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Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) essentially affects respiratory organs and tissues. SARS-CoV-2 RNAemia is often associated with more severe cases of coronavirus disease 2019 (COVID-19) compared to cases without RNAemia. To determine the impact of the pandemic on transfusion medicine, particularly transfusion-related infection, we examined the frequency of blood donation with RNAemia, the viral RNA (vRNA) concentration, and any possibility of transfusion-transmitted infection (TTI) among transfusion recipients., Study Design and Methods: vRNA was examined in plasma/serum samples from 496 of 513 blood donors who reported having been infected with SARS-CoV-2 within 2 weeks of donation among a total of ca. 9.9 million blood donations in Japan between January 15, 2020, and December 31, 2021. The clinical course of patients transfused with the blood component containing vRNA was also examined., Results: vRNA was detected in 23 of 496 samples. The median period from blood donation to COVID-19 onset was 1 day in 16 RNAemia-positive donors. Most samples had vRNA concentrations below the limit of quantification. Three patients were transfused with either a packed red blood cell or platelet concentrate that tested positive for vRNA, showing no COVID-19 symptoms and testing negative for vRNA in post-transfusion blood., Conclusion: The rate of RNAemia was 4.6% among blood donors who were found to be infected with SARS-CoV-2 shortly after donation, and vRNA concentrations in their donated blood were extremely low. There was no evidence of TTI in the recipients transfused with RNAemia-positive blood components. TTI risk in SARS-CoV-2 is negligible., (© 2023 AABB.)

Background: Transfusion-transmitted bacterial infections (TTBIs) in Japan have been largely prevented due to a short shelf life of 3.5 days after blood collection for platelet concentrate (PC) and washed PCs (WPCs; PC in which 95% plasma is replaced by platelet additive solution)., Case Presentation: Case 1: In January 2018, a woman in her 50s with aplastic anaemia who received WPC transfusion and developed a fever the next day and Streptococcus dysgalactiae subspecies equisimilis (SDSE) was detected in the residual WPC. Case 2: In May 2018, a man in his 60s with a haematologic malignancy who received PC transfusion and developed chills during the transfusion. SDSE was detected in the patient's blood and residual PC. The contaminated platelet products were both manufactured from blood donated by the same donor. The multi-locus sequencing typing revealed that SDSE detected in case 1 was identical to that from case 2; however, whole blood subsequently obtained from the donor was culture negative., Conclusion: WPC and PC produced from two blood donated 106 days apart by the same donor were contaminated with SDSE of the same strain and both caused TTBIs. Safety measures should be considered regarding blood collection from a donor with a history of bacterial contamination., (© 2023 International Society of Blood Transfusion.)

Background: In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg)., Study Design and Methods: B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein-Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG 1 expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry., Results: Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells., Discussion: We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful., (© 2023 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: The potential risk and association of bovine leukemia virus (BLV) with human remains controversial as it has been reported to be both positive and negative in human breast cancer and blood samples. Therefore, establishing the presence of BLV in comprehensive human clinical samples in different geographical locations is essential., Result: In this study, we examined the presence of BLV proviral DNA in human blood and breast cancer tissue specimens from Japan. PCR analysis of BLV provirus in 97 Japanese human blood samples and 23 breast cancer tissues showed negative result for all samples tested using long-fragment PCR and highly-sensitive short-fragment PCR amplification. No IgG and IgM antibodies were detected in any of the 97 human serum samples using BLV gp51 and p24 indirect ELISA test. Western blot analysis also showed negative result for IgG and IgM antibodies in all tested human serum samples., Conclusion: Our results indicate that Japanese human specimens including 97 human blood, 23 breast cancer tissues, and 97 serum samples were negative for BLV., (© 2022. The Author(s).)

Background: Bacterial contamination in platelet concentrates (PCs) is a major problem in transfusion medicine. Contamination with Staphylococcus aureus is occasionally missed, even with cultural screening., Study Design and Methods: Donors implicated in S. aureus-contaminated PC were followed up. Skin and nasal swab specimens from six donors and S. aureus isolated from PCs related to these donors were subjected to multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) to determine the identity of bacteria. To evaluate the validity of the screening method using BacT/ALERT 3D, we spiked S. aureus and three other bacterial species as comparisons into PCs and investigated their growth pattern., Results: S. aureus was isolated from all nasal specimens and from the arm skin specimens of three donors with atopic dermatitis. In all cases, the S. aureus strains isolated from the PC and those from the nasal and skin specimens of the same donor showed concordant results using MLST and PFGE. In the spiking study, S. aureus showed irregular detectability over 24 to 48 h post-spike periods, whereas the three other bacterial species were detected in all culture bottles after a 24-h post-spike period., Discussion: The strain identity of S. aureus between donor and PC suggests that the contaminants were derived from those colonized in the donor. Furthermore, S. aureus yielded false-negative results using BacT/ALERT 3D., (© 2022 AABB.)

Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that an Hn-reactive clone protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix (the mixture of Hc, Hn, and Lc proteins)-reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix-reactive antibodies provided enhanced protection compared to the use of each antibody alone.

The risk of sepsis through bacterial transmission is one of the most serious problems in platelet transfusion. In processing platelet concentrates (PCs), several methods have been put into practice to minimize the risk of bacterial transmission, such as stringent monitoring by cultivation assays and inactivation treatment by photoirradiation with or without chemical agents. As another potential option, we applied a light-emitting diode (LED) with a peak emission wavelength of 265 nm, which has been shown to be effective for water, to disinfect PCs. In a bench-scale UV-LED exposure setup, a 10-min irradiation, corresponding to an average fluence of 9.2 mJ/cm2, resulted in >2.0 log, 1.0 log, and 0.6 log inactivation (mean, n = 6) of Escherichia coli, Staphylococcus aureus, and Bacillus cereus, respectively, in non-diluted plasma PCs. After a 30-min exposure, platelet counts decreased slightly (18 ± 7%: mean ± SD, n = 7); however, platelet surface expressions of CD42b, CD61, CD62P, and PAC-1 binding did not change significantly (P>0.005), and agonist-induced aggregation and adhesion/aggregation under flow conditions were well maintained. Our findings indicated that the 265 nm UV-LED has high potential as a novel disinfection method to ensure the microbial safety of platelet transfusion., Competing Interests: The authors have declared that no competing interests exist.

Background and Objectives: Although HLA-eliminated platelets can facilitate transfusions to patients possessing HLA antibodies, no such products are currently available commercially perhaps because the platelet collection rate is not yet economically viable. We have improved this process' efficiency by employing a hollow-fibre system at the last step of the production process after an acid and a reaction buffer have been washed out conventionally by centrifugation., Materials and Methods: HLA-eliminated platelets were prepared via four distinct steps: chilled on ice, treated with an acid solution, diluted and finally washed using the hollow-fibre system. The efficiency of this platelet recovery process was determined. The resulting products' platelet characteristics, including a capacity for HLA expression, were evaluated in vitro and compared in detail to their corresponding originals., Results: The average efficiency of platelet recovery was 91%. Although the expression levels of CD62P, a molecular marker for platelet activation, were approximately threefold higher on new platelets than on the original platelets, their HLA expression levels were lower. The phagocytosis assay, with monoclonal antibodies and cognate HLA antibody-containing sera, suggested that HLA-ABC molecules on the cell surface were sufficiently removed. The platelet functions, including the agonist-induced aggregability and adherence/aggregability of the collagen-coated plates under certain conditions, were conserved and not significantly different from the original ones., Conclusion: We propose a novel preparation system for producing HLA-eliminated platelets without centrifugation, which ensures a highly efficient, and therefore, much more economical method of platelet recovery that also retains their key functionality., (© 2019 International Society of Blood Transfusion.)

The risk of transfusion-transmitted infection (TTI) for severe fever with thrombocytopenia syndrome virus (SFTSV) is a concern because person-to-person transmission resulting from contact with SFTSV-contaminated blood has been reported. To obtain information regarding the risk of TTI-SFTSV, antibody testing was performed for blood samples donated in an severe fever with thrombocytopenia syndrome-endemic area in Japan. No antibody-positive samples were detected among 3990 samples. This finding suggested that there were few cases of SFTSV infection among donors and that the risk of TTI-SFTSV was also estimated low in Japan., (© 2018 International Society of Blood Transfusion.)

Background: In previous studies, we demonstrated that the basophil activation test, which is performed using patient blood and the supernatants from transfused blood components, was able to elucidate not only the causative relationship between allergic transfusion reactions and the transfusion but also the mechanisms behind allergic transfusion reactions. However, for a large number of allergic transfusion reactions, patients are in a state of myelosuppression, and the basophil activation test cannot be performed for these patients because there are insufficient numbers of peripheral blood basophils., Study Design and Methods: To overcome this obstacle, we developed a passive immune basophil activation test, in which patient plasma and residually transfused blood are used as the patient's sources of immunoglobulin E and allergen, respectively, whereas healthy volunteer basophils serve as the responder cell source. The passive immune basophil activation test was performed for two patients who had severe allergic transfusion reactions, using supernatants of the residual platelet concentrates and the patients' own immunoglobulin E., Results: There were no differences in either surface immunoglobulin E or activation in response to allergens between untreated basophils and so-called quasi-basophils, in which immunoglobulin E was replaced by a third party's immunoglobulin E. In these patients, the supernatants of the residual platelet concentrates exclusively activated basophils in response to quasi-basophils onto which the patients' immunoglobulin E, but not a third party's immunoglobulin E, was bound., Conclusion: The passive immune basophil activation test may help clarify the causal relationship between allergic transfusion reactions and transfused blood, even when patients experience myelosuppression., (© 2017 AABB.)

Background: 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., Study Design and Methods: The amount of mitochondrial DAMPs was determined as an index of total copy numbers of mitochondrial DNA (mtDNA), including mtDNA itself and free mitochondria, using quantitative real-time polymerase chain reaction. To examine whether neutrophils, monocytes, and basophils were activated by mitochondrial DAMPs in vitro, an in vitro whole blood cell culture assay was performed., Results: In blood components associated with NHTRs, the mean total mtDNA concentration was highest in PCs followed in order by fresh-frozen plasma and red blood cells. The amount of mtDNA in NHTR PCs was higher than that in control PCs without NHTRs. The mitochondrial DAMPs present in NHTR PCs was high enough to activate neutrophils, monocytes, and basophils, when costimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or HLA antibodies., Conclusion: PLT-derived mitochondrial DAMPs are candidate risk factors for the onset of NHTRs., (© 2016 AABB.)

Background and Objectives: At Japanese Red Cross (JRC) Blood Centers, all donated blood is screened for hepatitis C virus (HCV) by serological and nucleic acid amplification testing. Donor plasma that tested reactive for anti-HCV by serological test is disqualified even if the donor tests negative for HCV RNA. These test results reflect both true-positive results because of past HCV infection and false-positive results because the cross-reactivity of plasma IgG, which current testing methods are unable to distinguish. To characterize these antibody test results, we examined the neutralizing activity of these plasma samples., Material and Methods: Donor plasma samples that tested reactive for anti-HCV by serological test but negative for HCV RNA (n = 43) were analysed for determining their neutralizing activities measured by the inhibition of the cellular entry of pseudoparticles harbouring HCV envelope glycoproteins (HCVpp)., Results: Strong and broad neutralizing activities against HCVpp entry similar to the samples that tested reactive for anti-HCV serological test and positive for HCV RNA (considered to be derived from individuals with chronic HCV infection) were observed in three of 43 plasma samples from donors who tested anti-HCV reactive but HCV RNA negative., Conclusion: By examining the neutralizing activities of plasma samples, we identified individuals with a past HCV infection from those in whom we were unable to confirm HCV infection according to the current testing algorithms of JRC, which do not perform anti-HCV confirmatory tests., (© 2015 International Society of Blood Transfusion.)

Background: In Japanese Red Cross (JRC) blood centers, blood collected from donors with serum alanine aminotransferase (ALT) levels of more than 60 U/L are disqualified even if serologically negative for transfusion-transmitted infections (TTIs). To assess potential risks of TTIs in plasma with elevated serum ALT levels in the current donor screening program of the JRC, we conducted a metagenomic analysis (MGA) of virome profiles in the plasma of blood donors with or without elevated serum ALT levels., Study Design and Methods: Based on serum ALT levels, donors were classified into three groups: "high," more than 79 U/L; "middle," 61 to 79 U/L; and "low," less than 61 U/L. We individually analyzed 100 plasma samples from each group by MGA, employing shotgun sequencing. Viral sequences detected using MGA were partly confirmed using real-time polymerase chain reaction (PCR)., Results: Donors with high and middle ALT levels were significantly younger than those with low ALT levels, and more than 90% were males. Herpesviridae, Anelloviridae, Picornaviridae, and Flaviviridae sequences were identified in plasma samples, and their distribution and frequency were not significantly different among the three groups., Conclusion: The serum ALT test may be unsuitable for monitoring for additional risks of TTIs in blood donors who were negative for typical TTIs using serologic and nucleic acid tests. Although MGA is less sensitive than PCR, it remains the best technology to detect known viruses in these donors., (© 2015 AABB.)

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.

Background: Recently, Japanese Red Cross blood centers have changed the confirmatory test method from an indirect immunofluorescence (IF) technique to Western blotting (WB) for antibodies against human T-cell leukemia virus Type 1 (HTLV-1). In this study, these HTLV-1 tests were assessed using another sensitive method, that is, a luciferase immunoprecipitation system (LIPS), to identify a better confirmatory test for HTLV-1 infection., Study Design and Methods: Plasma samples from 54 qualified donors and 114 HTLV-1 screening-positive donors were tested by LIPS for antibodies against HTLV-1 Gag, Tax, Env, and HBZ recombinant proteins. The donors were categorized into six groups, namely, (Group I) qualified donors, screening positive; (Group II) IF positive; (Group III) IF negative; (Group IV) WB positive; (Group V) WB negative; and (Group VI) screening positive in the previous blood donation, but WB-indeterminate during this study period., Results: In Groups II and IV, all plasma samples tested positive by LIPS for antibodies against Gag and Env proteins. In Group V, all samples tested negative by LIPS, whereas some Group III samples reacted with single or double antigens in LIPS. In Group VI, the LIPS test identified a donor with suspected HTLV-1 infection. The first case of a blood donor with plasma that reacted with HBZ was identified by LIPS., Conclusion: Reevaluation of the current HTLV-1 screening method using the LIPS test showed that both confirmatory tests had similar sensitivity and specificity only when WB indeterminate results were eliminated. LIPS is a promising method for detecting and characterizing HTLV-1 antibodies., (© 2014 AABB.)

Human neutrophil antigen (HNA)-typed granulocyte panels are widely used to screen for the presence of HNA antibodies and to determine antibody specificity. Many laboratories screen donors for HNA genotypes using low-throughput methods such as allele-specific polymerase chain reaction (PCR), PCR-restriction fragment-length polymorphism, and multiplex PCR. In the present study, we used a high-resolution melting (HRM) analysis to determine HNA genotypes. For the HRM analysis, purified genomic DNA samples were amplified via PCR with HNA-specific primers. Nucleotide substitutions in genes encoding HNAs were differentiated on the basis of the HRM curves, and the results of HRM and DNA sequencing analyses were determined to be in complete agreement. The gene frequency of HNA-1a, -1b, -1c, -3a, -3b, -4a, -4b, -5a, and -5b in the Japanese population was consistent with the previous reports. Our results suggest that HRM analysis can be used for genotyping HNA antigens determined by single nucleotide substitutions.

Background: Antibodies against human platelet antigens (HPA) are a cause of thrombocytopenia. Detection of rare anti-HPA antibodies using platelet preparations is difficult and would be improved by an alternative method that does not require platelets. In the present study, we describe the establishment of cell lines that stably express specific HPA associated with integrin α2β1 and the application of these cell lines for detecting anti-HPA-5a and anti-HPA-5b antibodies., Materials and Methods: Complementary DNA of the integrin α2 variants HPA-5b, -13b and -18b were individually transfected into K562 cells using retroviral vectors. Expression of integrin α2 was confirmed by flow cytometric analysis, immunoprecipitation and western blotting analysis. To verify whether the cell line panel was suitable for clinical diagnosis, we analysed its properties using monoclonal antibody-specific immobilisation of platelet antigens (MAIPA) and well-characterised serum samples., Results: Exogenous integrin α2 expression was observed in the transfected cells for over 6 months. The cell line panel specifically detected previously characterised anti-HPA-5a and anti-HPA-5b antisera. No reactivity was observed with control sera, including normal sera and HLA antisera., Discussion: We successfully established a cell line panel to facilitate the sensitive and reliable detection of anti-HPA-5a and anti-HPA-5b antibodies.

Background: The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level., Study Design and Methods: We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes., Results: We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses., Conclusion: We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system., (© 2013 American Association of Blood Banks.)

The xenotropic murine leukemia virus-related virus (XMRV) was first described as a novel human gammaretrovirus in prostate tumor tissues and was reported to be found in blood, suggesting the possibility of XMRV transmission via blood transfusion. The gag and env regions of the XMRV proviral DNA that were detected 1,030 blood samples collected from the greater Tokyo area were examined by real-time PCR analysis. However, XMRV infection was not found in the samples; this suggested that the risk of XMRV transmission via transfusion is very low in Japan.

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., Methods: Wild-type β3, HPA-1b, -6b, -7b and -7 variant cDNA as well as wild-type αIIb and HPA-3b cDNA were individually co-transduced with wild-type αIIb and β3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test., Results and Conclusion: Of the 12 sera containing HPA-1a (n = 2), HPA-3a (n = 6), HPA-6b (n = 3) or HPA-7 variant (n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA-3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay., (© 2011 The Authors. Transfusion Medicine © 2011 British Blood Transfusion Society.)

Background: The 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., Results: Antibody 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., Conclusion: Our 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.

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.

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 x 10(9)/L., Study Design and Methods: To elucidate the maternal cause of NAIT in the infant, serologic and genetic studies, including PLT genotyping and sequence-based analysis, were conducted. Additionally, serologic screening for the new PLT antigen was performed., Results: Serum from the NAIT infant's mother contained antibodies directed against a human PLT antigen (HPA) of the newborn. Using five-cell-lineage flow cytometry, we localized the antigen to a PLT glycoprotein (GP). Subsequent monoclonal antibody immobilization of PLT antigen assay and PLT immunofluorescence inhibition experiments localized the antigen to the GPIIIa subunit of the GPIIb/IIIa complex. GPIIIa localization was confirmed by sequence-based typing studies, which identified a 1297C>T (407proline>serine substitution) mutation on the ninth exon of the GPIIIa gene. This mutation identified the third allele of HPA-7. Anti-Hit(a) reacted with mutated GPIIIa-transfected cells but not with stable transfectants expressing wild-type GPIIIa. Serologic screening for Hit(a) in the Japanese population revealed a phenotypic frequency of approximately 0.0015., Conclusions: We identified a new third allele of HPA-7, which is characterized by a 1297C>T mutation in the GPIIIa gene. This 1297C>T allele was found in 0.15% of the Japanese population. An antibody against this antigen could be the cause of severe NAIT.

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes adult T -cell leukemia (ATL) but the expression of HTLV-1 is strongly suppressed in the peripheral blood of infected people. However, such suppression, which may explain the long latency in the development of ATL, is readily reversible, and viral expression resumes quickly with ex vivo culture of infected T -cells. To investigate the mechanism of in vivo -specific transcriptional suppression, we established a mouse model in which mice were intraperitoneally administered syngeneic EL4 T -lymphoma cells transduced with a recombinant retrovirus expressing a GFP-Tax fusion protein, Gax, under the control of the HTLV-1 enhancer (EL4-Gax)., Results: Gax gene transcription was silenced in vivo but quickly up-regulated in ex vivo culture. Analysis of integrated Gax reporter gene demonstrated that neither CpG methylation of the promoter DNA nor histone modification was associated with the reversible suppression. ChIP-analysis of LTR under suppression revealed reduced promoter binding of TFIIB and Pol-II, but no change in the binding of CREB or CBP/p300 to the viral enhancer sequence. However, the expression of TORC2, a co-activator of CREB, decreased substantially in the EL4-Gax cells in vivo, and this returned to normal levels in ex vivo culture. The reduced expression of TORC2 was associated with translocation from the nucleus to the cytoplasm. A knock-down experiment with siRNA confirmed that TORC2 was the major functional protein of the three TORC-family proteins (TORC1, 2, 3) in EL4-Gax cells., Conclusion: These results suggest that the TORC2 may play an important role in the in vivo -specific transcriptional control of HTLV-1. This study provides a new model for the reversible mechanism that suppresses HTLV-1 expression in vivo without the DNA methylation or hypoacetylated histones that is observed in the primary cells of most HTLV-1 -infected carriers and a substantial number of ATL cases.

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.

Background: Antibodies to neutrophil antigens have been implicated in neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. Most often, neutrophil-specific antibodies are directed toward human neutrophil antigen (HNA)-1 (Fcgamma receptor 3b) and HNA-2a (CD177) in these disorders., Study Design and Methods: To detect the alloantibodies in the serum samples, a panel of cell lines was established in which the HNA-1a, HNA-1b (polymorphisms of HNA-1), or HNA-2a gene was transduced with a retrovirus vector to confer stable transgene expression in K562 cells that exhibited low background reactivity to human serum samples obtained from healthy donors in flow cytometric analysis., Results: It was shown that several well-characterized human serum samples containing antibodies against HNA-1a, -1b, and -2a were unambiguously identified by the established panel cell lines and observed a lower background reactivity and longer shelf life of the K562 panel cell lines compared with isolated neutrophils, which have been used for the cell panel to identify antibodies against HNA in human serum samples., Conclusion: These results indicate that the K562 panel cell lines provide a good panel for detecting HNA-reactive neutrophil antibodies in human serum samples.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-mediated membrane fusion occurs as a sequence of events that is triggered by CD4 binding to the Env gp120 subunit. In this study, we analyzed the dynamics of Env-mediated membrane fusion at the single-cell level using fluorescent fusion proteins and confocal laser fluorescent microscopy. Either enhanced cyan or yellow fluorescent protein (CFP and YFP, respectively) was fused to the end of the cytoplasmic regions of the HIV-1 receptors (CD4 and CCR5) and Env proteins. Real-time imaging of membrane fusion mediated by these recombinant proteins revealed that the kinetics of fusion in our system was faster than that previously reported. Analysis of the receptor interaction by fluorescence resonance energy transfer (FRET) at the single-cell level demonstrated a tendency for oligomerization of CD4-CD4, but not of CD4-CCR5, in the absence of Env-expressing cells. However, when Env-expressing cells attached to the receptor cells, FRET produced by CD4-CCR5 interaction was increased; the FRET intensity began to decline before the formation of the fusion pore. These changes in FRET may represent the temporal association of these receptors, triggered by gp120 binding, and their dissociation during the formation of the fusion pore. In addition, the FRET analysis of receptor interactions in the presence of fusion inhibitors showed that not only inhibitors acting on CCR5 but also the gp41-derived peptide T-20 interfered with CD4-CCR5 interaction during fusion. These data suggest that T-20 could affect the formation of Env-receptors complexes during the membrane fusion.

G protein-coupled receptor CCR5 is the main coreceptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1), and various small-molecule CCR5 antagonists are being developed to treat HIV-1 infection. It has been reported that such CCR5 antagonists, including TAK-779, bind to a putative binding pocket formed by transmembrane domains (TMs) 1, 2, 3 and 7 of CCR5, indicating the importance of the conformational changes of the TMs during virus entry. In this report, using a single-round infection assay with human CCR5 and its substitution mutants, we demonstrated that a new CCR5 antagonist, TAK-220, shares the putative interacting amino acid residues Asn252 and Leu255 in TM6 with TAK-779 but also requires the distinct residues Gly163 and Ile198 in TMs 4 and 5, respectively, for its inhibitory effect. We suggested that, together with molecular models of the interactions between the drugs and CCR5, the inhibitory activity of TAK-220 could involve direct interactions with amino acid residues in TMs 4, 5, and 6 in addition to those in the previously postulated binding pocket. The possible interaction of drugs with additional regions of the CCR5 molecule would help to develop a new small-molecule CCR5 antagonist.

Pluripotent, self-renewing, hematopoietic stem cells are considered good targets for gene modification to treat a wide variety of disorders. However, as many genes are expressed in a stage-specific manner during the course of hematopoietic development, it is necessary to establish a lineage-specific gene expression system to ensure the proper expression of transduced genes in hematopoietic stem cells. In this study, we constructed a VSV-G-pseudotyped, human immunodeficiency virus type 1-based, self-inactivating lentivirus vector that expressed green fluorescent protein (GFP) under the control of the human CD41 (glycoprotein 2b; GP2b) promoter; this activity is restricted to megakaryocytic lineage cells. The recombinant virus was used to infect human peripheral blood CD34+ (hematopoietic stem/progenitor) cells, and lineage-specific gene expression was monitored with GFP measurements. The analysis by FACS determined that GFP expression driven by the GP2b promoter was restricted to megakaryocytic progenitors and was not present in erythrocytes. Furthermore, in the hematopoietic colony-forming assay, GFP expression was restricted to colony-forming units-megakaryocyte (CFU-Meg) colonies under the control of the GP2b promoter, whereas all myeloid colonies (burst-forming units-erythroid, colony-forming units-granulocyte-macrophage, and CFU-Meg) expressed GFP when the transgene was regulated by the cytomegalovirus promoter. These results demonstrated lineage-specific expression after gene transduction of hematopoietic stem cells. The application of this vector system should provide a useful tool for gene therapy to treat disorders associated with megakaryocyte (platelet) dysfunction.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.

To study the involvement of immune responses against Tax of human T-cell leukemia virus type 1 (HTLV-1) in the growth of and gene suppression in Tax-expressing tumor cells in vivo, we established a model system involving C57BL/6J mice and a syngeneic lymphoma cell line, EL4. When mice were immunized by DNA-based immunization with Tax expression plasmids, solid tumor formation upon subcutaneous inoculation of EL4 cells expressing green fluorescent protein-fused Tax (Gax) under the control of the HTLV-1 enhancer was strongly inhibited, and in vitro analysis showed that DNA immunization elicited cytotoxic T-lymphocyte (CTL) responses but not production of antibodies to Tax protein. Since EL4/Gax cells inoculated into DNA-immunized mice were not completely eradicated but were maintained as small solid tumors for a long period, there appeared to be a certain equilibrium between CTL activity and the growth of Gax-expressing cells. With such a balance, expression of the Gax gene in EL4/Gax cells was strongly suppressed. These results suggested that gene expression under the control of the HTLV-1 long terminal repeat and Tax is silenced in vivo, resulting in an equilibrium between viral expression and the host immune system. Such a balance would represent a status of persistent infection by HTLV-1 in virus-infected individuals during the latency period.

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 beta-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 (IC(50)s) for membrane fusion and viral replication were 0.87 +/- 0.11 and 1.4 +/- 0.1 nM, respectively. These values corresponded well to the IC(50) for (125)I-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 IC(50)s 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.

Using an inhibitory synthetic peptide (DP-178) from HIV-1 gp41, we have trapped HIV-1 envelope glycoprotein (Env) undergoing conformational changes during virus entry. Our data show that DP-178 binds gp41 and inhibits Env-mediated membrane fusion after gp120 interacts with cellular receptors, indicating that conformational changes involving the coiled coil domain of gp41 are required for entry. Capture of this fusion-active conformation of Env provides insights into the early events leading to Env-mediated membrane fusion.

The 5'-RU5 portion of human T-lymphocyte virus type I (HTLV-I) long terminal repeat (LTR) had been reported to contain cis-acting elements for the controlled viral gene expression by the rex gene product. In this study, the human immunodeficiency virus type I (HIV-1) Rev protein was found to enhance gene expression, acting through the 5'-RU5 portion of HTLV-I, while the Rex-responsive element (RxRE)-mediated activation by Rev was reconfirmed to be negative. This positive action of HIV-1 Rev on HTLV-I gene expression seemed to be distinct from the widely accepted Rex or Rev function to facilitate the nuclear export of RxRE-containing unspliced viral mRNAs, since a trans-dominant, nuclear export-deficient mutant (RevM10) still retained the RU5-mediated effector function. Analyses of the functional aspects of Tat/Rev fusion proteins on the HTLV-I RU5 suggested a specific interaction of Rev and RU5, but lacked evidence for the binding of Rev to the RU5 at the RNA level. These results suggest an answer to the controversy regarding a Rex-like function occasionally observed with HIV-1 Rev and its related proteins. It may also be suggested that particular care should be taken when such a trans-dominant Rev mutant is considered to be used as a genetic therapy against HIV-I infection, in individuals infected with both HIV-I and HTLV-1.

A Gag capsid mutant of human immunodeficiency virus type 1 (HIV-1) designated C6b was biologically and biochemically characterized with respect to its ability to suppress the replication of wild-type (wt) HIV. The C6b efficiently interfered with the replication of wt HIV-1 in the cleavage of Gag precursor, and also in the early replication process before or during viral DNA synthesis after viral penetration. The C6b Gag appeared to be unable to form chimeric multimers with HIV-2 Gag and failed to inhibit the replication of wt HIV-2.

In-frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV-1) gag gene, and potentials of the mutants to suppress the replication of wild-type HIV-1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV-1 replication completely in single-round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV-1 reported to date, and to be effective at both early and late viral replication phases. T-cells, which are engineered to express the C6b Gag in response to HIV-1 infection, were perfectly resistant to HIV-1.

Rev, a major regulatory protein of human immunodeficiency virus type 1, has been demonstrated to shuttle between the nucleus and cytoplasm of infected cells. The fate of the Rev protein in living cells was evaluated by pulse-chase experiments using a transient Rev expression system. Sixteen hours after chasing with unlabelled amino acids, 45% of the labelled Rev was still present, which clearly indicates a long half-life of Rev in living cells. A Rev mutant which is deficient in the ability to migrate from the nucleus to the cytoplasm was degraded more slowly than the wild-type Rev protein. As well, another Rev mutant protein, which lacks a functional nucleolar targeting signal (NOS) and is unable to enter the cell nucleus, was rapidly degraded and undetectable 16 h after chasing. Nuclear-nucleolar targeting properties provided by a divergent NOS from a related retrovirus, which was used to substitute for the NOS of Rev, increased the intracellular half-life of this Rev mutant. Moreover, coexpression of an intracellular anti-Rev single-chain antibody (SFv), which has been shown to interfere with the nuclear translocation of Rev, accelerated the degradation of the wild-type Rev protein. Differential degradation of Rev in the nucleus and cytoplasm may play a critical role in determining and maintaining different stages of human immunodeficiency virus type 1 infection, in conjunction with the shuttling properties of the Rev protein.

HTLV-I generally integrates at least one full-length copy in adult T-cell leukemia (ATL) cells. A group of patients without full-length provirus have a unique conserved truncation of the provirus which retains env-pX-3'LTR. Tumor cells of a patient from this group were genetically analyzed. Analysis of the 5' and 3' cellular flanking region adjacent to the provirus suggest that the defective provirus was integrated immediately downstream of a promoter of an unknown cellular gene. The activity of the promoter was weak but was responsive to Tax-like HTLV-I LTR. The provirus may have utilized it as a substitute for the 5'LTR and thus 3'LTR may have become an alternative promoter for the cellular gene, which may give similar viral-cellular interactions to that of general cases with full-length proviruses. Surprisingly, the 3' cellular flanking region which is thought to be controlled originally by the promoter is constitutively expressed specifically in an HTLV-I producing ATL cell line HUT1O2G, in which the corresponding region is not modified by provirus. The detection of this HTLV-I-induced transcript provides a probe to find an HTLV-I inducible unknown cellular gene that may be related to the pathogenesis of ATL.

Structural gene expression of human immunodeficiency virus type 1 (HIV-1) requires a viral regulatory protein, Rev transactivator. We investigated Rev-dependency of HIV-1 gene expression by various reporter systems. Expression of unspliced and single-spliced viral mRNAs was demonstrated to be differentially dependent on the Rev function. This difference of Rev-dependency was found not to be determined by cis-elements in gag, pol, and env coding sequences reported so far, and was lost when the reporter constructs containing minimum elements for Rev-responsiveness such as splice signals and rev responsive element were used for experiments. These findings indicated that the fundamental structure of HIV-1 mRNA was critical for the differential regulation of gene expression by Rev transactivator.

Applications of transdominant mutants of human immunodeficiency virus type 1 (HIV-1) regulatory proteins, especially Rev mutant, have been attempted for gene therapy against AIDS, because the Rev protein is essential for viral replication. We have previously reported that a mutant Rev protein (dRev) lacking its nucleolar targeting signal remained out of nuclei in expressed cells and strongly inhibited the function of Rev. To investigate the effects of dRev on HIV-1 replication, we established several dRev-expressing human cell lines with two different vector systems and examined virus production in these cells. An HIV-1-derived vector containing drev cDNA was constructed and introduced into CD4-positive HeLa cells and cells of the human T-cell line CCRF-CEM (CEM). In dRev-expressing HeLa cells, virus replication, syncytium formation, and cell death caused by HIV-1 infection were remarkably suppressed, and the same vector also conferred a resistant phenotype on CEM cells. The production was also suppressed in CEM cells containing the drev gene driven by a cytomegalovirus promoter. In addition, we found that dRev did not cause nucleolar dysfunction in a transient assay, in contrast to other transdominant mutants and wild-type Rev. Since dRev cannot migrate into the nuclei, it is expected not to interfere with nuclear/nucleolar functions of the host cell. We conclude that dRev is one promising candidate as an antiviral molecule for gene therapy against AIDS.