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Biomedical subjects

Toshiro Nagasawa

Publications and source records attributed to Toshiro Nagasawa.

At least 19 recordsLinked to original sources

Granulocyte colony-stimulating factor prevents progression of monocrotaline-induced pulmonary arterial hypertension in rats.

BACKGROUND: Regeneration of the lung microvasculature and replacing pulmonary artery lesions with functional endothelial cells could be a novel and effective therapeutic strategy for treating advanced pulmonary arterial hypertension (PAH). In the present study it was postulated that granulocyte colony-stimulating factor (G-CFS), which induces the proliferation of endothelial cells, would stimulate endothelial regeneration in situ at sites of impaired lung vasculature and prevent the development of PAH. METHODS AND RESULTS: Daily administration of G-CSF for 48 days did not affect the hemodynamism of normal Fischer 344 rats. PAH was induced with monocrotaline (60 mg/kg) and G-CSF was administered daily (100 microg/kg per day). Echocardiographic findings and an invasive catheter study indicated a significant decrease in the progression of PAH in rats given G-CSF. Furthermore, G-CSF increased Ki-67 positivity in the pulmonary arteries of PAH rats but did not accelerate c-kit positive cell recruitment into peripheral blood. Daily doses of G-CSF at both 2 and 100 microg/kg improved the survival and body weight gain of PAH rats. CONCLUSIONS: G-CSF improved the progression of PAH in a rat model, possibly by stimulating pulmonary endothelial cells to proliferate at sites of impaired lung vasculature. These findings show that cytokine therapy for PAH is valid based on the concept of vascular regeneration.

Animals↗

Stable transgene expression in mice generated from retrovirally transduced embryonic stem cells.

Silencing of transduced genes hampers production of transgenic mice using retroviral vectors. We show stable expression of the enhanced green fluorescent protein (EGFP) gene in chimeric mice generated from retrovirally transduced embryonic stem cells. The vector was a murine stem cell virus-typed retroviral vector (GCDsap) in which the long terminal repeat and primer-binding site were derived from a PCC4 cell-passaged myeloproliferative sarcoma virus and the endogenous retrovirus dl587rev, respectively. To increase the viral titer, the vector was packaged with vesicular stomatitis virus G protein, which allowed concentration of the virus into pellets followed by resuspension in serum-free medium. In chimeric mice, EGFP was detected in various tissues including hematopoietic cells, neurons, cardiac muscle, and intestine. Furthermore, high expression was maintained in the progeny of these mice, suggesting successful germline transmission of active proviruses. Although the proportion of EGFP-expressing cells and the mean intensity of EGFP expression varied among tissues and mice, 100% of peripheral blood leukocytes expressed EGFP in mice carrying a single provirus copy, as well as in their progeny. Therefore, the gene transfer system described here provides a useful tool not only to generate transgenic animals but also to manipulate human embryonic stem cells..

Animals↗

Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells.

Erythroblast enucleation is thought to be largely dependent on signals mediated by other cells, such as macrophages. In an attempt to improve the in vitro production of red blood cells (RBCs) from immature hematopoietic progenitor cells, we have developed a method to produce enucleated RBCs efficiently in the absence of feeder cells. Our method may represent an efficient way to produce transfusable RBCs on a large scale from hematopoietic progenitors.

Antigens, CD↗

Centrosome amplification in adult T-cell leukemia and human T-cell leukemia virus type 1 Tax-induced human T cells.

Centrosomes play pivotal roles in cell polarity, regulation of the cell cycle and chromosomal segregation. Centrosome amplification was recently described as a possible cause of aneuploidy in certain solid tumors and leukemias. ATL is a T-cell malignancy caused by HTLV-1. Although the precise mechanism of cell transformation is unclear, the HTLV-1-encoded protein, Tax, is thought to play a crucial role in leukemogenesis. Here we demonstrate that lymphocytes isolated from patients with ATL show centrosome amplification and that a human T cell line shows centrosome amplification after induction of Tax, which was suppressed by CDK inhibitors. Micronuclei formation was also observed after centrosome amplification in Tax-induced human T cells. These findings suggest that Tax deregulates CDK activity and induces centrosome amplification, which might be associated with cellular transformation by HTLV-1 and chromosomal instability in HTLV-1-infected human T cells.

Cell Transformation, Viral↗

2-Oxoglutarate downregulates expression of vascular endothelial growth factor and erythropoietin through decreasing hypoxia-inducible factor-1alpha and inhibits angiogenesis.

In oxygenated cells, hypoxia-inducible factor-1 (HIF-1) alpha subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel-Lindau tumor suppressor E3 ligase complex using 2-oxoglutarate as a substrate. We examined the effect of 2-oxoglutarate on the production of erythropoietin and vascular endothelial growth factor (VEGF). The expression of erythropoietin and VEGF protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The promoter activity of VEGF-luciferase was dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited the HIF-1alpha protein level in Hep3B cells in hypoxic conditions. However, MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1alpha protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of angiogenesis.

Cell Hypoxia↗

Exposure of medical staff to Strongyloides stercolaris from a patient with disseminated strongyloidiasis.

We examined whether medical staff were infected with Strongyloides stercolaris through exposure to the body substances of a patient with disseminated strongyloidiasis. The patient excreted a large number of S. stercolaris organisms in respiratory secretions and stool-like excretions from a nasogastric tube. Blood tests in six physicians and three nurses, who were highly suspected of having had contact with the substances without appropriate protection during medical care of the patient for about 1 week, showed no increase of eosinophiles or IgG antibodies against S. stercolaris. We conclude that adherence to the standard precautions is sufficient for preventing the nosocomial transmission of this organism.

Animals↗

Long-lasting in vitro hematopoiesis derived from primate embryonic stem cells.

OBJECTIVE: Induction of hematopoietic cells from human embryonic stem (ES) cells has been reported recently. However, before cells derived from human ES cells can be used in the clinic, preclinical studies using these cells in experimental primates will be necessary. Therefore, we attempted to establish a method to induce hematopoietic cells robustly and abundantly from primate ES cells. METHODS: A primate ES cell line, CMK-6, derived from the cynomolgus monkey was used in this study. We adapted a method to induce hematopoiesis from CMK-6 cells on feeder cells, and tested the effectiveness of three kinds of feeder cell lines (OP9, C2C12, and C3H10T1/2). In addition, we tested the effect of vascular endothelial growth factor (VEGF) and insulin-like growth factor-II (IGF-II) on hematopoiesis induction from CMK-6 cells. RESULTS: VEGF and IGF-II showed an extremely strong synergistic effect to induce hematopoiesis from CMK-6 cells. C3H10T1/2 cells proved to be very useful for the induction of hematopoiesis from CMK-6 cells, and the production of blood cells on C3H10T1/2 cells has been maintained as long as 5 months. During this long period, ES cell derivatives continuously produced mature blood cells, including terminally differentiated cells. CONCLUSION: We have developed an original method to produce enriched blood cells abundantly from primate ES cells for an extremely long period. This method may represent a good in vitro model for studying primate hematopoiesis and related diseases. Furthermore, our method may be useful for preclinical studies of transfusion therapy using blood cells derived from ES cells in experimental primate systems.

Animals↗

Refinement of cytokine use in the in vitro expansion of erythroid cells.

Blood transfusion is indispensable for many clinical applications. However, the supply of transfusable material is insufficient in many countries. Human cord blood contains many hematopoietic stem and progenitor cells, providing a promising resource for the production of transfusable material in vitro. In this study, we have refined a protocol to produce abundant red blood cells (RBC) from human cord blood in an in vitro culture system. We found that erythropoietin and interleukin-3 were most effective when they were added to the culture medium sequentially rather than simultaneously. Although insulin-like growth factor-I (IGF-1) has been reported to function as a positive regulator of RBC production in some in vitro culture systems, we found that IGF-1 had a negative effect upon RBC production. However, IGF-II appeared to function as a positive regulator of RBC production. Finally, stem cell factor functioned to both expand and accelerate the differentiation of immature erythroid cells.

Animals↗

Therapy-related acute myeloid leukemia 6 years after clonal detection of inv(11)(q21q23) and MLL gene rearrangement.

Results of recent studies with animal models suggest that expression of MLL fusion proteins promotes acute leukemogenesis. However, the most potent MLL fusion proteins are not sufficient for the development of acute myeloid leukemia (AML). The clinical data on the pathogenesis of this type of leukemia are limited. We analyzed the case of a patient with therapy-related AML with MLL rearrangement. The patient initially developed AML with t(8;21). Although the patient achieved complete remission with chemotherapy, an abnormal karyotype, inv(11)(q21q23), was detected. After 6-year persistence of a clone with the inversion 11 karyotype in the bone marrow, secondary AML developed. Results of fluorescence in situ hybridization analysis combined with magnet-activated cell sorting analysis showed that MLL rearrangement was detected in CD34+ and CD13+ fractions but not in a CD3+ fraction of the bone marrow. There were 2 important clinical findings. One was that MLL rearrangement was not sufficient for the development of leukemia. The other was that MLL rearrangement targets specific lineages.

Antigens, CD34↗

Successful treatment of a patient with POEMS syndrome by tandem high-dose chemotherapy with autologous CD34+ purged stem cell rescue.

A 36-year-old man was admitted because of numbness and muscle weakness in the lower extremities. He had gait disturbance, malaise, and body weight loss. Based on the existence of monoclonal gammopathy, the proliferation of abnormal plasma cells in the bone marrow, the presence of sclerotic bone lesion, polycythemia, mild splenomegaly, and an elevated level of serum vascular endothelial growth factor (VEGF) (14,900 pg/mL; normal, 62-707), he was diagnosed as having peripheral neuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome. Following 2 courses of conventional chemotherapy with doxorubicin and dexamethasone, peripheral blood stem cells were mobilized by high-dose etoposide (500 mg/m(2) x 3 days) and granulocyte colony-stimulating factor. After purging by CD34+ selection using the CliniMACS device, the selected cells (12.4 x 10(6)/kg) were cryopreserved. He was then treated with tandem high-dose chemotherapy (HDC) (melphalan 100 mg/m(2) x 2 days) with autologous stem cell rescue. After the first course of HDC, the serum level of VEGF normalized and the minimal residual disease in the bone marrow was reduced below the detection limit of CDR3 analysis by polymerase chain reaction. The patient has been in remission for more than 20 months. He has gradually recovered from the neurological symptoms and now has no impairments of daily living. Our experience suggests that autologous purged stem cell transplantation should be considered as the treatment of choice for POEMS syndrome.

Adult↗

Reticulocyte-gated flow cytometric analysis of red blood cells in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by the deficiency of glycosyl phosphatidylinositol (GPI)-anchored proteins in the affected blood cell membranes. Analysis of blood cells by flow cytometry is useful to identify the affected blood cells with PNH-specific phenotypes. Because PNH-affected red blood cells (RBC) have shortened life-spans in the circulation, ratios of PNH-affected populations analyzed by flow cytometry in whole RBC are lower than those in PNH-affected erythropoiesis. We developed a reticulocyte-gated 2-color flow cytometry of RBC and revealed that the percentages of PNH-affected, CD59--populations in reticulocytes were higher than in whole RBC in patients with PNH. A serial analysis of a patient with PNH who underwent peripheral blood stem cell transplantation confirmed the usefulness of this method to evaluate PNH-affected RBC populations with high sensitivity; ie, the presence of CD59- reticulocytes in the circulation could be a sensitive marker for PNH-affected erythropoiesis.

CD59 Antigens↗

Potent vaccine therapy with dendritic cells genetically modified by the gene-silencing-resistant retroviral vector GCDNsap.

Dendritic cells (DCs) genetically modified to express tumor-associated antigens (TAAs) would be promising tools in cancer immunotherapy. However, the use of retroviral vectors for such modifications is still a challenge because of low transduction efficiency and gene silencing in DCs. We have established an efficient method to prepare such DCs by in vitro differentiation of hematopoietic progenitor cells transduced with chicken ovalbumin (OVA) cDNA via the gene-silencing-resistant retroviral vector GCDNsap packaged in vesicular stomatitis virus G protein. When c-KIT(+)/lineage(-) cells were transduced with OVA followed by expansion and differentiation, more than 90% of mature DCs expressed the transgene. Mice inoculated with those cells completely rejected the OVA-expressing tumor E.G7-OVA, and the anti-tumor effects were stronger than those observed in mice inoculated with the same number of OVA peptide-pulsed DCs. The mice harbored more cytotoxic T lymphocytes (CTLs) against E.G7-OVA and produced antibody against OVA, suggesting the generation of multiple CTLs recognizing different OVA epitopes and OVA-specific CD4(+) T cells. Successive inoculations of the transduced DCs in a therapeutic setting eradicated preexisting E.G7-OVA and prevented the progression of retransplanted tumors. Thus, this vaccine therapy may represent a potent immunotherapeutic approach for various malignant tumors that express suitable TAAs.

Animals↗

Lipocalin 2 functions as a negative regulator of red blood cell production in an autocrine fashion.

Members of the lipocalin protein family are typically small, secreted proteins that possess a variety of functions. Although the physiological role of lipocalin 2 remains to be fully elucidated, a few pivotal functions have recently been reported, e.g., regulation of the apoptosis of leukocytes. Unexpectedly, lipocalin 2 is abundantly expressed in erythroid progenitor cells. An in vitro culture experiment demonstrated that lipocalin 2 induces apoptosis and inhibits differentiation of erythroid progenitor cells. During acute anemia the expression of lipocalin 2 was reduced in erythroid cells by a feedback system. Furthermore, injection of recombinant lipocalin 2 into mice suffering from acute anemia retarded the recovery of red blood cell (RBC) numbers, suggesting the importance of reduced expression of lipocalin 2 for the efficient recovery of RBC numbers. These results indicate that lipocalin 2 suppresses RBC production in an autocrine fashion. Hence, anemia arising from pathological conditions, such as chronic inflammation, might be partly due to increased levels of lipocalin 2 secreted from expanded leukocytes and/or macrophages. Also, anemia arising from malignancies might be partly due to the abundant secretion of lipocalin 2 from tumor cells. Thus, lipocalin 2 may represent an attractive therapeutic target for anemia under certain pathological conditions.

Acute-Phase Proteins↗

An in vivo assay for retrovirally transduced human peripheral T lymphocytes using nonobese diabetic/severe combined immunodeficiency mice.

OBJECTIVE: Availability of a mouse model to analyze human peripheral lymphocytes genetically modified with retroviral vectors would be useful in T-cell-directed gene transfer studies. To address this issue, we assessed the ability of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to maintain such cells in their peripheral blood. MATERIALS AND METHODS: Human peripheral lymphocytes stimulated with recombinant human interleukin-2 (rhIL-2) and anti-CD3 and CD28 antibodies were transduced with the enhanced green fluorescent protein (EGFP) gene using the retroviral vector GCsap(MSCV) and then transplanted into NOD/SCID mice at 1 x 10(8) cells per mouse. RESULTS: Transplanted human peripheral lymphocytes survived and expressed EGFP in the mice over the 6- to 8-week posttransplant period without any signs of graft-vs-host disease. Of importance was that these cells remained at the G(0)/G(1) stage and again proliferated in response to cytokines when cultured in vitro. Interestingly, the mice in which the transduced T lymphocytes remained at the resting stage clearly elucidated the superiority of the murine stem cell virus (MSCV) LTR to maintain the transgene expression by nonproliferating T lymphocytes over the Moloney murine leukemia virus (MoMLV)- and myeloproliferative sarcoma virus (MPSV)-derived LTRs, which was obscure in in vitro culture where the transduced lymphocytes was being stimulated with rhIL-2. CONCLUSIONS: The mouse model and GCsap(MSCV) vector described herein comprise a simple and reliable in vivo assay system for studies of gene and cell therapies employing human peripheral lymphocytes.

Animals↗

Effects of hematocrit value on microparticle enzyme immunoassay of tacrolimus concentration in therapeutic drug monitoring.

The effects of hematocrit (Ht) value on microparticle enzyme immunoassay (MEIA) of tacrolimus concentration were examined in 1063 whole-blood samples from 42 transplant recipients (13 liver, 20 kidney, and 9 bone marrow transplantations). MEIA guarantees the test's assay quality for blood tacrolimus in samples with Ht values of 25% to 45%. However, 129 samples (29.3%) obtained from liver transplant recipients and 107 samples (61.5%) from bone marrow transplant recipients had lower Ht (<25%). Further, 81 blood samples (18.1%) with Ht > 45% were observed in kidney transplant patients. Twenty-five whole-blood samples with low Ht were tested by 3 assay methods for tacrolimus: MEIA, modified, corrected MEIA (cMEIA), and enzyme-linked immunosorbent assay (ELISA). MEIA gave higher blood concentrations of tacrolimus than ELISA (16.1 versus 11.0 ng/mL, P < 0.001). This difference was generated by overestimation in MEIA and was not observed in samples with normal Ht. This overestimation was eliminated by using cMEIA on samples with low Ht values: there was no difference in blood tacrolimus concentration between cMEIA and ELISA (12.3 versus 11.0 ng/mL). ELISA or cMEIA should be used for tacrolimus assay in samples obtained from bone marrow transplant recipients with anemia and from liver and kidney transplant recipients with unstable Ht values.

Bone Marrow Transplantation↗

Erythropoietin-resistant anaemia in a predialysis patient with Klinefelter syndrome.

A diabetic predialysis patient who had significantly reduced sensitivity to erythropoietin therapy was admitted to Tsukuba University Hospital. Many factors that might have been the cause of the erythropoietin resistance were examined, and a diagnosis of refractory anaemia was made based on a bone marrow aspiration biopsy. A cytogenetic abnormality (47, XXY) was also detected in the bone marrow biopsy specimen, and hence the patient was also diagnosed with Klinefelter syndrome. It was suspected that the sex chromosome abnormality influenced glucose intolerance, renal insufficiency, and erythropoietin resistance due to myelodysplastic changes in the bone marrow.

Anemia↗

Aggressive Lennert's lymphoma: report of three cases in comparison to non-aggressive Lennert's lymphoma.

The present article describes three cases of Lennert's lymphoma exhibiting aggressive clinical courses. These cases were accompanied by disseminated intravascular coagulation (DIC) or hemophagocytic syndrome (HPS). These cases were compared to non-aggressive type of Lennert's lymphoma. Of the three cases, two demonstrated involvement of the liver and the other possessed bone marrow involvement. In one patient, while a lymph node biopsy revealed Lennert's lymphoma histologically, a liver biopsy obtained 2 months later revealed a high-grade large cell cytotoxic T-cell lymphoma. Two of these cases showed HPS and the other exhibited DIC. All patients died within 1 year of diagnosis, with the shortest survival period being 1.5 months. Immunohistochemically, lymphoma cells were CD8+, CD4-, granzyme B+, and T-cell intracellular antigen-1 (TIA-1)+, showing a cytotoxic T-cell phenotype. Two cases demonstrated positive reactivity for Epstein-Barr virus in lymphoma cells by in situ hybridization. These cases were compared with eight cases of non-aggressive Lennert's lymphoma. In comparison to non-aggressive disease, these three cases displayed a higher percentage of Ki-67-positive cells. In conclusion it was found that a subset of Lennert's lymphoma cases share common features with high-grade cytotoxic T-cell lymphoma, indicating that Lennert's lymphoma may be part of the spectrum of cytotoxic T-cell lymphoma.

Aged↗

Characterization of a myeloma patient with a life-threatening hemorrhagic diathesis: presence of a lambda dimer protein inhibiting shear-induced platelet aggregation by binding to the A1 domain of von Willebrand factor.

We have identified a patient with IgD lambda-type multiple myeloma who was characterized by a severe bleeding tendency, especially after puncture of arterial vessels. Both the bleeding time (>25 min) and activated partial thromboplastin time (APTT) were prolonged. To clarify the underlying pathogenesis, we purified the APTT-prolonging activity from the patient's serum. The purified protein was a highly negatively-charged homodimer of the lambda light chain. The lambda dimer protein (M-protein) inhibited ristocetinand high shear-induced platelet aggregation, dependent on platelet glycoprotein Ibalpha (GPIbalpha), but not epinephrine-, collagen-, ADP-, thrombin-, or botrocetin-induced platelet aggregation. The lambda dimer protein inhibited the binding of platelets to immobilized or ristocetin-treated von Willebrand factor (VWF). Furthermore, a 39/34 kD fragment of VWF encompassing the A1 domain specifically bound to the immobilized lambda dimer protein in the presence of ristocetin, suggesting that the lambda dimer protein directly binds to the A1 domain of VWF. To help elucidate the binding site within the A1 domain, binding of ristocetin-treated VWF to the immobilized lambda dimer protein was assayed in the presence of various anti-A1 domain monoclonal antibodies. Based on these data, we conclude that the lambda dimer protein binds to the region of the A1 domain composed of helices alpha3 and alpha4 and thus interferes with VWF-GPIbalpha interaction. The existence of a protein that inhibits high shear-induced platelet aggregation in acquired von Willebrand disease (VWD) has only rarely been reported. The results suggest that the hemostatic function in arteries with high shear force is profoundly disrupted if the binding of GPIbalpha to VWF is abrogated, supporting the relevance of shear-induced VWF interaction with GPIbalpha in the initiation of the hemostatic process.

Adenosine Diphosphate↗