RESUMO
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/normasAssuntos
Pesquisa Biomédica , Computação em Nuvem , Bancos de Tecidos , Criança , Humanos , PediatriaRESUMO
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
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
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
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 ProspectivosRESUMO
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 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
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 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
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.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos/imunologia , Dicroísmo Circular , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Cobaias , Anticorpos Anti-HIV/sangue , Antígenos HIV/química , Antígenos HIV/genética , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Ressonância de Plasmônio de Superfície , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMO
OBJECTIVE: To assess responses to indinavir (IDV)-ritonavir (RTV)-based regimens among HIV-1 infected patients with prior failure of protease inhibitors, and to assess the effects of adherence to therapy and pre-existing genotypic and phenotypic resistance on this response. METHODS: Twenty-eight patients initiating salvage regimens with IDV-RTV (800 mg and 200 mg twice daily, respectively) plus one or more reverse transcriptase inhibitor (RTI) were identified retrospectively. Genotypic and phenotypic susceptibilities to multiple antiretroviral agents were determined on viral samples collected at initiation of the salvage regimens, and adherence to therapy was determined through patient self-reporting. Response to therapy (viral RNA = 400 copies/ml) was assessed at the end of and beyond 6 months of follow-up. RESULTS: Based on responses measured in the first 6 months of follow-up, 16 responders and 12 non-responders were identified without differences in baseline demographic factors, laboratory parameters, extent of prior antiretroviral therapy, or characteristics of the RTI components of the new IDV-RTV-based regimens. Adequate adherence was associated with virologic responses (P = 0.005). There were trends for genotypic and phenotypic resistance to be associated with adequate adherence, and, surprisingly, phenotypic resistance to IDV was associated with virologic response rather than with therapeutic failure (P = 0.02). Beyond 6 months of follow-up (mean follow-up 69 weeks), adequate adherence was still associated with virologic response (P = 0.001), but genotypic or phenotypic resistance to IDV were not associated with therapeutic failure. CONCLUSIONS: These results suggest that IDV-RTV-based regimens may be able to overcome IDV resistance. This underscores the importance of drug adherence, potency, and exposure in determining virologic responses to antiretroviral therapy.
Assuntos
Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/uso terapêutico , HIV-1 , Indinavir/uso terapêutico , Ritonavir/uso terapêutico , Farmacorresistência Viral , Quimioterapia Combinada , Feminino , Seguimentos , Genótipo , Infecções por HIV/virologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Masculino , Cooperação do Paciente , Fenótipo , Estudos Retrospectivos , Terapia de Salvação/métodos , Falha de Tratamento , Carga ViralRESUMO
The blockade of the HIV protease has become a major target in the search for an effective therapy for AIDS.1 While many reports of potent HIV-1 inhibitors have appeared recently, the compound Ro 31-8959 remains the least selective for the HIV-1 and HIV-2 proteases.2 This property may result in reduced susceptibility to resistance since these represent the genetically most divergent strains of HIV presently known to exist.
RESUMO
A fluorescence-based, multiplexed, antibody-binding and mapping assay was developed to characterize antibody responses in HIV-1-infected individuals to the ectodomain of the HIV-1 gp41 envelope glycoprotein. The antigen panel included intact recombinant gp41, the fusion peptide region, the polar region, the N-heptad region, the C-heptad region as well as overlapping epitopes in the 2F5 and 4E10 monoclonal antibody-binding regions. The panel included both native and constrained peptides specifically designed to mimic putative gp41 prefusion and fusion intermediates. The results of these analyses revealed a broad pattern of immune responses against the test antigens, suggesting that none of these gp41 regions are immunologically silent. The HIV-1-positive sera were also evaluated using infectivity inhibition assays. No correlation was evident between the breadth or magnitude of specific anti-gp41 reactivities and virus neutralization potency. These evaluations demonstrated the substantial potential of the multiplexed antibody binding and mapping assay for rapid and sensitive analysis of complex antibody responses.
Assuntos
Mapeamento de Epitopos , Imunofluorescência/métodos , Anticorpos Anti-HIV/análise , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos/métodos , Citometria de Fluxo/métodos , Proteína gp41 do Envelope de HIV/química , Infecções por HIV/sangue , Infecções por HIV/imunologia , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Conformação Proteica , Sensibilidade e EspecificidadeRESUMO
The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors [(-)-6, (-)-7, (-)-23, (+)-24] based upon the 3,5,5-trisubstituted pyrrolin-4-one scaffold is described. Use of a monopyrrolinone scaffold leads to inhibitors with improved cellular transport properties relative to the earlier inhibitors based on bispyrrolinones and their peptide counterparts. The most potent inhibitor (-)-7 displayed 13% oral bioavailability in dogs. X-ray structure analysis of the monopyrrolinone compounds cocrystallized with the wild-type HIV-1 protease provided valuable information on the interactions between the inhibitors and the HIV-1 enzyme. In each case, the inhibitors assumed similar orientations for the P2'-P1 substituents, along with an unexpected hydrogen bond of the pyrrolinone NH with Asp225. Interactions with the S2 pocket, however, were not optimal, as illustrated by the inclusion of a water molecule in two of the three inhibitor-enzyme complexes. Efforts to increase affinity by displacing the water molecule with second and third generation inhibitors did not prove successful. Lack of success with this venture is a testament to the difficulty of accurately predicting the many variables that influence and build binding affinity. Comparison of the inhibitor positions in three complexes with that of Indinavir revealed displacements of the protease backbones in the enzyme flap region, accompanied by variations in hydrogen bonding to accommodate the monopyrrolinone ring. The binding orientation of the pyrrolinone-based inhibitors may explain their sustained efficacy against mutant strains of the HIV-1 protease enzyme as compared to Indinavir.
Assuntos
Carbamatos/síntese química , Inibidores da Protease de HIV/síntese química , Protease de HIV/química , Pirróis/síntese química , Animais , Disponibilidade Biológica , Carbamatos/química , Carbamatos/farmacocinética , Cristalografia por Raios X , Cães , Desenho de Fármacos , Protease de HIV/genética , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacocinética , Modelos Moleculares , Mutação , Ligação Proteica , Pirróis/química , Pirróis/farmacocinética , Pirróis/farmacologiaRESUMO
Naphthyridine 7 inhibits the strand transfer of the integration process catalyzed by integrase with an IC50 of 10 nM and inhibits 95% of the spread of HIV-1 infection in cell culture at 0.39 microM. It does not exhibit cytotoxicity in cell culture at < or =12.5 microM and shows a good pharmacokinetic profile when dosed orally to rats. The antiviral activity of 7 and its effect on integration were confirmed using viruses with specific integrase mutations.
Assuntos
Fármacos Anti-HIV/síntese química , Inibidores de Integrase de HIV/síntese química , HIV-1/efeitos dos fármacos , Naftiridinas/síntese química , Administração Oral , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , Humanos , Injeções Intravenosas , Naftiridinas/química , Naftiridinas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
[reaction: see text] A novel approach to alpha,alpha-disubstituted-beta-amino acids (beta(2,2)-amino acids) was employed in the synthesis of a series of 3-(pyrrolidin-1-yl)propionic acids possessing high affinity for the CCR5 receptor and potent anti-HIV activity. The rat pharmacokinetics for these new analogues featured higher bioavailabilities and lower rates of clearance as compared to cyclopentane 1.