RESUMO
INTRODUCTION: Eptacog beta is a new recombinant activated human factor VII bypassing agent approved in the United States for the treatment and control of bleeding in patients with haemophilia A or B with inhibitors 12 years of age or older. AIM: To prospectively assess in a phase 3 clinical trial (PERSEPT 2) eptacog beta efficacy and safety for treatment of bleeding in children <12 years of age with haemophilia A or B with inhibitors. METHODS: Using a randomised crossover design, subjects received initial doses of 75 or 225 µg/kg eptacog beta followed by 75 µg/kg dosing at predefined intervals (as determined by clinical response) to treat bleeding episodes (BEs). Treatment success criteria included a haemostasis evaluation of 'excellent' or 'good' without use of additional eptacog beta, alternative haemostatic agent or blood product, and no increase in pain following the first 'excellent' or 'good' assessment. RESULTS: Treatment success proportions in 25 subjects (1-11 years) who experienced 546 mild or moderate BEs were 65% in the 75 µg/kg initial dose regimen (IDR) and 60% in the 225 µg/kg IDR 12 h following initial eptacog beta infusion. By 24 h, the treatment success proportions were 97% for the 75 µg/kg IDR and 98% for the 225 µg/kg IDR. No thrombotic events, allergic reactions, neutralising antibodies or treatment-related adverse events were reported. CONCLUSION: Both 75 and 225 µg/kg eptacog beta IDRs provided safe and effective treatment and control of bleeding in children <12 years of age.
Assuntos
Fator VIIa , Hemofilia A , Proteínas Recombinantes , Criança , Estudos Cross-Over , Fator VIIa/efeitos adversos , Hemofilia A/tratamento farmacológico , Hemorragia/etiologia , Hemorragia/prevenção & controle , Humanos , Proteínas Recombinantes/efeitos adversosRESUMO
INTRODUCTION: Surgical procedures in persons with haemophilia A or B with inhibitors (PwHABI) require the use of bypassing agents (BPA) and carry a high risk of complications. Historically, only two BPAs have been available; these are reported to have variable responses. AIM: To prospectively evaluate the efficacy and safety of a new bypassing agent, human recombinant factor VIIa (eptacog beta) in elective surgical procedures in PwHABI in a phase 3 clinical trial, PERSEPT 3. METHODS: Subjects were administered 200 µg/kg (major procedures) or 75 µg/kg eptacog beta (minor procedures) immediately prior to the initial surgical incision; subsequent 75 µg/kg doses were administered to achieve postoperative haemostasis and wound healing. Efficacy was assessed on a 4-point haemostatic scale during the intra- and postoperative periods. Anti-drug antibodies, thrombotic events and changes in clinical/laboratory parameters were monitored throughout the perioperative period. RESULTS: Twelve subjects underwent six major and six minor procedures. The primary efficacy endpoint success proportion was 100% (95% CI: 47.8%-100%) for minor procedures and 66.7% (95% CI: 22.3%-95.7%) for major procedures; 81.8% (95% CI: 48.2%-97.7%) of the procedures were considered successful using eptacog beta. There was one death due to bleeding from a nonsurgical site; this was assessed as unlikely related to eptacog beta. No thrombotic events or anti-eptacog beta antibodies were reported. CONCLUSION: Two eptacog beta dosing regimens in PwHABI undergoing major and minor surgical procedures were well-tolerated, and the majority of procedures were successful based on surgeon/investigator assessments. Eptacog beta offers clinicians a new potential therapeutic option for procedures in PwHABI.
Assuntos
Hemofilia A , Hemostáticos , Fator VIIa , Hemofilia A/tratamento farmacológico , Hemostasia , Hemostáticos/uso terapêutico , Humanos , Assistência Perioperatória , Proteínas RecombinantesRESUMO
INTRODUCTION: Haemophilia patients with inhibitors often require a bypassing agent (BPA) for bleeding episode management. Eptacog beta (EB) is a new FDA-approved recombinant activated human factor VII BPA for the treatment and control of bleeding in haemophilia A or B patients with inhibitors (≥12 years of age). We describe here the EB safety profile from the three prospective Phase 3 clinical trials performed to date. AIM: To assess EB safety, immunogenicity and thrombotic potential in children and adults who received EB for treatment of bleeding and perioperative care. METHODS: Using a randomized crossover design, 27 subjects in PERSEPT 1 (12-54 years) and 25 subjects in PERSEPT 2 (1-11 years) treated bleeding episodes with 75 or 225 µg/kg EB initially followed by 75 µg/kg dosing at predefined intervals as determined by clinical response. Twelve PERSEPT 3 subjects (2-56 years) received an initial preoperative infusion of 75 µg/kg (minor procedures) or 200 µg/kg EB (major surgeries) with subsequent 75 µg/kg doses administered intraoperatively and post-operatively as indicated. Descriptive statistics were used for data analyses. RESULTS: Sixty subjects who received 3388 EB doses in three trials were evaluated. EB was well tolerated, with no allergic, hypersensitivity, anaphylactic or thrombotic events reported and no neutralizing anti-EB antibodies detected. A death occurred during PERSEPT 3 and was determined to be unlikely related to EB treatment by the data monitoring committee. CONCLUSION: Results from all three Phase 3 trials establish an excellent safety profile of EB in haemophilia A or B patients with inhibitors for treatment of bleeding and perioperative use.
Assuntos
Hemofilia A , Adulto , Criança , Estudos Cross-Over , Fator VIIa/efeitos adversos , Hemofilia A/tratamento farmacológico , Hemostasia , Humanos , Estudos Prospectivos , Proteínas RecombinantesRESUMO
Molecular and functional characterization of alveolar epithelial type I (AT1) cells has been challenging due to difficulty in isolating sufficient numbers of viable cells. Here we performed single-cell RNA-sequencing (scRNA-seq) of tdTomato+ cells from lungs of AT1 cell-specific Aqp5-Cre-IRES-DsRed (ACID);R26tdTomato reporter mice. Following enzymatic digestion, CD31-CD45-E-cadherin+tdTomato+ cells were subjected to fluorescence-activated cell sorting (FACS) followed by scRNA-seq. Cell identity was confirmed by immunofluorescence using cell type-specific antibodies. After quality control, 92 cells were analyzed. Most cells expressed 'conventional' AT1 cell markers (Aqp5, Pdpn, Hopx, Ager), with heterogeneous expression within this population. The remaining cells expressed AT2, club, basal or ciliated cell markers. Integration with public datasets identified three robust AT1 cell- and lung-enriched genes, Ager, Rtkn2 and Gprc5a, that were conserved across species. GPRC5A co-localized with HOPX and was not expressed in AT2 or airway cells in mouse, rat and human lung. GPRC5A co-localized with AQP5 but not pro-SPC or CC10 in mouse lung epithelial cell cytospins. We enriched mouse AT1 cells to perform molecular phenotyping using scRNA-seq. Further characterization of putative AT1 cell-enriched genes revealed GPRC5A as a conserved AT1 cell surface marker that may be useful for AT1 cell isolation.
Assuntos
Células Epiteliais Alveolares/metabolismo , Aquaporina 5/metabolismo , Membrana Celular/metabolismo , Pulmão/citologia , Receptores Acoplados a Proteínas G/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Animais , Biomarcadores/metabolismo , Separação Celular , Humanos , Camundongos Transgênicos , Ratos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Recent advances in sequencing technologies and bioinformatics tools have allowed for large-scale microbiome studies that are rapidly advancing medical research. However, small changes in technique or analysis can significantly alter the results and lead to conflicting findings. Quantifying the technical versus biological variation expected in targeted 16S rRNA gene sequencing studies and how this variation changes with input biomass is critical to guide meaningful interpretation of the current literature and plan future research. RESULTS: Data were compiled from 469 sequencing libraries across 19 separate targeted 16S rRNA gene sequencing runs over a 2.5-year time period. Following removal of contaminant sequences identified from negative controls, 244 samples retained sufficient reads for further analysis. Coefficients of variation for intra- and inter-assay variation from repeated measurements of a bacterial mock community ranged from 8.7 to 37.6% (intra) and 15.6 to 80.5% (inter) for all but one genus of bacteria whose relative abundance was greater than 1%. Intra- versus inter-assay Bray-Curtis pairwise distances for a single stool sample were 0.11 versus 0.31, whereas intra-assay variation from repeat stool samples from the same donor was greater at 0.38 (Wilcoxon p = 0.001). A dilution series of the bacterial mock community was used to assess the effect of input biomass on variability. Pairwise distances increased with more dilute samples, and estimates of relative abundance became unreliable below approximately 100 copies of the 16S rRNA gene per microliter. Using this data, we created a prediction model to estimate the expected variation in microbiome measurements for given input biomass and relative abundance values. CONCLUSIONS: Well-controlled microbiome studies are sufficiently robust to capture small biological effects and can achieve levels of variability consistent with clinical assays. Relative abundance is negatively associated with measures of variability and has a stronger effect on variability than does absolute biomass, suggesting that it is feasible to detect differences in bacterial populations in very low-biomass samples. Further, by quantifying the effect of biomass and relative abundance on compositional variability, we developed a tool for defining the expected variance in a given microbiome study.
Assuntos
Bactérias/isolamento & purificação , Microbioma Gastrointestinal , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Biomassa , DNA Bacteriano/genética , Estudos de Avaliação como Assunto , Fezes/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , FilogeniaRESUMO
BACKGROUND: Mother-to-child transmission of human immunodeficiency virus-type 1 (HIV-1) poses a serious health threat in developing countries, and adequate interventions are as yet unrealized. HIV-1 infection is frequently initiated by a single founder viral variant, but the factors that influence particular variant selection are poorly understood. RESULTS: Our analysis of 647 full-length HIV-1 subtype C and G viral envelope sequences from 22 mother-infant pairs reveals unique genotypic and phenotypic signatures that depend upon transmission route. Relative to maternal strains, intrauterine HIV transmission selects infant variants that have shorter, less-glycosylated V1 loops that are more resistant to soluble CD4 (sCD4) neutralization. Transmission through breastfeeding selects for variants with fewer potential glycosylation sites in gp41, are more sensitive to the broadly neutralizing antibodies PG9 and PG16, and that bind sCD4 with reduced cooperativity. Furthermore, experiments with Affinofile cells indicate that infant viruses, regardless of transmission route, require increased levels of surface CD4 receptor for productive infection. CONCLUSIONS: These data provide the first evidence for transmission route-specific selection of HIV-1 variants, potentially informing therapeutic strategies and vaccine designs that can be tailored to specific modes of vertical HIV transmission.
Assuntos
Genótipo , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/isolamento & purificação , Transmissão Vertical de Doenças Infecciosas , Seleção Genética , Aleitamento Materno , Estudos de Coortes , Feminino , HIV-1/genética , Humanos , Lactente , Recém-Nascido , Masculino , Troca Materno-Fetal , Gravidez , Análise de Sequência de DNA , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) replication requires reverse transcription of its RNA genome into a double-stranded cDNA copy, which is then integrated into the host cell chromosome. The essential steps of reverse transcription and integration are catalyzed by the viral enzymes reverse transcriptase (RT) and integrase (IN), respectively. In vitro, HIV-1 RT can bind with IN, and the C-terminal domain (CTD) of IN is necessary and sufficient for this binding. To better define the RT-IN interaction, we performed nuclear magnetic resonance (NMR) spectroscopy experiments to map a binding surface on the IN CTD in the presence of RT prebound to a duplex DNA construct that mimics the primer-binding site in the HIV-1 genome. To determine the biological significance of the RT-IN interaction during viral replication, we used the NMR chemical shift mapping information as a guide to introduce single amino acid substitutions of nine different residues on the putative RT-binding surface in the IN CTD. We found that six viral clones bearing such IN substitutions (R231E, W243E, G247E, A248E, V250E, and I251E) were noninfectious. Further analyses of the replication-defective IN mutants indicated that the block in replication took place specifically during early reverse transcription. The recombinant INs purified from these mutants, though retaining enzymatic activities, had diminished ability to bind RT in a cosedimentation assay. The results indicate that the RT-IN interaction is functionally relevant during the reverse transcription step of the HIV-1 life cycle. IMPORTANCE: To establish a productive infection, human immunodeficiency virus type 1 (HIV-1) needs to reverse transcribe its RNA genome to create a double-stranded DNA copy and then integrate this viral DNA genome into the chromosome of the host cell. These two essential steps are catalyzed by the HIV-1 enzymes reverse transcriptase (RT) and integrase (IN), respectively. We have shown previously that IN physically interacts with RT, but the importance of this interaction during HIV-1 replication has not been fully characterized. In this study, we have established the biological significance of the HIV-1 RT-IN interaction during the viral life cycle by demonstrating that altering the RT-binding surface on IN disrupts both reverse transcription and viral replication. These findings contribute to our understanding of the RT-IN binding mechanism, as well as indicate that the RT-IN interaction can be exploited as a new antiviral drug target.
Assuntos
Integrase de HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/fisiologia , Transcrição Reversa/fisiologia , Replicação Viral/fisiologia , Substituição de Aminoácidos/genética , Western Blotting , Primers do DNA/genética , Escherichia coli , Integrase de HIV/genética , Transcriptase Reversa do HIV/genética , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Integrase (IN) from human immunodeficiency virus, type 1 (HIV-1) exerts pleiotropic effects in the viral replication cycle. Besides integration, IN mutations can impact nuclear import, viral maturation, and reverse transcription. IN and reverse transcriptase (RT) interact in vitro, and the IN C-terminal domain (CTD) is both necessary and sufficient for binding RT. We used nuclear magnetic resonance spectroscopy to identify a putative RT-binding surface on the IN CTD, and surface plasmon resonance to obtain kinetic parameters and the binding affinity for the IN-RT interaction. An IN K258A substitution that disrupts reverse transcription in infected cells is located at the putative RT-binding surface, and we found that this substitution substantially weakens IN CTD-RT interactions. We also identified two additional IN amino acid substitutions located at the putative RT-binding surface (W243E and V250E) that significantly impair viral replication in tissue culture. These results strengthen the notion that IN-RT interactions are biologically relevant during HIV-1 replication and also provide insights into this interaction at the molecular level.
Assuntos
Integrase de HIV/química , Transcriptase Reversa do HIV/química , HIV-1/fisiologia , Transcrição Reversa/fisiologia , Replicação Viral/fisiologia , Substituição de Aminoácidos , Sítios de Ligação/fisiologia , Linhagem Celular , Integrase de HIV/genética , Integrase de HIV/metabolismo , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Humanos , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Mapeamento de Peptídeos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
Unlike activated T cells, quiescent CD4+ T cells have shown resistance to human immunodeficiency virus (HIV) infection due to a block in the early events of the viral life cycle. To further investigate the nature of this block, we infected quiescent CD4+ T cells with HIV-1(NL4-3) and immediately stimulated them. Compared to activated (prestimulated) cells, these poststimulated cells showed slightly decreased viral entry and delays in the completion of reverse transcription. However, the relative efficiency of integration was similar to that of prestimulated cells. Together, this resulted in decreased expression of tat/rev mRNA and synthesis of viral protein. Furthermore, based on cell cycle staining and BrdU incorporation, poststimulated cells expressing viral protein failed to initiate a second round of their cell cycle, independently of Vpr-mediated arrest. Together, these data demonstrate that the early stages of the HIV life cycle are inefficient in these poststimulated cells and that efficient replication cannot be induced by subsequent activation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , HIV-1/fisiologia , Ativação Linfocitária/imunologia , Replicação Viral , Linfócitos T CD4-Positivos/citologia , Linhagem Celular , Proliferação de Células , Regulação Viral da Expressão Gênica , Produtos do Gene gag/metabolismo , Produtos do Gene vpr/metabolismo , Humanos , Cinética , RNA Viral/genética , Transcrição Reversa/genética , Internalização do Vírus , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
In vitro studies using fusion proteins consisting of human immunodeficiency virus type 1 integrase (IN) and a synthetic polydactyl zinc finger protein E2C, a sequence-specific DNA-binding protein, showed that integration of retroviral DNA can be biased towards a contiguous 18-bp E2C-recognition site. To determine whether the fusion protein strategy can achieve site-specific integration in vivo, viruses were prepared by cotransfection and various IN-E2C fusion proteins were packaged in trans into virions. The resulting viruses incorporated with the IN-E2C fusion proteins were functional and capable of performing integration at a level ranging from 1 to 24% of that of viruses containing wild-type (WT) IN. Two of the more infectious viruses, which contained E2C fused to either the N (E2C/IN) or to the C (IN/E2C) terminus of IN, were tested for their ability to direct integration into a unique E2C-binding site present within the 5' untranslated region of erbB-2 gene on human chromosome 17. The copy number of proviral DNA was measured using a quantitative real-time nested-PCR assay, and the specificity of directed integration was determined by comparing the number of proviruses within the vicinity of the E2C-binding site to that in the whole genome. Viruses containing IN/E2C fusion proteins had sevenfold higher preference for integrating near the E2C-binding site than those viruses containing WT IN, whereas viruses containing E2C/IN had 10-fold higher preference. The results indicated that the IN-E2C fusion protein strategy is capable of directing integration of retroviral DNA into a predetermined chromosomal region in the human genome.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Integrase de HIV/metabolismo , HIV-1/genética , Integração Viral , Sítios de Ligação , Linhagem Celular , Cromossomos Humanos Par 17 , DNA Viral/análise , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Genes erbB-2 , Integrase de HIV/genética , HIV-1/fisiologia , Humanos , Reação em Cadeia da Polimerase , Provírus/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A late stage in assembly of alphaviruses within infected cells is thought to be directed by interactions between the nucleocapsid and the cytoplasmic domain of the E2 protein, a component of the viral E1/E2 glycoprotein complex that is embedded in the plasma membrane. Recognition between the nucleocapsid protein and the E2 protein was explored in solution using NMR spectroscopy, as well as in binding assays using a model phospholipid membrane system that incorporated a variety of Sindbis virus E2 cytoplasmic domain (cdE2) and capsid protein constructs. In these binding assays, synthetic cdE2 peptides were reconstituted into phospholipid vesicles to simulate the presentation of cdE2 on the inner leaflet of the plasma membrane. Results from these binding assays showed a direct interaction between a peptide containing the C-terminal 16 amino acids of the cdE2 sequence and a Sindbis virus capsid protein construct containing amino acids 19-264. Additional experiments that probed the sequence specificity of this cdE2-capsid interaction are also described. Further binding assays demonstrated an interaction between the 19-264 capsid protein and artificial vesicles containing neutral or negatively charged phospholipids, while capsid protein constructs with N-terminal truncations displayed either little or no affinity for such vesicles. The membrane-binding property of the capsid protein suggests that the membrane may play an active role in alphavirus assembly. The results are consistent with an assembly process involving an initial membrane association, whereby an association with E2 glycoprotein further enhances capsid binding to facilitate membrane envelopment of the nucleocapsid for budding. Collectively, these experiments elucidate certain requirements for the binding of Sindbis virus capsid protein to the cytoplasmic domain of the E2 glycoprotein, a critical event in the alphavirus maturation pathway.
Assuntos
Alphavirus/química , Capsídeo/química , Lipídeos de Membrana/química , Fosfolipídeos/química , Sindbis virus/química , Sequência de Aminoácidos , Sítios de Ligação , Capsídeo/metabolismo , Sequência Conservada , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sindbis virus/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Sequence-specific recognition between HIV-1 Rev and viral RNA mediates the nuclear export of the viral mRNA and thus is important for the viral life cycle. HIV Rev binds to its viral RNA target with high affinity and specificity and also binds to an in vitro selected RNA aptamer that has a significantly different sequence from the viral RNA target with a 6-fold higher affinity than its natural target. The high-resolution structures of HIV Rev Arg-rich motif (ARM) in complexes with the wild-type RNA and the RNA aptamer reveal that, despite the significantly different RNA sequences, the two complexes share similar structural features and the protein-RNA interactions are mediated mostly by the Arg side chains in Rev ARM. To gain further insight into the role of these Arg side chains in the sequence-specific protein-RNA recognition, we have characterized the flexibility of these Arg side chains at the interfaces of the two high-affinity complexes using (15)N R(1), R(2), nuclear Overhauser effect, and chemical-shift anisotropy dipolar cross-correlation relaxation measurements. The ARM peptide contains uniformly (13)C/(15)N-labeled Arg residues, and the RNA samples were unlabeled. Despite the apparently similar roles of Arg side chains in both complexes, most of them display a different dynamic behavior in the context of different RNA molecules, and extensive and highly diverse motions have been observed for all of these side chains that interact with RNA. Most of the differences in side-chain dynamics between the complexes cannot be inferred from the three-dimensional structures. Additionally, more than half of the residues have increased flexibility in the Rev-RNA aptamer complex that has a higher affinity. This study provides new insights into ARM-RNA recognition and indicates that retention of conformational flexibility is likely important in high-affinity ARM-RNA recognition.
Assuntos
Produtos do Gene rev/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , RNA/metabolismo , Arginina , Interpretação Estatística de Dados , Humanos , Conformação de Ácido Nucleico , Conformação Proteica , Eletricidade Estática , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
Posttranslational modification by the ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), has been established as an important regulatory mechanism. However, in most cases it is not clear how sumoylation regulates various cellular functions. Emerging evidence suggests that sumoylation may play a general role in regulating protein-protein interactions, as shown in RanBP2/Nup358 and RanGAP1 interaction. In this study, we have defined an amino acid sequence motif that binds SUMO. This motif, V/I-X-V/I-V/I, was identified by NMR spectroscopic characterization of interactions among SUMO-1 and peptides derived from proteins that are known to bind SUMO or sumoylated proteins. This motif binds all SUMO paralogues (SUMO-1-3). Using site-directed mutagenesis, we also show that this SUMO-binding motif in RanBP2/Nup358 is responsible for the interaction between RanBP2/Nup358 and sumoylated RanGAP1. The SUMO-binding motif exists in nearly all proteins known to be involved in SUMO-dependent processes, suggesting its general role in sumoylation-dependent cellular functions.