RESUMO
BACKGROUND: Alloantibodies against human platelet antigen (HPA)-15 are sometimes detected in patients with platelet transfusion refractoriness (PTR); however, little is known about their impact on PTR. STUDY DESIGN AND METHODS: Two patients who possessed HPA-15 alloantibodies (Patient 1, anti-HPA-15b; Patient 2, anti-HPA-15a) and human leukocyte antigen (HLA) antibodies were enrolled. The efficacy of HPA-15-compatible vs -incompatible platelet transfusion was compared by focusing on ABO- and HLA-matched transfusions on the basis of the 24-hour corrected count increment (CCI-24 hours) for platelets. The titers of HPA-15 antibodies in the patients' sera were also monitored. RESULTS: The patients received 71 and 12 ABO-compatible, HLA-matched platelet transfusions, respectively, during the monitoring periods. Among these transfusions, CCI-24 hours could be calculated in 27 and 10 transfusions, respectively, and the HPA-15 genotype of the donors was determined. There were no significant differences in the CCI-24 hours between the HPA-15 compatible and incompatible transfusions in both patients (P = .30 and .56, respectively, Mann-Whitney U test). There was no significant change in the HPA-15b antibody titer in Patient 1 during the monitoring period, while the HPA-15a antibody level in Patient 2 was undetectable at the end of the monitoring period, although the titer was low at the beginning. CONCLUSION: The efficacy of HPA-15-incompatible platelet transfusions was not necessarily inferior to that of HPA-15 compatible ones. Although the case number was limited, our results suggest that HPA-15 antibodies do not have a significant impact on the effects of platelet transfusion.
Assuntos
Antígenos CD/imunologia , Antígenos de Plaquetas Humanas/imunologia , Isoanticorpos/sangue , Leucemia Mieloide Aguda/imunologia , Síndromes Mielodisplásicas/imunologia , Proteínas de Neoplasias/imunologia , Transfusão de Plaquetas , Idoso , Antígenos CD/sangue , Incompatibilidade de Grupos Sanguíneos , Feminino , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/imunologia , Humanos , Isoanticorpos/imunologia , Japão , Leucemia Mieloide Aguda/sangue , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Proteínas de Neoplasias/sangue , Projetos Piloto , Transfusão de Plaquetas/efeitos adversos , Estatísticas não ParamétricasRESUMO
BACKGROUND AND OBJECTIVES: To detect HPA-15 alloantibodies, we previously developed a human platelet antigen 15 (HPA-15)-expressing cell line-based modified rapid monoclonal antibody immobilization of platelet antigen (CL-MR-MAIPA) assay. In this study, the protocol was modified for easier performance by introducing the mixed-passive haemagglutination (MPHA) principle. MATERIAL AND METHODS: In total, 20 samples that tested negative for HPA alloantibodies and eight that tested positive for HPA-15 alloantibodies (two and six positive for HPA-15a and HPA-15b antibodies, respectively) by CL-MR-MAIPA assay were used in this study. HPA-15 cell lines were incubated with serum/plasma and then solubilized. The lysate was transferred to a round-bottom well, which was coated with anti-human CD109 monoclonal antibodies. After incubation and repeated washings, sheep red blood cells, coated with anti-human IgG, were added to the wells. Haemagglutination was assessed the next day. RESULTS: The proposed cell line-based immune complex capture-dependent mixed-passive haemagglutination (CL-IC-MPHA) assay consisted of four steps, but required only 2 h to perform, except for overnight incubation for haemagglutination. Two HPA-15a alloantibody samples were reactive only for HPA-15a cells, and six HPA-15b alloantibody samples were reactive only for HPA-15b cells with the CL-IC-MPHA assay. The 20 samples that tested negative for HPA alloantibodies did not react with HPA-15a or HPA-15b cells. These data indicated that the CL-IC-MPHA assay was highly specific and sensitive. Unfortunately, the CL-IC-MPHA assay's analytic sensitivity was twofold to eightfold lower than that of the CL-MR-MAIPA assay. CONCLUSION: A novel, easy-to-perform protocol was successfully developed to detect HPA-15 alloantibodies with high specificity and sensitivity.
Assuntos
Antígenos CD/imunologia , Testes de Hemaglutinação/métodos , Técnicas de Imunoadsorção/normas , Proteínas de Neoplasias/imunologia , Antígenos CD/genética , Antígenos CD/metabolismo , Plaquetas/imunologia , Linhagem Celular , Células Cultivadas , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Testes de Hemaglutinação/normas , Humanos , Isoanticorpos/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Plaquetas/ultraestrutura , DNA Mitocondrial/análise , Mitocôndrias/genética , Transfusão de Plaquetas/efeitos adversos , Reação Transfusional/etiologia , Basófilos/imunologia , Segurança do Sangue , DNA Mitocondrial/genética , Humanos , Fenômenos do Sistema Imunitário , Mediadores da Inflamação , Japão , Monócitos/imunologia , Neutrófilos/imunologiaRESUMO
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.
Assuntos
Frequência do Gene , Técnicas de Genotipagem , Isoantígenos/genética , Neutrófilos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Povo Asiático , Feminino , Humanos , Japão , MasculinoRESUMO
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.
Assuntos
Alérgenos/imunologia , Hipersensibilidade Imediata/imunologia , Reação Transfusional/imunologia , Basófilos/efeitos dos fármacos , Basófilos/imunologia , Basófilos/metabolismo , Antígenos de Grupos Sanguíneos/imunologia , Proteínas Sanguíneas/imunologia , Dasatinibe/farmacologia , Humanos , Hipersensibilidade Imediata/diagnóstico , Transfusão de Plaquetas/efeitos adversos , Inibidores de Proteínas Quinases/farmacologia , Reação Transfusional/diagnósticoRESUMO
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.
Assuntos
Alanina Transaminase/sangue , Doadores de Sangue , Segurança do Sangue , Genoma Viral , Metagenômica/métodos , Plasma/virologia , Adolescente , Adulto , Idoso , Anelloviridae/genética , Anelloviridae/isolamento & purificação , Patógenos Transmitidos pelo Sangue , DNA Viral/genética , Feminino , Flaviviridae/genética , Flaviviridae/isolamento & purificação , Herpesviridae/genética , Herpesviridae/isolamento & purificação , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Picornaviridae/genética , Picornaviridae/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise de Sequência de RNA , Viremia/enzimologia , Viremia/virologia , Adulto JovemRESUMO
Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire vif from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116(th) position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus.
Assuntos
HIV-1/fisiologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral , Desenvelopamento do Vírus , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Ordem dos Genes , Vetores Genéticos/genética , Células HEK293 , Transcriptase Reversa do HIV/metabolismo , Humanos , Mutação , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Fatores de TempoRESUMO
Human immunodeficiency virus type 1 (HIV-1) hardly replicates in Old World monkeys. Recently, a mutant HIV-1 clone, NL-DT5R, in which a small part of gag and the entire vif gene are replaced with SIVmac239-derived ones, was shown to be able to replicate in pigtail monkeys but not in rhesus monkeys (RM). In the present study, we found that a modified monkey-tropic HIV-1 (HIV-1mt), MN4-5S, acquired the ability to replicate efficiently in cynomolgus monkeys as compared with the NL-DT5R, while neither NL-DT5R nor MN4-5S replicated in RM cells. These results suggest that multiple determinants may be involved in the restriction of HIV-1 replication in macaques, depending on the species of macaques. The new HIV-1mt clone will be useful for studying molecular mechanisms by which anti-viral host factors regulate HIV-1 replication in macaques.
Assuntos
HIV-1/genética , Especificidade de Hospedeiro/fisiologia , Macaca fascicularis/virologia , Replicação Viral , Animais , Infecções por HIV , Humanos , Depleção Linfocítica , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , ViremiaRESUMO
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously reported that efficient replication of HIV-1 in CM cells was achieved after we replaced the loop between alpha-helices 6 and 7 (L6/7) of the capsid protein (CA) with that of SIVmac239 in addition to the loop between alpha-helices 4 and 5 (L4/5) and vif. This virus (NL-4/5S6/7SvifS) was supposed to escape from host restriction factors cyclophilin A, CM TRIM5 alpha, and APOBEC3G. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. RESULTS: By long-term cultivation of human CEMss cells infected with NL-4/5S6/7SvifS, we succeeded in rescuing the impaired replicative capability of the virus in human cells. Sequence analysis of the CA region of the adapted virus revealed a G-to-E substitution at the 116 th position of the CA (G116E). Introduction of this substitution into the molecular DNA clone of NL-4/5S6/7SvifS indeed improved the virus' replicative capability in human cells. Although the G116E substitution occurred during long-term cultivation of human cells infected with NL-4/5S6/7SvifS, the viruses with G116E unexpectedly became resistant to CM, but not human TRIM5 alpha-mediated restriction. The 3-D model showed that position 116 is located in the 6th helix near L4/5 and L6/7 and is apparently exposed to the protein surface. The amino acid substitution at the 116 th position caused a change in the structure of the protein surface because of the replacement of G (which has no side chain) with E (which has a long negatively charged side chain). CONCLUSIONS: We succeeded in rescuing the impaired replicative capability of NL-4/5S6/7SvifS and report a mutation that improved the replicative capability of the virus. Unexpectedly, HIV-1 with this mutation became resistant to CM TRIM5 alpha-mediated restriction.
Assuntos
Substituição de Aminoácidos/genética , Proteínas de Transporte/imunologia , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Proteínas/imunologia , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Análise Mutacional de DNA , Proteína do Núcleo p24 do HIV/metabolismo , Humanos , Macaca fascicularis , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , Linfócitos T/virologia , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees but not cynomolgus or rhesus monkeys while simian immunodeficiency virus isolated from macaque (SIVmac) readily establishes infection in those monkeys. Several HIV-1 and SIVmac chimeric viruses have been constructed in order to develop an animal model for HIV-1 infection. Construction of an HIV-1 derivative which contains sequences of a SIVmac239 loop between alpha-helices 4 and 5 (L4/5) of capsid protein (CA) and the entire SIVmac239 vif gene was previously reported. Although this chimeric virus could grow in cynomolgus monkey cells, it did so much more slowly than did SIVmac. It was also reported that intrinsic TRIM5alpha restricts the post-entry step of HIV-1 replication in rhesus and cynomolgus monkey cells, and we previously demonstrated that a single amino acid in a loop between alpha-helices 6 and 7 (L6/7) of HIV type 2 (HIV-2) CA determines the susceptibility of HIV-2 to cynomolgus monkey TRIM5alpha. RESULTS: In the study presented here, we replaced L6/7 of HIV-1 CA in addition to L4/5 and vif with the corresponding segments of SIVmac. The resultant HIV-1 derivatives showed enhanced replication capability in established T cell lines as well as in CD8+ cell-depleted primary peripheral blood mononuclear cells from cynomolgus monkey. Compared with the wild type HIV-1 particles, the viral particles produced from a chimeric HIV-1 genome with those two SIVmac loops were less able to saturate the intrinsic restriction in rhesus monkey cells. CONCLUSION: We have succeeded in making the replication of simian-tropic HIV-1 in cynomolgus monkey cells more efficient by introducing into HIV-1 the L6/7 CA loop from SIVmac. It would be of interest to determine whether HIV-1 derivatives with SIVmac CA L4/5 and L6/7 can establish infection of cynomolgus monkeys in vivo.
Assuntos
Proteínas do Capsídeo/genética , HIV-1/genética , Recombinação Genética , Vírus da Imunodeficiência Símia/genética , Proteínas não Estruturais Virais/genética , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/virologia , Macaca fascicularis , Linfócitos T/virologiaRESUMO
To test requirement for apoptin in the replication of chicken anemia virus (CAV), an apoptin-knockout clone, pCAV/Ap(-), was constructed. DNA replication was completely abolished in cells transfected with replicative form of CAV/Ap(-). A reverse mutant competent in apoptin production regained the full level of DNA replication. DNA replication and virus-like particle (VLP) production of CAV/Ap(-) was fully complemented by supplementation of the wild-type apoptin. The virus yield of a point mutant, CAV/ApT(108)I, was 1/40 that of the wild type, even though its DNA replication level was full. The infectious titer of CAV was fully complemented by supplementing apoptin. Progeny virus was free from reverse mutation for T(108)I. To localize the domain within apoptin molecule inevitable for CAV replication, apoptin-mutant expressing plasmids, pAp1, pAp2, pAp3, and pAp4, were constructed by deleting amino acids 10-36, 31-59, 59-88 and 80-112, respectively. While Ap1 and Ap2 were preferentially localized in nuclei, Ap3 and Ap4 were mainly present in cytoplasm. Although complementation capacity of Ap3 and Ap4 was 1/10 of the wild type, neither of them completely lost its activity. VP3 of TTV did fully complement the DNA replication and VLP of CAV/Ap(-). These data suggest that apoptin is inevitable not only for DNA replication but also VLP of CAV. The common feature of apoptin and TTV-VP3 presented another evidence for close relatedness of CAV and TTV.
Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Anemia da Galinha/fisiologia , Torque teno virus/metabolismo , Replicação Viral/fisiologia , Animais , Proteínas do Capsídeo/fisiologia , Linhagem Celular , Vírus da Anemia da Galinha/classificação , Vírus da Anemia da Galinha/genética , Galinhas/virologia , Genoma Viral , Humanos , Proteínas Associadas a Pancreatite , Torque teno virus/genéticaRESUMO
Tripartite motif protein (TRIM) 5alpha is a restriction factor of human immunodeficiency virus type 1 in Old World monkey cell. It was found that both naturally occurring and artificial TRIM5alpha variants lacking the SPRY domain could silence TRIM5alpha activity. Specifically, the artificial TRIM5alpha mutant could suppress TRIM5alpha activity of various primate species with even higher efficiency than could small interfering RNAs. The findings indicate that TRIM5alpha variants lacking the SPRY domain are useful for silencing TRIM5alpha activity.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas de Transporte/genética , HIV-1/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Fatores de Restrição Antivirais , Sequência de Bases , Western Blotting , Linhagem Celular , Chlorocebus aethiops , Primers do DNA , Inativação Gênica , Vetores Genéticos , Haplorrinos , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Deleção de Sequência , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
The existence of spliced mRNA in Chicken anemia virus (CAV) was investigated, as three proteins appeared to be derived from a single 2.0 kb mRNA species. Human Torque teno virus (TTV), which displays a number of genomic similarities to CAV, is known to transcribe three mRNA species, suggesting that CAV may also have multiple mRNAs. Northern analysis of infected chicken MDCC-MSB1 cells revealed a 2.0 kb mRNA 3 h post-infection (p.i.) and additional 1.6, 1.3 and 1.2 kb bands visible at 48 and 72 h p.i. MDCC-MSB1 or COS1 cells transfected with a CAV clone showed similar results. The poly(A)+ RNA of infected cells was subjected to RT-PCR using a suite of CAV-specific primers. The major 2.0 kb RNA reacted with every primer, but the 1.3 and 1.2 kb RNAs only annealed to certain primers. The 2.0 kb mRNA had no deletions or mutations and was capable of encoding all three known CAV proteins. The 1.3 kb RNA had a splice site joining nt 1222 to nt 1814 and encoded head/tail viral protein 1 (VP1) without a frameshift. In addition, the 1.2 kb RNA possessed a splice site joining nt 994 to nt 1095 and encoded several putative, novel proteins with frameshift mutations. These splice sites conformed to the previously described GT-AG splicing rule. One further 0.8 kb RNA species appeared to be derived from a homologous recombination event. Discovery of the presence of spliced mRNA in CAV strengthens the similarity between CAV and TTV.