RESUMO
Thymic involution and proliferation of naive T cells both contribute to shaping the naive T-cell repertoire as humans age, but a clear understanding of the roles of each throughout a human life span has been difficult to determine. By measuring nuclear bomb test-derived 14C in genomic DNA, we determined the turnover rates of CD4+ and CD8+ naive T-cell populations and defined their dynamics in healthy individuals ranging from 20 to 65 years of age. We demonstrate that naive T-cell generation decreases with age because of a combination of declining peripheral division and thymic production during adulthood. Concomitant decline in T-cell loss compensates for decreased generation rates. We investigated putative mechanisms underlying age-related changes in homeostatic regulation of CD4+ naive T-cell turnover, using mass cytometry to profile candidate signaling pathways involved in T-cell activation and proliferation relative to CD31 expression, a marker of thymic proximity for the CD4+ naive T-cell population. We show that basal nuclear factor κB (NF-κB) phosphorylation positively correlated with CD31 expression and thus is decreased in peripherally expanded naive T-cell clones. Functionally, we found that NF-κB signaling was essential for naive T-cell proliferation to the homeostatic growth factor interleukin (IL)-7, and reduced NF-κB phosphorylation in CD4+CD31- naive T cells is linked to reduced homeostatic proliferation potential. Our results reveal an age-related decline in naive T-cell turnover as a putative regulator of naive T-cell diversity and identify a molecular pathway that restricts proliferation of peripherally expanded naive T-cell clones that accumulate with age.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem da Célula/imunologia , Homeostase/imunologia , Timo/imunologia , Adulto , Idoso , Envelhecimento/genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Linhagem da Célula/genética , Proliferação de Células , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/genética , Humanos , Imunofenotipagem , Interleucina-7/genética , Interleucina-7/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/imunologia , Fosforilação , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Transdução de Sinais , Timo/citologia , Timo/crescimento & desenvolvimentoRESUMO
The realization of electrochemical nucleic acid amplification tests (NAATs) at the point of care (POC) is highly desirable, but it remains a challenge given their high cost and lack of true portability/miniaturization. Here we show that mass-produced, industrial standardized, printed circuit boards (PCBs) can be repurposed to act as near-zero cost electrodes for self-assembled monolayer-based DNA biosensing, and further integration with a custom-designed and low-cost portable potentiostat. To show the analytical capability of this system, we developed a NAAT using isothermal recombinase polymerase amplification, bypassing the need of thermal cyclers, followed by an electrochemical readout relying on a sandwich hybridization assay. We used our sensor and device for analytical detection of the toxic microalgae Ostreopsis cf. ovata as a proof of concept. This work shows the potential of PCBs and open-source electronics to be used as powerful POC DNA biosensors at a low-cost.
Assuntos
Técnicas Biossensoriais , Técnicas de Amplificação de Ácido Nucleico , DNA/genética , Eletrônica , RecombinasesRESUMO
Respiratory viral infections often mimic the symptoms of infections caused by bacteria; however, restricted and targeted administration of antibiotics is needed to combat growing antimicrobial resistance. This is particularly relevant in low-income settings. In this work, we describe the use of isothermal amplification of viral DNA at 37 °C coupled to a paper-based vertical flow microarray (VFM) setup that utilizes a colorimetric detection of amplicons using functionalized gold nanoparticles. Two oligonucleotide probes, one in-house designed and one known adenoviral probe were tested and validated for microarray detection down to 50 nM using a synthetic target template. Furthermore, primers were shown to function in a recombinase polymerase amplification reaction using both synthetic template and viral DNA. As a proof-of-concept, we demonstrate adenoviral detection with four different adenoviral species associated with respiratory infections using the paper-based VFM format. The presented assay was validated with selected adenoviral species using the in-house probe, enabling detection at 1 ng of starting material with intra- and inter-assay %CV of ≤ 9% and ≤ 13%. Finally, we validate our overall method using clinical samples. Based on the results, the combination of recombinase polymerase amplification, paper microarray analysis, and nanoparticle-based colorimetric detection could thus be a useful strategy towards rapid and affordable multiplexed viral diagnostics.
Assuntos
Adenoviridae/isolamento & purificação , DNA Viral/análise , DNA Polimerase Dirigida por DNA/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/metabolismo , Adenoviridae/genética , Colorimetria , Primers do DNA , Ouro/química , Temperatura Alta , Humanos , Nanopartículas Metálicas/química , Análise de Sequência com Séries de Oligonucleotídeos , Sondas de Oligonucleotídeos , Papel , Estudo de Prova de Conceito , Infecções Respiratórias/virologia , Moldes GenéticosRESUMO
Analytical systems based on isothermal nucleic acid amplification tests (NAATs) paired with electroanalytical detection enable cost-effective, sensitive, and specific digital pathogen detection for various in situ applications such as point-of-care medical diagnostics, food safety monitoring, and environmental surveillance. Self-assembled monolayers (SAMs) on gold surfaces are reliable platforms for electroanalytical DNA biosensors. However, the lack of automation and scalability often limits traditional chip-based systems. To address these challenges, we propose a continuous thread-based device that enables multiple electrochemical readings on a functionalized working electrode Au thread with a single connection point. We demonstrate the possibility of rolling the thread on a spool, which enables easy manipulation in a roll-to-roll architecture for high-throughput applications. As a proof of concept, we have demonstrated the detection of recombinase polymerase amplification (RPA) isothermally amplified DNA from the two toxic microalgae species Ostreopsis cf. ovata and Ostreopsis cf. siamensis by performing a sandwich hybridization assay (SHA) with electrochemical readout.
Assuntos
Bioensaio , Ouro , Automação , Eletrodos , Técnicas de Diagnóstico MolecularRESUMO
Enzymes are the cornerstone of modern biotechnology. Achromopeptidase (ACP) is a well-known enzyme that hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria. It is therefore used for sample preparation in nucleic acid tests. However, ACP inhibits DNA amplification which makes its integration difficult. Heat is commonly used to inactivate ACP, but it can be challenging to integrate heating into point-of-care devices. Here, we use recombinase polymerase amplification (RPA) together with ACP, and show that when ACP is immobilized on nitrocellulose paper, it retains its enzymatic function and can easily and rapidly be activated using agitation. The nitrocellulose-bound ACP does, however, not leak into the solution, preventing the need for deactivation through heat or by other means. Nitrocellulose-bound ACP thus opens new possibilities for paper-based Point-of-Care (POC) devices.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Testes Imediatos , Infecções Estafilocócicas , Staphylococcus epidermidis/isolamento & purificação , Humanos , Sondas Moleculares/genética , Serina Endopeptidases/química , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologiaRESUMO
Fiber-based biosensors enable a new approach in analytical diagnostic devices. The majority of textile-based biosensors, however, rely on colorimetric detection. Here a woven biosensor that integrates microfluidics structures in combination with an electroanalytical readout based on a thiol-self-assembled monolayer (SAM) for Nucleic Acid Amplification Testing, NAATs is shown. Two types of fiber-based electrodes are systematically characterized: pure gold microwires (bond wire) and off-the-shelf plasma gold-coated polyester multifilament threads to evaluate their potential to form SAMs on their surface and their electrochemical performance in woven textile. A woven electrochemical DNA (E-DNA) sensor using a SAM-based stem-loop probe-modified gold microwire is fabricated. These sensors can specifically detect unpurified, isothermally amplified genomic DNA of Staphylococcus epidermidis (10 copies/µL) by recombinase polymerase amplification (RPA). This work demonstrates that textile-based biosensors have the potential for integrating and being employed as automated, sample-to-answer analytical devices for point-of-care (POC) diagnostics.
Assuntos
Técnicas Biossensoriais , Técnicas de Amplificação de Ácido Nucleico , DNA , Eletrodos , OuroRESUMO
The CD8+ T cell response to an antigen is composed of many T cell clones with unique T cell receptors, together forming a heterogeneous repertoire of effector and memory cells. How individual T cell clones contribute to this heterogeneity throughout immune responses remains largely unknown. In this study, we longitudinally track human CD8+ T cell clones expanding in response to yellow fever virus (YFV) vaccination at the single-cell level. We observed a drop in clonal diversity in blood from the acute to memory phase, suggesting that clonal selection shapes the circulating memory repertoire. Clones in the memory phase display biased differentiation trajectories along a gradient from stem cell to terminally differentiated effector memory fates. In secondary responses, YFV- and influenza-specific CD8+ T cell clones are poised to recapitulate skewed differentiation trajectories. Collectively, we show that the sum of distinct clonal phenotypes results in the multifaceted human T cell response to acute viral infections.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Viroses/virologia , Febre Amarela/virologia , Doença Aguda , Diferenciação Celular , Células Cultivadas , HumanosRESUMO
BACKGROUND: A timely differential diagnostic is essential to identify the etiology of central nervous system (CNS) infections in children, in order to facilitate targeted treatment, manage patients, and improve clinical outcome. OBJECTIVE: The Pediatric Infection-Point-of-Care (PI-POC) trial is investigating novel methods to improve and strengthen the differential diagnostics of suspected childhood CNS infections in low-income health systems such as those in Southwestern Uganda. This will be achieved by evaluating (1) a novel DNA-based diagnostic assay for CNS infections, (2) a commercially available multiplex PCR-based meningitis/encephalitis (ME) panel for clinical use in a facility-limited laboratory setting, (3) proteomics profiling of blood from children with severe CNS infection as compared to outpatient controls with fever yet not severely ill, and (4) Myxovirus resistance protein A (MxA) as a biomarker in blood for viral CNS infection. Further changes in the etiology of childhood CNS infections after the introduction of the pneumococcal conjugate vaccine against Streptococcus pneumoniae will be investigated. In addition, the carriage and invasive rate of Neisseria meningitidis will be recorded and serotyped, and the expression of its major virulence factor (polysaccharide capsule) will be investigated. METHODS: The PI-POC trial is a prospective observational study of children including newborns up to 12 years of age with clinical features of CNS infection, and age-/sex-matched outpatient controls with fever yet not severely ill. Participants are recruited at 2 Pediatric clinics in Mbarara, Uganda. Cerebrospinal fluid (for cases only), blood, and nasopharyngeal (NP) swabs (for both cases and controls) sampled at both clinics are analyzed at the Epicentre Research Laboratory through gold-standard methods for CNS infection diagnosis (microscopy, biochemistry, and culture) and a commercially available ME panel for multiplex PCR analyses of the cerebrospinal fluid. An additional blood sample from cases is collected on day 3 after admission. After initial clinical analyses in Mbarara, samples will be transported to Stockholm, Sweden for (1) validation analyses of a novel nucleic acid-based POC test, (2) biomarker research, and (3) serotyping and molecular characterization of S. pneumoniae and N. meningitidis. RESULTS: A pilot study was performed from January to April 2019. The PI-POC trial enrollment of patients begun in April 2019 and will continue until September 2020, to include up to 300 cases and controls. Preliminary results from the PI-POC study are expected by the end of 2020. CONCLUSIONS: The findings from the PI-POC study can potentially facilitate rapid etiological diagnosis of CNS infections in low-resource settings and allow for novel methods for determination of the severity of CNS infection in such environment. TRIAL REGISTRATION: ClinicalTrials.gov NCT03900091; https://clinicaltrials.gov/ct2/show/NCT03900091. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/21430.
RESUMO
Cell culture is a ubiquitous and flexible research method. However, it heavily relies on plastic consumables generating millions of tonnes of plastic waste yearly. Plastic waste is a major and growing global concern. Here we describe a new cell culture dish that offers a culture area equivalent to three petri dishes but that is on average 61% lighter and occupies 67% less volume. Our dish is composed of a lid and three thin containers surrounded by a light outer shell. Cell culture can be performed in each of the containers sequentially. The outer shell provides the appropriate structure for the manipulation of the dish as a whole. The prototype was tested by sequentially growing cells in each of its containers. As a control, sequential cultures in groups of 3 petri dishes were performed. No statistical differences were found between the prototype and the control in terms of cell number, cell viability or cell distribution.
Assuntos
Técnicas de Cultura de Células/instrumentação , Plásticos , Resíduos , Células HeLa , Humanos , Poliésteres/químicaRESUMO
Accurate variant calling and genotyping represent major limiting factors for downstream applications of single-cell genomics. Here, we report Conbase for the identification of somatic mutations in single-cell DNA sequencing data. Conbase leverages phased read data from multiple samples in a dataset to achieve increased confidence in somatic variant calls and genotype predictions. Comparing the performance of Conbase to three other methods, we find that Conbase performs best in terms of false discovery rate and specificity and provides superior robustness on simulated data, in vitro expanded fibroblasts and clonal lymphocyte populations isolated directly from a healthy human donor.
Assuntos
Mutação , Análise de Célula Única , Software , Linfócitos T CD8-Positivos , Fibroblastos , Humanos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
The hematopoietic system seeds the CNS with microglial progenitor cells during the fetal period, but the subsequent cell generation dynamics and maintenance of this population have been poorly understood. We report that microglia, unlike most other hematopoietic lineages, renew slowly at a median rate of 28% per year, and some microglia last for more than two decades. Furthermore, we find no evidence for the existence of a substantial population of quiescent long-lived cells, meaning that the microglia population in the human brain is sustained by continuous slow turnover throughout adult life.
Assuntos
Encéfalo/citologia , Encéfalo/fisiologia , Microglia/citologia , Microglia/fisiologia , Adolescente , Adulto , Encéfalo/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Humanos , Microglia/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/fisiologiaRESUMO
Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19+ PC subset was dynamically exchanged, whereas of two CD19- PC subsets, CD45+ PCs exhibited little and CD45- PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45- PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19- PC subsets support selection and maintenance of protective PCs for life in human intestine.
Assuntos
Intestinos/imunologia , Plasmócitos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Produtoras de Anticorpos/imunologia , Antígenos CD19/análise , Criança , Feminino , Humanos , Antígenos Comuns de Leucócito/análise , Masculino , Pessoa de Meia-IdadeRESUMO
Central nervous system injuries are accompanied by scar formation. It has been difficult to delineate the precise role of the scar, as it is made by several different cell types, which may limit the damage but also inhibit axonal regrowth. We show that scarring by neural stem cell-derived astrocytes is required to restrict secondary enlargement of the lesion and further axonal loss after spinal cord injury. Moreover, neural stem cell progeny exerts a neurotrophic effect required for survival of neurons adjacent to the lesion. One distinct component of the glial scar, deriving from resident neural stem cells, is required for maintaining the integrity of the injured spinal cord.