RESUMO
Background: Epigenetic clocks are emerging as a useful tool in many areas of research. Many epigenetic clocks have been developed for adults; however, there are fewer clocks focused on newborns and most are trained using blood from European ancestry populations. In this study, we built an epigenetic clock based on primary human umbilical vein endothelial cells from a racially and ethnically diverse population. Results: Using human umbilical vein endothelial cell [HUVEC]-derived DNA, we calculated epigenetic gestational age using 83 CpG sites selected through elastic net regression. In this study with newborns from different racial/ethnic identities, epigenetic gestational age and clinical gestational age were more highly correlated (r = 0.85), than epigenetic clocks built from adult and other pediatric populations. The correlation was also higher than clocks based on blood samples from newborns with European ancestry. We also found that birth weight was positively associated with epigenetic gestational age acceleration (EGAA), while NICU admission was associated with lower EGAA. Newborns self-identified as Hispanic or non-Hispanic Black had lower EGAA than self-identified as non-Hispanic White. Conclusions: Epigenetic gestational age can be used to estimate clinical gestational age and may help index neonatal development. Caution should be exercised when using epigenetic clocks built from adults with children, especially newborns. We highlight the importance of cell type-specific epigenetic clocks and general pan tissue epigenetic clocks derived from a large racially and ethnically diverse population.
RESUMO
A frequent topic of biomedical research is the potential clinical use of non-coding (nc) RNAs as quantitative biomarkers for a broad spectrum of health and disease. However, ncRNA analyses have not been pressed into widespread diagnostic use. Strong preclinical evidence suggests obstacles in the translation and reproducibility of this type of biomarker which may result from preanalytical and analytical variation in the non-standardized processes used to collect, process, and store samples, as well as the substantive differences between small and long ncRNA. We performed a narrative review of selected literature, through the lens of key laboratory-developed test (LDT) regulations under the Clinical Laboratory Improvement Amendments (CLIA) in the United States, to study critical gaps in ncRNA validation studies. This review describes the leading candidate ncRNA subclasses, their biogenesis and cellular function, and identifies specific pre-analytical variables with disproportionate impact on testing performance. We summarize these findings with strategic recommendations to clinicians and biomedical scientists involved in the design, conduct, and translation of ncRNA biomarker development.
Assuntos
Pesquisa Biomédica , RNA Longo não Codificante , Humanos , Estados Unidos , Reprodutibilidade dos Testes , RNA não Traduzido/genética , Biomarcadores , RNA Longo não Codificante/genéticaRESUMO
Newborn screening using dried plasma spots offers preanalytical advantages over conventional cards for plasma-associated targets of interest. Herein we present dried plasma spot-based methods for measuring metabolites using a 250+ compound liquid chromatography tandem mass spectrometry library. Quality assurance reduced this library to 134, and from these, 30 compounds determined the normal newborn reference ranges.
Assuntos
Biomarcadores/sangue , Cromatografia Líquida , Teste em Amostras de Sangue Seco/métodos , Metaboloma , Triagem Neonatal/métodos , Espectrometria de Massas em Tandem , Preservação de Sangue/métodos , Preservação de Sangue/normas , Teste em Amostras de Sangue Seco/normas , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Triagem Neonatal/normas , Estudos Prospectivos , Valores de Referência , Manejo de Espécimes/métodos , Manejo de Espécimes/normasRESUMO
Repurposing biological samples collected for required diagnostic purposes into suitable biobanking projects is a particularly useful method for enabling research in vulnerable populations. This approach is especially appropriate for the neonate in the neonatal intensive care unit (NICU), where blood volume reductions can quickly increase beyond minimal risk for adverse events, such as iatrogenic anemia, and proxy consent provided by parents or guardians is required. The method described in this study provides a framework to prospectively collect and store blood-derived clinical samples after all clinical and regulatory requirements are fulfilled. The consent approach incorporated a 30-day window to allow parents and guardians ample consideration time with follow-up involvement with NICU embedded study team members. The study enrolled 875 participants over a 3-year period. This established a critically needed biobank to support investigator-initiated research with explicit study aims requiring samples at defined day of life frequencies within the NICU and created a normative control reference bank for case comparisons for premature and full-term neonates with brain injury.
Assuntos
Bancos de Espécimes Biológicos , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Unidades de Terapia Intensiva Neonatal , Pais , Estudos ProspectivosAssuntos
Pesquisa Biomédica , Computação em Nuvem , Bancos de Tecidos , Criança , Humanos , PediatriaRESUMO
The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env approximately Gag/Pol > Gag approximately Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.
Assuntos
Adenoviridae/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia/imunologia , Vacinas contra a AIDS/imunologia , Adenoviridae/genética , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Ensaios Clínicos Fase II como Assunto , Humanos , Macaca mulatta/imunologia , Macaca mulatta/virologia , Testes de Neutralização , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Carga Viral , Viremia/imunologiaRESUMO
We sought to determine how effectively a CD8+ T cell inducing vaccine controls SHIV-89.6P infection in rhesus macaques at a range of challenge times post-vaccination. To this end, twenty eight Mamu-A*01+ rhesus macaques were given replication incompetent human serotype 5 adenovirus vector expressing SIVmac239 gag DNA and boosted 24 weeks later. Groups of 4 monkeys were then challenged with SHIV-89.6P at 1, 3, 6, 12, and 24 weeks after the boost. We compared the kinetics of viral load, CD4+ and virus-specific CD8+ T cells in these macaques. Measurements of CD8+ T cells taken before challenge show an exponential decay between 1 and 12 weeks following vaccination (p<0.0001). After week 12, no further decay was observed. Twenty of 24 vaccinated animals maintained more CD4+ T cells and kept their viral load at least one order of magnitude lower than the control animals throughout the chronic phase of the study. All 24 vaccinated animals survived the duration of the study. The viral and T cell kinetics over the first two weeks differed between the vaccinated groups, with more recent vaccination improving the early control of virus (p-value=0.027). The rates of virus specific CD8+ T cell expansion were greater in animals having higher viral loads at one week (r=0.45, p=0.029), suggesting that the kinetics of early viral load may have a role in virus specific CD8+ T cell generation, although these early differences did not lead to different clinical outcomes within the vaccinated animals.
Assuntos
Macaca mulatta/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Carga Viral , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Fatores de Tempo , VacinaçãoRESUMO
HIV-1 integrase catalyzes the insertion of viral DNA into the genome of the host cell. Integrase inhibitor N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide selectively inhibits the strand transfer process of integration. 4-Substituted pyrrolidinones possessing various groups on the pyrrolidinone nitrogen were introduced at the 5-position of the naphthyridine scaffold. These analogs exhibit excellent activity against viral replication in a cell-based assay. The preparation of these compounds was enabled by a three-step, two-pot reaction sequence from a common butenolide intermediate.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , Naftiridinas/síntese química , Naftiridinas/farmacologia , Administração Oral , Animais , Fármacos Anti-HIV/química , Inibidores de Integrase de HIV/química , Estrutura Molecular , Naftiridinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
A series of 10-hydroxy-7,8-dihydropyrazino[1',2':1,5]pyrrolo[2,3-d]pyridazine-1,9(2H,6H)-diones was synthesized and tested for their inhibition of HIV-1 replication in cell culture. Structure-activity studies indicated that high antiviral potency against wild-type virus as well as viruses containing integrase mutations that confer resistance to three different structural classes of integrase inhibitors could be achieved by incorporation of small aliphatic groups at certain positions on the core template. An optimal compound from this study, 16, inhibits integrase strand-transfer activity with an IC(50) value of 10 nM, inhibits HIV-1 replication in cell culture with an IC(95) value of 35 nM in the presence of 50% normal human serum, and displays modest pharmacokinetic properties in rats (i.v. t(1/2)=5.3 h, F=17%).
Assuntos
Química Farmacêutica/métodos , Integrase de HIV/síntese química , Integrase de HIV/farmacologia , Integrases/genética , Mutação , Administração Oral , Animais , Antivirais/farmacologia , Disponibilidade Biológica , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Modelos Químicos , Ratos , Relação Estrutura-Atividade , Replicação ViralRESUMO
A series of 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides was synthesized and tested for their inhibition of HIV-1 integrase catalytic activity and HIV-1 replication in cells. Structure-activity studies around lead compound 5 indicated that a coplanar relationship of metal-binding heteroatoms provides optimal binding to the integrase active site. Identification of potency-enhancing substituents and adjustments in lipophilicity provided 17b which inhibits integrase-catalyzed strand transfer with an IC(50) value of 74 nM and inhibits HIV-1 replication in cell culture in the presence of 50% normal human serum with an IC(95) value of 63 nM.
Assuntos
Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/efeitos dos fármacos , Pirazinas/química , Pirazinas/farmacologia , Catálise , Linhagem Celular , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/síntese química , HIV-1/enzimologia , HIV-1/fisiologia , Pirazinas/síntese química , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Behavioral testing of transgenic mouse models of Alzheimer's disease (AD) is the functional endpoint for determining the effectiveness of therapeutic interventions and elucidating AD pathogenesis. Utilizing these mouse models, there have been remarkably few attempts to analyze multiple behavioral measures/tasks with higher-level computation techniques, either to distinguish performance between transgenic groups or to reveal any "overall" cognitive benefit of a given therapeutic. The present study compared the classificatory accuracy of artificial neural networks (ANNs) versus more traditional discriminant function analysis (DFA) using multiple behavioral measures/tasks from two AD transgenic mouse investigations. These investigations were to determine if AD transgenic mice could be cognitively-protected by either long-term caffeine administration (CA) or by a cognitively-stimulating environment (SE). Both the entire set of behavioral measures and a subset of 8 cognitive-based measures were analyzed. Both classifiers revealed a beneficial "overall" effect of CA and SE to protect AD transgenic mice across multiple cognitive measures/tasks. However, for both CA and SE datasets, the ANN was superior to DFA for discerning transgenicity (non-transgenic vs. transgenic-controls) across multiple behavioral measures. These results indicate that ANNs have an excellent capacity to discriminate cognitive impairment in AD transgenic mice and thus designate ANNs as a novel, sensitive method for cognitive assessment in Alzheimer's research.
Assuntos
Doença de Alzheimer/complicações , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/etiologia , Redes Neurais de Computação , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Cafeína/administração & dosagem , Estimulantes do Sistema Nervoso Central/administração & dosagem , Transtornos Cognitivos/tratamento farmacológico , Discriminação Psicológica/efeitos dos fármacos , Discriminação Psicológica/fisiologia , Modelos Animais de Doenças , Meio Ambiente , Camundongos , Camundongos Transgênicos , Testes Neuropsicológicos , Presenilina-1/genéticaRESUMO
A series of potent novel 8-hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 12 is active against replication of HIV-1 in cell culture with a CIC(95) of 0.31microM. Further SAR exploration led to the preparation of pseudosymmetrical tricyclic pyrrolopyrazine inhibitors 23 and 24 with further improvement in antiviral activity.
Assuntos
Inibidores de Integrase de HIV/química , Integrase de HIV , Pirazinas/química , Linhagem Celular Tumoral , Integrase de HIV/fisiologia , Inibidores de Integrase de HIV/farmacologia , Humanos , Pirazinas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/virologiaRESUMO
A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of HIV with a CIC(95) of 0.31 microM and exhibits no shift in potency in the presence of 50% normal human serum. It displays a good pharmacokinetic profile when dosed in rats and no covalent binding with microsomal proteins in both in vitro and in vivo models.
Assuntos
Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , Pirazinas/química , Pirazinas/farmacologia , Animais , Benzeno/química , Linhagem Celular , HIV/efeitos dos fármacos , HIV/enzimologia , HIV/fisiologia , Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/farmacocinética , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Pirazinas/síntese química , Pirazinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
A series of HIV protease inhibitors with modifications on the P3 position have been designed and synthesized. These compounds exhibit excellent antiviral activity against both the wild type enzyme and PI-resistant clinical viral isolates. The synthesis and biological activity of the compounds are described.
Assuntos
Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Linhagem Celular , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Indicadores e Reagentes , Indinavir/síntese química , Indinavir/farmacologia , Relação Estrutura-AtividadeRESUMO
Lack of virus specific antibody response is commonly observed in both HIV-1-infected humans and SIV-infected monkeys with rapid disease progression. However, the mechanisms underlying this important observation still remain unclear. In a titration study of a SIVmac239 viral stock, three out of six animals with viral inoculation rapidly progressed to AIDS within 5 months. Unexpectedly, there was no obvious depletion of CD4(+) T cells in both peripheral and lymph node (LN) compartments in these animals. Instead, progressive depletion of proliferating B cells and disruption of the follicular dendritic cell (FDC) network in germinal centers (GC) was evident in the samples collected at as early as 20 days after viral challenge. This coincided with undetectable, or weak and transient, virus-specific antibody responses over the course of infection. In situ hybridization of SIV RNA in the LN samples revealed a high frequency of SIV productively infected cells and large amounts of accumulated viral RNA in the GCs in these animals. Early severe depletion of GC proliferating B cells and disruption of the FDC network may thus result in an inability to mount a virus-specific antibody response in rapid progressors, which has been shown to contribute to accelerated disease progression of SIV infection.
Assuntos
Linfonodos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia , Animais , Anticorpos Antivirais/sangue , Linfócitos B/citologia , Biópsia , Contagem de Células , Progressão da Doença , Centro Germinativo/imunologia , Centro Germinativo/virologia , Hiperplasia/patologia , Linfonodos/patologia , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/diagnóstico , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/imunologia , Carga ViralRESUMO
A 1,6-naphthyridine inhibitor of HIV-1 integrase has been discovered with excellent inhibitory activity in cells, good pharmacokinetics, and an excellent ability to inhibit virus with mutant enzyme.
Assuntos
Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/farmacocinética , Naftiridinas/síntese química , Administração Oral , Animais , Células Cultivadas , Integrase de HIV/efeitos dos fármacos , Integrase de HIV/genética , Inibidores de Integrase de HIV/farmacologia , Humanos , Concentração Inibidora 50 , Mutação , Naftiridinas/farmacocinética , Naftiridinas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
gp120 is a subunit of the envelope glycoprotein of HIV-1. The third variable loop region of gp120 (V3 loop) contains multiple immunodominant epitopes and is also functionally important for deciding cell-tropism of the virus. 447-52D is a monoclonal antibody that recognizes the conserved tip of the V3 loop in a beta-turn conformation. This antibody has previously been shown to neutralize diverse strains of the virus. In an attempt to generate an immunogen competent to generate 447-52D-like antibodies, the known epitope of 447-52D was inserted at three different surface loop locations in the small, stable protein Escherichia coli Trx (thioredoxin). At one of the three locations (between residues 74 and 75), the insertion was tolerated, the resulting protein was stable and soluble, and bound 447-52D with an affinity similar to that of intact gp120. Upon immunization, the V3 peptide-inserted Trx scaffold was able to generate anti-V3 antibodies that could compete out 447-52D binding to gp120. Epitope mapping studies demonstrated that these anti-V3 antibodies recognized the same epitope as 447-52D. Although the 447-52D-type antibodies were estimated to be present at concentrations of 50-400 microg/ml of serum, these were not able to effect neutralization of strains like JRFL and BAL but could neutralize the sensitive MN strain. The data suggest that because of the low accessibility of the V3 loop on primary isolates such as JRFL, it will be difficult to elicit a V3-specific, 447-52D-like antibody response to effectively neutralize such isolates.