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The leukaemic cells of 11 patients with Japanese adult T-cell leukaemia (ATL) were characterized in terms of phenotype employing various monoclonal antibodies against T-cell surface antigens. The leukaemic cells of all ATL patients except patient no. 11 displayed the phenotype OKT4+/OKT8-. However, the % of OKT3+cells varied from 3 to 98. In 1 case, cells with phenotype OKT4+/OKT8+, which is characteristic of T-cells at an early stage of differentiation, were found. These results suggest that some ATL cells may be in an immature differentiation stage. Among the 11 patients, 2 with unusual ATL cell phenotypes (1 case with OKT3-cells and 1 case with Ia+/EAC+cells) showed spontaneous regression of leukaemic cells. Characterization of leukaemic cells from a larger number of ATL patients may demonstrate a significant relationship between cell phenotype and prognosis.

Hairy cell leukemia (HCL) is a rare type of chronic B-cell leukemia characterized by the hairy morphology of the leukemia cells. All of 5 HCL samples and an HCL-derived cell line, BNBH-I, showed serrated edges and hairlike projections in May-Grünwald Giemsa stain and protruding actin spikes and lamellipodia in phalloidin stain. These structures were hardly detected on B-cell chronic lymphocytic leukemia (B-CLL) and precursor B-cell acute lymphocytic leukemia (B-ALL) cells. Because Rho guanosine triphosphatases (GTPases) regulate the formation of these structures, we examined the expression levels and activation states of Rho GTPases in HCL cells. RhoA, Rac1, and Cdc42 were overexpressed and constitutively activated in HCL samples and BNBH-I cells but not in B-CLL or precursor B-ALL cells. Next we overexpressed dominant-negative (DN)-RhoA, DN-Rac1, and DN-Cdc42 in BNBH-I. As a result, each DN mutant repressed the growth of BNBH-I cells by more than 50% and inhibited actin spike formation, but only DN-Racl suppressed lamellipodia formation. We also found that enforced expression of constitutively active-RhoA, Rac, or Cdc42 in the proB-cell line Ba/F3 was sufficient to induce actin spike formation, whereas none of these molecules produced lamellipodia. These results indicated that constitutively activated Rho GTPases regulate the growth and unique morphology of HCL cells.

ABSTRACT Although paroxysmal nocturnal hemoglobinuria (PNH) is often associated with aplastic anemia (AA), the nature of the pathogenetic link between PNH and AA remains unclear. Moreover, the PIG-A mutation appears to be necessary but not sufficient for the development of PNH, suggesting other factors are involved. The ability of PNH marrow cells to form in vitro hematopoietic colonies and the ability of PNH marrow to generate stroma that could support hematopoiesis of normal or PNH marrow in cross culture were investigated. PNH marrow from both post-Ficoll and post-lineage depleted hematopoietic progenitor cells grew similarly significantly fewer colonies than normal marrow. Sorting of CD59 + and CD59 − CD34 + CD38 − cells from patients with PNH showed similarly impaired clonogenic efficiency, indicating that the hematopoietic defect in PNH does not directly relate to GPI-anchored protein expression. PNH marrow readily grew stroma similar to marrow from normal donors. Cross culture experiments revealed that PNH stroma appears to function normally in vitro; it can support growth of normal marrow cells as well as normal stroma does, but neither PNH nor normal stroma could support the growth of PNH marrow cells. The hematopoietic defect in PNH is not due to defective stroma, but is due to defective progenitor cell growth related to additional unknown factors.

Venous thromboembolism (VTE) is one of the common manifestations in the anti-phospholipid (aPL) syndrome. We examined the levels of IgG antibodies (Abs) to beta2-glycoprotein I (beta2-GP I) and prothrombin, lupus anticoagulant (LA) activity, activated protein C resistance (APC-R), and factor V Leiden in 96 patients with systemic lupus erythematosus (SLE); 19 with VTE and 77 without VTE. Acquired APC-R, which was not found in any patient with the factor V Leiden mutation, was present in 33 (34.4%) out of the 96 patients with SLE. The presence of acquired APC-R was a strong risk factor for VTE. The SLE patients were divided into four groups according to the results of enzyme-linked immunosorbent assay (ELISA) and LA activity for each aPL Abs: ELISA+, LA+; ELISA+, LA-; ELISA-, LA+; and ELISA-, LA-. A significant association was observed between APC-R and the co-existence of anti-beta2-GP I Abs and LA activity or of anti-prothrombin Abs and LA activity. There was no association between APC-R and the presence of anti-beta2-GP I Abs, anti-prothrombin Abs, or LA activity alone. However, when multivariate logistical regression analysis was performed, it was clear that only the co-existence of anti-prothrombin and LA activity was a significant risk factor for APC-R. These findings indicate that the co-existence of anti-prothrombin Abs and LA activity may be an important factor in the pathogenesis of acquired APC-R in patients with SLE.

Stem cell factor (SCF) has crucial roles in proliferation, survival, and differentiation of hematopoietic stem cells and mast cells through binding to c-Kit receptor (KIT). Chemotaxis is another unique function of SCF. However, little is known about the intracellular signaling pathway of SCF/KIT-mediated cell migration. To investigate the signaling cascade, we made a series of 22 KIT mutants, in which tyrosine (Y) residue was substituted for phenylalanine (F) in the cytoplasmic domain, and introduced into BAF3 cells or 293T cells. On stimulation with SCF, BAF3 expressing KITWT(WT) showed cell migration and Ca2+ mobilization. Among 22 YF mutants, Y567F, Y569F, and Y719F showed significantly reduced cell migration and Ca2+ mobilization compared to WT. In Y567F, Lyn activation on SCF stimulation decreased and C-terminal Src kinase (Csk) suppressed KIT-mediated Ca2+ influx and cell migration, suggesting that Y567-mediated Src family kinase (SFK) activation leads to Ca2+ influx and migration. Furthermore, we found that p38 mitogen-activated protein kinase (p38 MAPK) and Erk1/2 were also regulated by Y567/SFK and involved in cell migration, and that p38 MAPK induced Ca2+ influx, thereby leading to Erk1/2 activation. In Y719F, the binding of phosphatidylinositol 3′-kinase (PI3K) to KIT was lost and KIT-mediated cell migration and Ca2+ mobilization were suppressed by PI3K chemical inhibitors or dominant-negative PI3K, suggesting the involvement of Y719-mediated PI3K pathway in cell migration. Combination of Csk and the PI3K inhibitor synergistically reduced cell migration, suggesting the cooperation of SFK and PI3K. Taken together, these results indicate that 2 major KIT signaling pathways lead to cell migration, one is Y567-SFK-p38 MAPK-Ca2+ influx-Erk and the other is Y719-PI3K-Ca2+ influx.

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Here, we found that overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA was induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibited NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb was also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1(+/+) and E2F1(-/-) murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity.

SummaryAnti-phospholipid (aPL) antibodies (Abs) are well known to be associated with thromboembolic events in patients with systemic lupus erythematosus (SLE). However, the clinical relevance of aPL Abs in patients without SLE (non-SLE) who have venous thromboembolism remains unclear. We evaluated 143 non-SLE patients with a first episode of clinically suspected deep vein thrombosis (DVT) by using objective tests for diagnosing DVT and laboratory tests including the activated protein C resistance (APC-R) test, the factor V Leiden test, and various aPL Abs. The prevalence of acquired APC-R, in which case there was no factor V Leiden mutation, was significantly higher in patients with DVT (15/58 cases, 25.9%, p

Anti-prothrombin antibodies (anti-prothrombin) and anti-beta2-glycoprotein I antibodies (anti-beta2-GP I) are the most common and characterized anti-phospholipid antibodies (aPL) detected using specific enzyme-linked immunosorbent assay (ELISA) systems. Recently, lupus anti-coagulant (LA) activity detected by a phospholipid-dependent coagulation assay was reported to be associated with anti-prothrombin and/or anti-beta2-GP I. Here we show that the co-existence of IgG anti-prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti-beta2-GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russell's viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA-; group C, ELISA- and LA+ group D, ELISA- and LA-. Regarding IgG anti-prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45.7%, P < 0.001, Fisher's exact probability test) than in the other groups (B, 2/30, 6.7%; C, 1/22, 4.5%; D, 1/37, 2.7%). With respect to IgM anti-prothrombin and IgG or IgM anti-beta2-GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co-existence of IgG anti-prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19.13; 95% confidence intervals, 4.74-77.18).

Background: Anti-phospholipid (aPL) antibodies (Abs) frequently found in the plasma of patients with systemic lupus erythematosus (SLE) have been associated with thrombotic complications. Our aim was to clarify the roles in thrombosis of aPL Abs that react with complexes of phospholipids and plasma proteins such as β2-glycoprotein I (β2-GPI), prothrombin, protein C, protein S, and annexin V. Methods: We determined the prevalence of aPL Abs to various phospholipid-binding plasma proteins in SLE patients with arterial thrombosis (30 cases), venous thrombosis (19 cases), thrombocytopenia (14 cases), fetal loss (14 cases), and patients without complications (91 cases). The aPL Abs were measured by an ELISA system in which human plasma proteins (β2-GPI, prothrombin, protein C, protein S, and annexin V) were immobilized on γ-irradiated or plain polystyrene plates. Results: All types of aPL Abs were frequently observed in the patients with SLE when γ-irradiated polystyrene plates were used (51 of 168 cases positive for anti-β2-GPI, 94 of 168 cases positive for anti-prothrombin, 36 of 168 cases positive for anti-protein C, 47 of 168 cases positive for anti-protein S, and 50 of 168 cases positive for anti-annexin V), whereas no Abs to these plasma proteins were detected when plain polystyrene plates were used. Multivariate analysis confirmed that both anti-β2-GPI and anti-prothrombin Abs were significant risk factors for arterial thrombosis [odds ratios (ORs), 8.8 and 14.5, respectively; 95% confidence intervals (CIs), 3.2–25 and 1.8–116, respectively] but not for venous thrombosis. The presence of anti-protein S Abs was a significant risk factor for venous thrombosis (OR, 30.4; CI, 3.3–281) but not for arterial thrombosis. The only significant risk factor for fetal loss was the presence of anti-annexin V Abs (OR, 5.9; CI, 1.4–14.8). Conclusions: Patients with SLE frequently have some aPL Abs to β2-GPI, prothrombin, protein C, protein S, and annexin V. Thrombotic complications in SLE may depend on the antigenic specificities of these Abs, alone or in combination.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan–class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A–deficient B-cell line (JY5), a PIG-A–deficient K562 cell line, an Epstein-Barr virus–transformed B-cell line (TK-14−) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A–deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34+ cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.

ABSTRACT To detect a small population of blood cells with a deficiency of glycosyl phosphatidylinositol (GPI)-anchored protein, we evaluated the expression of CD59 by flow cytometry on one million erythrocytes, which is about 100 times more than the number of erythrocytes tested by our standard immunoassay. Blood samples from healthy volunteers, patients with aplastic anemia (AA), and patients with myelodysplastic syndrome (MDS), who all showed no detectable GPI deficiency by the standard assay, were investigated. The numbers of CD59-deficient erythrocytes were 5 to 145/10 6 erythrocytes in the healthy volunteers (mean 29.2), and one of the volunteers had an increase in the deficient cells exceeding the mean + 3 SD (141.7), a normal limit. A CD59-deficient population was detected in 6 of the 21 (28.6%) patients with AA and 5 of the 18 (27.8%) patients with MDS. The new assay was performed again in 5 of these 11 patients and the normal individual who had the CD59-deficient populations at 6 and 12 months after the initial study. The number of deficient cells gradually increased in 1 patient with MDS (from 511 to 2892/10 6 erythrocytes), while the numbers of the other 4 patients showed a tendency to decline, although the deficient populations were repeatedly detected on most of the occasions. Changes in the number of the deficient cells were also seen in the healthy volunteer, but they were rather rapid; the numbers changed from 145 to 5661 and then to 18/10 6 erythrocytes within 3 months. The CD59 assay used in this study is easy to perform and enabled us to detect less than 1% GPI-deficient cells.

Cytokines exert pleiotropic effects on target cells in a manner dependent on the cell type or stage of differentiation. To determine how instinctive cell properties affect biological effects of cytokine, we introduced an erythroid/megakaryocyte lineage-specific transcription factor, GATA-1, into a murine myeloid cell line M1, which is known to undergo macrophage differentiation in response to interleukin 6 (IL-6). Overexpression of GATA-1 changed the phenotype of M1 cells from myeloid to megakaryocytic lineage. Furthermore, GATA-1 blocked both IL-6-induced macrophage differentiation and apoptosis of M1 cells. Although STAT3 is essential for IL-6-induced macrophage differentiation of M1 cells, GATA-1 had little or no effect on tyrosine phosphorylation, DNA binding, and transcriptional activities of STAT3 in Western blot analysis, electropholic mobility shift assay (EMSA), and luciferase assays. During IL-6-induced macrophage differentiation of M1 cells, IL-6 down-regulated cyclin D1 expression and induced p19INK4D expression, leading to reduction in cdk4 activities. In contrast, sustained expression of cyclin D1 and a significantly lesser amount of p19INK4D induction were observed in IL-6-treated M1 cells overexpressing GATA-1. Furthermore, although bcl-2 expression was severely reduced by IL-6 in M1 cells, it was sustained in GATA-1-introduced M1 cells during the culture with IL-6. Both IL-6-induced macrophage differentiation and apoptosis were significantly abrogated by coexpression of cyclin D1 and bcl-2, whereas overexpressions of cyclin D1 or bcl-2 inhibited only differentiation or apoptosis, respectively. These results suggested that GATA-1 may not only reprogram the lineage phenotype of M1 cells but also disrupt the biologic effects of IL-6 through the sustained expression of cyclin D1 and bcl-2.

Patients with aplastic anaemia (AA) frequently develop paroxysmal nocturnal haemoglobinuria (PNH) as a late complication. We investigated the frequency of the development of PNH features including a glycosyl phosphatidylinositol (GPI) anchoring defect in 73 Japanese patients with AA. A deficient expression of CD59 was found on erythrocytes and/or granulocytes in 21/73 (28.8%) of the patients. A Ham/sugar water test was positive in 13/21 patients. We also examined mutations of the PIG-A gene in 11 patients with CD59 deficiency. A heteroduplex analysis detected PIG-A gene abnormality in 10/11 patients tested. Nucleotide sequencing was performed in six patients and identified eight mutations including three mutations in one patient. The mutations of the PIG-A gene were all different and included two single-base insertions, one single-base deletion, two two-base deletions, and one each of eight-base insertion and nine- and ten-base deletions. All mutations but one caused frameshifts. Our findings indicate that a high proportion of Japanese patients with severe AA have a GPI-anchoring defect and that the PIG-A gene is mutated in the AA patients who had a GPI deficiency. We found no significant difference in the pattern of the PIG-A gene mutation between the AA patients with a GPI deficiency and those with de novo PNH.

SummaryAntiphospholipid antibodies (aPL) are well known to be associated with arterial and venous thrombosis. In a series of 180 patients with systemic lupus erythematosus (SLE), the prevalence of arterial thrombosis was obviously higher in the patients who had both anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) (17/35, 48.6%, p

The relationship between thrombocytopenia and the level of anticardiolipin antibodies (aCL) and/or the existence of lupus anticoagulant (LA) ware studied in 146 patients with systemic lupus erythematosus (SLE). These patients were divided into six groups: A, those LA positive with a high level of aCL (>10 U/ml) (10 cases); B, those LA positive with a low level of aCL (3–10 U/ml) (15 cases); C, those LA positive but aCL negative (

Chronic T lymphoid leukemias are defined as leukemias of post-thymic T cells. The CD4+CD8+ double-positive (DP) phenotype is seen in a few cases. Since DP generally occurs in thymic T cells, whether the DP T leukemia cells represent thymic or peripheral T cells has been a matter of controversy. To address this issue, we studied phenotypical features in eight cases of DP T cell leukemia. Thymic DP T cells and peripheral CD8+ T cells have CD8 of alphabeta subunit, while CD8alphaalpha is induced in CD4+ T cells on activation with IL-4. We found that two patients with DP T large granular lymphocyte leukemia (LGLL) showed dim expression of CD8alphaalpha, identical to the phenotype on IL-4-activated DP-T cells. The leukemic cells of these patients expressed IL-4 mRNA and produced high levels of IL-4. These findings suggest that they may be derived from peripheral CD4+ T cells. Three patients with adult T cell leukemia/lymphoma (ATLL) showed CD8alphaalpha, suggestive of an activated peripheral T cell origin. One case expressed CD8alphaalpha dim and IL-4 mRNA, while the other two cases expressed no IL-4 mRNA and showed CD8alphaalpha bright phenotype, features not found in normal T cell populations. Three patients with T-prolymphocytic leukemia (T-PLL) expressed CD8alphabeta. The DP phenotype is relatively common in T-PLL, and CD4+CD8alphabeta+ is characteristic of thymic T cells. The DP T-PLL cells did not express TdT,CD1 or recombination activating gene-1 (RAG-1), which is down-regulated at the late stage of thymic T cell development. On the basis of these findings, we propose a late thymic origin for DP T-PLL. The phenotype of DP T cells differed for each entity and appeared to correlate with minor normal DP T cell population.

Paroxysmal nocturnal haemoglobinuria (PNH), aplastic anaemia (AA) and myelodysplastic syndrome (MDS) are haemopoietic stem cell disorders. These disorders have some features in common, and a percentage of cases progress to acute leukaemia. We speculated that changes in gene stability are involved in the pathogenesis of these haemopoietic stem cell disorders. Therefore we investigated in vivo mutation frequencies in these disorders by erythrocyte glycophorin A (GPA) mutation assay. The assay enumerates NO or NN variant cells in 106 erythrocytes of the MN type using a flowcytometric technique. Patients undergoing chemotherapy known to be at risk of hypermutageneity were also studied. Events exceeding the 95th percentile of healthy donors (> or = 32 and 34 events, respectively for NO and NN variants) were defined as abnormal. Abnormal events in the NO variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, two out of seven patients with MDS, and four out of nine patients with PNH. Abnormal events in the NN variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, one out of seven patients with MDS, and two out of nine patients with PNH. These results suggest that not only PIG-A, but also other genes including the GPA gene, are hypermutable in haemopoietic stem cell disorders, and that mutagenic pressure and/or gene instability can contribute to the pathogenesis of these disorders.

Abnormalities of chromosome 14 involving band q32.33 are among the most commonly observed cytogenetic alterations in B-cell malignancies. To assess the incidence and pathogenetic implications of 14q32.33 translocation in chronic B-cell leukemia/lymphomas, we performed fluorescence in situ hybridization (FISH) analysis with variable region (VH) and gamma constant region (Cγ) gene probes in 37 patients with these disorders. Chromosome 14q32.33 translocation was detected in 2 of 18 patients with chronic lymphocytic leukemia (CLL), 1 of 2 with CLL of mixed cell types (CLL/PL), 1 of 2 with pro-lymphocytic leukemia (PLL), 5 of 6 with leukemic mantle-cell lymphoma (MCL), 2 of 7 with splenic B-cell leukemia/lymphoma of possible marginal zone origin (SBLL) and 2 with leukemic follicular lymphoma (FL). To further characterize 14q32.33 translocations in these patients, we developed a new procedure using double-color FISH with PRAD1, BCL2, VH and Cγ gene probes. Chromosome t(11;14) was detected in 1 patient with CLL/PL, 1 with PLL and 5 with MCL. Chromosome t(14;18) was detected in 2 patients with FL. In a PLL patient with t(11;14), the cosmid CPP29 containing the PRAD1 gene and its 5′-flanking region split and co-localized with both Cγ and VH gene probes, thus spanning the breakpoint. In CLL and SBLL patients, donor chromosomes were other than chromosomes 2, 11, 18 and 19, suggesting the involvement of a novel oncogene(s) in the pathogenesis of these diseases. Interphase FISH rapidly detected 14q32.33 translocation, t(11;14) and t(14;18) in B-cell malignancies with low mitotic activity at the single-cell level, facilitating the correlation of the molecular features of these translocations with clinical characteristics. Int. J. Cancer 72:31–38, 1997. © 1997 Wiley-Liss Inc.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by clonal blood cells that are deficient in the surface expression of glycosylphosphatidylinositol-anchored proteins due to somatic mutation in the X-linked gene PIG-A. In some patients, more than one abnormal clone may be present. Analysis of bulk DNA/RNA from granulocytes has been useful in identifying the predominant PIG-A mutation in each patient. However, it is often not useful in determining the presence of minor clones. Many patients have cells with partial deficiency. Here, we analyzed the PIG-A gene in two B-cell lines bearing complete or partial deficiencies, cells of hematopoietic progenitor colonies and peripheral blood granulocytes from the same patient. We found that two B-cell lines had different mutations, the granulocytes contained at least two mutants, and the hematopoietic progenitors contained four mutants. Three of the four were shared by B cells and/or granulocytes whereas the other one was found only in the hematopoietic progenitors. The partial deficiency was caused by a point mutation near an alternative splice site within exon 2 that resulted in partial decreases of activity and quantity of the full-length transcript. These results further show the oligoclonal nature of PNH and differences in extent of expansion among mutant clones.

Polyclonal B lymphocytosis was found in four patients having clinical and hematologic features resembling those of hairy cell leukemia (HCL). All four patients were women between 37 and 67 years of age. Three patients had splenomegaly. Lymphadenopthy was absent or slight. Persistent lymphocytosis was seen in all the patients, and anemia and/or thrombopenia was observed in three of the patients. Abnormal lymphocytes have long microvilli and prominent membranous ruffles on their surfaces. Bone marrow aspirates and biopsy specimens showed increased numbers of abnormal lymphocytes with round nuclei and abundant pale cytoplasm. Although these findings were similar to those of HCL, studies of Ig gene rearrangements and expression showed the polyclonal proliferation of B cells. We called this new disease hairy B-cell lymphoproliferative disorder (HBLD). All four patients exhibited a polyclonal increase in serum IgG. The morphology of the cells in HBLD was more similar to that of leukemia cells of a variant form of HCL (HCL-Japanese variant) than to typical HCL cells. The surface IgG+, CD5−, CD11c+, CD22+, CD24−, CD25− phenotype and the weak tartrate-resistant acid phosphatase activity in the cells were identical to those of HCL cells of the Japanese variant. Our findings suggest that the B cells in HBLD are the nonmalignant counterpart of leukemic B cells in HCL-Japanese variant.

The relationship between arterial or venous thrombosis and the levels of anticardiolipin antibodies (aCL) and/or existence of lupus anticoagulant (LA) was studied. The 141 patients with systemic lupus erythematosus (SLE) were divided into four groups: aCL single positive (25 cases), LA single positive (11 cases), aCL and LA double positive (25 cases), aCL and LA double negative (80 cases). The prevalence of thrombosis was higher in aCL and LA double positive patients (21/25 cases, 84.0%, P0.01) than that in aCL single positive patients (4/25 cases, 16.0%), LA single positive patients (1/11 cases, 9.1%) and double negative patients (3/80 cases, 3.8%). Furthermore, in these double positive patients, all patients (10/10 cases) with a high positive level of aCL (10 units/ml) had arterial thrombosis, whereas only 2/15 patients (13.3%) with a low positive level of aCL (3-10 units/ml) were affected. Venous thrombosis was frequently found in the low positive group (9/15 cases, 60.0%). On the contrary, none of 105 LA negative patients had arterial thrombosis and only seven (6.7%) had venous thrombosis. These findings indicate that a high aCL activity combined with a LA positive result might be a risk factor for arterial thrombosis.

A cell line, JHC-2, was established from the peripheral blood of a patient with hairy cell leukemia (HCL)-Japanese variant. The JHC-2 cells have cytologic features similar to those of the original tumor cells. They displayed hairy cytoplasmic projections by phase contrast and scanning electron microscopy. The tartrate-resistant acid phosphatase reaction was weakly positive. The immunophenotype of the JHC-2 cells was CD5-, CD10-, CD11c+/-, CD19+, CD21+, CD23+, CD24-, CD25+/-, CD38- and FMC-7+. The expression of surface immunoglobulin (IgG, kappa) and the configuration of Ig gene rearrangements in the JHC-2 cells were identical to those in the original leukemic cells, and the JHC-2 cells displayed trisomy 9 on cytogenetic examination. Southern blot analysis for the Epstein-Barr virus (EBV) genome showed that the JHC-2 cells contained the EBV genome, although the freshly isolated leukemic cells did not. These results indicate that the JHC-2 cell line is an EBV spontaneously transformed B cell line originating from HCL cells.

The relationships between paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA), and myelodysplastic syndrome (MDS) are not clear. Here we describe a patient, J20, who developed a reciprocal translocation of chromosome 12 and PNH during follow-up of AA. All metaphases in CD59-deficient bone marrow mononuclear cells had the translocation, whereas none of the CD59-deficient cells had it, indicating that the PNH clone coincided with a cell population bearing the chromosomal aberration. We found a somatic single-base deletion mutation in the PIG-A gene of this patient's peripheral blood cells. This is the first patient with PNH with a PNH clone containing a chromosomal translocation.

Prolymphocytic leukemia (PLL) was diagnosed by morphologic and immunophenotypical studies in a 72-year-old Japanese man. Massive splenomegaly was present but lymphadenopathy was minimal in this case. Chromosomal analysis of peripheral mononuclear cells showed t(11;14)(q13;q32) in all metaphases examined, except for one normal karyotype. Northern blot analysis of RNA prepared from leukemic cells obtained from the patient revealed overexpression of the PRAD1/cyclin D1 proto-oncogene, which has not been described previously in patients with PLL.

We studied the immunological function of hairy cells from hairy cell leukemia (HCL) patients presenting with pronounced polyclonal hypergammaglobulinemia (PPH). Hairy cell conditioned medium (HCCM) obtained from HCL patients with PPH augmented IgG production by normal peripheral blood mononuclear cells in a dose-dependent fashion, while HCCM from patients without PPH had no effect on IgG production. HCCM from the patients with PPH failed to enhance IgG synthesis by T cell-depleted monanuclear cells. Separation of T and B cells by a 0.4-μm membrane as well as monoclonal antibodies to HLA-DR and CD3 molecules prevented HCCM-dependent IgG synthesis. No B cell growth factor activity, interleukin-1, or interleukin-6 was detected in the HCCM. On examination by fractionation of the HCCM, IgG-inducing activity was detected in the fractions of 5000 to 8000 Da. These results indicate that hairy cells from HCL, patients with PPH secrete a factor inducing IgG synthesis, and that the induction of IgG synthesis by the factor requires T-B cell interactions involving T cell receptor/CD3 complex and MHC class II antigens. This factor may play an important role in the development of PPH.

We report a 37-year-old female HTLV-I carrier with complicating primary biliary cirrhosis (PBC) and mixed connective tissue disease (MCTD). Serum anti-HTLV-I antibody titer was ×256. Flower cells (4.5%) were found in the peripheral blood. Southern blot analysis showed no clonal integration in peripheral blood lymphocyte (PBL) DNA. Polymerase chain reaction showed the HTLV-I genome in PBLDNA. As cholestatic liver dysfunction and serum titer of anti-mitochondrial antibody were found, a clinical diagnosis of PBC was made. This patient later developed MCTD. These diseases responded well to prednisone. The pathogenetic relationship of HTLV-I infection with various autoimmune diseases is discussed.(Internal Medicine 32: 449-454, 1993)

A 64-year-old male suffered from splenomegaly without lymphadenopathy. His WBC count on admission was 6.1×l09/l with 55% abnormal lymphocytes. No monoclonal gammopathy was detected. Abnormal cells shown in films usually had relatively abundant cytoplasm with serrated edges. Under phase-contrast microscopy, the cells displayed short, needle-like processes. The immunophenotype of peripheral blood mononuclear cells were CD19+, CD20+, CDllc+, FMC7+, CD5+, CD10-, CD25- and SIg+. The spleen histology showed a distinctive pattern of white pulp infiltration by abnormal lymphocytes with features of plasma cell differentiation. These findings were compatible with the features of splenic lymphoma with villous lymphocytes.(Internal Medicine 32: 472-475, 1993)

The clinical and cytological features of 73 patients with chronic (mature) B-cell leukemias were studied. Large lymphocytes or cleft cells were often seen in patients referred to us as chronic lymphocytic leukemia (CLL). Immunophenotypical and histopathological studies were performed and the diagnosis of leukemic phase of non-Hodgkin's lymphoma (follicular lymphoma; FL and intermediate lymphocytic lymphoma; ILL) was made in a significant portion of the patients and only ten cases were diagnosed as CLL including mixed cell type.Although hairy cell leukemia (HCL) is quite rare in Japan, a disproportionally large number of patients were referred to our laboratory. Typical HCL seen in Western countries, was found in nine cases. On the other hand, most (29 cases) showed cytologic features distinct from typical HCL and were classified into HCL-Japanese Variant (HCL-J). Morphology of HCL-J cells in May-Giemsa stained films resembled large CLL lymphocytes, but several features shared by typical HCL and HCL-J including typical hairy morphology under phase contrast microscopy, CD11c+, L30- phenotype and the histology distinguished the disease from CLL and other B-cell leukemias. In two cases, the cells displayed prominent nucleoli and surface villi, the features consistent with those described for HCL Variant (prolymphocytic variant). Some patients were differentiated from HCL and were diagnosed as leukemic phase of ILL with large spleens or splenic lymphoma with villous lymphocytes (SLVL). Under phase contrast, fine cytoplasmic processes were obseved in ILL cells. Surface morphology of SLVL cells considerably varied from cell to cell; some displayed prominent cytoplasmic projections similar to those of hairy cells, while others had a few, short villi or lacked villi even by phase contrast microscopy. The spleen histology showed prominent involvement in the white pulp with the features of lymphoplasmacytic lymphoma.

T-prolymphocytic leukaemia (T-PLL) is a rare dis-order with a poor prognosis. A 69-year-old man was diagnosed as having a small-cell variant of T-PLL according to the French–American–British classification by haematological, immunological and ultrastructural studies, although the cells had a CD7− phenotype and no chromosomal abnormality. He had no symptoms or organomegaly. The number of his lymphocytes, 53.7 × 109/l at the time of diagnosis, gradually decreased without therapy, and he was in complete remission 39 months later. A rearranged band in the T-cell antigen receptor-β gene, which was detected at the time of diagnosis, decreased or disappeared. This is the first report of a T-PLL case with spontaneous complete remission.

10年以上もの長期間にわたり全身倦怠感,微熱,関節痛などを主とする諸症状に苦しむ30歳の女性が来院した.他院では,ステロイドや免疫抑制剤なども投与されていたが,寛解・増悪を繰り返していた. 6ヵ月以上続く全身倦怠感,微熱,咽頭痛,リンパ節腫脹,筋力の低下,筋痛,頭痛,関節痛,思考力・集中力の低下などが認められ, Holmesらの診断基準に従い, chronic fatigue syndrome (CFS)と診断した.ウイルス抗体価の検索では,麻疹,水痘ヘルペス,単純ヘルペス, EBウイルスが軽度高値で,ヒトヘルペスウイルス-6 (HHV-6)の抗体価は, 2,560倍ときわめて高値を示した.しかし,末梢血単核球を用いたPCRの検索ではHHV-6特異的配列は認められず,本例におけるHHV-6の関与は明らかでない.リムルステストは陽性で,エンドスペーシー,トキシカラーともに高値を示した.また, NK活性は, E/T比10対1, 20対1ともにきわめて低値を示した.一方, NK細胞の指標であるCD57は末梢血単核球中19.7%と正常であったが, CD16は2.3%と明らかに減少していた.このCD16異常とNK活性の障害に関しては今後の検討が必要である.治療としては,ステロイドを減量し,精神療法を中心に行ったところ,約2週間後より解熱傾向があり, 1ヵ月後には頭痛,嘔気,全身倦怠感などの諸症状とともに微熱も軽減し退院した.以後約1年間,症状は軽減したまま外来に通院していたが最近,頭痛と意識障害をきたして近医に緊急入院した.近年,欧米ではCFSに強い関心がもたれ,患者の支援組織も結成され,広くこの病態の克服に取り組んでいる.ところが,本邦ではほとんど注目されておらず,このような病態は膠原病や更年期障害,心身症などと診断されることが多いと思われる.したがって,今後適切な診断・治療を行うためにも正しい理解が必要である.

Hairy cell leukemia complicating hemolytic anemia developed in a 46-year-old woman. Morphologically and cytochemically typical hairy cells were found to express both CD20 and CD2 antigens. Expression of surface IgG of kappa-chain type and the rearrangement of Ig but not T-cell receptor beta genes confirmed a B-cell origin of the leukemia. Blood transfusion was followed by disappearance of the hemolysis and a marked improvement of the leukemia. However, the patient developed progressive spastic spinal paraplegia about seven months after transfusion and was diagnosed as having HTLV-I associated myelopathy (HAM) by the demonstration of HTLV-I antibodies in serum and cerebrospinal fluid. HTLV-I infection via the transfusion may have been involved in the hematologic improvement seen in this patient. Autopsy showed demyelination, vacuolar degeneration, gliosis, and perivascular cuffing in the white matter of spinal cord without evidence of leukemic infiltration.

We studied 25 Japanese patients with hairy cell leukemia (HCL) and found a manner in which HCL can be divided into two subtypes. In each patient, hairy cells (HC) showed striking surface hairs and reacted with HC-specific antibodies (alpha Leu-M5 and alpha HC-M). Twenty of the 25 patients had HC characterized by round nuclei with dense nuclear chromatin, weak tartrate-resistant acid phosphatase (TRAP) activity and the phenotype of low density surface immunoglobulin (SIg)+, Tac-. In this group of 20 patients, the male to female ratio was low, and there was frequent leukocytosis. On the other hand, the remaining 5 patients showed a high male to female ratio and a normal or decreased leukocyte count. HC had folded nuclei, strong TRAP activity and the phenotype of high density SIg+, Tac+. The features of the latter patients are consistent with those of HCL in Western countries, while those of the former group appear to indicate a unique variant of HCL.

Somatic mutation of PIGA in hematopoietic stem cells causes deficiency of glycosyl phosphatidylinositol–anchored proteins in paroxysmal nocturnal hemoglobinuria (PNH) that underlies the intravascular hemolysis but does not account for expansion of the PNH clone. Immune mechanisms may mediate clonal selection but appear insufficient to account for the clonal dominance necessary for PNH to become clinically apparent. Herein, we report 2 patients with PNH whose PIGA-mutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3′ untranslated region of HMGA2, the architectural transcription factor gene deregulated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression, in concert with mutant PIGA, accounts for clonal hematopoiesis in these 2 patients and suggest the concept of PNH as a benign tumor of the bone marrow.

We investigated the effects of extracellular ubiquitin on the colony formation of human hematopoietic progenitor cells (CFU-GM, CFU-E and BFU-E) and a granulocyte-colony stimulating factor (G-CSF)-dependent human myeloblastic leukemic cell line, OCI/AML/1a. Ubiquitin exhibited inhibitory effects on CFU-GM, CFU-E, BFU-E and OCI/AML/1a colony formation. Extracellular ubiquitin also inhibited the growth of KT3 (T lymphoblast), K562 (erythroleukemia) and Daudi (Burkitt's lymphoma) cells, stimulated the growth of HL60 (promyelocytic leukemia) and Jurkat (T-ALL) cells and showed no effect on the growth of U937 cells (monocytic leukemia). These results indicate that another, previously unrecognized function of extracellular ubiquitin is to regulate hematopoiesis.

This case report describes a hairy B cell lymphoproliferative disorder (HBLD) with clinical and hematological features resembling hairy cell leukemia. The patient was a 29-year-old female who demonstrated atypical lymphocytes in her peripheral blood. Physical examination demonstrated splenomegaly, but there were no palpable superficial lymph nodes. Hematological examination showed a leukocyte count of 10.6 x 10(3)/mm3 with 41% atypical lymphocytes. Bone marrow examination showed a normal cellular and an atypical lymphocyte count of 42%. The atypical lymphocytes had microvilli and prominent membranous ruffles on their surfaces. Atypical lymphocytes expressed CD5- CD10- CD11c+ CD19+ CD20+ CD23- CD25- on the surface of the cells on examination by with a fluorescence activated cell sorter. Although these findings were similar to hairy cell leukemia, Japanese variant, the surface marker of the kappa chain and lambda chain was unbiased and studies of immunoglobulin gene rearrangements and expression showed polyclonal proliferation of B cells. Therefore, we diagnosed this patient as having HBLD. Because she did not demonstrate anemia or thrombocytopenia, she is not currently receiving medication. To date, the atypical lymphocyte count has not changed.

The c-kit receptor tyrosine kinase (KIT) is constitutively activated by 2 types of naturally occurring mutations, the Val559→Gly (G559) mutation in the juxtamembrane domain and the Asp814→Val (V814) mutation in the catalytic domain. We evaluated the effects of the tyrosine kinase inhibitors STI571 and AG1296 on BaF3 cells expressing wild-type KIT (KITWT) or activating mutants of KIT (KITG559 and KITV814) in the presence or absence of the KIT ligand, stem cell factor (SCF). Both STI571 and AG1296 inhibited SCF-dependent activation of KITWT and SCF-independent activation of KITG559 more efficiently, whereas SCF-independent activation of KITV814 was scarcely affected. Furthermore, both inhibitors inhibited SCF-dependent growth of BaF3-KITWT cells and, with higher potencies, SCF-independent growth of BaF3-KITG559 cells through the induction of apoptosis. In contrast, the inhibitors had little or no effect on SCF-independent growth of BaF3-KITV814 cells or on IL-3-dependent growth of BaF3-Mock cells. These results suggested that both inhibitors may be effective therapeutic agents for oncogenic KIT with the juxtamembrane domain mutation, but not with the catalytic domain mutation, and that the activation mechanism of the catalytic domain mutant KIT is complex and entirely different from that of the wild-type KIT or the juxtamembrane domain mutant KIT.Int J Hematol. 2002; 76: 427-435.

Paroxysmal noctumal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder that causes clonal expansion of Glycosylphosphaticfylinositol (GPI)-anchor deficient cells. A somatic mutation of PIG-A that encodes a subunit of the enzyme complex in the GPI-anchor synthesis results in deficiency of GPI-anchored proteins. Two lines of evidence suggest that second abnormality is involved in clonal expansion of GPI-anchor deficient cells. Pig-a dismpted hematopoietic stem cells in mouse and PIG-A deficient cells in normal human individuals did not clonally expand Two possible mechanisms for clonal expansion have been proposed One is an immunoselection mechanism in that PNH cells are more resistant to immunological attack than normal cells because of the deficiency of GPI-anchored proteins. The other is an intrinsic mechanism in that PNH cells have a second mutation responsible for clonal expansion like benign tumors.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder caused by PIG-A mutations. Many patients with PNH have more than one PNH clone, but it is unclear v^hether a single PNH clone remains dominant or if minor clones eventually become dominant. Furthermore, it is unknown how many hematopoietic stem cells (HSCs) sustain hematopoiesis and how long a single HSC can support hematopoiesis in humans. To understand the dynamics of HSCs, we reanalyzed the PIG-A gene mutations in 9 patients with PNH 6 to 10 years after their initial analysis. The proportions of affected peripheral blood PMNs in individual patients were highly variable; 2 patients increased, 3 patients decreased, and 4 had stable populations (changed less than 20%). In all cases, the previously predominant clone was still dominant and present as assessed by DNA analysis. Two patients whose affected PMNs decreased and who have been followed significantly longer than others, now have an aplastic condition, suggesting that aplasia is a terminal feature of PNH. This is the first report directly demonstrating that one stem cell clone can sustain human hematopoiesis for 6 to 10 years. The complexity of these results reflects the marked variability of the clinical course of PNH.

Fatigue is an indispensable sense for ordering rest. However, the neuronal and molecular mechanisms of fatigue remain unclear. Chronic fatigue syndrome (CFS) with long-lasting fatigue sensation seems to be a good model for studying these mechanisms underlying fatigue sensation. Recently, we found that most patients with CFS showed a low level of serum acetylcarnitine, which well correlated with the rating score of fatigue, and that a considerable amount of acetyl moiety of serum acetylcarnitine is taken up into the brain. Here we show by metabolite analysis of the mouse brain that an acetyl moiety taken up into the brain through acetylcarnitine is mainly utilized for the biosynthesis of glutamate. When we studied the cerebral uptake of acetylcarnitine by using [2-(11)C]acetyl-L-carnitine in 8 patients with CFS and in 8 normal age- and sex-matched controls, a significant decrease was found in several regions of the brains of the patient group, namely, in the prefrontal (Brodmann's area 9/46d) and temporal (BA21 and 41) cortices, anterior cingulate (BA24 and 33), and cerebellum. These findings suggest that the levels of biosynthesis of neurotransmitters through acetylcarnitine might be reduced in some brain regions of chronic fatigue patients and that this abnormality might be one of the keys to unveiling the mechanisms of the chronic fatigue sensation.

GATA-2 is considered to be essential for the development, maintenance, and function of hematopoietic stem cells (HSCs). However, it was also reported that GATA-2 inhibits the growth of HSCs. To examine the role of GATA-2 in the growth of hematopoietic cells, we introduced an estradiol-inducible form of GATA-2 (GATA-2/estrogen receptor [ER]) into interleukin 3 (IL-3)-dependent cell lines, Ba/F3, 32D, and FDC-P1. Estradiol-induced GATA-2 suppressed c-myc mRNA expression and inhibited IL-3-dependent growth in these clones. As for this mechanism, GATA-2 was found to inhibit ubiquitin/proteasome-dependent degradation of p21(WAF1) and p27(Kip1) and to induce their accumulation by repressing the expression of Skp2 and Cul1, both of which are components of the ubiquitin ligase for p21(WAF1) and p27(Kip1). Overexpression of c-myc restored the expression of Skp2 and Cul1 mRNA, reduced the amounts of p21(WAF1) and p27(Kip1) proteins, and canceled GATA-2-induced growth suppression, suggesting that down-regulation of c-myc expression may be primarily responsible for GATA-2-induced growth suppression. Next, we transduced retrovirus containing GATA-2/ER into murine bone marrow mononuclear cells (MNCs) and stem/progenitor (Sca-1(+)Lin(-)) cells. GATA-2/ER suppressed cytokine-dependent growth of MNCs and Sca-1(+)Lin(-) cells by about 70%, which was also accompanied by the reduced expression of c-myc, Skp2, and Cul1 mRNA and the accumulation of p21(WAF1) and p27(Kip1) proteins. In addition, the amount of GATA-2 protein was found to decline in hematopoietic stem/progenitor cells that were promoted to enter cell cycle by the stimulation with cytokines. These results suggest that GATA-2 may regulate expression levels of p21(WAF1) and p27(Kip1), thereby contributing to the quiescence of hematopoietic stem/progenitor cells.

The hairy cell leukemia prolymphocytic variant, a subtype of hairy cell leukemia, is an extremely rare disease, especially in Japan. We report a case in which treatment with 2'-deoxycoformycin (DCF) improved the clinical features of the disease. The patient, a 70-year-old female, was first treated with 2-chlorodeoxyadenosine, but showed only transient improvement in the hematological findings. DCF was then administered every week. Following the start of this treatment, the leukemia cell count rapidly decreased and the platelet count simultaneously increased. This effect of DCF has so far been long term. More clinical studies are needed to confirm the therapeutic value of DCF.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by clonal blood cells that are deficient in glycosylphosphatidylinositol-anchored proteins because of somatic mutations of the PIG-A gene. Many patients with PNH have more than one PNH clone, but it is unclear whether a single PNH clone remains dominant or minor clones eventually become dominant. Furthermore, it is unknown how many hematopoietic stem cells (HSCs) sustain hematopoiesis and how long a single HSC can support hematopoiesis in humans. To understand dynamics of HSCs, we reanalyzed the PIG-A gene mutations in 9 patients 6 to 10 years after the previous analyses. The proportion of affected peripheral blood polymorphonuclear cells (PMNs) in each patient was highly variable; it increased in 2 (from 50% and 65% to 98% and 97%, respectively), was stable in 4 (changed less than 20%), and diminished in 3 (94%, 99%, and 98% to 33%, 57%, and 43%, respectively) patients. The complexity of these results reflects the high variability of the clinical course of PNH. In all patients, the previously predominant clone was still present and dominant. Therefore, one stem cell clone can sustain hematopoiesis for 6 to 10 years in patients with PNH. Two patients whose affected PMNs decreased because of a decline of the predominant PNH clone and who have been followed up for 24 and 31 years now have an aplastic condition, suggesting that aplasia is a terminal feature of PNH.

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3–dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-rasG12V), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

The ubiquitin–proteasome pathway is responsible for selective degradation of short-lived cellular proteins and is critical for the regulation of many cellular processes. We previously showed that ubiquitin (Ub) secreted from hairy cell leukemia cells had inhibitory effects on clonogenic growth of normal hematopoietic progenitor cells. In this study, we examined the effects of exogenous Ub on the growth and survival of a series of human hematopoietic cells, including myeloid cell lines (HL-60 and U937), a B-cell line (Daudi), and T-cell lines (KT-3, MT-4, YTC-3, and MOLT-4). Exogenous Ub inhibited the growth of various hematopoietic cell lines tested, especially of KT-3 and HL-60 cells. The growth-suppressive effects of Ub on KT-3 and HL-60 cells were almost completely abrogated by the proteasome inhibitor PSI or MG132, suggesting the involvement of the proteasome pathway in this process. Furthermore, exogenous Ub evoked severe apoptosis of KT-3 and HL-60 cells through the activation of caspase-3. In interleukin-6 (IL-6)-dependent KT-3 cells, STAT3 was found to be conjugated by exogenous biotinylated Ub and to be degraded in a proteasome-dependent manner, whereas expression levels of STAT1, STAT5, or mitogen-activated protein kinase were not affected. Moreover, IL-6-induced the up-regulation of Bcl-2 and c-myc, and JunB was impaired in Ub-treated KT-3 cells, suggesting that the anti-apoptotic and mitogenic effects of IL-6 were disrupted by Ub. These results suggest that extracellular Ub was incorporated into hematopoietic cells and mediated their growth suppression and apoptosis through proteasome-dependent degradation of selective cellular proteins such as STAT3.

A high rate of Borna disease virus (BDV) infection has been demonstrated in patients with chronic fatigue syndrome (CFS). Herein, we focused on BDV infection in two family clusters of patients with CFS: a father, mother, two sons and one daughter (family #1); and a father, mother, two daughters and one son (family #2). All members, except for the elder son in family #1 and the father and son in family #2, were diagnosed with CFS. The results supported that all the family members with CFS were infected with BDV, as evidenced by the presence of antibodies to viral p40, p24 and/or gp18 and BDV p24 RNA in peripheral blood mononuclear cells. The healthy members, except for the father of family #2 who was positive for antibody to p24, were all negative by both assays. Follow-up studies in family #1 continued to reveal BDV antibodies and BDV RNA, except in the mother, who lost the RNA upon slight recovery from the disease.

Expression of alkaline phosphatase (ALP) on the surface membrane of neutrophils (mNAP) was studied by immunofluorescence using an anti-ALP monoclonal antibody. Fluorescent intensity distribution of mNAP was analyzed using FACS (fluorescence-activated cell sorter). The mean fluorescent intensity (MFI) of the mNAP in this assay was well correlated with the neutrophil ALP (NAP) score demonstrated cytochemically (r = 0.832). mNAP levels in various hematological disorders were evaluated by % mNAP+ cells and MFI. The levels in aplastic anemia and polycythemia vera were significantly higher, and in chronic myelocytic leukemia and paroxysmal nocturnal hemoglobinuria (PNH), the levels were significantly lower compared with the levels in healthy volunteers. Two-color immunofluorescence with anti-ALP and anti-CD16 showed that the PNH clone was essentially negative for mNAP, whereas residual normal neutrophils (CD16+) had levels slightly higher than those in normal individuals. Highly reproducible results were obtained in the blood samples which were stored at 4 degrees C for at least 24 hr without any treatment prior to immunofluorescent staining. No degradation of fluorescent intensity was seen 4 days after staining and fixation. The mNAP assay is simple, without subjective evaluation for quantification, and is useful for differential diagnosis of hematological disorders.

Recently, we found a serum acylcarnitine (ACR) deficiency in Japanese patients with chronic fatigue syndrome (CFS). To clarify whether this ACR abnormality is a characteristic of CFS or not, we also studied the levels of serum carnitine in Swedish subjects. Both serum ACR and free carnitine (FCR) levels in normal healthy subjects were quite different between Japanese (n=131) and Swedish people (n=46) (p

The chronic fatigue syndrome (CFS) is a condition of unknown etiology, characterized by a persistent debilitating fatigue, the muscle-related symptoms and the neuropsychiatric symptoms. Recently, it has been reported that the patients with CFS might have impaired activation of the hypothalamic-pituitary-adrenal axis, and suggested that a part of the patho-genesis of CFS might be associated with abnormalities of the endocrine system. Herein, we show that the majority of Japanese patients with CFS had a serum dehydroepiandrosterone sulfate (DHEA-S) deficiency. Serum DHEA-S is one of the most abundantly produced hormones which is secreted from the adrenal glands, and its physiological function is thought to be a precursor of sex steroids. DHEA-S has recently been shown to have physiological properties, such as neurosteroids, which are associated with such psychophysiological phenomena as memory, stress, anxiety, sleep and depression. Therefore, the deficiency of DHEA-S might be related to the neuropsychiatric symptoms in patients with CFS.

A 17-year-old female developed natural killer (NK) cell-derived large granular lymphocyte (LGL) lymphoma of the lung. She had a past history of hypersensitivity to mosquito bites (HMB). After an eight-year chronic, active Epstein-Barr virus (EBV) infection, she developed multiple lung lesions and pleural effusion. In the effusion, 60% of the cells were LGL. They were CD2+, 3-, 16+, 56+, 57+, 45RO+/RA + weak, and possessed strong NK activity. No rearrangement of T-cell-receptor genes was detected. From all these results, a diagnosis of NK-LGL lymphoma of the lung was made. EB virus DNA was detected in cells infiltrating the pleural effusion. The clonality of the LGLs was determined by Southern blot hybridization with the terminal repeat sequence of EB virus as a probe, and by chromosomal abnormalities. The patient died from respiratory failure. Necropsy of the lung revealed diffuse lymphoma composed of polymorphic cells with typical angiocentric lesions. Reportedly, lymphomas of NK lineage show predominantly extranodal involvement, and primary lung lesions are rare. In the pleural effusion of the present case, abnormally high levels of soluble Fas ligand, interleukin-10 and interferon gamma were detected. This hypercytokinemia, reflecting the microenvironment of lymphoma cells, may play a role in the progression of the lymphoma and organ injury in the lung.