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The solution-state fluxional behavior of bullvalene has fascinated physical organic and supramolecular chemists alike. Little effort, however, has been put into investigating bullvalene applications in bulk, partially due to difficulties in characterizing such dynamic systems. To address this knowledge gap, we herein probe whether bullvalene Hardy-Cope rearrangements can be mechanically perturbed in bulk polymer networks. We use dynamic mechanical analysis to demonstrate that the activation barrier to the glass transition process is significantly elevated for bullvalene-containing materials relative to "static" control networks. Furthermore, bullvalene rearrangements can be mechanically perturbed at low temperatures in the glassy region; such behavior facilitates energy dissipation (i.e., increased hysteresis energy) and polymer chain alignment to stiffen the material (i.e., increased Young's modulus) under load. Computational simulations corroborate our work that showcases bullvalene as a reversible "low-force" covalent mechanophore in the modulation of viscoelastic behavior.
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Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) possess mutations that prevent antibody therapeutics from maintaining antiviral binding and neutralizing efficacy. Monoclonal antibodies (mAbs) shown to neutralize Wuhan-Hu-1 SARS-CoV-2 (ancestral) strain have reduced potency against newer variants. Plasma-derived polyclonal hyperimmune drugs have improved neutralization breadth compared with mAbs, but lower titers against SARS-CoV-2 require higher dosages for treatment. We previously developed a highly diverse, recombinant polyclonal antibody therapeutic anti-SARS-CoV-2 immunoglobulin hyperimmune (rCIG). rCIG was compared with plasma-derived or mAb standards and showed improved neutralization of SARS-CoV-2 across World Health Organization variants; however, its potency was reduced against some variants relative to ancestral, particularly omicron. Omicron-specific antibody sequences were enriched from yeast expressing rCIG-scFv and exhibited increased binding and neutralization to omicron BA.2 while maintaining ancestral strain binding and neutralization. Polyclonal antibody libraries such as rCIG can be utilized to develop antibody therapeutics against present and future SARS-CoV-2 threats.
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COVID-19 , Humanos , SARS-CoV-2/genética , Anticorpos Monoclonais/uso terapêutico , Antivirais , Saccharomyces cerevisiae , Anticorpos Neutralizantes/uso terapêutico , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Antivirais/uso terapêuticoRESUMO
OBJECTIVE: Early life trauma (ELT) and HIV are associated with social processing deficits. In people with HIV (PWH), we examined whether facial emotion identification accuracy differs by ELT and whether neuroendocrine factors including cortisol, oxytocin (OT), and arginine vasopressin, and/or immune system measures play a role in the ELT-performance association. METHODS: We used secondary data from the placebo condition of a pharmacologic challenge study in PWH. Presence of ELT was measured with the Childhood Trauma Questionnaire (at least moderate experiences of sexual, physical, and/or emotional abuse). Social processing was measured with the Facial Emotion Perception Test (FEPT). Salivary immune system measures and cortisol were sampled across a 5-hour study session. Blood was collected at study session start (12 pm ) to measure OT and arginine vasopressin. We examined the association of ELT with FEPT and five biological moderators (from principal components analysis of 12 biomarkers) of ELT-FEPT associations. RESULTS: Of 58 PWH (42 men; mean [standard deviation] age = 33.7 [8.9] years), 50% endorsed ELT. ELT-exposed PWH demonstrated lower identification accuracy across all emotional expressions (unstandardized ß [ B ] = 0.13; standard error [SE] = 0.05; p = .021, d = 0.63) and had higher OT levels compared with ELT-unexposed PWH ( t(1,56) = 2.12, p = .039; d = 0.57). For total accuracy, an OT/C-reactive protein factor moderated the ELT-FEPT association ( B = 0.14; SE = 0.05; p = .014); accuracy was lower in ELT-exposed PWH versus ELT-unexposed PWH when the factor was low but not when high. Similar results were obtained for fearful, neutral, and happy faces ( p values < .05). Regardless of ELT, a myeloid migration (MCP-1/MMP-9) factor was associated with reduced accuracy ( p values < .05). CONCLUSIONS: Our pilot findings suggest that ELT may alter social processing in PWH, and OT and C-reactive protein may be a target for improving social processing in ELT-exposed PWH, and myeloid migration markers may be a target in PWH more generally.
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Infecções por HIV , Ocitocina , Adulto , Arginina Vasopressina , Proteína C-Reativa , Feminino , Infecções por HIV/complicações , Humanos , Hidrocortisona , Inflamação , Masculino , Metaloproteinase 9 da Matriz , Percepção SocialRESUMO
BACKGROUND: Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The "classic" quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 "next-generation" viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. METHODS: Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy-suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples. RESULTS: Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95% credible interval [CI], 2.1-3.5-fold) for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold (95% CI, 2.6-5.4-fold). Frozen storage did not substantially alter infectious units per million values (-18%; 95% CI, -52% to 39%). CONCLUSIONS: The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays.
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Infecções por HIV , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Latência Viral , Replicação Viral , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos , Estudos de Casos e Controles , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV , HIV-1/isolamento & purificação , Humanos , Leucaférese , Carga Viral , Replicação Viral/fisiologiaRESUMO
BACKGROUND: Removal of cytokines, chemokines, and microvesicles from the supernatant of allogeneic erythrocytes may help mitigate adverse transfusion reactions. Blood bank-based washing procedures present logistical difficulties; therefore, we tested the hypothesis that on-demand bedside washing of allogeneic erythrocyte units is capable of removing soluble factors and is feasible in a clinical setting. METHODS: There were in vitro and prospective, observation cohort components to this a priori planned substudy evaluating bedside allogeneic erythrocyte washing, with a cell saver, during cardiac surgery. Laboratory data were collected from the first 75 washed units given to a subset of patients nested in the intervention arm of a parent clinical trial. Paired pre- and postwash samples from the blood unit bags were centrifuged. The supernatant was aspirated and frozen at -70°C, then batch-tested for cell-derived microvesicles, soluble CD40 ligand, chemokine ligand 5, and neutral lipids (all previously associated with transfusion reactions) and cell-free hemoglobin (possibly increased by washing). From the entire cohort randomized to the intervention arm of the trial, bedside washing was defined as feasible if at least 75% of prescribed units were washed per protocol. RESULTS: Paired data were available for 74 units. Washing reduced soluble CD40 ligand (median [interquartile range]; from 143 [1 to 338] ng/ml to zero), chemokine ligand 5 (from 1,314 [715 to 2,551] to 305 [179 to 488] ng/ml), and microvesicle numbers (from 6.90 [4.10 to 20.0] to 0.83 [0.33 to 2.80] × 106), while cell-free hemoglobin concentration increased from 72.6 (53.6 to 171.6) mg/dl to 210.5 (126.6 to 479.6) mg/dl (P < 0.0001 for each). There was no effect on neutral lipids. Bedside washing was determined as feasible for 80 of 81 patients (99%); overall, 293 of 314 (93%) units were washed per protocol. CONCLUSIONS: Bedside erythrocyte washing was clinically feasible and greatly reduced concentrations of soluble factors thought to be associated with transfusion-related adverse reactions, increasing concentrations of cell-free hemoglobin while maintaining acceptable (less than 0.8%) hemolysis.
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Remoção de Componentes Sanguíneos/métodos , Quimiocinas , Citocinas , Transfusão de Eritrócitos/métodos , Eritrócitos/química , Reação Transfusional/prevenção & controle , Preservação de Sangue , Estudos de Coortes , Eritrócitos/citologia , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Estudos ProspectivosRESUMO
BACKGROUND: In April 2015, the government of Georgia (country) initiated the world's first national hepatitis C elimination program. An analysis of blood donor infectious screening data was conducted to inform a strategic plan to advance blood transfusion safety in Georgia. STUDY DESIGN AND METHODS: Descriptive analysis of blood donation records (2015-2017) was performed to elucidate differences in demographics, donor type, remuneration status, and seroprevalence for infectious markers (hepatitis C virus antibody [anti-HCV], human immunodeficiency virus [HIV], hepatitis B virus surface antigen [HBsAg], and Treponema pallidum). For regression analysis, final models included all variables associated with the outcome in bivariate analysis (chi-square) with a p value of less than 0.05. RESULTS: During 2015 to 2017, there were 251,428 donations in Georgia, representing 112,093 unique donors; 68.5% were from male donors, and 51.2% of donors were paid or replacement (friends or family of intended recipient). The overall seroprevalence significantly declined from 2015 to 2017 for anti-HCV (2.3%-1.4%), HBsAg (1.5%-1.1%), and T. pallidum (1.1%-0.7%) [p < 0.0001]; the decline was not significant for HIV (0.2%-0.1%). Only 41.0% of anti-HCV seropositive donors underwent additional testing to confirm viremia. Infectious marker seroprevalence varied by age, sex, and geography. In multivariable analysis, first-time and paid donor status were associated with seropositivity for all four infectious markers. CONCLUSION: A decline during the study period in infectious markers suggests improvement in blood safety in Georgia. Areas that need further improvement are donor recruitment, standardization of screening and diagnostic follow-up, quality assurance, and posttransfusion surveillance.
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Segurança do Sangue , Transfusão de Sangue , Hepacivirus , Anticorpos Anti-Hepatite C/sangue , Hepatite C/sangue , Adolescente , Adulto , Biomarcadores/sangue , Seleção do Doador , Feminino , República da Geórgia/epidemiologia , Antígenos de Superfície da Hepatite B/sangue , Hepatite C/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Sífilis/sangue , Sífilis/epidemiologia , Treponema pallidumRESUMO
Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.
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Infecções por HIV/virologia , HIV-1 , Carga Viral/métodos , Latência Viral , Fármacos Anti-HIV/uso terapêutico , Teorema de Bayes , Linfócitos T CD4-Positivos/virologia , Biologia Computacional , Simulação por Computador , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/virologia , Funções Verossimilhança , Cadeias de Markov , Método de Monte Carlo , Reprodutibilidade dos Testes , Carga Viral/estatística & dados numéricos , Replicação ViralRESUMO
Early treatment of HIV infection with antiretroviral therapy in recently identified HIV-infected individuals reduces viral replication and decreases the risk of transmission. The screening and supplemental, confirmatory assays used to identify infection are influenced by early treatment and may obscure a clear diagnosis of HIV infection. In this issue of the Journal of Clinical Microbiology, Manak et al. demonstrate the impact of antiretroviral therapy on the evolution of biomarkers that have traditionally been used for identifying HIV infection (M. M. Manak, L. L. Jagodzinski, A. Shutt, J. A. Malia, et al., J Clin Microbiol 57:e00757-19, 2019, https://doi.org/10.1128/JCM.00757-19).
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Infecções por HIV , HIV , Humanos , Programas de RastreamentoRESUMO
BACKGROUND: Several retrospective studies have suggested that transfusion with red blood cells (RBCs) stored for longer periods is associated with increased mortality. The Age of Blood Evaluation (ABLE) study randomized subjects to receive fresh vs. standard issue RBC units and showed no difference in the primary or secondary endpoints of mortality or change in multi-organ dysfunction syndrome (MODS) score. METHODS: In this study a subset of 100 ABLE subjects were enrolled to measure coagulation and immune parameters. Samples were collected pre-transfusion and on days 2, 6, 28, and 180 post-transfusion. Levels of 16 coagulation parameters, regulatory and functional T cells, 25 cytokines, and 16 markers of extracellular vesicles (EVs) were determined. RESULTS: Changes from baseline in levels of protein C, factor V, and EVs expressing phosphatidyl serine and CTLA-4 (CD152) differed between recipients of fresh and standard storage age RBC units, with the vast majority of coagulation and EV markers and all cytokines tested showing no difference between study arms. Although most analytes showed no difference between subjects in the fresh and standard arms of the study, 6 coagulation parameters, 15 cytokines, and 7 EV parameters changed significantly in the period post-transfusion. DISCUSSION: Transfusion of fresh vs. standard issue RBC units does not result in substantial changes in coagulation or immune parameters, up to day 35 of RBC storage. Furthermore, significant changes in multiple coagulation and immune parameters are detectable post-transfusion, though causality cannot be determined based on the current study.
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Coagulação Sanguínea/imunologia , Preservação de Sangue , Citocinas , Transfusão de Eritrócitos , Eritrócitos , Vesículas Extracelulares , Biomarcadores/sangue , Estado Terminal , Citocinas/sangue , Citocinas/imunologia , Eritrócitos/imunologia , Eritrócitos/metabolismo , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
BACKGROUND: Retrospective studies suggested that storage age of RBCs is associated with inflammation and thromboembolism. The Red Cell Storage Duration Study (RECESS) trial randomized subjects undergoing complex cardiac surgery to receive RBCs stored for shorter versus longer periods, and no difference was seen in the primary outcome of change in multiple organ dysfunction score. STUDY DESIGN AND METHODS: In the current study, 90 subjects from the RECESS trial were studied intensively using a range of hemostasis, immunologic, and nitric oxide parameters. Samples were collected before transfusion and on Days 2, 6, 28, and 180 after transfusion. RESULTS: Of 71 parameters tested, only 4 showed a significant difference after transfusion between study arms: CD8+ T-cell interferon-γ secretion and the concentration of extracellular vesicles bearing the B-cell marker CD19 were higher, and plasma endothelial growth factor levels were lower in recipients of fresh versus aged RBCs. Plasma interleukin-6 was higher at Day 2 and lower at Days 6 and 28 in recipients of fresh versus aged RBCs. Multiple parameters showed significant modulation after surgery and transfusion. Most analytes that changed after surgery did not differ based on transfusion status. Several extracellular vesicle markers, including two associated with platelets (CD41a and CD62P), decreased in transfused patients more than in those who underwent surgery without transfusion. CONCLUSIONS: Transfusion of fresh versus aged RBCs does not result in substantial changes in hemostasis, immune, or nitric oxide parameters. It is possible that transfusion modulates the level of platelet-derived extracellular vesicles, which will require study of patients randomly assigned to receipt of transfusion to define.
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Antígenos CD , Coagulação Sanguínea/imunologia , Preservação de Sangue , Transfusão de Eritrócitos , Eritrócitos/metabolismo , Interleucina-6 , Óxido Nítrico , Idoso , Antígenos CD/sangue , Antígenos CD/imunologia , Feminino , Humanos , Interleucina-6/sangue , Interleucina-6/imunologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/sangue , Óxido Nítrico/imunologia , Estudos Retrospectivos , Fatores de TempoRESUMO
BACKGROUND: Patients with cancer or other diagnoses associated with chronic anemia often receive red blood cell (RBC) transfusion as outpatients, but the effect of transfusion on functional status is not well demonstrated. STUDY DESIGN AND METHODS: To estimate the effect of transfusion on functional status and quality of life, we measured 6-minute walk test distance and fatigue- and dyspnea-related quality-of-life scores before and 1 week after RBC transfusion in 208 outpatients age ≥50 with at least one benign or malignant hematology/oncology diagnosis. To account for potential confounding effects of cancer treatment, patients were classified into two groups based on cancer treatment within 4 weeks of the study transfusion. Minimum clinically important improvements over baseline were 20 meters in walk test distance, 3 points in fatigue score, and 2 points in dyspnea score. RESULTS: The median improvement in unadjusted walk test distance was 20 meters overall and 30 meters in patients not receiving recent cancer treatment. Fatigue scores improved overall by a median of 3 points and by 4 points in patients without cancer treatment. There was no clinically important change in dyspnea scores. In multiple linear regression analysis, patients who maintained hemoglobin (Hb) levels of 8 g/dL or greater at 1 week posttransfusion, who had not received recent cancer treatment, and who did not require hospitalization during the study showed clinically important increases in mean walk test distance. CONCLUSIONS: Red blood cell transfusion is associated with a modest, but clinically important improvement in walk test distance and fatigue score outcomes in adult hematology/oncology outpatients.
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Assistência Ambulatorial/métodos , Anemia/terapia , Transfusão de Eritrócitos , Idoso , Anemia/sangue , Dispneia/etiologia , Transfusão de Eritrócitos/efeitos adversos , Transfusão de Eritrócitos/métodos , Teste de Esforço , Fadiga/etiologia , Feminino , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Qualidade de Vida , Resultado do TratamentoRESUMO
BACKGROUND: The major aims of the RBC-Omics study were to evaluate the genomic and metabolomic determinants of spontaneous and stress-induced hemolysis during RBC storage. This study was unique in scale and design to allow evaluation of RBC donations from a sufficient number of donors across the spectrum of race, ethnicity, sex, and donation intensity. Study procedures were carefully piloted, optimized, and controlled to enable high-quality data collection. METHODS: The enrollment goal of 14,000 RBC donors across four centers, with characterization of RBC hemolysis across two testing laboratories, required rigorous piloting and optimization and establishment of a quality assurance (QA) and quality control (QC) program. Optimization of WBC elution from leukoreduction (LR) filters, development and validation of small-volume transfer bags, impact of manufacturing and sample-handling procedures on hemolysis parameters, and testing consistency across laboratories and technicians and over time were part of this quality assurance/quality control program. RESULTS: LR filter elution procedures were optimized for obtaining DNA for analysis. Significant differences between standard and pediatric storage bags led to use of an alternative LR-RBC transfer bag. The impact of sample preparation and freezing methods on metabolomics analyses was evaluated. Proficiency testing monitored and documented testing consistency across laboratories and technicians. CONCLUSION: Piloting and optimization, and establishment of a robust quality assurance/quality control program documented process consistency throughout the study and was essential in executing this large-scale multicenter study. This program supports the validity of the RBC-Omics study results and a sample repository that can be used in future studies.
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Preservação de Sangue/métodos , Hemólise/fisiologia , Trifosfato de Adenosina/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Humanos , Controle de QualidadeRESUMO
BACKGROUND: Genetic determinants may underlie the susceptibility of red blood cells (RBCs) to hemolyze in vivo and during routine storage. This study characterized the reproducibility and dynamics of in vitro hemolysis variables from a subset of the 13,403 blood donors enrolled in the RBC-Omics study. STUDY DESIGN AND METHODS: RBC-Omics donors with either low or high hemolysis results on 4°C-stored leukoreduced (LR)-RBC samples from enrollment donations stored for 39 to 42 days were recalled 2 to 12 months later to donate LR-RBCs. Samples of stored LR-RBCs from the unit and from transfer bags were evaluated for spontaneous and stress-induced hemolysis at selected storage time points. Intradonor reproducibility of hemolysis variables was evaluated in transfer bags over two donations. Hemolysis data at serial storage time points were generated on LR-RBCs from parent bags and analyzed by site, sex, race/ethnicity, and donation frequency. RESULTS: A total of 664 donors were successfully recalled. Analysis of intradonor reproducibility revealed that osmotic and oxidative hemolysis demonstrated good and moderate reproducibility (Pearson's r = 0.85 and r = 0.53, respectively), while spontaneous hemolysis reproducibility was poor (r = 0.40). Longitudinal hemolysis in parent bags showed large increases over time in spontaneous (508.6%) and oxidative hemolysis (399.8%) and smaller increases in osmotic (9.4%) and mechanical fragility (3.4%; all p < 0.0001). CONCLUSION: Spontaneous hemolysis is poorly reproducible in donors over time and may depend on site processing methods, while oxidative and osmotic hemolysis were reproducible in donors and hence could reflect consistent heritable phenotypes attributable to genetic traits. Spontaneous and oxidative hemolysis increased over time of storage, whereas osmotic and mechanical hemolysis remained relatively stable.
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Eritrócitos/citologia , Doadores de Sangue/estatística & dados numéricos , Preservação de Sangue , Eritrócitos/metabolismo , Feminino , Hemólise/fisiologia , Humanos , Cinética , Masculino , Osmose/fisiologia , Oxirredução , Reprodutibilidade dos TestesRESUMO
Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical fourth-generation antigen (Ag)/antibody (Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab-alone assays but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening, and next-generation assays. Three-hundred-member panels of 20 serially diluted well-characterized antibody-negative HIV isolates for which the researchers were blind to the results (blind panels) were distributed to manufacturers and end-user labs to assess the relative analytic sensitivity of currently approved and preapproved clinical HIV fourth-generation Ag/Ab combo or p24 Ag-alone immunoassays for the detection of diverse subtypes. The limits of detection (LODs) of virus were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blind panel. On the basis of the proportion of positive results on 300 observations, all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half-log LODs, illustrating the similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo assays performed poorly. The similar performance of the different commercially available fourth-generation assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next-generation preclinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while rapid fourth-generation assays performed poorly for p24 Ag detection.
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Sorodiagnóstico da AIDS/métodos , Sorodiagnóstico da AIDS/normas , Proteína do Núcleo p24 do HIV/sangue , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/diagnóstico , HIV/isolamento & purificação , Imunoensaio/normas , Carga Viral/normas , Benchmarking , HIV/imunologia , Anticorpos Anti-HIV/sangue , Antígenos HIV/sangue , Antígenos HIV/imunologia , Infecções por HIV/sangue , Humanos , Limite de Detecção , Sensibilidade e EspecificidadeRESUMO
A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1ß, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1ß, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies.IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells.
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Citocinas/metabolismo , Infecções por HIV/imunologia , HIV/imunologia , HIV/fisiologia , Replicação Viral/efeitos dos fármacos , Adulto , Antígenos de Diferenciação/biossíntese , Linfócitos T CD4-Positivos/virologia , Feminino , Regulação da Expressão Gênica , Sobreviventes de Longo Prazo ao HIV , Humanos , Proteínas de Membrana/biossíntese , Pessoa de Meia-Idade , Plasma/química , Receptores de HIV/biossínteseRESUMO
Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART), and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9) potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002), more potently than vorinostat (p = 0.02). rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p<0.05). rGal-9 signals through N-linked oligosaccharides and O-linked hexasaccharides on the T cell surface, modulating the gene expression levels of key transcription initiation, promoter proximal-pausing, and chromatin remodeling factors that regulate HIV latency. Beyond latent viral reactivation, rGal-9 induces robust expression of the host antiviral deaminase APOBEC3G in vitro and ex vivo (FDR<0.006) and significantly reduces infectivity of progeny virus, decreasing the probability that the HIV reservoir will be replenished when latency is reversed therapeutically. Lastly, endogenous levels of soluble galectin-9 in the plasma of 72 HIV-infected ART-suppressed individuals were associated with levels of HIV RNA in CD4+ T cells (p<0.02) and with the quantity and binding avidity of circulating anti-HIV antibodies (p<0.009), suggesting a role of galectin-9 in regulating HIV transcription and viral production in vivo during therapy. Our data suggest that galectin-9 and the host glycosylation machinery should be explored as foundations for novel HIV cure strategies.
Assuntos
Linfócitos T CD4-Positivos/virologia , Galectinas/metabolismo , Infecções por HIV/metabolismo , Ativação Viral/fisiologia , Latência Viral/fisiologia , Fármacos Anti-HIV/uso terapêutico , Western Blotting , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Reação em Cadeia da Polimerase , Transcrição Gênica/fisiologia , TranscriptomaRESUMO
Despite the availability of effective antiretroviral therapies, cognitive impairment (CI) remains prevalent in HIV-infected (HIV+) individuals. Evidence from primarily cross-sectional studies, in predominantly male samples, implicates monocyte- and macrophage-driven inflammatory processes linked to HIV-associated CI. Thus, peripheral systemic inflammatory markers may be clinically useful biomarkers in tracking HIV-associated CI. Given sex differences in immune function, we focused here on whether mean and intra-individual variability in inflammatory marker-predicted CI in HIV+ and HIV- women. Seventy-two HIV+ (36 with CI) and 58 HIV- (29 with CI) propensity-matched women participating in the Women's Interagency HIV Study completed a neuropsychological battery once between 2009 and 2011, and performance was used to determine CI status. Analysis of 13 peripheral immune markers was conducted on stored biospecimens at three time points (7 and 3.5 years before neuropsychological data collection and concurrent with data collection). HIV+ women showed alterations in 8 immune markers compared to HIV- women. The strongest predictors of CI across HIV+ and HIV- women were lower mean soluble tumor necrosis factor receptor I (sTNFRI) levels, higher mean interleukin (IL)-6 levels, and greater variability in C-reactive protein (CRP) and matrix metalloproteinase (MMP)-9 (p values < 0.05). Stratified by HIV, the only significant predictor of CI was greater variability in CRP for both HIV+ and HIV- women (p values < 0.05). This variability predicted lower executive function, attention/working memory, and psychomotor speed in HIV+ but only learning in HIV- women (p values < 0.05). Intra-individual variability in CRP levels over time may be a good predictor of CI in predominately minority low-socioeconomic status midlife women.
Assuntos
Complexo AIDS Demência/diagnóstico , Proteína C-Reativa/metabolismo , Complexo AIDS Demência/sangue , Complexo AIDS Demência/imunologia , Complexo AIDS Demência/fisiopatologia , Adulto , Idoso , Atenção/fisiologia , Biomarcadores/sangue , Proteína C-Reativa/imunologia , Estudos de Casos e Controles , Função Executiva/fisiologia , Feminino , HIV-1/patogenicidade , Humanos , Interleucina-6/sangue , Interleucina-6/imunologia , Estudos Longitudinais , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/imunologia , Memória de Curto Prazo/fisiologia , Pessoa de Meia-Idade , Testes Neuropsicológicos , Desempenho Psicomotor/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Receptores Tipo I de Fatores de Necrose Tumoral/imunologiaRESUMO
The development of the fetal immune system during pregnancy is a well-orchestrated process with important consequences for fetal and neonatal health, but prenatal factors that affect immune activation are poorly understood. We hypothesized that chronic fetal inflammation may lead to alterations in development of the fetal immune system. To test this hypothesis, we examined neonates with gastroschisis, a congenital abdominal wall defect that leads to exposure of the fetal intestines to amniotic fluid, with resultant intestinal inflammation. We determined that patients with gastroschisis show high systemic levels of inflammatory cytokines and chemokines such as eotaxin, as well as earlier activation of CD4(+) and CD8(+) effector and memory T cells in the cord blood compared with controls. Additionally, increased numbers of T cells and eosinophils infiltrate the serosa and mucosa of the inflamed intestines. Using a mouse model of gastroschisis, we observed higher numbers of eosinophils and both type 2 and type 3 innate lymphoid cells (ILC2 and ILC3), specifically in the portion of organs exposed to the amniotic fluid. Given the role of IL-5 produced by ILC2 in regulating eosinophil development and survival, we determined that maternal or fetal administration of the anti-IL-5 neutralizing Ab, or a depleting Ab against ILCs, can both effectively reduce intestinal eosinophilia. Thus, a congenital anomaly causing chronic inflammation can alter the composition of circulating and tissue-resident fetal immune cells. Given the high rate of prenatal and neonatal complications in these patients, such changes have clinical significance and might become targets for fetal therapy.
Assuntos
Doenças Fetais/imunologia , Doenças Fetais/terapia , Gastrosquise/imunologia , Gastrosquise/terapia , Interleucina-5/imunologia , Intestinos/efeitos dos fármacos , Líquido Amniótico/imunologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Quimiocina CCL11/sangue , Citocinas/sangue , Modelos Animais de Doenças , Eosinofilia/terapia , Eosinófilos/imunologia , Eosinófilos/fisiologia , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Terapias Fetais , Humanos , Memória Imunológica , Recém-Nascido , Inflamação/terapia , Interleucina-5/antagonistas & inibidores , Interleucina-5/sangue , Intestinos/imunologia , Intestinos/patologia , Linfócitos/imunologia , Camundongos , Mães , GravidezRESUMO
Background: Human immunodeficiency virus (HIV) antibodies are generated and maintained by ongoing systemic expression of HIV antigen. We investigated whether HIV antibody responses as measured by high-throughput quantitative and qualitative assays could be used to indirectly measure persistent HIV replication in individuals receiving antiretroviral therapy (ART). Methods: HIV antibody responses were measured over time in the presence or absence of suppressive ART and were compared to the HIV reservoir size and expression of antiviral restriction factors. Results: Among untreated individuals, including both elite controllers (ie, persons with a viral load of ≤40 copies/mL) and noncontrollers, antibody parameters were stable over time and correlated with the individual viral load. Viral suppression with ART led to a progressive decline in antibody responses after treatment induction that persisted for 5-7 years. Higher levels of HIV antibodies during suppressive therapy were associated with later initiation of ART after infection, with higher DNA and cell-associated RNA levels, and with lower expression of multiple anti-HIV host restriction factors. Discussion: These findings suggest that declining antibody levels during ART reflect lower levels of antigen production and/or viral replication in the persistent HIV reservoir. Results of relatively inexpensive and quantitative HIV antibody assays may be useful indirect markers that enable efficient monitoring of the viral reservoir and suppression during functional-cure interventions.
Assuntos
Anticorpos Anti-HIV/sangue , Infecções por HIV/sangue , HIV-1/fisiologia , Replicação Viral , Terapia Antirretroviral de Alta Atividade , Biomarcadores/sangue , Perfilação da Expressão Gênica , Antígenos HIV/sangue , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Estudos Longitudinais , RNA Viral/isolamento & purificação , Manejo de Espécimes , Carga ViralRESUMO
BACKGROUND: Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. METHODS AND FINDINGS: We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures-including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue-the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. CONCLUSIONS: allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs.