Kazuta Yasui

No association of xenotropic murine leukemia virus-related virus with prostate cancer or chronic fatigue syndrome in Japan

BackgroundThe involvement of xenotropic murine leukemia virus-related virus (XMRV) in prostate cancer (PC) and chronic fatigue syndrome (CFS) is disputed as its reported prevalence ranges from 0% to 25% in PC cases and from 0% to more than 80% in CFS cases. To evaluate the risk of XMRV infection during blood transfusion in Japan, we screened three populations--healthy donors (n = 500), patients with PC (n = 67), and patients with CFS (n = 100)--for antibodies against XMRV proteins in freshly collected blood samples. We also examined blood samples of viral antibody-positive patients with PC and all (both antibody-positive and antibody-negative) patients with CFS for XMRV DNA.ResultsAntibody screening by immunoblot analysis showed that a fraction of the cases (1.6-3.0%) possessed anti-Gag antibodies regardless of their gender or disease condition. Most of these antibodies were highly specific to XMRV Gag capsid protein, but none of the individuals in the three tested populations retained strong antibody responses to multiple XMRV proteins. In the viral antibody-positive PC patients, we occasionally detected XMRV genes in plasma and peripheral blood mononuclear cells but failed to isolate an infectious or full-length XMRV. Further, all CFS patients tested negative for XMRV DNA in peripheral blood mononuclear cells.ConclusionOur data show no solid evidence of XMRV infection in any of the three populations tested, implying that there is no association between the onset of PC or CFS and XMRV infection in Japan. However, the lack of adequate human specimens as a positive control in Ab screening and the limited sample size do not allow us to draw a firm conclusion.

Mitochondrial damage-associated molecular patterns as potential proinflammatory mediators in post–platelet transfusion adverse effects

Platelet concentrates (PCs) are the most common blood components eliciting nonhemolytic transfusion reactions (NHTRs), such as allergic transfusion reactions and febrile reactions. However, the precise mechanisms of NHTRs in PC transfusion remain largely unknown. Previous studies reported that mitochondria‐derived damage‐associated molecular patterns (DAMPs) could be important mediators of innate cell inflammation. Platelets (PLTs) represent a major reservoir of mitochondria in the blood circulation. The aim of this study was to determine the possible involvement of mitochondrial DAMPs in NHTRs.

Application of the basophil activation test in the analysis of allergic transfusion reactions

Allergic reactions, which are mediated by mast cells and/or basophils, are the most common adverse effects associated with transfusion, particularly with use of platelet concentrates (PCs). In general, the activation of mast cells/basophils and their subsequent histamine release are mediated via two pathways: the allergendependent and allergen-independent pathways. In the latter, biologic response modifiers (BRMs), such as bacterial components, activated complement components, cytokines and chemokines directly activate mast cells/basophils. Although allergens in blood components may include food or medications ingested by the donor immediately before blood collection, such specific allergens have not been identified. Indeed, the only allergens identified in blood components are plasma proteins including IgA, haptoglobin and C4; transfusion to patients who are deficient in those plasma proteins reportedly causes serious allergic reactions. Besides such cases with plasma protein deficiency, allergic reactions after PC transfusion are likely to be elicited mainly via the allergen-independent pathway by BRMs that are present or accumulated in PCs, although the roles of BRMs in this pathway remain largely unknown (Cognasse et al., 2006; Blumberg et al., 2006; Wakamoto et al., 2003). In addition, because allergic transfusion reactions are diagnosed based on symptoms, it is often difficult to determine whether the transfused PCs are causative or whether the reactions are, in actuality, allergic in nature. These difficulties are partly because of the lack of an assay system with which activation of mast cells/basophils can be assessed. Fortunately, however, a simple test called the basophil activation test (BAT) has recently been developed for managing allergic diseases. In the test, whole blood is incubated with an allergen, and subsequent basophil activation is assessed using flow cytometry based on upregulation of cell degranulation/activation markers, CD63 or CD203c (Boumiza et al., 2005; Bühring et al., 1999). In this study, using

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti-human platelet antibodies

Background: A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies.

Detection of anti–Siglec-14 alloantibodies in blood components implicated in nonhaemolytic transfusion reactions

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Possible involvement of heparin-binding protein in transfusion-related acute lung injury

BACKGROUND In antibody-mediated nonhemolytic transfusion reactions, transfusion-related acute lung injury (TRALI) tends to occur typically within 2 hours after a blood transfusion. White cell antibodies or immune complexes have been frequently shown to be associated with the syndrome, although the mechanisms by which they induce TRALI are poorly understood. The aim of this study was to characterize soluble mediators that are released from cells at an early stage after immune stimulation. STUDY DESIGN AND METHODS To explore the mechanism of TRALI, an in vitro whole-blood cell culture assay was established in which cells were stimulated by human antibodies and the activation of neutrophils was monitored by a cell surface marker (Mac-1) with flow cytometry and by measurement of the release of soluble factors, including perforin, interleukin-6, tumor necrosis factor-alpha, and heparin-binding protein (HBP) with enzyme-linked immunosorbent assays. In addition, the involvement of two neutrophil FcgammaRs (FcgammaRIIIb and FcgammaRIIa, also known as CD16 and CD32, respectively) was examined during antibody-induced cell activation with anti-FcgammaR blocking antibodies. RESULTS Substantial amounts of HBP were released within 30 minutes of stimulation by human antibodies, although other soluble mediators were not released within the same period. Furthermore, the release of HBP was mediated via signals through both FcgammaRIIIb and FcgammaRIIa. CONCLUSION HBP appears to be one of the primary effector molecules of antibody-mediated nonhemolytic transfusion reactions including TRALI.

Spontaneous and rapid reexpression of functional CXCR4 by human steady-state peripheral blood CD34+ cells

Although only 5% of steady-state peripheral blood (PB) CD34+ cells were found to express chemokine receptor CXCR4, 45% of the cells became CXCR4+ after incubation at 37°C for 4 hours. In contrast, there were no remarkable differences between PB CD34+ cells before and after the 37°C incubation in their expression of selectin ligand, VLA-4, and VLA-5 or in their affinity for VCAM-1 or fibronectin.This increase in CXCR4 expression level was inhibited by the addition of brefeldin A, actinomycin D, or cycloheximide.When PB CD34+ cells with CXCR4 expression levels enhanced by a 4-hour preincubation at 37°C or bone marrow (BM) CD34+ cells were exposed overnight to stromal cell-derived factor 1 (SDF-1), the expression levels of CXCR4 were greatly reduced, and when SDF-1 was removed, CXCR4 levels were thereafter up-regulated.The reexpressed CXCR4 was able to elicit integrin-dependent migration of hematopoietic progenitor cells.There was no difference in the severe combined immunodeficient mouse repopulating cell activity between PB CD34+ cells with and cells without a 37°C preincubation.

Down-regulation of ASY/Nogo transcription associated with progression of adult T-cell leukemia/lymphoma

Adult T‐cell leukemia/lymphoma (ATLL) is an aggressive form of human leukemia/lymphoma. Although this disease is initiated by infection with human T‐lymphotropic virus type 1 (HTLV‐1), many HTLV‐1 carriers survive for a long period without aggressive illness, suggesting that other factors may play roles in the progression of ATLL to an aggressive state. However, the mechanism involved in this progression still remains unclear. Previously, we have reported that ASY/Nogo mRNA was markedly down‐regulated in human small‐cell lung carcinomas, whereas it was expressed in normal tissues and other lung carcinomas, such as adenocarcinoma and squamous cell carcinoma. To understand whether or not ASY/Nogo gene is involved in the progression of ATLL, we examined the expression of ASY/Nogo mRNA in smoldering, chronic and aggressive ATLL, and found that the expression level of ASY/Nogo mRNA was markedly reduced in clinically aggressive ATLL. HTLV‐1 Tax expression was not affected by the down‐regulation of ASY/Nogo mRNA. These results indicate that the ASY/Nogo gene may act as a suppressor against ATLL progression, independent of Tax expression.

The IgE‑dependent pathway in allergic transfusion reactions: involvement of donor blood allergens other than plasma proteins

On transfusion, several plasma proteins can cause anaphylaxis in patients deficient in the corresponding plasma proteins. However, little is known about other allergens, which are encountered much more infrequently. Although it has been speculated that an allergen-independent pathway underlying allergic transfusion reactions (ATRs) is elicited by biological response modifiers accumulated in blood components during storage, the exact mechanisms remain unresolved. Furthermore, it is difficult even to determine whether ATRs are induced via allergen-dependent or allergen-independent pathways. To distinguish these two pathways in ATR cases, we established a basophil activation test, in which the basophil-activating ability of supernatants of residual transfused blood of ATR cases to whole blood basophils was assessed in the presence or absence of dasatinib, an inhibitor of IgE-mediated basophil activation. Three of 37 supernatants from the platelet concentrates with ATRs activated panel blood basophils in the absence, but not in the presence, of dasatinib. The basophil activation was inhibited by treatment of anti-fish collagen I MoAb in one case, suggesting that the involvement of fish allergens may have been present in donor plasma. We concluded that unknown non-plasma proteins, some of which had epitopes similar to fish antigens, in blood component may be involved in ATRs via an allergen/IgE-dependent pathway.

Multiple ETS Family Proteins Regulate PF4 Gene Expression by Binding to the Same ETS Binding Site

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the −51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a −51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the −51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the −51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.