In vitro activation and maturation of human mononuclear phagocytes by stimulation with liposomes coated with a neoglycolipid containing α1-3, α1-6-mannotriose.

In this study, we assessed the potential of liposomes coated with a neoglycolipid containing α1-3,α1-6-mannotriose residues (Man3-DPPE; Manα1-6(Manα1-3)Manitol-DPPE) for in vitro activation and maturation of human mononuclear phagocytes. In response to treatment with Man3-DPPE-coated liposomes (Man3-OMLs), PMA-stimulated human THP-1 cells showed enhanced expression of CD40, CD80 and HLA-DR and secreted significant levels of IL-12p40. Among various linkages of Man2-DPPE-coated liposomes, only liposomes coated with Manα1-6Manitol-DPPE (α1-6Man2-DPPE) induced these cellular responses similarly to Man3-OML treatment. Liposomes coated with Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manitol-DPPE (Man5-DPPE) failed to activate the cells. These results suggest that an unsubstituted α1-6Man branch bound to a mannitol unit at the reducing end in Man3-DPPE is required for in vitro activation of human mononuclear phagocytes. Man3-OML-induced IL-12p40 production was not inhibited by BAY11-7082, an inhibitor of the MyD88-dependent signaling network, suggesting that TLRs are not involved in activation of human mononuclear phagocytes by Man3-OMLs. Stimulation of inflammatory monocytes or monocyte-derived dendritic cells (moDCs) with Man3-OMLs also induced enhanced expression of co-stimulatory molecules, HLA-DR, and CCR7, and IL-12p40 production from both types of cells. In response to Man3-OML treatment, moDCs but not inflammatory monocytes produced bioactive IL-12p70, which was enhanced by CD40 ligation. Thus, Man3-OMLs can activate naïve human mononuclear phagocytes and lead human moDCs to a fully matured status in vitro to elicit CTLs and a Th1 response without addition of inflammatory cytokines or TLR agonists.

[BCL2, BCL6, and MYC-positive intravascular large B-cell lymphoma presenting with bilateral adrenal gland lesions].

Primary adrenal lymphoma is a rare lymphoma, accounting for <0.2% of non-Hodgkin lymphoma. The leading histopathological subtype of adrenal lymphoma is diffuse large B-cell lymphoma, and intravascular large B-cell lymphoma (IVLBCL) is rare. Here, we report a case of IVLBCL occurrence as a bilateral adrenal gland tumor, which was diagnosed by CT-guided biopsy. Tumor cells were positive for CD20 and MUM-1 but not for CD10 on immunostaining, suggesting non-germinal center B-cell subtype lymphoma. In addition, the triple expression of BCL2, BCL6, and MYC was demonstrated on tumor cells. The bone marrow examination revealed the involvement of lymphoma cells but not hemophagocytosis. The chromosomal analysis revealed complex karyotypic abnormalities without a rearrangement of BCL2 or MYC using FISH analysis. Although the patient responded to R-CHOP chemotherapy, he developed central nervous system involvement by lymphoma. To date, the significance of the triple expression of BCL2, BCL6, and MYC without gene translocation remains partially elucidated. Therefore, an accumulation of similar cases is needed to elucidate the pathogenesis and clinical significance of the triple expression of these oncoproteins.

Successful long-term management with low-dose prednisolone in an adult patient with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare congenital disease caused by mutations in ribosomal protein genes and is characterized by pure red cell aplasia. While the prognosis is relatively favorable, quality of life (QOL) among DBA patients is negatively impacted by the adverse effects of long-term prednisolone (PSL) therapy and blood transfusions. We describe a 43-year-old man who was diagnosed with DBA (Hb of 2.18 g/dl) at the age of two months. He was initially treated with PSL and blood transfusions, followed by cyclosporine and low-dose (6 mg/day) PSL, which resulted in a sustained hemoglobin level of 9 g/dl without severe adverse events or loss of QOL. High levels of eADA and GSH as well as a RPS19 gene mutation were confirmed. The only curative therapy is hematopoietic stem cell transplantation, which is associated with significant mortality. However, using low-dose PSL to maintain a stable hemoglobin level may improve QOL for patients who receive curative treatment.

Establishment and characterization of a novel VEGF-producing HHV-8-unrelated PEL-like lymphoma cell line, OGU1.

Primary effusion lymphoma (PEL) is a rare B-cell lymphoma subtype that is characterized by lymphomatous effusion without the presence of masses, and it typically occurs in human immunodeficiency virus (HIV)-infected individuals. Lymphoma cells are universally positive for human herpesvirus 8 (HHV-8). Recently, a cavity-based effusion lymphoma that is similar to PEL without HHV-8 infection, called HHV-8-unrelated PEL-like lymphoma, has been reported in non-HIV-infected individuals. However, the pathophysiology of this lymphoma is largely undefined. We established a novel B-cell line OGU1 derived from a patient with HHV-8-unrelated PEL-like lymphoma. Notably, OGU1 cells produced vascular endothelial growth factor (VEGF) and expressed VEGF receptor 1, whose inhibitors retarded cell growth. Because VEGF acts as a vascular permeability and growth factor, it could play a role, at least in part, in the pathogenesis of this unique lymphoma. Thus, the OGU1 cell line is useful for the investigation of HHV-8-unrelated PEL-like lymphoma.

[Successful treatment with rituximab in a patient with splenic marginal zone B-cell lymphoma accompanied by cold agglutinin disease].

An 81-year-old man was admitted to our hospital due to dyspnea in July 2008. A physical examination revealed marked splenomegaly, and the results of laboratory tests were as follows: hemoglobin (Hb)=7.0 g/dL, Ret=6.4%, WBC=24,100/µL (Ly: 20,003/µL), indirect bilirubin=3.6 mg/dL, LDH=232 IU/L. The cold agglutinin titer was 1 : 8,192, and a direct antiglobulin test was positive. A PET scan showed abnormal accumulation in the spleen and bone marrow. A bone marrow aspirate examination and biopsy demonstrated diffuse involvement of abnormal lymphocytes that were found to be positive for CD20 and negative for CD5, CD10, and cyclin D1. The immunoglobulin genes were clonally rearranged. Based on these findings, splenic marginal zone B-cell lymphoma (SMZL) associated with cold agglutinin disease (CAD) was diagnosed. Because the patient refused splenectomy, he was treated with four cycles of rituximab therapy (375 mg/kg, once a week). The Hb level and lymphocyte count subsequently normalized and the splenomegaly resolved. One year later, he relapsed and was again treated with rituximab therapy with complete remission. CAD accompanied by SMZL is very rare. Rituximab may be chosen as an alternative and effective therapeutic option in patients with SMZL-particularly those with autoimmune hemolytic anemia.

[Analysis of molecular mechanism involved in development of acute myeloid leukemia].

We examined the role of molecules related to drug resistance, such as P-glycoprotein (P-gp) and telomerase (TERT), signaling molecules of STATs and FLT3 in leukemia pathogenesis in de novo acute myeloid leukemia (AML), and myelodysplastic syndrome in the phase of overt leukemia (MDS-OL). Subjects were 18 patients with de novo AML, in which expression of P-gp, TERT, STAT3, STAT5, and FLT3 was observed in 11, 14, 16, 18, and 14 of patients, respectively. Phosphorylation of STAT3, STAT5, and FLT3 in patients with de novo AML was observed in 10 out of 14, 14 out of 18, and 10 out of 14 patients, respectively. Phosphorylation of STAT5 was associated with expression of both P-gp and TERT, suggesting that STAT5 is one of the transcription factors for these genes. On the other hand, P-gp, TERT, STAT3, STAT5, and FLT3 were expressed in 3, 1, 1, 6, and 1 of the 7 patients with MDS-OL, respectively. While phosphorylation of STAT5 was observed in 4 out of 7 patients, phosphorylation of STAT3 or FLT3 was not detected in all cases examined. Telomere length varied from 2.7 kb to 6.0 kb in de novo AML, accompanied by an increased level of telomerase activity in 4 of 5 patients with de novo AML. In contrast, all MDS-OL cases showed a similar telomere length of 4-5 kb. These results indicate that consideration should be given to the differences of molecular mechanisms in the pathogenesis of de novo AML and MDS-OL for the treatment strategy of AML.

Akt and PKC are involved not only in upregulation of telomerase activity but also in cell differentiation-related function via mTORC2 in leukemia cells.

We have shown previously that PI3K/Akt pathway is active after cell differentiation in HL60 cells. In the present study, we have investigated whether additional molecules, such as protein kinase C (PKC), are involved in the regulation, not only of telomerase, but also of leukemia cell differentiation. We show that PKC activates telomerase and is, itself, activated following VD3- or ATRA-induced differentiation of HL60 cells, as was observed for PI3K/Akt. To clarify the significance of PI3K/Akt and PKC pathway activation in leukemia cell differentiation, we examined the active proteins in either the downstream or upstream regulation of these pathways. In conjunction with the activation of Akt or PKC, mTOR and S6K were phosphorylated and the protein expression levels of Rictor were increased, compared with Raptor, following cell differentiation. Silencing by Rictor siRNA resulted in the attenuation of Akt phosphorylation on Ser473 and PKCα/ßII phosphorylation, as well as the inhibition of Rictor itself, suggesting that Rictor is an upstream regulator of both Akt and PKC. In addition, in cells induced to differentiate by ATRA or VD3, Nitroblue-tetrazolium (NBT) reduction and esterase activity, were blocked either by LY294002, a PI3K inhibitor, or by BIM, a PKC inhibitor, without affecting cell surface markers such as CD11b or CD14. Intriguingly, the silencing of Rictor by its siRNA also suppressed the reducing ability of NBT following VD3-induced cell differentiation. Taken together, our results show that Rictor associated with mTOR (mTORC2) regulates the activity of both Akt and PKC that are involved in cell functions such as NBT reduction and esterase activity induced by leukemia cell differentiation.

JAK2-negative acute monocytic leukemia with TET2 mutation in essential thrombocythemia with JAK2 mutation with literature review.

Essential thrombocythemia (ET) is an indolent myeloproliferative neoplasm (MPN) with a transformation to acute myeloid leukemia in <5% of patients. A 79-year-old man with JAK2V617F-positive ET exhibited leukocytosis with an increase in monoblastic cells, leading to a diagnosis of acute monoblastic and monocytic leukemia. Leukemic cells carried a TET2 mutation but not JAK2V617F mutation. We concluded that the TET2 mutation occurred in MPN-initiating cells and overcame JAK2-mutated cells. The absence of a JAK2 mutation in the leukemic cells in this case suggests the leukemia emerged from a JAK2-negative MPN cell clone carrying the TET2 mutation.

Multistep regulation of telomerase during differentiation of HL60 cells.

Using three different differentiation agents (1alpha, 25 dihydroxyvitamin D3, all-trans-retinoic acid, and Am80), down-regulation of telomerase activity was found to be a common response during the monocytic or granulocytic differentiation of human acute myeloblastic leukemia cell line 60 (HL60) cells. Rapid down-regulation of telomerase transcription occurred during early differentiation of HL60 cells prior to G(1) arrest. Akt kinase activity was suppressed after 6 h of differentiation along with inhibition of telomerase activity, and the extent of the suppression that occurred while maintaining telomerase protein expression suggested the post-translational regulation of telomerase activity. Recombinant Akt dose-dependently increased telomerase activity, and telomerase was inhibited at the transcriptional and post-translational levels by LY294002, suggesting that PI-3K/Akt is one of the key signaling proteins involved in telomerase regulation. Each of the three differentiation agents caused a significant increase of signaling proteins (including Akt) at 3 days after the initiation of differentiation. Changes of acetyl-histone H4, which regulates transcription of the telomerase gene, were observed before the activation of Akt. This finding suggests that epigenetic control of telomerase transcription occurs before activation of Akt during the late stage of differentiation. These results indicate that telomerase activity is regulated by at least two mechanisms during granulocytic and monocytic differentiation, with one mechanism being transcriptional and the other being post-translational.

In vitro uptake of oligomannose-coated liposomes leads to differentiation of inflammatory monocytes into mature antigen-presenting cells that can activate T cells.

Oligomannose-coated liposomes (OMLs), containing entrapped antigens, serve as effective antigen delivery vehicles and as a novel adjuvant to induce antigen-specific cellular immune responses. However, in vitro activation of antigen-presenting cells (APCs) by OMLs has not yet been demonstrated. In this paper, we found that OMLs can deliver the antigens and the stimulatory signals into inflammatory monocytes in vitro, leading to differentiation of the cells to mature APCs. When OMLs were co-cultured with peripheral blood mononuclear cells from C57BL/6 mice in the presence of mouse serum, OMLs were preferentially incorporated into both Ly6Chigh monocytes and Ly6Clow monocytes, which are referred to as murine inflammatory and resident monocytes, respectively. The expression of CD11c, CD80, CD86, CCR7, and MHC class II on the Ly6Chigh monocytes was significantly enhanced during the 24 h after OML uptake, whereas upregulation of these molecules on the Ly6Clow monocytes was limited. In addition, the antigenic peptide of OVA encased in OMLs was presented on MHC class I of only Ly6Chigh monocytes. Furthermore, OVA-encasing OML-ingesting monocytes can activate CD8+ T cells from OT-1 mice, suggesting that antigens encapsulated in OMLs were cross-presented in inflammatory monocytes. Adoptive transfer of the monocytes that engulf OVA-encasing OMLs led to induction of an antigen-specific Th1 immune response in mice. Taken together, mature APCs can be generated from inflammatory monocytes in peripheral blood by ex vivo treatment of the cells with OMLs without any additional stimuli.

Production of vascular endothelial growth factor in T-cell prolymphocytic leukemia.

We describe a 79-year-old man who had massive pleural effusion and a proliferation of prolymphocytic leukemia cells in the peripheral blood, bone marrow, and pleural effusion fluid. Immunophenotyping of leukemia cells revealed either CD3+CD4+CD8-CD25+ or CD3+CD4+CD8+CD25+. The antibody against human T-cell lymphotropic virus type I was negative. A diagnosis of T-PLL was made. The level of VEGF in the plasma or pleural effusion fluid was very high. Moreover, polymerase chain reaction analysis demonstrated an expression of VEGF mRNA in the leukemia cells, indicating a production of VEGF from leukemia cells and its involvement in the pathogenesis of T-PLL.

[2-year complete remission after withdrawal of methotrexate in a rectal methotrexate-associated diffuse large B-cell lymphoma].

A 71-year-old man, who had been receiving methotrexate (MTX) and prednisolone for the treatment of rheumatoid arthritis, was admitted to our hospital in August of 2004 with rectal hemorrhage. Histological examination of an ulcerative lesion of the rectum revealed diffuse large B-cell lymphoma (DLBCL). Chemotherapy with the CHOP regimen with dose reduction following cessation of MTX was initiated. However, the patient experienced septic shock secondary to febrile neutropenia and was then followed up without chemotherapy. The DLBCL rectal lesion regressed spontaneously thereafter and had resolved completely without treatment 2 years after the initial presentation, suggesting that the withdrawal of MTX led to regression of the DLBCL. The DLBCL in our patient is compatible with MTX-associated lymphoproliferative disorders. Immunoglobulin gene rearrangement and Epstein-Barr virus (EBV) infection found in tumor cells indicated that the EBV was involved in the monoclonal proliferation of B-cells in this patient whose immune function was suppressed by MTX therapy.

Overexpression of telomerase confers a survival advantage through suppression of TRF1 gene expression while maintaining differentiation characteristics in K562 cells.

Leukemic stem cells that expressed endogenous telomerase activity were induced to show overexpression of exogenous hTERT and were analyzed for biological changes in order to assess the possible influence of telomerase gene therapy on the transplantation of normal hematopoietic stem cells. Introduction of hTERT into K562, a telomerase-positive immortal cell line, resulted in a 2.5-fold elevation of telomerase activity and the lengthening of telomeres by 6 kb to 23 kb. Real-time fluorescent PCR, which could perform quantitative analysis of transcripts, revealed a 175-fold increase in hTERT expression, suggesting the posttranscriptional regulation of telomerase. Ectopic expression of hTERT in K562 cells showed a survival advantage during culture in the absence of serum. Expression of mRNA for the telomeric-repeat binding factor 1 (TRF1) and caspase-3 activity were both decreased in hTERT-transfected K562 cells. Transduced cells retained their usual phenotypic characteristics, differentiation ability, and signal transduction response to TPA. These data suggest that ectopic expression of hTERT by normal hematopoietic stem cells may confer a survival advantage without changing their innate biological characteristics.

Activation of extracellular signal-regulated kinase through B-cell antigen receptor in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) cells express on their surface membranes immunoglobulin (Ig) M or IgD, both of which normally function as B-cell antigen receptors (BCRs). However, in contrast to normal B-cells, in B-CLL cells several important signaling pathways, such as the activation of protein tyrosine kinase via BCR, are defective. We have examined whether the activities of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38 MAPK, and Akt kinase, are functional in B-CLL cells, because these kinases play critical roles in activation in response to BCR stimulation, tumor cell growth, and survival. In B-CLL cells, BCR cross-linking neither induced activation nor enhanced the activities of Lyn, Syk, p21ras, JNK, p38 MAPK, or Akt kinases, whereas p38 MAPK and Akt were constitutively active. In contrast, BCR cross-linking resulted in ERK activation, although the activation in quiescent cells was case dependent. These results suggest that some signaling pathways, such as the activation of ERK through BCR, are functional in B-CLL cells despite the extensive impairment of signaling pathways.

Constitutive activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in B-cell lymphoproliferative disorders.

Signaling molecules such as p21(ras) (Ras), mitogen-activated protein kinase (MAPK), and Akt kinase play pivotal roles in the proliferation and survival of lymphoid cells in response to many kinds of stimulation. It is not fully understood, however, how these molecules participate in the growth of malignant lymphoid cells. We determined whether Ras, MAPKs such as extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 MAPK, and Akt kinase are activated in B-cell tumors, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt-like lymphoma, diffuse large B-cell lymphoma, and plasma cell leukemia. We found that Lyn protein tyrosine kinase was constitutively phosphorylated on tyrosine, and that ERK and p38 MAPK were constitutively active in all cases of the B-cell tumor. In contrast, activation of Ras and Akt kinase was found in limited cases, and JNK kinase activity was not observed in any case. These results suggest that ERK and p38 play roles in the oncogenesis of B-cell tumors.

[Pure red cell aplasia occurring during the course of chronic myelogenous leukemia].

The patient was a 47-year-old man who was diagnosed in 1989 as having chronic myelogenous leukemia (CML). He had been treated with interferon-alpha (IFN-alpha) and hydroxyurea. In August 1999, he was admitted to our hospital for examination of severe anemia and increased platelet count. On admission, his hemoglobin level was 6.3 g/dl, reticulocyte count was 0.7%, WBC count was 5,100/microliter, and platelet count was 57.3 x 10(4)/microliter. Bone marrow aspiration showed myeloid hyperplasia and near absence of erythroblasts. Bone marrow karyotype analysis showed a Ph chromosome with additional abnormalities. Pure red cell aplasia (PRCA) with accelerated-phase CML was considered. The IFN-alpha therapy was discontinued. Hydroxyurea at an increased dosage was effective in controlling the CML. In contrast, administration of cyclosporin A was not effective for the PRCA. The patient's condition was later complicated by acute hepatitis C virus infection. The IFN-alpha was restarted to control the CML and hepatitis. The patient remained erythroblastopenic and transfusion-dependent for more than 2 years. Association of CML and PRCA is rare. We discuss the mechanisms underlying PRCA occurring during the course of CML.

[Biclonal light chain gammopathy in multiple myeloma--a case report].

A 74-year-old woman was admitted to our hospital in March 1998 for low-back pain. In 1990, she had a chemotherapy for diffuse mixed cell lymphoma. Biochemical and serologic assays revealed a total protein level of 9.7 g/dl and an IgG level of 4,530 mg/dl. Immunoelectrophoresis showed monoclonal IgG protein associated with two monoclonal kappa and lambda light chain components. Bone marrow examination showed proliferation of myeloma cells comprising up to 25% of all nucleated cells. Myeloma cells were immunohistochemically positive for IgG and kappa and lambda light chains. IgG contained equal amounts of IgG 1 and IgG 2 subtypes and the complementarity determining region 3 (CDR 3) of myeloma cells showed oligoclonality by polymerase chain reaction, suggesting the myeloma cells may have two components. The patient received melphalan and prednisone in combination, resulting in only a minor response. She eventually developed angioimmunoblastic T-cell lymphoma. Biclonal gammopathy associated with malignant lymphoma is rare in case of multiple myeloma and may provide some insight into the pathogenesis of plasma cell tumors.

[Successful use of etoposide in an elderly patient with chronic recurrent hemophagocytic syndrome].

A 66-year-old man was admitted to our hospital for fever on January 19, 1998. He began showing periodic high fever in June 1997 and an increased serum LDH in August 1997. His history included surgery for esophageal cancer in 1993. On admission, the patient's body temperature was 38.5 degrees C. Physical examination was negative for lymphadenopathy, hepatosplenomegaly, and skin rash. Peripheral blood revealed a hemoglobin level of 8.6 g/dl and a platelet count of 7.9 x 10(4)/microliter. Bone marrow examination showed hypocellularity with marked histiocytic hemophagocytosis. The various bacterial cultures were negative. Serum LDH was elevated to 1,606 IU/l, and ferritin was greater than 3,000 ng/ml. Antinuclear antibodies were negative. No significant elevation of viral antibody titers including that to Epstein-Barr virus was found. Hemophagocytic syndrome (HPS) was diagnosed, but no underlying diseases was identified. The patient's condition was complicated by interstitial pneumonia and pleural effusion. gamma-globulin and pulse methylprednisolone both proved ineffective for the HPS; however, complete remission was achieved with cyclic intravenous administration of etoposide (VP-16, 150 mg/day). Interestingly, the interstitial pneumonia resolved promptly with etoposide therapy. The patient relapsed, in July 2001, exhibiting high fever, cytopenia, and marrow hemophagocytosis. His condition was ameliorated by administration of etoposide. This was a rare case of chronic and recurrent HPS of unknown etiology accompanied by interstitial pneumonia. Etoposide should be considered as a primary therapy for HPS and its complications in cases such as our patients.

The role of the JAK-STAT pathway and related signal cascades in telomerase activation during the development of hematologic malignancies.

Telomerase, comprising a reverse transcriptase protein (TERT) and an RNA template, plays a critical role during senescence and carcinogenesis; however, the mechanisms by which telomerase is regulated remain to be elucidated. Several signaling pathways are involved in the activation of TERT at multistep levels. The JAK-STAT pathway is indispensable for mediating signals through growth factor and cytokine receptors during the development of hematopoietic cells, and its activity is frequently upregulated in hematological malignancies. Here, we review the role of the JAK-STAT pathway and related signaling cascades in the regulation of telomerase in hematological malignancies.

Telomerase activation as a repair response to radiation-induced DNA damage in Y79 retinoblastoma cells.

The molecular mechanism of telomerase activation induced by ionizing radiation (IR) remains poorly understood. We demonstrate that DNA damage induced by IR at doses of 2-5 Gy triggers activation of Akt, predominant to that of protein phosphatase 2A (PP2A), resulting in human telomerase reverse transcriptase (hTERT) phosphorylation and increased telomerase activity in Y79 cells. DNA damage induced by IR at doses greater than 10 Gy might trigger PP2A activation, predominant to that of Akt, resulting in hTERT dephosphorylation and decreased telomerase activity. Our results suggest that differential activation of Akt and PP2A may be responsible for telomerase regulation.