ALBERT

Description: The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

Description: Abstract 2924 Poster Board II-900 Introduction : Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades the essential amino acid tryptophan along the kynurenine pathway. Pro-inflammatory cytokines, such as IFN-g, induce IDO during the inflammatory response in many human cell types. The induction of IDO is synergistic in the presence of TNF-a, IL-1 or IL-6, and might be mediated by a signaling pathway from NF-κB and/or MAPKs. Furthermore, some metabolites derived from tryptophan by IDO, such as L-kynurenine, block antigen-driven specific T-cell proliferation and induce T-cell death. Thus, IDO activity might play an important role in regulation of the immune response exerted by antigen presenting cells and also provide transformed cells with a potent tool to help escape from assault by the immune system. Indeed, we have previously reported that high serum L-kynurenine level is associated with poor prognosis of diffuse large B-cell lymphoma (DLBCL) (ASH 2008 abstract 2812). Here, we investigated the IDO expression of patients with DLBCL. Patients and methods : The study protocol comprised a prospective, consecutive entry design that was approved by our Institutional Review Board. We investigated 119 patients between December 2003 and June 2008 who were histologically diagnosed with DLBCL according to the WHO classification. We performed immunohistochemical (IHC) analysis for IDO expression by mouse anti-human IDO monoclonal antibody. Patients aged

Description: Background: A combination of rituximab and CHOP (R-CHOP) is currently the standard treatment for diffuse large B-cell lymphoma (DLBCL). We previously reported the utility of THP-COP regimen consisted of cyclophosphamide (CPA), pirarubicin (tetrahydropyranyl adriamycin: THP), vincristine (VCR), and prednisolone (PSL) in patients with DLBCL (Tsurumi H. et al. Hematol Oncol 2007). THP, a derivative of doxorubicin (DOX), is another anthracycline that has been reported to be less cardiotoxic than DOX. We conducted this phase II study to verify untreated patients with CD20 positive DLBCL. Patients and Methods: The primary objective was to assess the efficacy of R-THP-COP with response rates. Secondary objectives were to assess the overall survival and drug safety. The study was conducted with local ethics committee approval, and all patients gave written, informed consent prior to enrollment. Adverse effects were graded according to the National Cancer Institute Common Toxicity Criteria version 2.0. Over 18 were eligible for inclusion in the study if they had a diagnosis of untreated CD20 positive DLBCL. DLBCL was confirmed by biopsy. All patients had DLBCL at clinical stage (CS) II, III, or IV. Patients aged

Description: Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time- courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM- CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF, or FMLP at the inflammatory sites.

Description: Recent studies have indicated that patients who receive stem cell transplantation (SCT) and an adjuvant rituximab demonstrate an increased risk of developing hypogammaglobulinemia. We have found that such hypogammaglobulinemia were due to the delayed recovery of memory B cells with an abnormal cell marker expression and impaired immunoglobulin production in vitro. (Nishio et al. Eur J Haemtol, 2006, Nishio et al. Br J Haematol, 2007) However, no speculation has been made regarding what factor(s) determined the risk of developing hypogammaglobulinemia after autologous SCT with the identical conditioning regimen and rituximab. Accumulated evidences have shown that FCGR3A of valine (V) allele has a higher affinity to human IgG than the phenylalanine (F) allele, and that cells bearing the FCGR3A V allele mediate antibody dependent cellular cytotoxicity more effectively. Compatibly, previous clinical studies that have examined single nucleotide polymorphisms (SNPs) of Fc receptor genes demonstrated that FCGR3A gene SNPs are associated with the response to rituximab, as a single agent, in patients with follicular lymphoma or Waldenstrom’s macroglobulinemia. These findings suggest that FCGR3A SNPs may be related to the levels of immunoglobulin after SCT and an adjuvant rituximab. To clarify this hypothesis, the FCGR3A-158V/F genotype and the levels of serum immunoglobulin six months after SCT were tested in twenty non- Hodgkin’s lymphoma (NHL) patients having received autlogous peripheral blood stem cell transplantation (APBSCT) with an adjuvant rituximab. We also compared the levels of immunoglobulin in ten NHL patients who received an identical conditioning regimen and APBSCT, but no rituximab (control group). Of the twenty patients tested for the FCGR3A-158V/F polymorphism, seven patients (35%) had homozygous F/F (158 F/F), 12 (60%) had heterozygous V/F (158 V/F), and one (5%) had homozygous V/V (158 V/V). Since only one patient was found to have 158 V/V polymorphism in this study, we defined those patients who had 158 F/F as the low affinity group, while those who had at least one 158 V allele were defined as the high affinity group following the previous definition by Anolik et al (Arthritis Rheum 2003). The three groups were not different in terms of gender, age, the disease stage, bone marrow involvement or number of extranodal sites involved at diagnosis. Before starting induction therapy, there was no significant difference in the levels of immunoglobulin among three groups. However, after APBSCT, the levels of IgG were significantly lower in the low affinity group (6.87 ± 2.38 g/l) than those in the high affinity group (10.20 ± 2.43 g/l) and control group (10.64 ± 3.04 g/l; both P

Description: Introduction: We have previously reported serum concentration of sFas predict a clinical outcome of patients with DLBCL. Here, we added the number of patients and confirmed that a high serum sFas level was associated with unfavorable prognosis of patients with DLBCL. Patients and methods: We used a prospective, consecutive entry design, and the study protocol was approved by the Institution’s Review Board. Between October 1995 and September 2002 previously untreated 132 patients with DLBCL (Group A: patients treated without rituximab) and between December 2002 and March 2007 previously untreated 75 patients with DLBCL (Group B: patients treated with rituximab) confirmed by biopsy participated in this study. We considered eligible for this study all patients with histologically confirmed diagnosis of DLBCL, according to the Working-Formulation and the WHO classification. Serum sFas was determined using ELISA. Patients were assigned to receive eight cycles of CHOP or THP (tetrahydropyranyl-adriamycin) -COP therapy before September 2002. The patients who had been registered after October, 2002 received the R-CHOP or R-THP-COP therapy. A serum sFas level of 3.0ng /ml was used as the cut-off value in this study. All follow-up data were updated on March 1, 2007. Results: In Group A: 132 patients were enrolled in this category (82 males, 50 females, age; 14 to 92 years, median; 66 years). IPI scoring was available in all patients; accordingly 18.2% patients were classified as low risk, 25.8% as low-intermediate; 32.6% as high-intermediate, and 23.5% as high risk. Overall, the CR rate was 73.5%; PRs were observed in 7.6% and failures in 18.9%. The CR rates for patients with an sFas level of 3.0ng /ml and over and less than 3.0ng /ml were 51.1% and 81.6%, respectively (P

Description: Abstract 1926 Poster Board I-949 Purpose: Diffuse large B cell lymphoma (DLBCL) is a heterogeneous entity, with patients exhibiting a wide range of outcomes. The introduction of rituximab to CHOP (R-CHOP) has significantly altered improvement in survival. This raises concern regarding the utility of previously identified prognostic factors. Before rituximub era, some investigators have suggested that serum levels of some cytokines and their soluble receptors might reflect tumor growth and host tumor responses. Interleukin-18 (IL-18), originally designated as an interferon (IFN)-gamma inducing factor, is a cytokein which stimulates cytotoxic NK cell activity and T cells to produce IFN-gamma, IL-2, and GM-CSF. Increased IL-18 serum levels have been found in patients with some hematopoietic neoplasms. IL-18 is also immunostimulatory cytokine with antitumor activity in preclinical models, and a phase I study of recombinant human IL-18 was done to patients with advanced cancer. We have previously reported that IL-18 was strong prognostic factor in aggressive lymphoma patients who received CHOP without rituximab (ASH 2004, abstract #4543). The aim of the present study is to re-assess the prognostic significance of serum IL-18 in DLBCL treated with rituximab, and we also assessed IL-18 with subtype of DLBCL, GCB type and non GCB type. Meterials and methods: Consecutive 154 previously untreated patients with DLBCL prospectively participated in this study between 2002 and 2008. The patients were treated with 6-8 cycles of R-CHOP or R-THP (pirarubicin) -COP regimens. There was no difference with CHOP and THP-COP in our recent prospective randomized study (Tsurumi H et. al. JCRCO 2004). Serum IL-18 was determined by ELISA, and we classified subgroups of DLBCL according to Hans et al. Results: In all patients with DLBCL, the mean ± SD of serum IL-18 level was 829.5±1280.8 pg/ml (range 56 - 8697.5) with a median of 415.8 pg/ml. Various poor prognostic features, such as poor PS, many extranodal sites, advanced disease (CS III/IV) increased LDH and elderly people were strongly associated with a high serum IL-18 levels. The median serum IL-18 levels of the different IPI risk groups were as follows: 201pg/ml for the L risk; 361pg/ml for the LI risk; 440pg/ml for the HI risk; 691pg/ml for the H risk, respectively (P

Description: Resistance against retinoic acid (RA) is a serious problem of differentiation-induction therapy for acute promyelocytic leukemia (APL) in clinical practice. RA exerts its biological activities primarily through the nuclear receptor dimmer, consisting of retinoic acid receptor (RAR) and retinoid X receptor (RXR). Both receptors consist of three subtypes (α, β and γ) and form heterodimeric RAR/RXR and homodimeric RXR/RXR complexes. 9-cis RA, which is a high-affinity ligand for RXR but also binds to RAR, induces cell differentiation in wild type HL-60 human myeloid leukemia cells. However, in HL-60R cells, the RA-resistant subclone of HL-60, 9-cis RA can not induce cell differentiation and apoptosis. We recently reported that malfunction of RXRα due to posttranslational modification by phosphorylation to be associated with carcinogenesis of hepatocellular carcinoma (HCC). Phosphorylated form of RXRα (p-RXRα) at serine 260 by Ras/MAPK is resistant to ubiquitin/proteasome-mediated degradation and the accumulation of p-RXRα interferes with the function of remaining normal RXRα in a dominant negative manner, thereby promoting growth of HCC cells. We also found that in the presence of MEK inhibitor PD98059, 9-cis RA can induce the degradation of p-RXRα and thus restoring the function of this receptor in RXRα-phosphorylated human HCC cells. Based on the results as described above, we initiated this study to examine whether 9-cis RA can exert growth inhibitory effects on RA-resistant HL-60R cells when combined with MEK inhibitor, with focusing on the inhibition of expression of p-RXRα protein. We found that RXRα protein was originally expressed in both HL-60 and HL-60R cells, and that the expression level of RXRα protein was inhibited by about 60% and 20%, respectively, when those cells were treated with 0.5 μM 9-cis RA. Not only total RXRα protein, but also the level of p-RXRα protein was constitutively expressed in both HL-60 and HL-60R cells, and was significantly decreased in HL-60 cells by treatment with 9-cis RA alone. On the other hand, in HL-60R cells, 9-cis RA alone nor 20 μM PD98059 alone did not cause a down regulation of these proteins. However, when HL-60R cells were treated with the combination of 9-cis RA plus PD98059, the expression level of p-RXRα protein was markedly decreased. Moreover, the combination of these agents induced apoptosis in HL-60R cells, whereas similar effect was not obtained when the cells were treated with either agent alone. The combined treatment of these agents also cooperatively inhibited the growth of HL-60 R cells. The present findings suggest that the accumulation of p-RXRα might impair the function of normal RXRα as a master regulator of nuclear receptors and, thus, contributing to the resistance to RA in HL-60R cells. Combination of 9-cis RA plus MEK inhibitor might be an effective regimen for patients with RA resistant APL.

Description: The ubiquitin-proteasome pathway, which degrades intracellular proteins, is involved in numerous cellular processes, including the supply of immunocompetent peptides to the antigen presenting machinery. Proteolysis by proteasomes is conducted by three β subunits, β1, β2, and β5, of the 20S proteasome. Recently, a novel β subunit expressed exclusively in cortical thymic epithelial cells was discovered in mice. This subunit, designated β5t, is a component of the thymoproteasome, a specialized type of proteasomes implicated in thymic positive selection. In this study, we show that, like its mouse counterpart, human β5t is expressed exclusively in the thymic cortex. Human β5t was expressed in approximately 80% of cortical thymic epithelial cells and some cortical dendritic cells. Human β5t was incorporated into proteasomes with two other catalytically active β subunits β1i and β2i, forming 20S proteasomes with subunit compositions characteristic of thymoproteasomes. The present study demonstrates, for the first time, the existence of thymoproteasomes in the human thymic cortex, indicating that thymoproteasome function is likely conserved between humans and mice.

Description: We and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5-mediated survival. In the absence of human IL-5 (hIL-5) mature eosinophils succumbed after 4 days, while in the presence of hIL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hIL-5, while no significant DNA fragmentation was observed in the culture with hIL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hIL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hIL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis.