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The introduction of anti-inflammatory therapies has enabled substantial improvement of disease activity in patients with inflammatory bowel diseases (IBD). However, IBD can lead to serious complications such as intestinal fibrosis and colorectal cancer. Therefore, novel therapies reducing the development of these complications are needed. Angiotensin II (Ang II) promotes tissue inflammation by stimulating the production of monocyte chemoattractant protein-1 (MCP-1) or proinflammatory cytokines. It plays a pivotal role in IBD progression. Although blockade of Ang II has been reported to ameliorate experimental colitis and reduce colorectal cancer risk, the cellular and molecular mechanisms remain poorly understood. Our previous work showed that irbesartan, an Ang II type 1 receptor blocker, reduced the number of C–C chemokine receptor 2-positive (CCR2+) monocytic cells in the inflamed pancreas. This study aimed to investigate the possible antifibrotic and antitumour effects of irbesartan using the azoxymethane/dextran sodium sulphate mouse model. Irbesartan suppressed MCP-1 production and the accumulation of Ly6C+CCR2+ monocytes and fibrocytes in the inflamed colon, downregulated the expression of type 1 collagen and matrix metalloproteinase 9 and inhibited the development of intestinal fibrosis and tumours. Our observations suggest that blocking the MCP-1/CCR2 pathway using irbesartan might be beneficial in preventing colitis-associated colon tumours. [ABSTRACT FROM AUTHOR]

The Ten Eleven Translocation 1 (TET1) gene encodes an epigenetic modifying molecule that is involved in demethylation of 5-methylcytosine. In hematological malignancies, loss-of-function mutations of TET2, which is one of the TET family genes including TET1, are frequently found, while the mutations of TET1 are not. However, clinical studies have revealed that TET1 is highly expressed in some cases of the hematological malignancies including acute myeloid leukemia. Indeed, studies by mouse models using conventional Tet1 knockout mice demonstrated that Tet1 is involved in myeloid leukemogenesis by Mixed Lineage Leukemia (MLL) fusion gene or TET2 mutant. Meanwhile, the other study showed that Tet1 is highly expressed in hematopoietic stem cells (HSCs), and that deletion of Tet1 in HSCs enhances potential self-renewal capacity, which is potentially associated with myeloid leukemogenesis. To examine the role of Tet1 in myeloid leukemogenesis more precisely, we generated novel conditional Tet1-knockout mice, which were used to generate the compound mutant mice by crossing with the inducible MLL-ENL transgenic mice that we developed previously. The leukemic immortalization in vitro was not critically affected by conditional ablation of Tet1 in HSCs with the induced expression of MLL-ENL or in hematopoietic progenitor cells retrovirally transduced with MLL-ENL. In addition, the leukemic phenotypes caused by the induced expression of MLL-ENL in vivo was not also critically affected in the compound mutant mouse model by conditional ablation of Tet1, although we found that the expression of Evi1, which is one of critical target genes of MLL fusion gene, in tumor cells was remarkably low under Tet1-ablated condition. These results revealed that Tet1 was dispensable for the myeloid leukemogenesis by MLL-ENL, suggesting that the therapeutic application of Tet1 inhibition may need careful assessment. [ABSTRACT FROM AUTHOR]

Immune thrombocytopenic purpura (ITP) typically presents with bleeding due to immunologic thrombocytopenia. Severe hemorrhage due to ITP is sometimes lethal, and the urgent recovery of platelets is necessary. In addition to conventional therapeutic strategies, romiplostim shows promising efficacy for chronic ITP. However, there is little evidence for the utilization of this treatment for acute ITP or acute exacerbation of chronic ITP. We report the case details of three elderly ITP patients presenting with lethal diffuse alveolar hemorrhage. These patients had the following underlying pulmonary diseases: case 1, nontuberculous mycobacterial infection and sarcoidosis; case 2, cryptogenic organizing pneumonia; case 3, pulmonary emphysema. These patients recovered following treatment with romiplostim at a higher dose (10 μg/kg), in addition to conventional therapies including corticosteroids and high-dose intravenous immunoglobulin. In summary, the addition of romiplostim resulted in earlier recovery of thrombocytopenia than has been previously reported. Our three cases suggest that early romiplostim at a higher dose could be an efficacious therapeutic option for acute ITP patients with severe lethal bleeding. [ABSTRACT FROM AUTHOR]

Aim The spleen is not believed to contribute to hematopoiesis in healthy adults. However, several reports have demonstrated that the spleen in adults contains a large number of hematopoietic stem/progenitor cells ( HSC). Although splenectomy increases platelet and leukocyte counts, the effects of splenectomy on circulating HSC have not been elucidated. In this study, we evaluated the association between the number of circulating HSC and splenectomy in patients with hepatitis C virus ( HCV)-associated liver cirrhosis ( LC). Methods In 48 patients with various stages of HCV-associated chronic liver disease and seven patients with LC who underwent splenectomy, and 10 healthy volunteers, we determined the numbers of circulating CD34+ cells and colony-forming unit culture by flow cytometry and methylcellulose culture, respectively. Plasma stromal cell-derived factor-1α ( SDF-1α) concentrations were measured using an enzyme-linked immunosorbent assay. Results The numbers of circulating CD34+ cells and colony-forming unit culture decreased but the plasma SDF-1α concentration increased with the progression of liver disease. There was an inverse correlation between the number of circulating HSC and the plasma SDF-1α concentration. The numbers of circulating HSC and platelets were determined before and after splenectomy in seven patients with LC. In these patients, the numbers of circulating HSC and platelets increased significantly after splenectomy and the enhancing effect persisted for a long time. Conclusion Our data suggest that the spleen plays an important role in modulating HSC dynamics in patients with HCV-associated chronic liver disease. Our results also imply that splenectomy may improve liver function in patients with LC. [ABSTRACT FROM AUTHOR]

Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)+CD45– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl4). Because the vast majority of EGFP+CD45– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP+ PaSCs were also observed in CCl4-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl4-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6Chigh monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP+F4/80+CCR2+ monocytic cells and EGFP+ PaSCs in the pancreas of CCl4-treated chimeric mice receiving EGFP+ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP+ PaSCs in injured mice. We propose that CCR2+ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs. [ABSTRACT FROM AUTHOR]

Objective: We previously reported that hepatic stellate cells (HpSCs) are of hematopoietic origin in liver injury. However, the immediate precursors of HpSCs remain unknown. This study was conducted to elucidate whether terminally differentiated blood cells can differentiate into HpSCs. Materials and Methods: We adoptively transferred a variety of cells isolated from enhanced green fluorescent protein (EGFP)−transgenic mice into carbon tetrachloride (CCl4)−treated nontransgenic mice twice weekly for 2 weeks. We examined the presence of EGFP+ HpSCs in the injured liver using immunofluorescence analysis. Results: Monocytes, neutrophils, eosinophils, B cells, or T cells from EGFP mice were transferred into CCl4-treated mice. Thirty percent of EGFP+ cells in the livers of mice given Ly6Chighc-kit− monocytes were negative for CD45, but were positive for glial fibrillary acidic protein, desmin, CD146, ADAMTS13, and α-smooth muscle actin, well-known markers of HpSCs. EGFP+CD45− cells were predominantly positive for glial fibrillary acidic protein. Although 48% of EGFP+ cells were positive for procollagen type I, half of them were CD45−. In the livers of mice given neutrophils, eosinophils, B cells, or T cells, all of the EGFP+ cells were CD45+. The majority of EGFP+ cells in the nonparenchymal cell fraction purified from the livers of mice given Ly6Chighc-kit− monocytes contained lipid droplets and were positive for glial fibrillary acidic protein, desmin, ADAMTS13, and procollagen type I. When Ly6C+ monocyte-depleted peripheral blood total nucleated cells were adoptively transferred into CCl4-treated mice, we found no EGFP+CD45− cells in the liver. Conclusions: These results suggest that Ly6C+ monocytes can become HpSCs in the injured liver. [Copyright &y& Elsevier]

Objective: To develop an efficient method for single hematopoietic stem cell (HSC) transplantation for high-level hematopoietic engraftment. Materials and Methods: We combined single-cell sorting with short-term culture of putative HSCs. Mouse bone marrow cells that had been highly enriched for HSCs were individually deposited into a 96-well culture plate and incubated in the presence of mouse c-kit ligand and either mouse interleukin-11 or human recombinant granulocyte colony-stimulating factor. One week later, the resulting clones of cells were individually transplanted into lethally irradiated recipients. We also carried out time-course analysis of proliferation of the individual clones. Finally, we used micromanipulation of the paired progenies of the single cells and studied self-renewal and differentiation potentials of HSCs again in combination with transplantation. Results: There was a correlation between clone size at day 7 of culture and engraftment at 2 months post-transplantation. Small clones, such as those consisting of <15 cells, often showed high-level multilineage engraftment, while clones consisting of ≥40 cells showed very low levels of engraftment. Daily observation of cell divisions of individual clones revealed that some HSCs are in the G0 state for as long as 1 week, despite the presence of permissive cytokines. Studies using micromanipulation of paired progenies documented the ability of an HSC to generate two HSCs, as well as asymmetric cell divisions. Conclusions: Single-cell sorting combined with short-term culture of individual putative HSCs provides an efficient method for single HSC transplantation. Analyses of the kinetics of individual HSCs provided direct evidence for HSC cell-cycle dormancy, self-renewal, and expansion. [Copyright &y& Elsevier]

A 30-year-old Brazilian man hospitalized with AIDS developed a high-grade fever. Neither culture studies nor radiological examinations revealed the cause; small yet highly intense signals in the basal ganglia were detected upon gadolinium (Gd)-enhanced T1-weighted magnetic resonance imaging (MRI) of the head. This finding was equivocal at that time but obviously abnormal for his age. A week later, he developed a movement disorder in his right arm, speech apraxia, and a worsening disturbance of consciousness. Repeated Gd-enhanced T1-weighted MRI demonstrated incredible changes in the brain; enhanced lesions in the basal ganglia deteriorated over time, multiple nodular and ring-enhanced lesions were observed in almost the entire brain. A diagnosis of toxoplasma encephalitis (TE) was confirmed by the detection of Toxoplasma gondii DNA in the cerebrospinal fluid. After initiation of intravenous trimethoprim-sulfamethoxazole (TMP-SMX; 10 mg/kg/day of TMP and 50 mg/kg/day of SMX) treatment, his symptoms and radiological findings improved dramatically. Our case suggests that high-intensity signals seen in the basal ganglia of a Gd-enhanced T1-weighted MRI, even at the preclinical stage, is indicative of TE. Because the use of MRI in general has become more widespread, it is predicted that preclinical lesions of TE will be found in various clinical settings more frequently. [ABSTRACT FROM AUTHOR]

Abstract: Deletion of the long arm of chromosome 15 is known as a rare but recurrent chromosomal abnormality in myeloid malignancies. We report a novel case of minimally differentiated hypoplastic acute myeloid leukemia (AML M0) in a patient who initially had a normal karyotype, but clonal interstitial deletion of chromosome 15, del(15)(q11.2q22), coincided with increment of leukemic cells a year later. We also summarize 18 published cases with myeloid malignancies and this chromosomal abnormality. [Copyright &y& Elsevier]

Objective: Recent studies have reported that bone marrow cells can give rise to tissue fibroblasts. However, the bone marrow cell(s) that gives rise to fibroblasts has not yet been identified. In the present study, we tested the hypothesis that tissue fibroblasts are derived from hematopoietic stem cells (HSCs) in vivo. Methods: These studies were conducted using mice whose hematopoiesis had been reconstituted by transplantation of a clonal population of cells derived from a single enhanced green fluorescent protein (EGFP)-positive HSC in conjunction with murine tumor models. Results: When tumors propagated in the transplanted mice were evaluated for the presence of EGFP+ HSC-derived cells, two prominent populations of EGFP+ cells were found. The first were determined to be fibroblasts within the tumor stromal capsule, a subset of which expressed type I collagen mRNA and α-smooth muscle actin. The second population was a perivascular cell associated with the CD31+ tumor blood vessels. Conclusion: These in vivo findings establish an HSC origin of fibroblasts. [Copyright &y& Elsevier]

Objective: Using transplantation of a clonal population of cells derived from a single hematopoietic stem cell (HSC) of transgenic enhanced green fluorescent protein (EGFP) mice, we have documented the hematopoietic origin of myofibroblasts, such as kidney mesangial cells and brain microglial cells. Because myofibroblasts are thought to be an activated form of fibroblasts, we tested the hypothesis that fibroblasts are derived from HSCs. Materials and Methods: Clones of cells derived from single cells of EGFP Ly-5.2 C57Bl/6 mice were transplanted into lethally irradiated Ly-5.1 mice. Using bone marrow and peripheral blood cells from mice showing high-level multilineage hematopoietic reconstitution, we induced growth of fibroblasts in vitro. Results: Culture of EGFP+ bone marrow cells from clonally engrafted mice revealed adherent cells with morphology typical of fibroblasts. Flow cytometric analysis revealed that the majority of these cells are CD45− and express collagen-I and the collagen receptor, discoidin domain receptor 2 (DDR2). Reverse transcriptase polymerase chain reaction analysis of cultured cells demonstrated expression of procollagen 1-α1, DDR2, fibronectin, and vimentin mRNA. Fibroblast colonies consisting of EGFP+ cells were observed in cultures of bone marrow cells from clonally engrafted mice, indicating an HSC origin of fibroblast colony-forming units. Culture of peripheral blood nucleated cells from clonally engrafted mice revealed EGFP+ cells expressing collagen-I and DDR2, indicating that fibrocytes are also derived from HSCs. Conclusion: We conclude that a population of fibroblasts and their precursors are derived from HSCs. [Copyright &y& Elsevier]

Background: Hodgkin lymphoma usually presents with sequential enlargement of peripheral lymph nodes, and bone marrow invasion rarely occurs (approximately 3-5%). However, several cases have been reported as "primary" bone marrow Hodgkin lymphoma, especially among patients with human immunodeficiency virus and the elderly. This type of Hodgkin lymphoma is characterized by no peripheral lymphadenopathies and has been reported to have poorer prognosis.Case Presentation: A 38-year-old Japanese man was admitted to our hospital because of fever of unknown origin and pancytopenia without lymphadenopathies. Bone marrow examination revealed Hodgkin cells mimicking abnormal cells. These were positive for CD30, EBER-1, CD15, PAX-5, and Bob-1 and negative for Oct-2, CD3, CD20, surface immunoglobulin, CD56. On the basis of systemic evaluation and bone marrow examination, he was diagnosed with primary bone marrow Hodgkin lymphoma. We initiated therapy with DeVIC (dexamethasone, etoposide, ifosfamide, and carboplatin) therapy, but remission was not achieved. Then, the patient was treated with brentuximab vedotin combined with systemic chemotherapy (Adriamycin, vinblastine and dacarbazine), which was effective.Conclusions: There is no established treatment strategy for Hodgkin lymphoma, and therapeutic outcomes using ABVD (Adriamycin, bleomycin, vinblastine and dacarbazine)-like or CHOP (cyclophosphamide, Adriamycin, vincristine, and prednisone)-like regimens are reportedly poor. Only a few patients have been reported to achieve long-term remission. Through this case report, we suggest an alternative therapeutic option for primary bone marrow Hodgkin lymphoma. [ABSTRACT FROM AUTHOR]

ABSTRACT Background and objective: Interstitial lung diseases (ILD) are characterized by progressive interstitial pulmonary fibrosis and a decline in lung function. Fibrocytes are bone marrow-derived mesenchymal progenitor cells that may play a role in the pathogenesis of pulmonary fibrosis. Circulating fibrocyte numbers have been correlated with the prognosis of patients with idiopathic pulmonary fibrosis. The aim of the present study was to evaluate the relationship between circulating fibrocytes, and parameters of disease activity and progression in several groups of patients with ILD. Methods: The study population comprised 41 patients with ILD and seven healthy control subjects. Circulating CD45+ collagen-I+ fibrocytes were evaluated by flow cytometry. Results: The number of circulating fibrocytes was significantly increased in all patients with ILD and particularly in patients with idiopathic interstitial pneumonitis and interstitial pneumonitis associated with collagen vascular disease as compared with healthy control subjects. The numbers of circulating fibrocytes were significantly correlated with pulmonary function test parameters and with serum levels of sialylated carbohydrate antigen, a marker of disease activity. Temporal changes in circulating fibrocyte numbers were evaluated in two patients, and the results suggested that these changes correlated with the activity of ILD. Conclusions: The results from this study provide further evidence for the role of circulating fibrocytes in fibrotic lung diseases. [ABSTRACT FROM AUTHOR]

The appearance of monocytes before neutrophils in the blood during haematopoietic recovery in myelosuppressive patients is commonly observed, thus suggesting a difference in the cell division history between these two lineages in the differentiation from granulocyte–macrophage (GM) progenitors. We investigated the cell division histories of murine GM progenitors. When analysed by the dye dilution method, GM progenitors gave rise to Gr-1+Fms+ and Gr-1+Fms− cells that passed through similar rounds of cell division during initial 5 d of culture. The Gr-1+Fms+ cells showed morphological features of monocytes, while Gr-1+Fms− cells exhibited an immature morphology of neutrophils. In the subsequent culture, a decline in the number of Gr-1+Fms+ cells was observed, while Gr-1+Fms− cells increased. The proliferation of Gr-1+Fms− cells and no cell division of Gr-1+Fms+ cells were confirmed by DNA staining, Ki-67 expression, membrane dye staining and bromodeoxyuridine incorporation. These Gr-1+Fms− cells acquired mature neutrophil morphology, whereas Gr-1+Fms+ cells became macrophages. These results demonstrate that GM progenitors generate postmitotic monocytes earlier than mature neutrophils. Our data may also offer one explanation for the rapid recovery of monocytes in comparison with neutrophils in the early phase of haematopoietic regeneration. [ABSTRACT FROM AUTHOR]

CD25 is expressed on leukemic cells in 10–20% cases of acute myeloid leukemia (AML), and its expression is associated with poor prognosis. We reevaluated the relationship between CD25 expression and the leukemia-initiating cell (LIC) properties of AML using a patient-derived xenograft model. We divided lineage marker-negative (Lin–) CD34+CD38– or Lin–CD34+ cells from CD25-positive AML into CD25-positive and -negative populations, and then transplanted each population into NOD.Cg-PrkdcscidIl2rgtm1Wjl/Sz mice. Leukemic engraftment was observed with both CD25-positive and -negative populations from three of nine CD25-positive AML patients. In two of those three patients, CD25-positive and -negative Lin–CD34+ cells engrafted at the primary transplantation led to leukemic engraftment at the secondary transplantation, in which engrafted cells contained both CD25-positive and -negative Lin–CD34+ AML cells. In an in vitro culture system, expression of CD25 was considerably induced in the CD25-negative population of Lin–CD34+ cells from two cases of CD25-positive AML. In one case, CD25-positive Lin–CD34+ cells gave rise to CD25-negative as well as -positive CD34+ cells. These observations suggest that there exist CD25-positive and -negative populations that can reconstitute CD25-positive AML in a patient-derived xenograft model, and that CD25 expression fluctuates in the LICs of AML. [ABSTRACT FROM AUTHOR]

We identified a novel fusion gene, FOXP1-PDGFRA , in a patient with myeloproliferative neoplasm (MPN) with eosinophilia, harboring the chromosome abnormality t(3;4)(p13;q12). The patient responded well to imatinib and has remained in molecular remission for 3 years. This is the seventh fusion gene involving PDGFRA in MPN with eosinophilia. PDGFRA was truncated in its autoinhibitory domain, as in other PDGFRA -related MPNs, and was fused to FOXP1 at its functional forkhead domain. Comparing genomic DNA with mRNA sequences provides the possibility that the splicing process near the breakpoint junction in the FOXP1 - PDGFRA fusion gene may use the normal splice donor site for intron 23a of FOXP1 and the cryptic splice acceptor site in exon 12 of PDGFRA . This is the first report to describe the FOXP1-PDGFRA fusion gene in MPN. [ABSTRACT FROM AUTHOR]

Interleukin 17 (IL-17) is a pleiotropic cytokine that acts on both immune and non-immune cells and is generally implicated in inflammatory and autoimmune diseases. Although IL-17 as well as their source, mainly but not limited to Th17 cells, is also abundant in the inflamed intestine, the role of IL-17 in inflammatory bowel disease remains controversial. In the present study, by using IL-17 knockout (KO) mice, we investigated the role of IL-17 in colitis, with special focus on the macrophage subpopulations. Here we show that IL-17KO mice had increased susceptibility to DSS-induced colitis which was associated with decrease in expression of mRNAs implicated in M2 and/or wound healing macrophages, such as IL-10, IL-1 receptor antagonist, arginase 1, cyclooxygenase 2, and indoleamine 2,3-dioxygenase. Lamina propria leukocytes from inflamed colon of IL-17KO mice contained fewer CD11b+Ly6C+MHC Class II+ macrophages, which were derived, at least partly, from blood monocytes, as compared to those of WT mice. FACS-purified CD11b+ cells from WT mice, which were more abundant in Ly6C+MHC Class II+ cells, expressed increased levels of genes associated M2/wound healing macrophages and also M1/proinflammatory macrophages. Depletion of this population by topical administration of clodronate-liposome in the colon of WT mice resulted in the exacerbation of colitis. These results demonstrate that IL-17 confers protection against the development of severe colitis through the induction of an atypical M2-like macrophage subpopulation. Our findings reveal a previously unappreciated mechanism by which IL-17 exerts a protective function in colitis. [ABSTRACT FROM AUTHOR]

Objectives Invasive fungal diseases ( IFDs) are life-threatening events in patients with haematologic disorders, and the spectrum of the aetiological pathogens continues to expand. This study aimed to evaluate the clinical utility of a panfungal polymerase chain reaction ( PCR) assay for the management of IFDs in such patients. Methods We prospectively analysed 273 consecutive blood samples from 64 risk episodes in 51 patients with haematologic disorders at high risk for IFD who were treated at our hospital between April 2007 and October 2010. Results PCR-positive results were obtained in 18 of 64 risk episodes (35.3%). IFD was documented in 14 episodes (21.9%, 9 probable IFDs and 5 possible IFDs) according to the revised criteria of the European Organization for Research and Treatment of Cancer/ Mycoses Study Group. PCR was positive in all of these 14 episodes, and in 4 of the 50 episodes with no IFD category. Sensitivity, specificity, positive predictive value, and negative predictive value of our assay were 100%, 92%, 78% and 100% respectively. A considerable number of fungi (44.4%) that are less common than Aspergillus and Candida species were positive by PCR. Molecular diagnoses of Cunninghamella species, Aspergillus ustus, Fusarium species, Scedosporium apiospermum, Rhodotorula species and Rhizopus species were beneficial in selecting suitable treatments. Conclusions Our panfungal PCR approach allows for the highly sensitive and specific detection and identification of a wide spectrum of fungal pathogens, which provides indispensable information for managing IFDs, especially refractory or breakthrough IFDs during antifungal therapy in high-risk patients with haematologic disorders. [ABSTRACT FROM AUTHOR]

The regulation of human early lymphopoiesis remains unclear. B- and T-lineage cells cannot develop simultaneously with conventional stromal cultures. Here we show that telomerized human bone marrow stromal cells supported simultaneous generation of CD19+ CD34lo/− CD10+cy CD79a+ CD20+/−Vpre B− pro- B cells and CD7+ CD34+ CD45 RA+ CD56−cy CD3− early T/Natural Killer ( NK) cell precursors from human haematopoietic progenitors, and the generation of both lymphoid precursors was promoted by flt3 ligand (flt3 L). On the other hand, stem cell factor or thrombopoietin had little or no effect when used alone. However, both acted synergistically with flt3 L to augment the generation of both lymphoid precursors. Characteristics of these lymphoid precursors were evaluated by gene expression profiles, rearrangements of Ig H genes, or replating assays. Similar findings were observed with primary human bone marrow stromal cells. Notably, these two lymphoid-lineage precursors were generated without direct contact with stromal cells, indicating that early B and T/ NK development can occur, at least in part, by stromal cell-derived humoral factors. In serum-free cultures, flt3 L elicited similar effects and appeared particularly important for B cell development. The findings of this study identified the potential of human bone marrow stromal cells to support human early B and T lymphopoiesis and a principal role for flt3 L during early lymphopoiesis. [ABSTRACT FROM AUTHOR]

Ets1 is a member of the Ets transcription factor family. Alternative splicing of exon VII results in two naturally occurring protein isoforms: full-length Ets1 (p51-Ets1) and Ets1ΔVII (p42-Ets1). These isoforms bear key distinctions regarding protein-protein interactions, DNA binding kinetics, and transcriptional target specificity. Disruption of both Ets1 isoforms in mice results in the loss of detectable NK and NKT cell activity and defects in B and T lymphocytes. We generated mice that express only the Ets1ΔVII isoform. Ets1ΔVII homozygous mice express no p51-Ets1 and elevated levels of the p42-Ets1 protein relative to the wild type and display increased perinatal lethality, thymomegaly, and peripheral lymphopenia. Proliferation was increased in both the thymus and the spleen, while apoptosis was decreased in the thymus and increased in the spleen of homozygotes. Significant elevations of CD8+ and CD8+CD4+ thymocytes were observed. Lymphoid cell (CD19+, CD4+, and CD8+) reductions were predominantly responsible for diminished spleen cellularity, with fewer memory cells and a failure of homeostatic proliferation to maintain peripheral lymphocytes. Collectively, the Ets1ΔVII mutants demonstrate lymphocyte maturation defects associated with misregulation of p16Ink4a, p27Kip1, and CD44. Thus, a balance in the differential regulation of Ets1 isoforms represents a potential mechanism in the control of lymphoid maturation and homeostasis. [ABSTRACT FROM AUTHOR]

Objective: Although the anticancer activities of histone deacetylase (HDAC) inhibitors have been studied, a role for HDAC in normal hematopoiesis has not been clearly defined. Previous studies have shown that the potent HDAC inhibitor FK228 stimulates interleukin (IL)-3−mediated erythropoiesis. Here, we examined whether the widely used valproic acid (VPA) affects megakaryopoiesis as well as erythropoiesis. Materials and Methods: CD34+ cells were incubated in serum-free or serum-containing cultures with cytokines, with or without VPA. Results: In the serum-free cultures containing IL-3+stem cell factor (SCF), VPA significantly increased generation of CD61+GPA− megakaryocytic and a CD61+GPA+ mixture of megakaryocytic and erythroid precursors from CD34+ hematopoietic precursors at a pharmacological concentration (100 μg/mL). The increase in generation of megakaryocytic and erythroid precursors by VPA was confirmed by replating cultured cells with thrombopoietin+SCF and erythropoietin+SCF, respectively. VPA was as potent as FK228. In cultures with granulocyte-macrophage colony-stimulating factor+SCF, where CD61−GPA+ erythroid precursors were mostly developed, VPA mainly enhanced the generation of CD61−GPA+ erythroid precursors. In serum-containing cultures, only low numbers of CD61+ or GPA+ cells were developed with IL-3+SCF. Nevertheless, a substantial number of these cells were generated with VPA. Furthermore, these stimulating effects of VPA were observed by incubating CD34+ cells from patients with myelodysplastic syndrome. Quantitative reverse transcription polymerase chain reaction showed that VPA enhanced GATA-2, but not GATA-1, messenger RNA expression with IL-3+SCF. Conclusions: These results indicate a novel role for VPA in enhancing the potential of IL-3 to stimulate megakaryopoiesis as well as erythropoiesis and suggest a new therapeutic approach of epigenetic therapy for hematological disease. [Copyright &y& Elsevier]

Study Objective. To investigate whether a drug interaction exists between bortezomib and the cytochrome P450 (CYP) 3A4 inhibitor itraconazole and/or the CYP2C19 inhibitor lansoprazole that results in increased severity of bortezomib-induced peripheral neuropathy and thrombocytopenia. Design. Retrospective medical record review. Setting. Hematology-oncology ward of a university-affiliated hospital in Japan. Patients. Six adults with relapsed multiple myeloma who received intravenous bortezomib plus oral dexamethasone as the first course of a 21-day cycle between July 2007 and December 2008. Four of the six patients were treated concomitantly with itraconazole or lansoprazole: two with itraconazole, one with lansoprazole, and one with both itraconazole and lansoprazole. Measurements and Main Results. Using the National Cancer Institute's Common Terminology Criteria for Adverse Events, we identified the presence and graded the severity of peripheral neuropathy and thrombocytopenia before and during each patient's first 21-day course of bortezomib plus dexamethasone therapy. All three patients who received itraconazole experienced new or worsening peripheral neuropathy; they also experienced grade 4 thrombocytopenia. The patient who received lansoprazole alone, as well as the two patients who did not receive itraconazole or lansoprazole, had no changes in either adverse effect. We also evaluated the relationship between peripheral neuropathy and bortezomib plus dexamethasone therapy by using the Naranjo adverse drug reaction probability' scale, and a probable relationship was found. We further assessed whether a drug interaction between bortezomib and itraconazole and/or lansoprazole had occurred involving the CYP3A4 and/or the CYP2C19 pathways, respectively--resulting in increased severity of the bortezomib-induced peripheral neuropathy and thrombocytopenia by using the Horn drug interaction probability scale. We found that the occurrence of this drug interaction was strongly supported. Conclusions. Itraconazole appears to exacerbate peripheral neuropathy and thrombocytopenia induced by bortezomib; however, the mechanism of this drug interaction is unknown. Clinicians should closely monitor for bortezomib-induced adverse effects when itraconazole, or any other potent CYP3A4 inhibitor, is administered concomitantly with bortezomib. [ABSTRACT FROM AUTHOR]

Background: Although platelets, which contain large amounts of phospholipids, play an important role in blood coagulation, there is still no routine assay to examine the effects of platelets in blood coagulation. Methods: Hemostatic abnormalities in patients with thrombocytopenia, including those with idiopathic thrombocytopenic purpura (ITP), were examined using clot wave analysis (CWA)–small-amount tissue-factor-induced FIX activation (sTF/FIXa) and thrombin time (TT). Results: Although there were no marked differences in the three parameters of activated partial thromboplastin time (APTT) between normal healthy volunteers and typical patients with ITP, the peak heights of the CWA-sTF/FIXa were markedly low in patients with ITP. The three peak times of the CWA-sTF/FIXa in patients with a platelet count of ≤8.0 × 1010/L were significantly longer than those in patients with a platelet count > 8.0 × 1010/L and the peak heights of the CWA-sTF/FIXa in patients with a platelet count of ≤8.0 × 1010/L were significantly lower than those in patients with >8.0 × 1010/L. The peak heights of the CWA-APTT in patients with ITP were significantly lower than in patients with other types of thrombocytopenia. The three peak heights of the CWA-sTF/FIXa in ITP patients were significantly lower than those in patients with other types of thrombocytopenia. The CWA-TT showed lower peak heights and longer peak times in patients with ITP in comparison to patients with other types of thrombocytopenia. Conclusions: The CWA-sTF/FIXa and CWA-TT results showed that blood coagulation is enhanced by platelets and that the blood coagulation ability in ITP patients was low in comparison to healthy volunteers and patients with other types of thrombocytopenia. [ABSTRACT FROM AUTHOR]

Abstract: The ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type I domain 13) related markers were measured in the plasma of healthy volunteers and thrombotic microangiopathy (TMA) patients including thrombotic thrombocytopenic purpura (TTP) to examine their efficacy in the diagnosis of TTP. The plasma levels of the ADAMTS13 antigen and ADAMTS13-factor XI complex were significantly lower in TMA patients with a significant decreased ADAMTS13 activity (and these patients were considered to have TTP) than in the healthy volunteers. The plasma levels of ADAMTS13 antigens closely correlated with those of ADAMTS13-factor XI complex. Autoantibody for ADAMTS 13 was also positive in almost all TTP patients. In addition, the ratio of von Willebrand factor (VWF)/ADAMTS13 activity was significantly high in TTP suggesting that this ratio might be more useful for the differential diagnosis of TTP than the ADAMTS13 assay alone. These findings suggest that ADAMTS13 related markers are useful for the diagnosis and analysis of TTP. [Copyright &y& Elsevier]

Abstract: Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in various cancers and plays a crucial role in oncogensis, including the activation of genes encoding apoptosis inhibitors and cell-cycle regulators. We investigated the biological significance of the Janus kinase (Jak)-STAT pathway in human hepatocellular carcinoma (HCC). Constitutive activation of STAT3 was seen in 49.4% of human HCC specimens and in HCC cell lines. Jak inhibitor AG490 inhibited activation of STAT3 and markedly reduced cell viability without significant apoptosis. AG490 also induced S phase cell-cycle arrest with down-regulation of cyclin D1, A, E and up-regulation of p21, p27, phospho-Chk2. AG490 also inhibited caspase inhibitory proteins, such as XIAP and survivin, and augmented TRAIL-induced apoptosis. Our study suggests that the Jak-STAT pathway plays an important role in cell-cycle progression and resistance to apoptosis. Inhibition of the Jak-STAT pathway may thus be a therapeutic target for HCC. [Copyright &y& Elsevier]

Histone acetylation and deacetylation play fundamental roles in transcriptional regulation. We investigated the role of histone deacetylases (HDACs) in human adult haematopoiesis, using the structurally distinct HDAC inhibitors FK228 (depsipeptide) and Trichostatin A. When CD34+ cells were cultured with interleukin (IL)-3 or stem cell factor (SCF) + IL-3, FK228 (0·5 ng/ml) specifically enhanced the generation of immature erythroid cells with a CD36+ glycophorin A (GPA)low phenotype. In semisolid cultures, FK228 promoted the formation of erythroid colonies by CD34+ cells with IL-3 and SCF + IL-3. Furthermore, upon exposure to FK228, CD34+ cell-derived CD36+GPA− cells were induced to form erythroid colonies with IL-3 alone. Conversely, FK228 inhibited the generation of CD36+GPAhigh relatively mature erythroid cells from CD34+ cells in the presence of erythropoietin (EPO) and SCF + EPO. FK228 suppressed the EPO-mediated survival of CD36+GPAlow/-and CD36+GPAhigh cells and induced their apoptosis. Similar effects were observed for trichostatin A in the generation of erythroid cells in IL-3- and EPO-containing cultures. These data suggest that HDACs negatively regulate the IL-3-mediated growth of early erythroid precursors by suppressing their responsiveness to IL-3, while playing an important role in EPO-mediated differentiation and survival of erythroid precursors. Our data revealed that HDACs have diverse functions in human adult erythropoiesis. [ABSTRACT FROM AUTHOR]

Background: Bone marrow (BM)-derived cells differentiate into a wide variety of cell types. BM contains a heterogeneous population of stem and progenitor cells including hematopoietic stem cells, marrow stromal cells, and perhaps other progenitor cells. To establish unequivocally the transdifferentiation capability of a hematopoietic cell to a nonhematopoietic cell (endothelial cells, neurons, and glial cells), it is imperative to demonstrate that a single cell or clone of that single cell (clonal analysis) differentiates into cells comprising vessels or other cells in the brain. Methods: We generated mice that exhibited a high level of hematopoietic reconstitution from a single enhanced green fluorescent protein (EGFP) stem cell. To achieve this, we combined FACS sorting and cell culture to generate a population of cells derived from a single hematopoietic stem cell (Lin−, CD34−, c-kit+, and Sca-1+). Clonal populations of cells were then transplanted into lethally irradiated recipient mice. After 3–4 months of engraftment, some mice underwent middle cerebral artery (MCA) suture occlusion. EGFP immunocytochemistry and dual labeling was performed with cell-specific markers on tissue from various time points. Results: In all transplanted mice, EGFP+ highly ramified cells were seen in the brain parenchyma. These cells stained with RCA120 lectin and had the characteristics of parenchymal microglial cells. In brains without infarction and in uninfarcted brain regions of mice that underwent MCA occlusion, there were many EGFP+ cells in a perivascular distribution, associated with both small and larger blood vessels. The cells were tightly apposed to the vessel wall and some had long processes that enveloped the endothelial cells. After MCA occlusion, there was an influx of EGFP expressing cells in the ischemic tissue that colocalized with the “neovascularization.” These EGFP+ cells were wrapped around endothelial cells in an albuminal location and did not coexpress von Willebrand Factor or CD31. We detected rare dual-labeled EGFP and NeuN-expressing cells. We detected two staining patterns. The more frequent pattern was phagocytosis of NeuN cells by EGFP expressing cells. However, we also detected rarer cells where the EGFP and NeuN appeared to be colocalized by confocal microscopy. Conclusions: HSC differentiate into parenchymal microglial cells and perivascular cells in the brain. The numbers of these cells increase after cerebral ischemia. The HSC is therefore one source of parenchymal microglial cells and a source for perivascular cells. After a cerebral infarction, there are rare HSC-derived cells that stain with the neuronal marker, NeuN. However, the more common pattern appears to represent phagocytosis of damaged neurons by EGFP+ microglial cells. [Copyright &y& Elsevier]

Dendritic cells (DC) with the potential to induce anti-tumour immunity represent one of the promising candidates for cancer vaccines. Efficiency of ex vivo DC generation depends on culture conditions, especially protein components in the plasma or serum used. Using human serum albumin (HSA), we devised a constant and reproducible culture method for DC generation from peripheral blood CD14+ cells. The number of DC obtained with 2% HSA-supplemented cultures containing granulocyte-macrophage colony-stimulating factor and interleukin 4 were consistently higher than in cultures with various concentrations of autologous plasma or serum. The concentrations and time points tested for plasma or serum considerably affected the number of DC recovered. DC prepared with HSA acquired the ability to uptake dextran, and expressed high levels of major histocompatibility (MHC) and co-stimulatory molecules similar to DC cultured with autologous plasma or serum. Although DC cultured with autologous plasma or serum consisted of CD1a+ and CD1a- populations, DC differentiated in the presence of HSA expressed CD1a. DC obtained with HSA primed and induced immunogenic peptide-specific cytotoxic T lymphocytes against a tumour rejection antigen, HER2. These findings suggest that our method for preparation of DC with HSA should prove valuable in DC generation for immunotherapy. [ABSTRACT FROM AUTHOR]

Peripheral blood monocytes are common precursor cells of dendritic cells (DCs) and macrophages. We have searched for factors with the potential to regulate the differentiation of monocytes to DCs and macrophages. When CD14+ monocytes are cultured with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) 4, the CD14+CD1a- population, which consists of macrophages, was found in the serum-containing cultures but not in the serum-free cultures. Addition of IL-6 receptor-neutralizing monoclonal antibody (mAb) or gp130-neutralizing mAb to the serum-containing cultures resulted in a decreased population of CD14+CD1a- cells. An increase in the CD14+CD1a- population with reduction in CD14-CD1a+ DCs was observed with the addition of IL-6 to cultures, whereas IL-11, leukaemia inhibitory factor, oncostatin M or macrophage colony-stimulating factor did not affect the differentiation of monocytes in the presence of GM-CSF plus IL-4. This effect of IL-6 was blocked by tumour necrosis factor α (TNF-α), lipopolysaccharide (LPS), IL-1β, CD40 ligand (CD40L) and transforming growth factor β1 (TGF-β1). Among these factors, TNF-α was most potent in interfering with the action of IL-6. These results suggest that IL-6 inhibits the differentiation of monocytes to DCs by promoting their differentiation toward macrophages, which is modulated by factors such as TNF-α, LPS, IL-1β, CD40L and TGF-β1. [ABSTRACT FROM AUTHOR]

We searched for cytokines with the potential to support the survival of human B-cell precursor acute lymphoblastic leukaemia (pre-B ALL) cells. 47 patients with pre-B ALL were classified into four stages: stage I, CD19+CD10-CD20-; stage II, CD19+CD10+CD20-; stage III, CD19+CD10+CD20+cytoplasmic μ-heavy chain (cμ)-; stage IV, CD19+CD10+CD20+cμ+. Interleukin (IL)-3 receptor α chain (IL-3Rα) was expressed in all stages, whereas the expressions of IL-7Rα and IL-2Rα were pronounced in stages IV and II, respectively. Neither IL-3, IL-7 nor IL-2 supported the survival of pre-B ALL cells. When pre-B ALL cells were layered on stromal, MS-10, cells, viability of the pre-B ALL cells increased. Addition of IL-3 to culture containing MS-10 cells enhanced the survival of pre-B ALL cells in all cases, whereas addition of IL-7 augmented the survival of pre-B ALL cells of some cases of stage III and all cases of stage IV. The survival of pre-B ALL cells was also supported by the conditioned media of MS-10 cells. Stromal-cell-derived factor 1 (SDF-1) supported the survival of pre-B ALL cells. Effects of the conditioned media of MS-10 cells were abrogated by an anti-SDF-1 neutralizing antibody. The extent of survival of pre-B ALL cells supported by stromal cells and IL-3 and IL-7, correlated with the expression level of bcl-2 protein. The effects of stromal cells may be in part related to SDF-1. [ABSTRACT FROM AUTHOR]

Summary. Transforming growth factor-beta 1 (TGF-beta 1) acts directly on haemopoietic progenitor cells to regulate their growth. To investigate a possible link between the action of TGF-beta 1 and cell death regulators such as bcl-2, we utilized Ba/F3 cells, the interleukin-3 (IL-3)-dependent growth of which could be modulated by TGF-beta 1, as well as haemopoietic progenitor cells. We demonstrate here that upregulation of bcl-2 protein (Bcl-2) as well as that of an inhibitor of cyclin/cyclin-dependent kinase complex, p27, was associated with TGF-beta 1-induced deceleration of the cell-cycling of haemopoietic progenitor cells and Ba/F3 cells. The data from cell-cycle analysis of Ba/F3 cells showed that TGF-beta 1 retarded the G1 to S phase transition. Analysis of cells with the potential to express Bcl-2 in an inducible manner indicated that up-regulation of Bcl-2 was sufficient for not only an increase in the level of p27 but also to inhibit the cell growth. Using c-kit-overexpressing cells, we observed that the potential of TGF-beta 1 to up-regulate the expression of Bcl-2 and p27 could be counteracted by the c-kit ligand, stem cell factor. These results demonstrate that Bcl-2 exerts an essential function in the regulation of G1 to S phase transition of haemopoietic cells by TGF-beta 1. [ABSTRACT FROM AUTHOR]

T cell precursors are an attractive target for adoptive immunotherapy. We examined the regulation of human early T lymphopoiesis by human bone marrow stromal cells to explore in vitro manipulation of human T cell precursors in a human-only coculture system. The generation of CD7+CD56−cyCD3− proT cells from human hematopoietic progenitors on telomerized human bone marrow stromal cells was enhanced by stem cell factor, flt3 ligand, and thrombopoietin, but these stimulatory effects were suppressed by interleukin 3. Expression of Notch ligands Delta-1 and -4 on stromal cells additively promoted T cell differentiation into the CD7+cyCD3+ pre–T cell stage, while cell growth was strongly inhibited. By combining these coculture systems, we found that initial coculture with telomerized stromal cells in the presence of stem cell factor, flt3 ligand, and thrombopoietin, followed by coculture on Delta-1– and -4–coexpressing stromal cells led to a higher percentage and number of pre–T cells. Adoptive immunotherapy using peripheral blood T cells transduced with a tumor antigen-specific T cell receptor (TCR) is a promising strategy but has several limitations, such as the risk of forming a chimeric TCR with the endogenous TCR. We demonstrated that incubation of TCR-transduced hematopoietic progenitors with the combination of coculture systems gave rise to CD7+TCR+CD3+CD1a− T cell precursors that rapidly proliferated and differentiated under the culture condition to induce mature T cell differentiation. These data show the regulatory mechanism of early T lymphopoiesis on human stromal cells and the potential utility of engineered human stromal cells to manipulate early T cell development for clinical application. [ABSTRACT FROM AUTHOR]

Targeted disruption of the Fli1 gene results in embryonic lethality. To dissect the roles of functional domains in Fli1, we recently generated mutant Fli1 mice that express a truncated Fli1 protein (Fli1Δ CTA) that lacks the carboxy-terminal regulatory (CTA) domain. Heterozygous Fli1Δ CTA mice are viable, while homozygous mice have reduced viability. Early postnatal lethality accounts for 30% survival of homozygotes to adulthood. The peripheral blood of these viable Fli1Δ CTA/Fli1Δ CTA homozygous mice has reduced platelet numbers. Platelet aggregation and activation were also impaired and bleeding times significantly prolonged in these mutant mice. Analysis of mRNA from total bone marrow and purified megakaryocytes from Fli1Δ CTA/Fli1Δ CTA mice revealed downregulation of genes associated with megakaroyctic development, including c-mpl, gpIIb, gpIV, gpIX, PF4, NF-E2, MafG, and Rab27B. While Fli1 and GATA-1 synergistically regulate the expression of multiple megakaryocytic genes, the level of GATA-1 present on a subset of these promoters is reduced in vivo in the Fli1Δ CTA/Fli1Δ CTA mice, providing a possible mechanism for the impared transcription observed. Collectively, these data showed for the first time a hemostatic defect associated with the loss of a specific functional domain of the transcription factor Fli1 and suggest previously unknown in vivo roles in megakaryocytic cell differentiation. [ABSTRACT FROM AUTHOR]