Rika A. Furuta

An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR kit is amplified using standard primers for XMRV.

During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6%) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6%) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.

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.

Inhibitory Effects of Small-Molecule CCR5 Antagonists on Human Immunodeficiency Virus Type 1 Envelope-Mediated Membrane Fusion and Viral Replication

ABSTRACT We established a human immunodeficiency virus type 1 (HIV-1) envelope (Env)-mediated membrane fusion assay and examined the small-molecule CCR5 antagonist TAK-779 and its derivatives for their inhibitory effects on HIV-1 Env-mediated membrane fusion and viral replication. The membrane fusion assay is based on HIV-1 long terminal repeat-directed β-d-galactosidase reporter gene expression in CD4- and CCR5-expressed HeLa (MAGI-CCR5) cells after cocultivation with effector 293T cells expressing HIV-1 Env. Inhibition of HIV-1 replication was also determined in MAGI-CCR5 cells infected with the corresponding cell-free HIV-1. TAK-779 effectively suppressed R5 HIV-1 (strain JR-FL) Env-mediated membrane fusion as well as viral replication. Its 50% inhibitory concentrations (IC50s) for membrane fusion and viral replication were 0.87 ± 0.11 and 1.4 ± 0.1 nM, respectively. These values corresponded well to the IC50 for 125I-RANTES (regulated on activation, T cell expressed, and secreted) binding to CCR5 (1.4 nM). The inhibitory effects of 18 TAK-779 derivatives on membrane fusion differed from one compound to another. However, there was a close correlation among their inhibitory effects on membrane fusion, viral replication, and RANTES binding. The correlation coefficient between their IC50s for membrane fusion and viral replication was 0.881. Furthermore, since this assay depends on Env expressed in the effector cells, it is also applicable to the evaluation of CXCR4 antagonists. These results indicate that the HIV-1 Env-mediated membrane fusion assay is a useful tool for the evaluation of entry inhibitors.

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.

Persistent infection by human parvovirus B19 in qualified blood donors

Persistent parvovirus B19 infection with a low viral load has been reported in immunocompromised and in immunocompetent individuals (reviewed in Parsyan and Candotti). Large cross-sectional studies using highly sensitive DNA amplification methods have also demonstrated persistent B19 infection. Recently, Lefere and colleagues conducted a longitudinal study of nonimmunodeficient patients who were multitransfused with red blood cells, demonstrating that asymptomatic chronic B19 infections may persist for a long period. To characterize the natural course of persistent B19 infections, we conducted the following longitudinal study using an in-house TaqMan polymerase chain reaction method for B19 DNA and enzyme immunoassays to detect B19 immunoglobulin M (IgM) and immunoglobulin G (IgG; Denka Seiken, Tokyo, Japan). This study was approved by the ethical Committee of the Japanese Red Cross Osaka Blood Center. In Japan, all donated blood is tested for B19 infection with an in-house receptor-mediated hemagglutination method that detects B19 antigen as a marker of a high viremic stage of infection (cutoff, approx. 2.5 ¥ 10 IU/mL B19 DNA; data not shown). Using this method, we identified 102 cases of B19 infection among 979,052 blood donors in Osaka between 1997 and 1999. We were able to test the plasma samples of 20 of these 102 donors (15 male, 5 female; mean age, 34.3 years) when they returned for subsequent blood donations for viral load and B19-specific IgG and IgM. We did not examine the donors at their first visit because B19 antigen-positive blood was automatically disqualified and disposed. The mean duration of follow-up was 838 days (range, 101-1749 days). The results of sequential viral load testing for all donors are shown in Fig. 1A. In the first 6 months, we observed a rapid decline in plasma B19 DNA, which decreased continuously, but never became undetectable. Median plasma B19 viral loads for samples tested within every 6 months are shown in Fig. 1B. We analyzed the B19 antibody for all donors during the study period (Fig. 2A). For 9 donors (Donors 1-9) with both IgG and IgM, IgM became undetectable, while for 9 others (Donors 10-18), only B19 IgG was detected, presumably because B19 IgM had decreased to an undetectable level before the second visit. The remaining 2 donors (Donors 19 and 20) had B19 IgM– detectable until the last visit (at 729 and 743 days). Although the initial profile for B19 antibodies showed different patterns, once established, B19-specific IgG persisted in all 20 donors. Summaries for 3 representative cases corresponding to each of these patterns for IgM, IgG, and viral load are presented in Fig. 2B. Consistent with previous studies that suggest that B19 DNA may persist for a long period in immunocompetent individuals, we observed persistent B19 infection in healthy blood donors in the present longitudinal study. During the follow-up period, none of the 20 infected blood donors reported symptoms of B19 infection, although they retained high levels of B19 IgG and low viral load. Our data suggest that in healthy individuals, the B19 plasma viral load declines to below 10 IU per mL in approximately 1 year and to 10 IU per mL in approximately 2 years after an acute (high viremia) infection. The patterns of plasma B19 viral load observed in our study may be useful for identifying more suitable blood donors in circumstances where B19 NAT is unavailable. We encourage further studies with a larger sample size to validate these preliminary findings. Harumichi Matsukura, BS Sachiko Shibata, MT Yoshihiko Tani, MD, PhD Hirotoshi Shibata, MD, PhD Rika A. Furuta, PhD e-mail: furuta@osaka.bc.jrc.or.jp Japanese Red Cross Osaka Blood Center Osaka, Japan B A

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

Neonatal alloimmune thrombocytopenia caused by an antibody specific for a newly identified allele of human platelet antigen-7

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is a neonatal disorder characterized by maternal alloimmunization against fetal platelet (PLT) antigens inherited from the father. A healthy 30‐year‐old Japanese woman (Hit) gave birth to her second child after an uneventful pregnancy. Nine hours after birth, the infant presented with severe petechiae and a PLT count of 6 × 109/L.

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.