Dexamethasone Prolongs Cardiac Allograft Survival in a Murine Model Through Myeloid-derived Suppressor Cells.

BACKGROUND: Recently, myeloid-derived suppressor cells (MDSCs) have attracted considerable attention because of their cancer-promoting and immunosuppressive effects. The glucocorticoid dexamethasone (Dex) is an important immunosuppressive agent used to treat autoimmune diseases and organ transplant rejection. However, the mechanism by which it modulates the immune system is not completely understood. MATERIAL AND METHODS: In this study, we investigated the mechanisms by which Dex modulated the immune response in mice given an allogeneic cardiac transplant. RESULTS: Dex injection significantly prolonged heart graft survival compared with phosphate-buffered saline-injected controls. Dex treatment increased the number of splenic MDSCs. Moreover, Gr-1high/CD11b+ MDSCs and CD3+/CD4+/Foxp3+ regulatory T cells (Tregs) were significantly increased in the Dex group compared with controls. Administration of anti-Gr-1 antibody (Ab) to the Dex group significantly shortened mouse heart graft survival. In addition, anti-Gr-1 Ab treatment significantly reduced Tregs in the Dex + anti-Gr-1 co-treatment group compared with the Dex group. These observations suggest that Dex treatment increased both MDSCs and Tregs, and that MDSCs regulated the incidence of Tregs in this immunosuppressive pathway. CONCLUSION: An important role of Dex in the prevention of the rejection of cardiac grafts in mice is to expand MDSCs and Tregs.

C/EBPß promotes BCR-ABL-mediated myeloid expansion and leukemic stem cell exhaustion.

The BCR-ABL fusion oncoprotein accelerates differentiation and proliferation of myeloid cells during the chronic phase of chronic myeloid leukemia (CP-CML). Here, the role of CCAAT/enhancer binding protein ß (C/EBPß), a regulator for 'emergency granulopoiesis,' in the pathogenesis of CP-CML was examined. C/EBPß expression was upregulated in Lineage(-) CD34(+) CD38(-) hematopoietic stem cells (HSCs) and myeloid progenitors isolated from bone marrow of patients with CP-CML. In EML cells, a mouse HSC line, BCR-ABL upregulated C/EBPß, at least in part, through the activation of STAT5. Myeloid differentiation and proliferation induced by BCR-ABL was significantly impaired in C/EBPß-deficient bone marrow cells in vitro. Mice that were transplanted with BCR-ABL-transduced C/EBPß knockout bone marrow cells survived longer than mice that received BCR-ABL-transduced wild-type (WT) bone marrow cells. Significantly higher levels of leukemic stem cells were maintained in BCR-ABL-transduced C/EBPß-deficient cells than in BCR-ABL-transduced WT cells. These results suggest that C/EBPß is involved in BCR-ABL-mediated myeloid expansion. Further elucidation of the molecular mechanisms underlying the C/EBPß-mediated stem cell loss might reveal a novel therapeutic strategy for eradication of CML stem cells.

Combined effects of novel heat shock protein 90 inhibitor NVP-AUY922 and nilotinib in a random mutagenesis screen.

To overcome imatinib resistance, more potent ABL tyrosine kinase inhibitors (TKIs), such as nilotinib and dasatinib have been developed, with demonstrable preclinical activity against most imatinib-resistant BCR-ABL kinase domain mutations, with the exception of T315I. However, imatinib-resistant patients already harboring mutations have a higher likelihood of developing further mutations under the selective pressure of potent ABL TKIs. NVP-AUY922 (Novartis) is a novel 4,5-diaryloxazole adenosine triphosphate-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and deplete the levels of HSP90 client protein including BCR-ABL. In this study, we investigated the combined effects of AUY922 and nilotinib on random mutagenesis for BCR-ABL mutation (Blood, 109; 5011, 2007). Compared with single agents, combination with AUY922 and nilotinib was more effective at reducing the outgrowth of resistant cell clones. No outgrowth was observed in the presence of 2 µM of nilotinib and 20 nM of AUY922. The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to AUY922 and nilotinib even in BaF3 cells expressing BCR-ABL mutants including T315I. In vivo studies also demonstrated that the combination of AUY922 and nilotinib prolonged the survival of mice transplanted with mixture of BaF3 cells expressing wild-type BCR-ABL and mutant forms. Taken together, this study shows that the combination of AUY922 and nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells.

AV-65, a novel Wnt/ß-catenin signal inhibitor, successfully suppresses progression of multiple myeloma in a mouse model.

Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Although new molecular targeting agents against MM have been developed based on the better understanding of the underlying pathogenesis, MM still remains an incurable disease. We previously demonstrated that ß-catenin, a downstream effector in the Wnt pathway, is a potential target in MM using RNA interference in an in vivo experimental mouse model. In this study, we have screened a library of more than 100 000 small-molecule chemical compounds for novel Wnt/ß-catenin signaling inhibitors using a high-throughput transcriptional screening technology. We identified AV-65, which diminished ß-catenin protein levels and T-cell factor transcriptional activity. AV-65 then decreased c-myc, cyclin D1 and survivin expression, resulting in the inhibition of MM cell proliferation through the apoptotic pathway. AV-65 treatment prolonged the survival of MM-bearing mice. These findings indicate that this compound represents a novel and attractive therapeutic agent against MM. This study also illustrates the potential of high-throughput transcriptional screening to identify candidates for anticancer drug discovery.

Galectin-9 exhibits anti-myeloma activity through JNK and p38 MAP kinase pathways.

Galectins constitute a family of lectins that specifically exhibit the affinity for beta-galactosides and modulate various biological events. Galectin-9 is a tandem-repeat type galectin with two carbohydrate recognition domains and has recently been shown to have an anti-proliferative effect on cancer cells. We investigated the effect of recombinant protease-resistant galectin-9 (hGal9) on multiple myeloma (MM). In vitro, hGal9 inhibited the cell proliferation of five myeloma cell lines examined, including a bortezomib-resistant subcell line, with IC(50) between 75.1 and 280.0 nM, and this effect was mediated by the induction of apoptosis with the activation of caspase-8, -9, and -3. hGal9-activated Jun NH(2)-terminal kinase (JNK) and p38 MAPK signaling pathways followed by H2AX phosphorylation. Importantly, the inhibition of either JNK or p38 MAPK partly inhibited the anti-proliferative effect of hGal9, indicating the crucial role of these pathways in the anti-MM effect of hGal9. hGal9 also induced cell death in patient-derived myeloma cells, some with poor-risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. These suggest that hGal9 is a new therapeutic target for MM that may overcome resistance to conventional chemotherapy.

Glyoxalase-I is a novel target against Bcr-Abl+ leukemic cells acquiring stem-like characteristics in a hypoxic environment.

Abl tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib are ineffective against Bcr-Abl(+) leukemic stem cells. Thus, the identification of novel agents that are effective in eradicating quiescent Bcr-Abl(+) stem cells is needed to cure leukemias caused by Bcr-Abl(+) cells. Human Bcr-Abl(+) cells engrafted in the bone marrow of immunodeficient mice survive under severe hypoxia. We generated two hypoxia-adapted (HA)-Bcr-Abl(+) sublines by selection in long-term hypoxic cultures (1.0% O(2)). Interestingly, HA-Bcr-Abl(+) cells exhibited stem cell-like characteristics, including more cells in a dormant, increase of side population fraction, higher beta-catenin expression, resistance to Abl TKIs, and a higher transplantation efficiency. Compared with the respective parental cells, HA-Bcr-Abl(+) cells had higher levels of protein and higher enzyme activity of glyoxalase-I (Glo-I), an enzyme that detoxifies methylglyoxal, a cytotoxic by-product of glycolysis. In contrast to Abl TKIs, Glo-I inhibitors were much more effective in killing HA-Bcr-Abl(+) cells both in vitro and in vivo. These findings indicate that Glo-I is a novel molecular target for treatment of Bcr-Abl(+) leukemias, and, in particular, Abl TKI-resistant quiescent Bcr-Abl(+) leukemic cells that have acquired stem-like characteristics in the process of adapting to a hypoxic environment.

Risk-adjusted assessment of incidence and quantity of blood use in acute-care hospitals in Japan: an analysis using administrative data.

BACKGROUND AND OBJECTIVES: Continuous monitoring of blood use and feedback on transfusions are effective in decreasing inappropriate blood transfusions. However, traditional methods of monitoring have practical challenges, such as the limited availability of experts and funding. Administrative data including a patient classification system may be employed for risk-adjusted assessment of hospital-wide blood use. MATERIALS AND METHODS: We conducted an audit of blood use at two hospitals and determined proportions of appropriate blood use at each hospital. We then used administrative data of 587,045 cases provided by 73 hospitals to develop two mathematical models to calculate risk-adjusted use of blood products. The first model is a logistic regression model to predict the percentage of transfused patients. Patient demographics, surgery and diagnostic groups were utilized as predictors of transfusion. The second model is a case-mix adjusted model which predicts hospital-wide use of units of blood products from the distribution of diagnosis-related groups. For each model, the observed to expected (O/E) ratio of blood use in each hospital was calculated. We compared resultant ratios with proportions of appropriate blood use in two of the hospitals studied. RESULTS: Both models showed good prediction abilities. O/E ratios calculated using the two models were relevant to proportions of appropriate transfusions. CONCLUSIONS: Risk-adjusted assessments of blood product use based on administrative data allow hospital-wide evaluation of transfusion use. Comparing blood use between different hospitals contributes toward establishing appropriate transfusion practices.

Modified ELISPOT assay may predict T-cell hyporesponsiveness to non-inherited maternal antigens.

Clinical reports have suggested the existence of immunological tolerance to noninherited maternal antigens (NIMA) in human leukocyte antigen (HLA) mismatched allogeneic stem cell transplantation (allo-SCT). We studied the T-cell reactivity using IFN-gamma enzyme-linked immunospot (ELISPOT) assay in three HLA fully matched allo-SCT cases and one healthy volunteer family case. In HLA fully matched allo-SCT cases, ELISPOT assay could detect the hyporesponsiveness of T cells from donors to the B cells from recipients. Moreover, ELISPOT assay showed that the T cells from an individual responded to B cell from his mother significantly weakly than those from an unrelated HLA-haploidentical individual. These observations suggest that our IFN-gamma ELISPOT assay-based method may predict the presence of immunological tolerance to NIMA.

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia.

Bcr-Abl is the cause of Philadelphia-positive (Ph(+)) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph(+) leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-X(L), greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph(+) leukemic cells.

Successful non-T cell-depleted HLA haplo-identical three-loci mismatched hematopoietic stem cell transplantation from mother to son based on the feto-maternal microchimerism in chronic myelogenous leukemia.

A 17-year-old male with chronic myelogenous leukemia in blast crisis received a non-T cell-depleted (TCD) HLA haplo-identical three-loci mismatched hematopoietic stem cell transplant (HSCT) from his mother. Long-term feto-maternal microchimerism was detected by nested polymerase chain reaction with sequence-specific primer typing. The post-transplantation prophylaxis against graft-versus-host disease (GVHD) was tacrolimus with minidose methotrexate. Sustained engraftment was obtained. Acute GVHD (grade 2) developed, but improved rapidly. Bone marrow aspiration on day 120 showed complete remission. Non-TCD HLA haplo-identical HSCT based on feto-maternal microchimerism might be feasible and has important implications in the selection of alternative family donors in HSCT.

Early relapse after high-dose chemotherapy rescued by tumor-free autologous peripheral blood stem cells in acute lymphoblastic leukemia: importance of monitoring for WT1-mRNA quantitatively.

A 24-year-old woman who suffered from ALL with MLL gene rearrangement received high-dose chemotherapy followed by autologous PBSC transplantation during complete remission (CR). Reverse transcriptase-polymerase chain reaction (RT-PCR) used to detect MLL/LTG4 chimeric mRNA showed no minimal residual disease (MRD) in the graft or bone marrow at the transplantation. However, the leukemia relapsed four months after transplantation. Retrospective analysis of quantitative measurement of Wilms tumor gene (WT-1) mRNA showed an increased level in the bone marrow although it was within the normal range. These observations suggest that careful monitoring of MRD by quantitative measurement of WT-1 mRNA in addition to disease-specific chimeric mRNA is required to predict relapse.

Human herpes virus 8-negative primary effusion lymphoma in a patient with a ventriculoperitoneal shunt tube.

A 60-year-old woman was referred to our hospital in 1996 due to an abdominal distension in the right lower quadrant. She had undergone a partial resection of a cholesteatoma at the right temporal lobe of the cerebrum 30 years previously, and a ventriculoperitoneal shunt (VPS) tube had been placed with drainage into the right lower peritoneal cavity. The patient developed paralytic ileus in December 1966, and ultrasound and computed tomography of the abdomen revealed a cystic mass in the right lower quadrant without lymphadenopathies or masses. Cytologic examinations of the fluid in the cystic mass revealed signs of malignant lymphoma. After the resection of the cystic mass, lymphoma cells were detected in the fluid, but the wall of the cyst consisted of only fibrous tissues. Results of immunophenotypic analysis of the lymphoma cells by immunocytochemistry or flow cytometry were positive for CD19, CD20, CD22, CD45, and HLA-DR but negative for CD45RO, CD3, CD4, and CD8. The genome of human herpes virus (HHV)-8 was not detected in the lymphoma cells, but Epstein-Barr (EB) nuclear antigen 1 and EB virus (EBV)-encoded small nuclear RNAs were detected. Chromosome analysis by the G-banding method showed complicated abnormalities including der(8)t(2;8)(q31;q24), but Southern blotting analysis suggested that the c-myc oncogene did not participate in the lymphomagenesis. The patient's disease was diagnosed as HHV-8-negative primary effusion lymphoma (PEL). The long-standing inflammatory stimulation by a VPS tube might have contributed to the clonal evolution of EBV-infected lymphocytes. resulting in the development of PEL.

Subcutaneous infection with Mycobacterium fortuitum after allogeneic bone marrow transplantation.

Reports of cases of mycobacterial infections after SCT are rare. We report a 30-year-old female with a cutaneous infection of Mycobacterium fortuitum 30 months after allogeneic bone marrow transplantation for acute lymphoblastic leukemia. The patient was successfully treated with surgical debridement followed by oral minocycline and clarithromycin. Mycobacterial infections should be considered in SCT patients with undiagnosed refractory chronic cutaneous infection, and surgical debridement is useful for the diagnosis and treatment of such infections.

Clinical characteristics of B-cell lymphoma-associated hemophagocytic syndrome (B-LAHS): comparison of CD5+ with CD5- B-LAHS.

OBJECTIVE: B-cell lymphoma-associated hemophagocytic syndrome (B-LAHS) has been increasingly reported in Asia and is regarded as a variant of intravascular lymphomatosis (IVL). Recently CD5 was reported to be expressed in some cases of diffuse large cell lymphoma and IVL. We therefore examined the expression of CD5 on lymphoma cells in B-LAHS and compared the clinical and laboratory data between CD5+ and CD5- B-LAHS. METHODS: The expression of CD5 on lymphoma cells was examined using flow cytometry and immunohistochemical analysis of paraffin sections. The clinical records were reviewed to characterize clinical features. PATIENTS: Twelve patients with B-LAHS; ten men and two women, age ranging from 41 to 82 years (median, 63.5 years) were included in this study. RESULTS: B-LAHS is characterized by fever and hepatosplenomegaly without lymphadenopathy at the initial presentation. Histological examination showed hemophagocytosis and infiltration of lymphoma cells in the bone marrow, and in some cases intravascular proliferation of lymphoid cells characteristic of IVL. All patients showed increased levels of lactate dehydrogenase, C-reactive protein, ferritin and soluble interleukin-2 receptor. In eight of the twelve patients, lymphoma cells were positive for CD5. But no differences were observed in the clinical or laboratory findings between CD5+ B-LAHS and CD5- B-LAHS. CONCLUSION: No clinical differences were observed between CD5+ B-LAHS and CD5- B-LAHS. Further studies are required to elucidate the differences in pathogenesis between these two subgroups of B-LAHS.

[Philadelphia chromosome-positive acute lymphoblastic leukemia with monosomy 7 successfully treated with intermediate- and high-dose ara-C].

A 52-year-old man was admitted for treatment of acute lymphoblastic leukemia (ALL). The bone marrow was hypercellular with 67.2% blasts, which were negative for peroxidase, and expressed CD13, CD33, CD34, CD10 and CD7. Cytogenetic and molecular studies revealed t(9;22) and -7(Ph/-7) with major BCR/ABL rearrangement. The patient was treated with the L-AdVP regimen, but failed to achieve complete remission (CR). He then received two courses of chemotherapy consisting of intermediate- and high-dose cytarabine (ara-C), resulting in CR. This case suggests that Ph/-7 ALL with major BCR/ABL gene rearrangement showing coexpression of myeloid antigens may be sensitive to intermediate- and high-dose ara-C.