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Biomedical subjects

S Nagakura

Publications and source records attributed to S Nagakura.

At least 37 records · Page 2Linked to original sources
PubMed1996

Preferential hematopoiesis by paroxysmal nocturnal hemoglobinuria clone engrafted in SCID mice.

In paroxysmal nocturnal hemoglobinuria (PNH), little is known about the molecular events leading to the clinical manifestations except for the hemolysis. To unfold the complex pathophysiology, it is necessary to elucidate the nature of the PNH clone. PNH exhibits an acquired stem cell disorder, a clonal expansion of affected cells, concomitant depression of normal hematopoiesis in bone marrow (BM), and, although infrequently, the development of leukemia. The PNH clone is thus expected to exhibit some neoplastic features. We report here that CD34+ hematopoietic progenitor cells of PNH-BM yielded blood cells of three lineages with PNH phenotype alone when transplanted into sublethally irradiated severe combined immunedeficient mice. The hematopoiesis persisted for more than 10 months and did not always need human cytokines. In contrast, the hematopoiesis by control grafts obtained from healthy volunteers required an intense cytokine treatment. This in vivo model defines the preferential hematopoiesis of pluripotent PNH progenitor cells, indicating the intrinsic growth abnormality of PNH clone.

Adult↗
PubMed1995

Markedly high population of affected reticulocytes negative for decay-accelerating factor and CD59 in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) blood cells lack glycosylphosphatidylinositol-anchored membrane proteins such as decay-accelerating factor (DAF) and CD59. This lack is of diagnostic value in PNH. Because reticulocytes in PNH are not yet well characterized, we analyzed reticulocytes obtained from 12 patients with PNH and from 5 healthy volunteers by two-color flow cytometry with a membrane-permeable fluorescent dye, thiazole orange, to identify reticulocytes and monoclonal antibodies to DAF and CD59. Healthy individuals had no affected cells. In all patients, the population of affected reticulocytes negative for DAF and CD59 was markedly higher than the population of affected erythrocytes. Moreover, the population of affected erythrocytes became obviously low in patients who received transfusions and suffered from hemolytic precipitation, whereas the population of affected reticulocytes was unchanged. The persistently high population of affected reticulocytes, despite cytolytic exclusion and an inherently short lifetime, might possibly be explained by relative reticulocytosis caused by an anemia-induced feedback stimulation of erythropoiesis in PNH. Thus, affected reticulocytes could be a reliable marker for the diagnosis of PNH and for the evaluation of erythropoiesis by PNH stem cell.

Adolescent↗
PubMed1995

Paroxysmal nocturnal hemoglobinuria clone in bone marrow of patients with pancytopenia.

The lack of glycosylphosphatidylinositol (GPI)-anchored membrane proteins such as decay-accelerating factor (DAF) and CD59 on blood cells has a diagnostic value in paroxysmal nocturnal hemoglobinuria (PNH). Because PNH often develops in patients with aplastic anemia (AA), we attempted to detect a PNH clone in the bone marrow (BM) of patients with AA and pancytopenia before affected cells were evident in the peripheral blood (PB). We used flow cytometry with monoclonal antibodies against DAF and CD59 for the detection of the clone. Affected cells were observed in the BM of 3 of 7 patients with AA and 1 of 3 patients with pancytopenia of unknown origin, but not in their PB. All 8 patients with apparent PNH had affected cells in their BM and PB. On the basis of the early appearance of the PNH clone in the BM, a prospective 4-month follow-up study of the PB cells was performed. The study showed the release of affected mature cells first in granulocytes, then in monocytes, and finally in lymphocytes. Ham's test was positive before affected erythrocytes were detected by flow cytometry. Our findings indicate that detection of the PNH clone in BM could be predictive of the development of PNH in patients with AA and pancytopenia.

Anemia, Aplastic↗
PubMed1995

Expression of cryptantigen Th on paroxysmal nocturnal hemoglobinuria erythrocytes in association with a hemolytic exacerbation.

Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes lack complement regulatory membrane proteins and are susceptible to complement. Although the critical role of complement in intravascular hemolysis in PNH is accepted, the precise mechanism of complement activation in vivo is unknown. Accordingly, in a PNH patient who was suffering from a hemolytic precipitation soon after a common cold-like upper respiratory infection, we analyzed the erythrocytes with lectins and by flow cytometry to detect membrane alteration that lead to complement activation. The lectin reactivity of erythrocytes showed the expression of cryptantigen Th. The patient serum at the time of the hemolysis induced the expression of Th on erythrocytes from PNH patients and from healthy volunteers in vitro, whereas neither the patient serum after recovery from the hemolysis nor blood type-matched control serum from healthy donor showed this activity. Moreover, autologous serum selectively hemolyzed Th+ PNH erythrocytes, but not Th- PNH erythrocytes, or Th+ control erythrocytes. Hemolysis was not observed either in complement-inactivated serum or in blood type-matched cord blood serum, which lacks natural antibodies to cryptantigens. These findings indicate that the immunoreaction of infection-induced Th with natural antibody on PNH erythrocytes is a trigger of the complement activation, leading to intravascular hemolysis.

Adolescent↗
PubMed1994

Persistence of affected T lymphocytes in long-term clinical remission in paroxysmal nocturnal hemoglobinuria.

Long-term clinical remission of more than 10 years is rarely seen in paroxysmal nocturnal hemoglobinuria (PNH). Affected blood cells in PNH lack glycosylphosphatidylinositol (GPI)-anchored membrane proteins such as decay-accelerating factor (DAF) and CD59. We performed a flow cytometric analysis of circulating blood cells obtained from two patients with PNH who had been in clinical remission for more than 10 and 25 years, respectively. Affected cells with the PNH phenotype were demonstrated only among T-lymphocytes. Persistent affected T cells were negative for the CD52 protein only, this protein being a GPI-anchored lymphocyte marker without complement regulatory activity. The persistence of the affected T cells may be explained either by an inherently long life span after the disappearance of the PNH stem cell or by insidious production at a subclinical level by affected stem cell. In either event, detection of affected T cells, especially CD52-negative T cells, may be useful for the evaluation of long-term clinical remission in PNH.

Adrenal Cortex Hormones↗
PubMed1994

Interleukin-2-dependent T-cell lines established from paroxysmal nocturnal hemoglobinuria patients.

Peripheral blood T lymphocytes obtained from two patients with paroxysmal nocturnal hemoglobinuria (PNH) were immortalized with human T-lymphotropic virus type 1 (HTLV-1). These cells showed interleukin-2 (IL-2)-dependent cell growth in culture. Cell surface analysis showed that they had the phenotype of a helper/inducer T subset that was positive for CD2, CD3, and CD4, but negative for CD8, similar to adult T-cell leukemia cells induced by HTLV-1. These cell lines lacked glycosylphosphatidylinositol (GPI)-anchored proteins, CDw52, CD55 (decay-accelerating factor; DAF), and CD59 on the cell surface, whereas intracellular DAF protein was detected. These T-subset cell lines with a PNH phenotype did not synthesize GPI anchor, whereas a control cell line, similarly prepared from the T cells of a healthy volunteer, produced the anchor. The control cells expressed CDw52, DAF, and CD59 on the cell surface and showed the phenotype of a helper/inducer subset. Southern blot analysis confirmed the clonality of each cell line. These CD4+ T-cell lines with a PNH phenotype and a subset-matched control counterpart could be a useful model for PNH investigation.

Antigens, CD↗
PubMed1994

Establishment of a human T-cell line with deficient surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins from a patient with paroxysmal nocturnal haemoglobinuria.

A novel interleukin-2 dependent T-cell line, PMT-2Y, was established from the peripheral blood of a patient with paroxysmal nocturnal haemoglobinuria (PNH) by human T lymphotropic virus type I (HTLV-I)-mediated transformation. PMT-2Y cells are positive for CD2, CD3, CD4, CD25, T cell receptor alpha beta and HLA-DR, but negative for CD1, CD7, CD8, CD19 and CD20, indicating that the clone belongs to a helper/inducer subset of T cells. PMT-2Y cells have the monoclonal integration of HTLV-I proviral DNA, suggesting that they derived from a single clone. Moreover, they lack surface expression of complement regulatory proteins such as DAF (CD55) and CD59, that are the most important glycosylphosphatidylinositol (GPI)-anchored membrane proteins defective in haemopoietic cells of patients with PNH. Northern blot analysis, however, revealed the production of normal levels of DAF mRNAs. Thus, PMT-2Y is derived from a PNH T cell clone and may be a useful model to study PNH.

Antigens, CD↗
PubMed1993

Impaired glycosylation of glycosylphosphatidylinositol-anchor synthesis in paroxysmal nocturnal hemoglobinuria leucocytes.

Metabolic labeling with [3H]sugars in vivo or [3H]sugar nucleotides in vitro of glycosylphosphatidylinositol (GPI)-anchor precursors in peripheral blood granulocytes and cultured T lymphocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients showed a synthetic defect in the GPI-anchor. Among the GPI-anchor precursors, phosphatidylinositol (PI) was normally synthesized, while the synthesis of glucosaminylphosphatidylinositol (GlcN-PI) and subsequent mannosylation of GlcN-PI were inhibited in affected cells. The defect in the GPI-anchor synthesis in PNH is thus attributed to interrupted glycosylation at plural sites in the synthesis of the common carbohydrate structure of the anchor.

Carbohydrate Sequence↗
PubMed1993

Expression of decay-accelerating factor and CD59 in lymphocyte subsets of healthy individuals and paroxysmal nocturnal hemoglobinuria patients.

The expression of phosphatidylinositol (PI)-anchored complement-regulatory membrane proteins on circulating blood cells has been well clarified; however, the PI proteins on lymphocyte subsets have not been fully analyzed yet. We examined the expression of decay-accelerating factor (DAF) and CD59 on the T lymphocytes (CD2+, CD3+, CD4+, and CD8+) and CD20+ B lymphocytes in ten healthy volunteers and 12 paroxysmal nocturnal hemoglobinuria (PNH) patients by cytofluorometry. In healthy controls, each subset of lymphocytes showed a small population of cells weakly positive and a large population of cells strongly positive for DAF and CD59, while erythrocytes showed a single population of cells positive for the PI proteins. The two-population expression of DAF was most distinctive in CD8+ T cells among the subsets. In PNH, each subset of lymphocytes showed a moderately higher population of cells weakly positive and a smaller population of cells strongly positive for the membrane proteins compared with those in the healthy controls. Moreover, in some PNH cases, a negative population for the proteins was found in all subsets. Thus the analysis of PI-anchored proteins on lymphocytes subsets (especially CD8+ T cells) was considered to be of diagnostic value in PNH patients who receive blood transfusion after hemolytic attack of affected erythrocytes. Furthermore, the two-population expression of PI proteins in normal lymphocytes suggests that membrane PI protein would be a new subset marker of lymphocytes.

Adult↗
PubMed1992

Increased plasma decay-accelerating factor levels in paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a complement regulatory membrane protein that is often absent from the cell surface of blood cells in paroxysmal nocturnal hemoglobinuria (PNH). DAF has also recently been found in the body fluids of healthy individuals. However, its precise structure and biological significance are not yet clear. To clarify the clinical and pathological implications of free DAF, we measured plasma DAF levels in PNH patients, using a newly developed quantitative enzyme-linked immunosorbent assay (ELISA), with a measurable range between 0.2-12 ng, for soluble DAF. ELISA assays revealed significantly increased plasma DAF levels in PNH patients (258 +/- 150 ng/ml, mean +/- S.D., n = 9) as compared with healthy controls (80 +/- 41 ng/ml, n = 17) (p less than 0.01). Taken together with the finding that DAF is synthesized in and released extracellularly from affected PNH cells, plasma DAF levels would be useful for clinical diagnosis and for the quantitative evaluation of the clonal expansion of affected cells in PNH.

Adult↗