatment. Phosphate and tensin homologue deleted on chromosome 10 (PTEN) level was the same in both sensitive cells and resistant cells, but was quickly downregulated in sensitive cells after TRAIL treatment. Also, resistant cells expressed a high level of phosphorylated inactive form of PTEN than the sensitive cells. Expression levels of PH domain leucine-rich repeat protein phosphatase were slightly higher in sensitive than resistant cells. When resistant cells were treated with LY 294002 (a PI3K inhibitor), the expression level of phosphorylated Akt was distinctly downregulated, and there was induction of apoptosis when these cells were treated with a combination of TRAIL and LY 294002. When MB-IT-sensitive cells were treated with okadaic acid, a phosphatase inhibitor, TRAIL-induced apoptosis was significantly reduced. CONCLUSION These results suggest that cellular resistance to TRAIL could be developed through phosphorylation (activation) of Akt and phosphorylation (inactivation) of PTEN.