Yuji Yonemura

Characterization of monocyte/macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis

IntroductionRecent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.MethodsSkin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.ResultsIn the skin from SSc patients, the number of CD163+ cells or CD204+ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14+ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14+ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163+ cells belong to CD14brightCD204+ population.ConclusionsThis is the first report indicating CD163+ or CD204+ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14brightCD163+CD204+ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163+ or CD204+ macrophages in the skin.

Effect of recombinant human interleukin-11 on rat megakaryopoiesis and thrombopoiesis in vivo : comparative study with interleukin-6

Summary. The ability of recombinant human interleukin‐11 (IL‐11) to stimulate rat megakaryopoiesis and thrombopoiesis in vivo was investigated. Once daily subcutaneous injections of IL‐11 at doses of 2, 8 and 20 μg/rat for 5 d caused dose‐dependent increases in platelet counts. The chronic administration of 20 μg/rat/d for 14 d resulted in biphasic increases in platelet counts with peaks at days 8 and 15 of up to 30% over the control, continuing for more than 5 d after cessation of IL‐11 injections. Moreover, a striking increase in megakaryocytic size and ploidy in bone marrow in response to IL‐11 was elicited. IL‐11 induced a dose‐dependent elevation in bone marrow cell numbers but not in splenic weight and cell numbers. Modifications of these parameters were noted as soon as 24 h after the first IL‐11 injections. IL‐11 had a same potency of thrombopoietic effect in rats as compared with IL‐6. However, elevation of acute phase protein such as immunosuppressive acidic protein was 2–2‐fold in rats given 20 μg/d of IL‐6 over those receiving a same dose of IL‐11 (470 v 210 μg/ml). In addition, the rate of body‐weight increase in rats receiving IL‐11 for 5 d as well as 14 d did not differ from that in control animals. In IL‐6 treated rats, the increase in body weight was significantly slower than the controls, which was observed even in the group given 8 μg/d of IL‐6. These results suggest that IL‐11 may be an effective strategy for the treatment of thrombocytopenia.

Long-term responses and outcomes following immunosuppressive therapy in large granular lymphocyte leukemia-associated pure red cell aplasia: a Nationwide Cohort Study in Japan for the PRCA Collaborative Study Group

This report describes long-term responses following immunosuppressive therapy in large granular lymphocyte leukemia-associated pure red cell aplasia. Large granular lymphocyte leukemia-associated pure red cell aplasia accounts for a significant portion of secondary pure red cell aplasia cases. However, because of its rarity, long-term responses and relapse rates after immunosuppressive therapy are largely unknown. We conducted a nationwide survey in Japan and collected 185 evaluable patients. Fourteen patients with large granular lymphocyte leukemia-associated pure red cell aplasia were evaluated. Cyclophosphamide, cyclosporine A and prednisolone produced remissions in 6/8, 1/4 and 0/2 patients respectively. Seven and 5 patients were maintained on cyclophosphamide or cyclosporine A respectively. Two patients relapsed after stopping cyclophosphamide, and 2 patients relapsed during maintenance therapy with cyclosporine A. The median relapse-free survival in the cyclophosphamide - and the cyclosporine A groups was 53 and 123 months respectively. Large granular lymphocyte leukemia-associated pure red cell aplasia showed a good response to either cyclophosphamide or cyclosporine A. Most patients continued to receive maintenance therapy and it remains uncertain whether cyclophosphamide or cyclosporine A can induce a maintenance-free hematologic response in large granular lymphocyte leukemia-associated pure red cell aplasia.

Long-term response and outcome following immunosuppressive therapy in thymoma-associated pure red cell aplasia: a nationwide cohort study in Japan by the PRCA collaborative study group

This nationwide survey in Japan indicates that thymoma-associated pure red cell aplasia responds in most instances to cyclosporine A, and that this treatment is also effective in preventing relapse of anemia. Background Thymoma-associated pure red cell aplasia (PRCA) accounts for a significant proportion of cases of secondary PRCA and immunosuppressive therapy has been reported to be useful in this condition. However, because of its rarity, the long-term response and relapse rates after immunosuppressive therapy are largely unknown, and optimal management of this disorder remains unclear. The aim of this study was to collect more information on the outcome of patients with thymoma-associated PRCA. Design and Methods We conducted a nationwide survey in Japan. From a total of 185 patients, comprising 73 with idiopathic and 112 with secondary PRCA, 41 patients with thymoma were evaluated for this report. End-points of this study were the response rate, duration of the response after immunosuppressive therapy and overall survival. Results Surgical removal of thymoma was reported in 36 patients, 16 of whom developed PRCA at a median of 80 months post-thymectomy. First remission induction therapy was effective in 19 of 20 patients treated with cyclosporine, 6 of 13 patients treated with corticosteroids and 1 of 1 treated with cyclophosphamide. No cyclosporine-responders relapsed within a median observation period of 18 months (range; 1 to 118 months). Relapse of anemia was observed in three corticosteroid-responders who did not receive additional cyclosporine. Only two patients were in remission after stopping therapy for 19 and 67 months. The estimated median overall survival time of all patients was 142 months. Conclusions Thymoma-associated PRCA showed an excellent response to cyclosporine and cyclosporine-containing regimens were effective in preventing relapse of anemia. It does, however, remain uncertain whether cyclosporine can induce a maintenance-free hematologic response.

Paroxysmal nocturnal haemoglobinuria with coexisting deficiency of the ninth component of complement: lack of massive haemolytic attack

A 47‐year‐old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement‐sensitivity lysis tests, 80% of her erythrocytes were markedly complement‐sensitive (PNH‐III). Laser cytofluorimetry with a monoclonal antibody against decay‐accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF‐negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusions of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patients erythrocytes. When the patients serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.

Purification of megakaryocyte differentiation activity from a human fibrous histiocytoma cell line: N-terminal sequence homology with activin A

We purified a protein possessing a potent ability to induce the differentiation of a murine megakaryoblastic cell line, L8057, from the supernatant of a human fibrous histiocytoma cell line, KHM-5M. The protein, a homodimer of a molecular weight of 25-kDa, has an N-terminal sequence identical with that of the beta A-chain of inhibin, indicating that it is identical to or highly homologous with activin A. Thus, it is speculated that activin A or its homologue is involved in megakaryocytic differentiation.

NKG2D-mediated immunity underlying paroxysmal nocturnal haemoglobinuria and related bone marrow failure syndromes.

It is considered that a similar immune mechanism acts in the pathogenesis of bone marrow (BM) failure in paroxysmal nocturnal haemoglobinuria (PNH) and its related disorders, such as aplastic anaemia (AA) and myelodysplastic syndromes (MDS). However, the molecular events in immune‐mediated marrow injury have not been elucidated. We recently reported an abnormal expression of stress‐inducible NKG2D (natural‐killer group 2, member D) ligands, such as ULBP (UL16‐binding protein) and MICA/B (major histocompatibility complex class I chain‐related molecules A/B), on granulocytes in some PNH patients and the granulocyte killing by autologous lymphocytes in vitro. The present study found that the expression of NKG2D ligands was common to both granulocytes and BM cells of patients with PNH, AA, and MDS, indicating their exposure to some incitement to induce the ligands. The haematopoietic colony formation in vitro by the patients’ marrow cells significantly improved when their BM cells were pretreated with antibodies against NKG2D receptor, suggesting that the antibodies rescued haematopoietic cells expressing NKG2D ligands from damage by autologous lymphocytes with NKG2D. Clinical courses of patients with PNH and AA showed a close association of the expression of NKG2D ligands with BM failure and a favourable response to immunosuppressive therapy. We therefore propose that NKG2D‐mediated immunity may underlie the BM failure in PNH and its‐related marrow diseases.

Altered Expression of Gangliosides in Erythrocytes of Paroxysmal Nocturnal Hemoglobinuria

In paroxysmal nocturnal hemoglobinuria (PNH), impaired glycosyl-phosphatidylinositol (PI)-anchoring of membrane proteins such as decay-accelerating factor has been known to lead to increased susceptibility to complement. Moreover, abnormal expression of non-PI-anchoring glycoproteins such as C3b/C4b receptor (CR1) or glycophorin-alpha also has been shown in PNH. Therefore, we biochemically analyzed glycosphingolipids (GSL) as one of the membrane glycoconjugates of PNH erythrocytes. Erythrocytes of all seven PNH patients showed altered expression of sialosyl GSL (gangliosides) as compared with the control erythrocytes of healthy donors. Both a sialosylparagloboside (IV6NeuAc-nLc4Cer) among four major gangliosides and some minor gangliosides in normal erythrocytes variably disappeared in erythrocytes from the peripheral blood of PNH patients. As one of the possible mechanisms of altered expression of gangliosides in PNH erythrocytes, structural analysis suggested impaired sialylation of GSL. These results suggest not only the altered metabolism of gangliosides in PNH erythrocytes, but also a metabolic disorder of membrane glycoconjugates as a new feature of PNH.

Online reporting system for transfusion-related adverse events to enhance recipient haemovigilance in Japan: A pilot study

BACKGROUND A surveillance system for transfusion-related adverse reactions and infectious diseases in Japan was started at a national level in 1993, but current reporting of events in recipients is performed on a voluntary basis. A reporting system which can collect information on all transfusion-related events in recipients is required in Japan. METHODS We have developed an online reporting system for transfusion-related events and performed a pilot study in 12 hospitals from 2007 to 2010. RESULTS The overall incidence of adverse events per transfusion bag was 1.47%. Platelet concentrates gave rise to statistically more adverse events (4.16%) than red blood cells (0.66%) and fresh-frozen plasma (0.93%). In addition, we found that the incidence of adverse events varied between hospitals according to their size and patient characteristics. CONCLUSION This online reporting system is useful for collection and analysis of actual adverse events in recipients of blood transfusions and may contribute to enhancement of the existing surveillance system for recipients in Japan.

Smoldering γδT-cell granular lymphocytic leukemia associated with pure red cell aplasia

We present a case of granular lymphocytic leukemia (GLL) associated with pure red cell aplasia (PRCA). Twenty-seven percent of the peripheral blood mononuclear cells (PBMC) were positive for TCRδ1 but