High frequency of several PIG-A mutations in patients with aplastic anemia and myelodysplastic syndrome.

To clarify some characteristics of phosphatidylinositol glycan-class A gene (PIG-A) mutations in aplastic anemia (AA) and myelodysplastic syndrome (MDS) patients compared with those in paroxysmal nocturnal hemoglobinuria (PNH) patients, we investigated PIG-A mutations in CD59- granulocytes and CD48- monocytes from seven AA, eight MDS, and 11 PNH Japanese patients. The most frequent base or type abnormalities of the PIG-A gene in AA and MDS patients were base substitutions or missense mutations, respectively, and deletions or frameshift mutations, respectively, in PNH patients. Several PIG-A mutations, most of which were statistically minor, were found in glycosylphosphatidylinositol-negative cells from all AA and MDS patients but not from all PNH patients. However, the common PIG-A mutations during the clinical course between CD59- granulocytes and/or CD48- monocytes from each AA or MDS patient, except for Case 5, were not found. PIG-A mutations were different between the granulocytes and monocytes from five AA and five MDS patients. Our results indicate that there were some characteristics of PIG-A mutations in AA and MDS patients compared with PNH patients and that several minor PNH clones in these patients occurred at random during the clinical course. This partly explains the transformation of AA or MDS to PNH at intervals.

Ten-year survivor with multiple myeloma in first complete remission following treatment with conventional chemotherapy. Case report and a review of the literature.

A 62-year-old woman with multiple myeloma, who has been in complete remission (CR) for 10 years, is reported. The patient was treated with conventional chemotherapy, including nitrosourea derivatives. Five patients with myeloma, including the present case, who have survived for 10 years or more in CR and on whom detailed clinical descriptions were published, are reviewed. Their disease condition represented "a cure" or "a state extremely close to cure". The review indicates the following favorable prognostic factors common to these patients: age < or =65 years and a rapid response to treatment. Progressive bone destruction and/or lytic changes at disease onset is perhaps not a bad prognostic factor in myeloma patients with excellent outcome.

Complement and polymorphonuclear leukocyte activation each play a role in determining myocardial ischemia-reperfusion injury.

Cobra venom factor (CVF) transiently activates polymorphonuclear leukocytes (PMN) by complement activation, followed by rapid complement depletion and gradual reversal of PMN activation. Utilizing these sequential changes caused by CVF, the individual and combined effects of complement and PMNs on myocardial infarct size (IS) were investigated. Rats were treated with CVF, and/or anti-PMNs. Complement was depleted, but circulating PMNs were being activated at 4h after CVF administration, and at 36h after, complement was depleted, but PMNs were in a near basal condition. Under anesthesia, the rats had a 30-min coronary occlusion followed by 6h of reperfusion. The IS was assessed by tetrazolium staining. CVF, as well as anti-PMNs, reduced myeloperoxidase (MPO) activity in the risk area and the reduced MPO resulted in a reduced IS, which was also the effect of anti-PMNs, but complement depletion by CVF, during which circulating PMNs were activated, failed to reduce the IS despite low MPO activity. These results suggest that complement and the condition of PMNs each play a role in determining the IS, and ischemic reperfusion injury might be produced even by relatively low myocardial MPO activity.

Truncated mutant B subunit for factor XIII causes its deficiency due to impaired intracellular transportation.

Two Japanese patients were newly diagnosed as having B subunit (XIIIB) deficiency of factor XIII (former type I deficiency). Both patients have a previously described one-base deletion at the boundary between intron A/exon II in the XIIIB gene, heterozygously or homozygously. A founder effect was proposed for this mutation because 3 unrelated patients with XIIIB deficiency also share 2 3'-polymorphisms. In one patient heterozygous for the above mutation, a novel mutation was also identified: a deletion of guanosine in exon IX (delG) of the XIIIB gene. To understand the molecular and cellular pathology of the delG mutation, expression studies were performed using a cultured mammalian cell line. Pulse-chase experiments showed that a resultant truncated XIIIB remained inside the cells and could not be secreted into the culture medium. Furthermore, immunocytochemical examinations by epifluorescence, confocal, and electron microscopes indicated impaired intracellular transportation of the truncated XIIIB from the endoplasmic reticulum to the Golgi apparatus. No mutations in the gene for the A subunit (XIIIA) were identified in this patient. Therefore, secretion of the truncated XIIIB must also be impaired in vivo, leading to a secondary XIIIA deficiency. These results support a previous conclusion that genetic defects of XIIIB are the basis for the former type I factor XIII deficiency.

The distribution of PIG-A gene abnormalities in paroxysmal nocturnal hemoglobinuria granulocytes and cultured erythroblasts.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia that is characterized by a deficiency of glycosylphosphatidylinositol-anchored membrane proteins due to phosphatidylinositol glycan-class A (PIG-A) gene abnormalities in various lineages of peripheral blood cells and hematopoietic precursors. The purpose of our study was to clarify the distribution of PIG-A gene abnormalities among various cell lineages during differentiation and maturation in PNH patients. The expression of CD16b or CD59 in peripheral blood granulocytes or cultured erythroblasts from three Japanese PNH patients was analyzed using flow cytometry. PIG-A gene abnormalities in both cell types, including glycophorin A(+) bone marrow erythroblasts, were examined using nucleotide sequence analysis. The expression study of PIG-A genes from each patient was also performed using JY-5 cells.Flow cytometry revealed that the erythroblasts consisted of negative, intermediate, and positive populations in Cases 1 and 3 and negative and intermediate populations in Case 2. The granulocytes consisted of negative and positive populations in all three cases. DNA sequence analysis indicated that all the PNH cases had two or three types of PIG-A gene abnormalities, and that a predominant clone with an abnormal PIG-A gene was different in granulocytes and erythroblasts from Cases 2 and 3. Expression studies showed that all the mutations from the patients were responsible for the null phenotype.PIG-A gene abnormalities result in deficiencies of glycosylphosphatidylinositol-anchored proteins in PNH erythroblasts and granulocytes. The distribution of predominant PNH clones with PIG-A gene abnormalities is often heterogeneous between the cell types, suggesting that a clonal selection of PIG-A gene abnormalities occurs independently among various cell lineages during differentiation and maturation.

[Investigation on apoptosis in paroxysmal nocturnal hemoglobinuria (PNH) granulocytes].

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disordr characterized by increased susceptibility of erythrocytes to complement-mediated hemolysis. Recently, PNH is a stem cell disorder with all hematopoietic lineages affected, which have deficiencies of glycosylphosphtidylinositol-anchored membrane proteins due to the phosphatidylinositol glycan-class A (PIG-A) gene abnormalities. However, it is unknown how PNH clones with PIG-A gene abnormalities increase in bone marrow. The possibility has been suggested that resistance of PNH cells to apoptosis causes the increase. We studied two-color or single-color flow cytometric analysis using Annexin V and propidium iodide or 7-amino actinomycin D for evaluation of spontaneous apoptosis in peripheral blood granulocytes from PNH patient (n = 5) and healthy volunteers (n = 5), respectively. Apoptotic granulocytes were evaluated before and after 6-, 12-, 18- and 24-hour cultures without serum. Flow cytometric analyses showed that there were no significant differences of the number of proportion of apoptotic cells between them. This fact reveals that the sensitivity of PNH cells to apoptosis is similar to that of normal cells, suggesting that PNH clones should not be increased according to resistance to apoptosis as an intrinsic characteristic.

Immune thrombocytopenia in an elderly patient treated successfully by pulse therapy with cyclophosphamide.

Thrombocytopenia due to immune mechanisms is rare and difficult to manage in elderly patients. We describe a case of an 89-year-old female with severe immune thrombocytopenia (ITP) who rapidly improved by pulse therapy with cyclophosphamide. She was admitted to our hospital because she had arthralgia in both sides of her femoral region since January 1999, aphthous stomatitis and ecchymosis of the leg since April 1999, and bloody phlegm in July 1999. On admission, her peripheral blood count revealed severe thrombocytopenia (0.1 x 10(4)/microl). Her megakaryocyte count from bone marrow was increased to 512/microl without abnormal cells. Systemic lupus erythematosus was suspected because of strong positive protein in the urine in addition to the clinical and hematological findings described above, but she was negative for all the autoantibodies examined. Finally, she was diagnosed as having ITP on the basis of high platelet associated immunoglobulin G in addition to hematological and physical findings and she was treated with prednisolone. It was difficult to maintain her platelet count with only prednisolone, but 600 mg of cyclophosphamide rapidly increased her platelet count in spite of tapering the prednisolone. In September 2000, her platelet count was kept within normal limits by administration of 15 mg/day of prednisolon. It is suggested that immunosuppressive therapy for ITP using high-dose cyclophosphamide is useful in elderly patients as well as in juvenile adult patients.

Induction of apoptosis in vivo and in vitro in hairy cell leukemia treated by deoxycoformycin.

The leukemic cells of a patient with hairy cell leukemia were treated in vitro with 2'-deoxycoformycin (dCF), an inhibitor of adenosine deaminase, and deoxyadenosine (dAdo). Following this treatment, viability of the hairy cells progressively declined, and DNA fragmentation was observed. When the patient was treated with 4 mg/m2 dCF intravenously, hairy cells in his peripheral blood rapidly decreased, and a large number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP- biotin nick end-labeling)-positive cells were detected in a bone marrow biopsy specimen. These findings indicate that dCF induced apoptosis of hairy cells both in vivo and in vitro. Furthermore, bcl-2 mRNA was down-regulated by dCF and dAdo in the hairy cells in vitro. Our results suggest the importance of bcl-2 mRNA regulation in apoptotic cell death mediated by dCF in hairy cell leukemia.

Neutrophil-specific reduction in the expression of granulocyte--macrophage colony-stimulating factor receptor subunits in myelodysplastic syndromes.

The proliferative and differentiative response of neutrophils to granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be impaired in patients with myelodysplastic syndromes (MDS). To investigate the mechanisms of the defective response in MDS, we examined expression levels of GM-CSF receptor alpha (GMR alpha) and common beta (beta c) subunits on CD16(+) neutrophils, CD14(+) monocytes and CD3(+) T cells from 26 MDS patients and 10 healthy controls using flow cytometry. Expression of GMR alpha was significantly decreased on the neutrophils of five out of 26 patients and was not specific for any FAB subtype. In contrast, beta c expression on neutrophils was significantly reduced in 14 out of 26 patients with a higher proportion occurring in the advanced stages of MDS including refractory anaemia with excess of blasts (RAEB), RAEB in transformation (RAEBt) and overt leukaemia compared with refractory anaemia (RA)/RA with ringed sideroblasts (RARS) or healthy controls. Decreased beta c also correlated with the degree of hypogranular neutrophil morphology and increased infection. Expression of both subunits on T cells and monocytes in MDS was similar to normal controls. Polymerase chain reaction amplification of reverse-transcribed mRNA isolated from the affected neutrophils suggests that the reduction of beta c may result from decreased message levels. The observed reduction in GM-CSF receptor expression could account for the impaired proliferative and maturational responses in MDS.

T-prolymphocytic leukaemia with spontaneous remission.

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 x 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-beta 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.

A 92-year-old man with systemic lupus erythematosus who developed acute lupus pneumonitis.

We describe a 92-year-old male patient with systemic lupus erythematosus (SLE) who had sudden onset of thrombocytopenia and developed acute lupus pneumonitis (ALP). Although steroid pulse therapy was effective for ALP, he developed complicated bacterial pulmonary disease. This patient is the oldest ever reported to have contracted SLE.

[Effective treatment combining antithymocyte globulin, cyclosporin A, and granulocyte colony-stimulating factor for atypical paroxysmal nocturnal hemoglobinuria accompanied by bone marrow hypoplasia].

A 25-year-old man was admitted for evaluation of pancytopenia on May 2, 1997. On admission, he had pancytopenia with a normal reticulocyte count. Bone marrow aspirate specimens displayed a normal karyotype and hypocellularity without myelodysplasia. Although total bilirubin and lactate dehydrogenase levels were within their normal ranges, the haptoglobin level was low; additionally, two-color flow cytometric analysis determined that 3.3% of erythrocytes were double-negative for CD55 and CD59 expression. Atypical paroxysmal nocturnal hemoglobinuria with bone marrow hypoplasia was diagnosed. Because initial treatment with cyclosporin A was not effective, the patient was subsequently given a combination of antithymocyte globulin, cyclosporin A, and granulocyte colony-stimulating factor. Although the pancytopenia subsided, the percentage of double-negative erythrocytes in the patient's blood remained almost unchanged compared to findings obtained on admission.

Complement sensitivity of erythrocytes in a patient with inherited complete deficiency of CD59 or with the Inab phenotype.

We investigated the complement sensitivity of erythrocytes from three patients, one with inherited complete deficiency of CD59, one with the Inab phenotype, and one with paroxysmal nocturnal haemoglobinuria (PNH). The complement lysis sensitivity units on the erythrocytes were 11.7, 4.6, and 47.6 for inherited CD59 deficiency, Inab phenotype, and PNH, respectively. Two-colour flow cytometric analysis showed that the erythrocytes from the three patients consisted of a single population negative for CD59, negative for decay accelerating factor (DAF), and negative for both proteins, respectively. In addition, only the Inab phenotype patient had no haemolysis in vivo. These facts suggest that CD59 deficiency plays a more important role than DAF deficiency in complement-mediated haemolysis in vitro and in vivo, and that deficiency of both proteins, but not CD59 or DAF alone, causes complement sensitivity corresponding to that of PNH III erythrocytes in vitro.

[Myositis caused by hydroxyurea in a patient with chronic myelogenous leukemia].

A 59-year-old man with chronic myelogenous leukemia (CML) had a white-blood-cell (WBC) count of 55,400/microliter when admitted in July 1997, and was placed on oral hydroxyurea (HU) of 1,500 mg/day. Treatment with 600 MU/day of interferon alpha (IFN alpha) was started on August 5. HU was discontinued when the patient's WBC count dropped to 8,100/microliter on August 18. However, HU was resumed about a month later, after his WBC count increased to 10,100/microliter, but discontinued when the patient started to complain of chills, high fever, and bilateral femoral pain. HU treatment was initiated again one week later, after the patient's WBC count had begun rising but ceased again after he experienced chills, high fever, and bilateral femoral pain. The patient's myogenetic enzymes were found to be increasing the following day, and a lymphocyte stimulation test (LST) with HU showed a high stimulation index of 41.7%. The elevation of myogenetic enzymes and the results of the LST suggested that myositis due to HU was the cause of the patient's clinical manifestations. His myositis spontaneously disappeared after HU was discontinued. Although the patient is no longer receiving HU, IFN alpha has brought his CML under control. To our knowledge, this is the first reported case of myositis caused by HU.

[Fatal pulmonary thromboembolism in a patient with paroxysmal nocturnal hemoglobinuria].

A 38 year old woman with paroxysmal nocturnal hemoglobinuria was admitted to our hospital because of hemoptysis. Pulmonary infarction was diagnosed by the perfusion lung scan. In spite of the administration of prednisolone, dextran, low molecular weight heparin, and warfarin, she died of pulmonary infarction and secondary pulmonary hypertension. Autopsy revealed thromboembolism of both pulmonary arteries and hepatic central vein thrombosis. Recent understanding of the pathogenesis and incidence of thromboembolism in paroxysmal nocturnal hemoglobinuria was discussed.

Surface expression of complement regulatory proteins, decay-accelerating factor (DAF) and CD59, on cultured human endothelial cells.

To clarify the response of endothelial cells to complement, we studied not only the reaction of endothelial cells against complement lysis sensitivity (CLS) test, but also the expression of complement regulatory proteins by two-color flow cytometric analysis and backscattered scanning immunoelectron microscopic analysis using monoclonal antibodies to decay-accelerating factor (DAF) and/or CD59. Complement activation didn't lead to the cell death of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs). In control, two-color flow cytometric analysis indicated that HAECs consisted of a single double-positive population for these proteins as well as HUVECs. Then, HUVECs and HAECs after the CLS test resulted in having same distribution by flow cytometry. Moreover, the microscopic analysis showed that DAF or CD59 was expressed with a diffuse distribution. However, DAF on HUVECs and CD59 on HAECs were present at the margin of cell surfaces more than at the other places. These findings suggest that endothelial cells have a defense mechanism for complement activation in vitro by the changes of expression of complement regulatory proteins on the membrane surface, and that the mechanism of HAECs to complement is the same as that of HUVECs.