Shigeo Nomura

Relationship of microparticles withβ2-glycoprotein I and P-selectin positivity to anticardiolipin antibodies in immune thrombocytopenic purpura

We investigated the association ofβ2-glycoprotein I and P-selectin with platelet-derived microparticles in 48 patients with immune thrombocytopenic purpura and 20 normal controls using two-color flow cytometric analysis. In addition, anticardiolipin antibodies were detected by an enzyme-linked immunosorbent assay. Platelet microparticles from the patients showed a higher positivity forβ2-glycoprotein I than those from the normal controls (23.1±15.4% vs. 5.3±3.1%, p<0.01), but this positivity was not related to the presence of platelet-associated IgG or to the severity of thrombocytopenia. In the 18 patients with more than 20% P-selectin-positive microparticles,β2-glycoprotein I positivity was significantly higher than in the 30 patients with less than 20% P-selectin-positive microparticles (37.1±20.5% vs. 21.5±17.3%, p<0.01). In addition, anticardiolipin antibodies were detected in eight patients, and they had a significantly higher level ofβ2-glycoprotein I-positive microparticles than the patients without such antibodies (42.0±22.9% vs. 22.6±18.9%, p<0.05). Our results suggest that anticardiolipin antibodies activate platelets in immune throm bocytopenic purpura and cause the generation of microparticles rich inβ2-glycoprotein I and P-selectin. These microparticles may then act to regulate coagulation abnormalities in patients with anticardiolipin antibodies.

Anti-phospholipid antibodies bind to platelet microparticles in idiopathic (autoimmune) thrombocytopenic purpura.

SummaryWe recently reported that IgM antibody-related microparticles exist in some patients with idiopathic thrombocytopenic purpura (ITP) [14]. In this study, we investigated the relationship between antiphospholipid (cardiolipin and phosphatidylinositol) antibodies and microparticles in 56 ITP patients. We used an ELISA to detect anti-phospholipid antibodies. IgG antibodies against cardiolipin and phosphatidylinositol were detected in 13 and 12 patients, respectively. The titers of IgG antibodies against these phospholipids did not correlate with the platelet-associated IgG level or the platelet count. Next, we investigated the binding of anti-phospholipid antibodies to platelets and microparticles. Microparticles were obtained by incubating washed platelets with collagen plus thrombin. ITP plasma containing IgG-class anti-phospholipid antibodies showed significantly increase binding to microparticles compared with plasma without such antibodies (p<0.001). Our results suggest that anti-phospholipid antibodies could affect the function of platelet microparticles in ITP.

Intracellular formation of amyloid fibrils in Myeloma. Cytochemical, immunochemical, and electron microscopic observations

Needle‐like, rod‐shaped, and rhomboid intracytoplasmic crystalline inclusions were found in myeloma cells from the bone marrow of a patient with IgA‐κ and Bence Jones‐κ type multiple myeloma. Cytochemical, immunochemical, and electron microscopic studies revealed that these inclusions consisted of at least three kinds of components: kappa light chain crystals, lysosomal enzymes, and amyloid fibrils. The coexistence of these three kinds of inclusions in one myeloma cell suggests an intracellular site for the formation of amyloid fibrils. From these observations, the authors postulated that the excessively produced light chains were transported to the Golgi apparatus where they were transformed into crystalline inclusions, and thereafter, digested by lysosomal enzymes to form amyloid fibrils. The demonstration of crystalline inclusions which reacted exclusively with antisera against free light chains indicates that the Bence Jones protein is a precursor of amyloid light chain.

Clinical Evaluation of Transfusion of Prestorage-Leukoreduced Apheresis Platelets

With increased use of platelet concentrate (PC) in recent years, adverse reactions to PC transfusion have received much clinical attention. Most of these reactions stem from white blood cells (WBC) contaminating the transfused PC. Several are thought to be preventable by removing WBC before PC storage. Methods: We routinely filtered apheresis PC either during collection or immediately afterwards and monitored various indicators of platelet quality during storage. After transfusion to patients, transfusion efficacy and the type, severity, and frequency of posttransfusion side effects were compared with those of a control group in which PC was filtered at the bedside. Results: Prestorage-filtered PC contained an average of 3.1±0.7 × 1011 platelets and 0.9±1.2 × 106 contaminating leukocytes. Measurement of platelet function and metabolic indicators revealed normal values throughout the storage period. CD62 measurement revealed no undue platelet activation after filtration or during the storage period. Cytokine, histamine, bradykinin, and complement levels showed no significant increase after filtration or during storage. Transfusion efficacy and overall side effect incidence rates were similar for the prestorage- and bedside-filtered groups, but reactions of the bedside-filtered group included serious reactions such as breathing difficulties and shock. No serious reactions were noted in the prestorage-filtered group. Conclusion: Filtering PC before storage has no adverse effect on PC quality and may reduce the severity of post transfusion side effects.

New monoclonal anti‐human Fc gamma receptor II antibodies induce platelet aggregation

We developed two new monoclonal antibodies, designaled NNKY3‐2 and NNKY4‐7, that recognized a 40‐kD platelet protein. They appeared to be monoclonal anti‐Fc gamma receptor II (FC‐/RII) antibodies from the results of flow cytometric binding inhibition studies using another monoclonal anti‐FcγRII antibody (2E1). The addition of NNKY3‐2 or NNKY4‐7 to platetet‐rich plasma (PRP) led to a typical aggregation pattern preceded by a lag phase, but their addition to washed platelets did not induce aggregation. The aggregation of PRP by these antibodies was inhibited by prostaglandin E1 (PGE1) or staurosporine (protein kinase C inhibitor), whereas it was only slightly affected by a monoclonal anti‐GPIIb/IIIa antibody or Arg‐Gly‐Asp‐Ser, Furthermore, these antibodies induced the aggregation of washed platelets plus normal serum, but not that of washed platelets plus heat‐treatcd serum (destruction of complement activity). These results suggest that NNKY3‐2 or NNKY4‐7‐induced aggregation involves an unusual pathway independent of fibrinogen, and that the important factor is the participation of complement. NNKY3‐2 and NNKY4‐7 may be useful to study the relationship between autoantibodies, the Fc receptor, and complement in idiopathic thrombocytopenic purpura.

Effects of Nilvadipine on Cytokine-Levels and Soluble Factors in Collagen Disease Complicated with Essential Hypertension

We examined some immunological parameters, particularly cytokines and soluble factors in collagen diseases complicated with essential hypertension. We also investigated the effects of Nilvadipine on immunological parameters after treatment with this drug for six months. The frequency of helper/inducer T cells (CD4+ CD8- cells, CD4+ CD45RA- cells) decreased in the peripheral blood on a 6 month treatment with nilvadipine. There was a significant decrease of suppressor/inducer T cells (CD4+ 45RA+ cells), and an insignificant decrease of activated T cells (CD3+ HLA-DR+ cells) and memory T cells (CD45RA- CD45RO+ cells) after treatment. Before treatment with Nilvadipine, interleukin-1beta, tumor necrosis factor-a, and interleukin-6 levels increased higher in the patients than in healthy volunteers. However, interleukin-1beta and interleukin-6 concentrations tended to decrease after treatment with Nilvadipine. Besides, tumor necrosis factor-alpha decreased significantly after treatment. The soluble interleukin-2 receptor concentrations also showed a decreased tendency after treatment, although high concentrations were found in the patients before treatment. In contrast, soluble human leukocyte antigen-1 and soluble thrombomodulin levels showed no significant change after treatment. These results suggest that Nilvadipine inhibits the generation of cytokines derived from activated T lymphocytes. Nilvadipine, calcium antagonist, may be useful for inhibition of vascular complication in collagen diseases.

Characterization of a Novel Activation-Specific Antiplatelet Monoclonal Antibody

We developed a murine monoclonal antibody (KmT-50) which binds to a protein expressed on the surface of human platelets after activation. KmT-50 is an immunoglobulin G1 monoclonal antibody that recognizes a 50-kD antigen localized in intracellular organelles and the open canalicular system of unstimulated platelets. After platelets were stimulated, the antigen was secreted via the surface-connected canalicular system and exposed on the platelet membrane. KmT-50 bound not only to human platelets, but also to rabbit and monkey platelets. This new antibody may be useful for experimental studies on thrombosis and hemostasis.

Hypertension and diabetes mellitus increase the risk of haemorrhage in chronic idiopathic thrombocytopenic purpura.

The risk factors for haemorrhage in chronic idiopathic thrombocytopenic purpura (ITP) remain poorly understood. We classified 49 patients with chronic ITP into two groups on the basis of the presence (n = 11) or absence (n = 38) of hypertension and/or diabetes mellitus, and then analyzed their clinical and immunological characteristics. The patients with hypertension and/or diabetes were older than those without these complications. There were no significant differences between the two groups with regard to platelet count or the levels of platelet-associated immunoglobulin G, platelet-associated immunoglobulin M, and platelet-associated C3. Positivity for anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib autoantibodies was also similar. However, severe purpura and a poor response to prednisolone were far more common in the patients with hypertension and/or diabetes. We conclude that ITP complicated by hypertension and/or diabetes may be resistant to prednisolone and thus require more careful treatment.

Platelet Transfusion for Patients with Glanzmann's Thromboasthenia

We read with interest the recent paper by Ito et al. [l] on antibody removal therapy in a patient with Glanzmann’s thromboasthenia (GT). They described a successful antibody removal therapy at delivery in a patient with GT who had multiple antiHLA antibodies and an anti-glycoprotein (GP) IIb/IIIa antibody. GT is characterized by a bleeding tendency associated with abnormalities of GP IIb/IIIa. Even in patients with GT, if they have been bleeding severely, circumstances may allow therapy with transfusion of platelets. However, such patients may acquire antibodies against platelet GP IIb/IIIa. We recently attempted transfusion of platelets in a patient with GT who had severe and progressive anemia due to genital bleeding. Consequently, her genital bleeding stopped. However, the same symptoms reappeared after a few months. We again attempted transfusion of platelets, but this time bleeding persisted. On day 3 after transfusion, the serum completely inhibited aggregation of normal platelets upon ADP stimulation. Furthermore, it also blocked the binding of monoclonal anti-GP IIb/IIIa antibody to normal platelets using flow cytometric analysis. Thus, the patient seemed to have acquired antibodies against GP IIb/ IIIa. Alloimmune and isoimmune antibodies produced in patients with GT who have received multiple transfusions have been reported previously [2]. We think that platelet transfusion for patients with GT should be administered carefully. However, when the patient’s life is in peril, platelet transfusion may be necessary. The report by Ito et al. [l] provides a useful insight into the prognosis of such patients. .............................................. References

A Case of Malignant Pleural Mesothelioma with Cystic Formation in the Mediastinum

症例は77歳の女性.1995年4月頃より持続する咳嗽にて当院を受診, 胸部単純X線胸部造影CTでは右側に中等量の胸水を伴い, 縦隔には辺縁が造影されるとともに内部が不均一に造影される一部嚢胞状腫瘤と右側背側胸壁に基部を持つ半円型のよく造影される腫瘤を認めた.気管は縦隔の腫瘤により高度に圧迫をうけていた.胸水穿刺および背側の腫瘤に対して経皮的生検を行った.病理学的免疫組織化学的検索では腫瘍は上皮様構造と肉腫様構造を呈する部分を認め, ピアルロニダーゼ消化試験陽性, CEA陰性, ケラチン陽性であった.以上の画像ならびに病理学的, 組織化学的, 免疫組織学的所見より, びまん性悪性胸膜中皮腫 (biphasic type) と診断した.本症例ではCT上右側胸壁に半球形の腫瘤病変を認め, 縦隔には嚢胞状腫瘤が認められる特異な所見を示した.