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Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of human angiotensin converting enzyme 2 (hACE-2) binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using Wuhan-WT (vaccine strain), delta (B.1.167.2), omicron BA1 and BA2 variant viral strains showed strong correlation with cell-based pseudovirus neutralization activity (PNA) and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta and omicron variant resistance to neutralization in samples with paired vaccine strain and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. Importantly, this completely automated assay can be performed in 4 h to measure neutralizing antibody titers for 16 samples over 8 serial dilutions or, 128 samples at a single dilution with replicates. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.
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BACKGROUND AND AIMS: Dickkopf-1 (DKK1) is associated with poor prognosis in intrahepatic cholangiocarcinoma (iCCA), but the mechanisms behind this are unclear. Here, we show that DKK1 plays an immune regulatory role in vivo and inhibition reduces tumour growth. METHODS: Various in vivo GEMM mouse models and patient samples were utilized to assess the effects of tumour specific DKK1 overexpression in iCCA. DKK1-driven changes to the tumour immune microenvironment were characterized by immunostaining and gene expression analysis. DKK1 overexpressing and damage-induced models of iCCA were used to demonstrate the therapeutic efficacy of DKK1 inhibition in these contexts using the anti-DKK1 therapeutic, DKN-01. RESULTS: DKK1 overexpression in mouse models of iCCA drives an increase in chemokine and cytokine signalling, the recruitment of regulatory macrophages, and promotes the formation of a tolerogenic niche with higher numbers of regulatory T cells. We show a similar association of DKK1 with FOXP3 and regulatory T cells in patient tissue and gene expression data, demonstrating these effects are relevant to human iCCA. Finally, we demonstrate that inhibition of DKK1 with the monoclonal antibody mDKN-01 is effective at reducing tumour burden in two distinct mouse models of the disease. CONCLUSION: DKK1 promotes tumour immune evasion in iCCA through the recruitment of immune suppressive macrophages. Targeting DKK1 with a neutralizing antibody is effective at reducing tumour growth in vivo. As such, DKK1 targeted and immune modulatory therapies may be an effective strategy in iCCA patients with high DKK1 tumour expression or tolerogenic immune phenotypes.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Peptídeos e Proteínas de Sinalização Intercelular , Animais , Humanos , Camundongos , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fenótipo , Microambiente TumoralRESUMO
Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.
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Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.
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Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy of the bile ducts within the liver characterized by high levels of genetic heterogeneity. In the context of such genetic variability, determining which oncogenic mutations drive ICC growth has been difficult, and developing modes of patient stratification and targeted therapies remains challenging. Here we model the interactions between rare mutations with more common driver genes and combine in silico analysis of patient data with highly multiplexed in vivo CRISPR-spCas9 screens to perform a functional in vivo study into the role genetic heterogeneity plays in driving ICC. Novel tumor suppressors were uncovered, which, when lost, cooperate with the RAS oncoprotein to drive ICC growth. Focusing on a set of driver mutations that interact with KRAS to initiate aggressive, sarcomatoid-type ICC revealed that tumor growth relies on Wnt and PI3K signaling. Pharmacologic coinhibition of Wnt and PI3K in vivo impeded ICC growth regardless of mutational profile. Therefore, Wnt and PI3K activity should be considered as a signature by which patients can be stratified for treatment independent of tumor genotype, and inhibitors of these pathways should be levied to treat ICC. SIGNIFICANCE: This work shows that, despite significant genetic heterogeneity, intrahepatic cholangiocarcinoma relies on a limited number of signaling pathways to grow, suggesting common therapeutic vulnerabilities across patients.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Heterogeneidade Genética , Humanos , Fosfatidilinositol 3-Quinases/genéticaRESUMO
The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1-7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.
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Teste para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/sangue , SARS-CoV-2/metabolismo , Saliva/virologia , COVID-19/epidemiologia , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Epidemias , Serviços de Assistência Domiciliar , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Curva ROC , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Manejo de Espécimes/métodosRESUMO
Background: The purpose of this study was to investigate if admission levels of total tau (T-tau) and ß-amyloid isoforms 1-40 (Aß40) and 1-42 (Aß42) could predict clinical outcome in patients with mild traumatic brain injury (mTBI). Methods: A total of 105 patients with mTBI [Glasgow Coma Scale (GCS) ≥ 13] recruited in Turku University Hospital, Turku, Finland were included in this study. Blood samples were drawn within 24 h of admission for analysis of plasma T-tau, Aß40, and Aß42. Patients were divided into computed tomography (CT)-positive and CT-negative groups. The outcome was assessed 6-12 months after the injury using the Extended Glasgow Outcome Scale (GOSE). Outcomes were defined as complete (GOSE 8) or incomplete (GOSE < 8) recovery. The Rivermead Post Concussion Symptoms Questionnaire (RPCSQ) was also used to assess mTBI-related symptoms. Predictive values of the biomarkers were analyzed independently, in panels and together with clinical parameters. Results: The admission levels of plasma T-tau, Aß40, and Aß42 were not significantly different between patients with complete and incomplete recovery. The levels of T-tau, Aß40, and Aß42 could poorly predict complete recovery, with areas under the receiver operating characteristic curve 0.56, 0.52, and 0.54, respectively. For the whole cohort, there was a significant negative correlation between the levels of T-tau and ordinal GOSE score (Spearman ρ = -0.231, p = 0.018). In a multivariate logistic regression model including age, GCS, duration of posttraumatic amnesia, Injury Severity Score (ISS), time from injury to sampling, and CT findings, none of the biomarkers could predict complete recovery independently or together with the other two biomarkers. Plasma levels of T-tau, Aß40, and Aß42 did not significantly differ between the outcome groups either within the CT-positive or CT-negative subgroups. Levels of Aß40 and Aß42 did not significantly correlate with outcome, but in the CT-positive subgroup, the levels of T-tau significantly correlated with ordinal GOSE score (Spearman ρ = -0.288, p = 0.035). The levels of T-tau, Aß40, and Aß42 were not correlated with the RPCSQ scores. Conclusions: The early levels of T-tau are correlated with the outcome in patients with mTBI, but none of the biomarkers either alone or in any combinations could predict complete recovery in patients with mTBI.
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Toxinas Botulínicas/uso terapêutico , Mãos/fisiopatologia , Espasticidade Muscular/tratamento farmacológico , Úlcera por Pressão/tratamento farmacológico , Feminino , Humanos , Injeções Intramusculares , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/complicações , Manejo da Dor , Escala Visual AnalógicaRESUMO
Activated hepatic stellate cells (aHSCs) orchestrate scarring during liver injury, with putative quiescent precursor mesodermal derivation. Here we use lineage-tracing from development, through adult homoeostasis, to fibrosis, to define morphologically and transcriptionally discreet subpopulations of aHSCs by expression of WT1, a transcription factor controlling morphological transitions in organogenesis and adult homoeostasis. Two distinct populations of aHSCs express WT1 after injury, and both re-engage a transcriptional signature reflecting embryonic mesothelial origin of their discreet quiescent adult precursor. WT1-deletion enhances fibrogenesis after injury, through upregulated Wnt-signalling and modulation of genes central to matrix persistence in aHSCs, and augmentation of myofibroblastic transition. The mesothelial-derived lineage demonstrates punctuated phenotypic plasticity through bidirectional mesothelial-mesenchymal transitions. Our findings demonstrate functional heterogeneity of adult scar-orchestrating cells that can be whole-life traced back through specific quiescent adult precursors to differential origin in development, and define WT1 as a paradoxical regulator of aHSCs induced by injury but suppressing scarring.
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Cicatriz/genética , Epitélio/embriologia , Células Estreladas do Fígado/citologia , Cirrose Hepática/genética , Fígado/embriologia , Miofibroblastos/citologia , Proteínas WT1/genética , Animais , Linhagem da Célula , Cicatriz/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Camundongos , Miofibroblastos/metabolismo , Proteínas WT1/metabolismoRESUMO
The aim of the study was to examine the ability of eight protein biomarkers and their combinations in discriminating computed tomography (CT)-negative and CT-positive patients with traumatic brain injury (TBI), utilizing highly sensitive immunoassays in a well-characterized cohort. Blood samples were obtained from 160 patients with acute TBI within 24 h of admission. Levels of ß-amyloid isoforms 1-40 (Aß40) and 1-42 (Aß42), glial fibrillary acidic protein (GFAP), heart fatty-acid binding protein (H-FABP), interleukin 10 (IL-10), neurofilament light (NF-L), S100 calcium-binding protein B (S100B), and tau were measured. Patients were divided into CT-negative (n = 65) and CT-positive (n = 95), and analyses were conducted separately for TBIs of all severities (Glasgow Coma Scale [GCS] score 3-15) and mild TBIs (mTBIs; GCS 13-15). NF-L, GFAP, and tau were the best in discriminating CT-negative and CT-positive patients, both in patients with mTBI and with all severities. In patients with all severities, area under the curve of the receiver operating characteristic (AUC) was 0.822, 0.817, and 0.781 for GFAP, NF-L, and tau, respectively. In patients with mTBI, AUC was 0.720, 0.689, and 0.676, for GFAP, tau, and NF-L, respectively. The best panel of three biomarkers for discriminating CT-negative and CT-positive patients in the group of all severities was a combination of GFAP+H-FABP+IL-10, with a sensitivity of 100% and specificity of 38.5%. In patients with mTBI, the best panel of three biomarkers was H-FABP+S100B+tau, with a sensitivity of 100% and specificity of 46.4%. Panels of biomarkers outperform individual biomarkers in separating CT-negative and CT-positive patients. Panels consisted mainly of different biomarkers than those that performed best as an individual biomarker.
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Biomarcadores/sangue , Lesões Encefálicas Traumáticas/sangue , Lesões Encefálicas Traumáticas/diagnóstico , Adulto , Idoso , Lesões Encefálicas Traumáticas/patologia , Proteína 3 Ligante de Ácido Graxo/sangue , Feminino , Proteína Glial Fibrilar Ácida/sangue , Humanos , Interleucina-10/sangue , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios XRESUMO
The purpose of this study was to correlate the early levels of glial fibrillary acidic protein (GFAP) and neurofilament light protein (NF-L) with outcome in patients with mild traumatic brain injury (mTBI). A total of 107 patients with mTBI (Glasgow Coma Scale ≥13) who had blood samples for GFAP and NF-L available within 24 h of arrival were included. Patients with mTBI were divided into computed tomography (CT)-positive and CT-negative groups. Glasgow Outcome Scale-Extended (GOSE) was used to assess the outcome. Outcomes were defined as complete (GOSE 8) versus incomplete (GOSE <8), and favorable (GOSE 5-8) versus unfavorable (GOSE 1-4). GFAP and NF-L concentrations in blood were measured using ultrasensitive single molecule array technology. Patients with incomplete recovery had significantly higher levels of NF-L compared with those with complete recovery (p = 0.005). The levels of GFAP and NF-L were significantly higher in patients with unfavorable outcome than in patients with favorable outcome (p = 0.002 for GFAP and p < 0.001 for NF-L). For predicting favorable outcome, the area under the receiver operating characteristic curve for GFAP and NF-L was 0.755 and 0.826, respectively. In a multi-variate logistic regression model, the level of NF-L was still a significant predictor for complete recovery (odds ratio [OR] = 1.008; 95% confidence interval [CI], 1.000-1.016). Moreover, the level of NF-L was a significant predictor for complete recovery in CT-positive patients (OR = 1.009; 95% CI, 1.001-1.016). The early levels of GFAP and NF-L are significantly correlated with the outcome in patients with mTBI. The level of NF-L within 24 h from arrival has a significant predictive value in mTBI also in a multi-variate model.
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Biomarcadores/sangue , Concussão Encefálica/sangue , Proteína Glial Fibrilar Ácida/sangue , Proteínas de Neurofilamentos/sangue , Recuperação de Função Fisiológica/fisiologia , Adulto , Idoso , Feminino , Escala de Resultado de Glasgow , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosAssuntos
Toxinas Botulínicas/administração & dosagem , Desenvolvimento de Medicamentos/ética , Espasticidade Muscular/tratamento farmacológico , Doenças do Sistema Nervoso/tratamento farmacológico , Adolescente , Austrália/epidemiologia , Toxinas Botulínicas/história , Toxinas Botulínicas/uso terapêutico , Lesões Encefálicas Traumáticas/epidemiologia , Lesões Encefálicas Traumáticas/reabilitação , Criança , Pré-Escolar , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Espasticidade Muscular/economia , Espasticidade Muscular/epidemiologia , Espasticidade Muscular/reabilitação , Doenças do Sistema Nervoso/epidemiologia , Doenças do Sistema Nervoso/psicologia , Doenças do Sistema Nervoso/reabilitação , Qualidade de VidaRESUMO
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
Traumatic brain injury (TBI) results in heterogeneous pathology affecting multiple cells and tissue types in the brain. It is likely that assessment of such complexity will require simultaneous measurement of multiple molecular biomarkers in a single sample of biological fluid. We measured glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1), neurofilament light chain (NF-L) and total tau in plasma samples obtained from 107 subjects enrolled in the Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot (TRACK-TBI Pilot) Study using the Quanterix Simoa 4-Plex assay. We also measured NF-L using the Simoa singleplex assay. We computed the correlation between the different biomarkers and calculated the discriminative value of each biomarker for distinguishing between subjects with abnormal versus normal head computed tomography (CT). We found a strong correlation between NF-L values derived from the multiplex and singleplex assays (correlation coefficient = 0.997). Among biomarker values derived from the multiplex assay, the strongest correlation was between the axonal and neuronal markers, NF-L and UCH-L1 (coefficient = 0.71). The weakest correlation was between the glial marker GFAP and the axonal marker tau (coefficient = 0.06). The areas under the curves for distinguishing between subjects with/without abnormal head CT for multiplex GFAP, UCH-L1, NF-L, and total tau were: 0.88 (95% confidence interval 0.81-0.95), 0.86 (0.79-0.93), 0.84 (0.77-0.92), and 0.77 0.67-0.86), respectively. We conclude that the multiplex assay provides simultaneous quantification of GFAP, UCH-L1, NF-L, and tau, and may be clinically useful in the diagnosis of TBI as well as identifying different types of cellular injury.
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Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism.
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Envelhecimento/metabolismo , Fibronectinas/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Músculo Esquelético/metabolismo , Regeneração/genética , Nicho de Células-Tronco , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Citometria de Fluxo , Integrinas/metabolismo , Camundongos , Músculo Esquelético/citologia , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: To determine whether transient hypoxia during breath-hold diving causes neuronal damage or dysfunction or alters amyloid metabolism as measured by certain blood biomarkers. DESIGN: Sixteen divers competing in the national Swedish championship in breath-hold diving and five age-matched healthy control subjects were included. Blood samples were collected at baseline and over a course of 3 days where the divers competed in static apnea (STA), dynamic apnea without fins (DYN1) and dynamic apnea with fins (DYN2). MAIN OUTCOMES: Biomarkers reflecting brain injury and amyloid metabolism were analysed in serum (S-100ß, NFL) and plasma (T-tau, Aß42) using immunochemical methods. RESULTS: Compared to divers' baseline, Aß42 increased after the first event of static apnea (p = 0.0006). T-tau increased (p = 0.001) in STA vs baseline and decreased after one of the dynamic events, DYN2 (p = 0.03). Further, T-tau correlated with the length of the apneic time during STA (ρ = 0.7226, p = 0.004) and during DYN1 (ρ = 0.66, p = 0.01). CONCLUSION: The findings suggest that transient hypoxia may acutely increase the levels of Aß42 and T-tau in plasma of healthy adults, further supporting that general hypoxia may cause mild neuronal dysfunction or damage and stimulate Aß production.
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Peptídeos beta-Amiloides/sangue , Suspensão da Respiração , Mergulho/efeitos adversos , Hipóxia/sangue , Fragmentos de Peptídeos/sangue , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Proteínas tau/sangue , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neurofilamentos/sangue , Estatísticas não Paramétricas , Adulto JovemRESUMO
Disease detection at the molecular level is driving the emerging revolution of early diagnosis and treatment. A challenge facing the field is that protein biomarkers for early diagnosis can be present in very low abundance. The lower limit of detection with conventional immunoassay technology is the upper femtomolar range (10(-13) M). Digital immunoassay technology has improved detection sensitivity three logs, to the attomolar range (10(-16) M). This capability has the potential to open new advances in diagnostics and therapeutics, but such technologies have been relegated to manual procedures that are not well suited for efficient routine use. We describe a new laboratory instrument that provides full automation of single-molecule array (Simoa) technology for digital immunoassays. The instrument is capable of single-molecule sensitivity and multiplexing with short turnaround times and a throughput of 66 samples/h. Singleplex and multiplexed digital immunoassays were developed for 16 proteins of interest in cardiovascular, cancer, infectious disease, neurology, and inflammation research. The average sensitivity improvement of the Simoa immunoassays versus conventional ELISA was >1200-fold, with coefficients of variation of <10%. The potential of digital immunoassays to advance human diagnostics was illustrated in two clinical areas: traumatic brain injury and early detection of infectious disease.
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Automação Laboratorial/instrumentação , Automação Laboratorial/métodos , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Imunoensaio/instrumentação , Imunoensaio/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Processamento de Sinais Assistido por Computador/instrumentação , Fatores de TempoRESUMO
BACKGROUND: Nucleic acid testing (NAT) has become the standard for high sensitivity in detecting low levels of virus. However, adoption of NAT can be cost prohibitive in low-resource settings where access to extreme sensitivity could be clinically advantageous for early detection of infection. We report development and preliminary validation of a simple, low-cost, fully automated digital p24 antigen immunoassay with the sensitivity of quantitative NAT viral load (NAT-VL) methods for detection of acute HIV infection. METHODS: We developed an investigational 69-min immunoassay for p24 capsid protein for use on a novel digital analyzer on the basis of single-molecule-array technology. We evaluated the assay for sensitivity by dilution of standardized preparations of p24, cultured HIV, and preseroconversion samples. We characterized analytical performance and concordance with 2 NAT-VL methods and 2 contemporary p24 Ag/Ab combination immunoassays with dilutions of viral isolates and samples from the earliest stages of HIV infection. RESULTS: Analytical sensitivity was 0.0025 ng/L p24, equivalent to 60 HIV RNA copies/mL. The limit of quantification was 0.0076 ng/L, and imprecision across 10 runs was <10% for samples as low as 0.09 ng/L. Clinical specificity was 95.1%. Sensitivity concordance vs NAT-VL on dilutions of preseroconversion samples and Group M viral isolates was 100%. CONCLUSIONS: The digital immunoassay exhibited >4000-fold greater sensitivity than contemporary immunoassays for p24 and sensitivity equivalent to that of NAT methods for early detection of HIV. The data indicate that NAT-level sensitivity for acute HIV infection is possible with a simple, low-cost digital immunoassay.
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Proteína do Núcleo p24 do HIV/análise , Infecções por HIV/diagnóstico , HIV/isolamento & purificação , Imunoensaio/métodos , Linhagem Celular , Proteína do Núcleo p24 do HIV/sangue , Infecções por HIV/sangue , Humanos , Imunoensaio/economia , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/análise , Sensibilidade e Especificidade , Carga Viral/economia , Carga Viral/métodosRESUMO
BACKGROUND: The association between increases in cardiac troponin and adverse cardiac outcomes is well established. There is a growing interest in exploring routine cardiac troponin monitoring as a potential early indicator of adverse heart health trends. Prognostic use of cardiac troponin measurements requires an assay with very high sensitivity and outstanding analytical performance. We report development and preliminary validation of an investigational assay meeting these requirements and demonstrate its applicability to cohorts of healthy individuals and patients with heart failure. METHODS: On the basis of single molecule array technology, we developed a 45-min immunoassay for cardiac troponin I (cTnI) for use on a novel, fully automated digital analyzer. We characterized its analytical performance and measured cTnI in healthy individuals and heart failure patients in a preliminary study of assay analytical efficacy. RESULTS: The assay exhibited a limit of detection of 0.01 ng/L, a limit of quantification of 0.08 ng/L, and a total CV of 10% at 2.0 ng/L. cTnI concentrations were well above the assay limit of detection for all samples tested, including samples from healthy individuals. cTnI was significantly higher in heart failure patients, and exhibited increasing median and interquartile concentrations with increasing New York Heart Association classification of heart failure severity. CONCLUSIONS: The robust 2-log increase in sensitivity relative to contemporary high-sensitivity cardiac troponin immunoassays, combined with full automation, make this assay suitable for exploring cTnI concentrations in cohorts of healthy individuals and for the potential prognostic application of serial cardiac troponin measurements in both apparently healthy and diseased individuals.