RESUMO
Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.
Assuntos
Infecções por HIV , HIV-1 , RNA Viral , Tropismo Viral , Ativação Viral , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/uso terapêutico , Antígenos Virais/análise , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linfócitos T CD4-Positivos , Proteína do Núcleo p24 do HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Humanos , Imunoensaio , Hibridização in Situ Fluorescente , RNA Mensageiro/análise , RNA Viral/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1+ adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/106 CD4+ T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (â¼1/106 CD4+ T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.
Assuntos
DNA Viral/sangue , Infecções por HIV , Provírus/genética , Latência Viral/genética , Adulto , Feminino , Infecções por HIV/sangue , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodosRESUMO
BACKGROUND: Biological sex and the estrogen receptor alpha (ESR1) modulate human immunodeficiency virus (HIV) activity. Few women have enrolled in clinical trials of latency reversal agents (LRAs); their effectiveness in women is unknown. We hypothesized that ESR1 antagonism would augment induction of HIV expression by the LRA vorinostat. METHODS: AIDS Clinical Trials Group A5366 enrolled 31 virologically suppressed, postmenopausal women on antiretroviral therapy. Participants were randomized 2:1 to receive tamoxifen (arm A, TAMOX/VOR) or observation (arm B, VOR) for 5 weeks followed by 2 doses of vorinostat. Primary end points were safety and the difference between arms in HIV RNA induction after vorinostat. Secondary analyses included histone 4 acetylation, HIV DNA, and plasma viremia by single copy assay (SCA). RESULTS: No significant adverse events were attributed to study treatments. Tamoxifen did not enhance vorinostat-induced HIV transcription (between-arm ratio, 0.8; 95% confidence interval [CI], .2-2.4). Vorinostat-induced HIV transcription was higher in participants with increases in H4Ac (fold increase, 2.78; 95% CI, 1.34-5.79) vs those 9 who did not (fold increase, 1.04; 95% CI, .25-4.29). HIV DNA and SCA plasma viremia did not substantially change. CONCLUSIONS: Tamoxifen did not augment vorinostat-induced HIV RNA expression in postmenopausal women. The modest latency reversal activity of vorinostat, postmenopausal status, and low level of HIV RNA expression near the limits of quantification limited assessment of the impact of tamoxifen. This study is the first HIV cure trial done exclusively in women and establishes both the feasibility and necessity of investigating novel HIV cure strategies in women living with HIV. CLINICAL TRIALS REGISTRATION: NCT03382834.
Assuntos
Síndrome da Imunodeficiência Adquirida , Infecções por HIV , HIV-1 , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Linfócitos T CD4-Positivos , DNA/uso terapêutico , Receptor alfa de Estrogênio/metabolismo , Feminino , HIV-1/genética , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Histonas/uso terapêutico , Humanos , RNA/metabolismo , RNA/uso terapêutico , Tamoxifeno/efeitos adversos , Tamoxifeno/metabolismo , Viremia/tratamento farmacológico , Latência Viral , Vorinostat/metabolismo , Vorinostat/farmacologia , Vorinostat/uso terapêuticoRESUMO
We demonstrate that human immunodeficiency virus (HIV) gag p24 protein is more readily detected in gut and lymph node tissues than in blood CD4+ T cells and correlates better with CD4 count during antiretroviral therapy (ART). Gut p24 levels also measurably decline with ART in natural controllers. During ART, gut p24 expression is more strongly associated both with HIV-specific CD8+ T-cell frequency and plasma soluble CD14 levels than gut HIV RNA expression. This study supports using gag p24 as a marker of HIV expression in HIV+ tissues to study effects of viral persistence and to monitor efficacy of treatment in HIV-based clearance studies.
Assuntos
Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Biomarcadores/sangue , Biópsia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Feminino , Proteína do Núcleo p24 do HIV/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Humanos , Ativação LinfocitáriaRESUMO
Accurate characterization of the human immunodeficiency virus (HIV) reservoir is imperative to develop an effective cure. HIV was measured in antiretroviral therapy-suppressed individuals using the intact proviral DNA assay (IPDA), along with assays for total or integrated HIV DNA, and inducible HIV RNA or p24. Intact provirus correlated with total and integrated HIV.
Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD4-Positivos , DNA Viral/genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Provírus/genética , Latência ViralRESUMO
Histone deacetylase inhibitors (HDACi) are the most widely studied HIV latency-reversing agents (LRAs). The HDACi suberoylanilide hydroxamic acid (vorinostat [VOR]) has been employed in several clinical HIV latency reversal studies, as well as in vitro models of HIV latency, and has been shown to effectively induce HIV RNA and protein expression. Despite these findings, response to HDACi can vary, particularly with intermittent dosing, and information is lacking on the relationship between the host transcriptional response and HIV latency reversal. Here, we report on global gene expression responses to VOR and examine the longevity of the transcriptional response in various cellular models. We found that many genes are modulated at 6 h post-VOR treatment in HCT116, Jurkat, and primary resting CD4 T cells, yet return to baseline levels after an 18-h VOR-free period. With repeat exposure to VOR in resting CD4 T cells, we found similar and consistent transcriptional changes at 6 h following each serial treatment. In addition, serial exposure in HIV-infected suppressed donor CD4 T cells showed consistent transcriptional changes after each exposure to VOR. We identified five host genes that were strongly and consistently modulated following histone deacetylase (HDAC) inhibition; three (H1F0, IRGM, and WIPI49) were upregulated, and two (PHF15 and PRDM10) were downregulated. These genes demonstrated consistent modulation in peripheral blood mononuclear cell (PBMC) samples from HIV-positive (HIV+) participants who received either single or multiple doses of 400 mg of VOR. Interestingly, the host transcriptional response did not predict induction of cell-associated HIV RNA, suggesting that other cellular factors play key roles in HIV latency reversal in vivo despite robust HDACi pharmacological activity.IMPORTANCE Histone deacetylase inhibitors are widely studied HIV latency-reversing agents (LRAs). VOR, an HDACi, induces histone acetylation and chromatin remodeling and modulates host and HIV gene expression. However, the relationship between these events is poorly defined, and clinical studies suggest diminished HIV reactivation in resting CD4 T cells with daily exposure to VOR. Our study provides evidence that VOR induces a consistent level of host cell gene transcription following intermittent exposure. In addition, in response to VOR exposure a gene signature that was conserved across single and serial exposures both in vitro and in vivo was identified, indicating that VOR can consistently and reproducibly modulate transcriptional host responses. However, as the HIV response to HDACi declines over time, other factors modulate viral reactivation in vivo despite robust HDAC activity. The identified host gene VOR biomarkers can be used for monitoring the pharmacodynamic activity of HDAC inhibitors.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/tratamento farmacológico , Vorinostat/farmacologia , Acetilação , Linfócitos T CD4-Positivos/efeitos dos fármacos , HIV-1/metabolismo , HIV-1/patogenicidade , Inibidores de Histona Desacetilases/farmacologia , Humanos , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Cultura Primária de Células , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Vorinostat/metabolismoRESUMO
A series of unique macrocyclic HDACs 1, 2, and 3 selective inhibitors were identified with good enzymatic activity and high selectivity over HDACs 6 and 8. These macrocyclic HDAC inhibitors used an ethyl ketone as the zinc-binding group. Compounds 25 and 26 stood out as leads due to their low double-digit nM EC50s in the 2C4 cell-based HIV latency reactivation assay. The PK profiles of these macrocyclic HDAC inhibitors still needed improvement.
Assuntos
Fármacos Anti-HIV/farmacologia , Descoberta de Drogas , HIV/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Clearance of HIV-infected germinal center (GC) CD4+ follicular helper T cells (Tfh) after combination antiretroviral therapy (ART) is essential to an HIV cure. Blocking B cell lymphoma 6 (BCL6; the master transcription factor for Tfh cells) represses HIV infection of tonsillar CD4+ Tfh ex vivo, reduces GC formation, and limits immune activation in vivo We assessed the anti-HIV activity of a novel BCL6 inhibitor, FX1, in Tfh/non-Tfh CD4+ T cells and its impact on T cell activation and SAMHD1 phosphorylation (Thr592). FX1 repressed HIV-1 infection of peripheral CD4+ T cells and tonsillar Tfh/non-Tfh CD4+ T cells (P < 0.05) and total elongated and multispliced HIV-1 RNA production during the first round of viral life cycle (P < 0.01). Using purified circulating CD4+ T cells from uninfected donors, we demonstrate that FX1 treatment resulted in downregulation pSAMHD1 expression (P < 0.05) and T cell activation (HLA-DR, CD25, and Ki67; P < 0.01) ex vivo corresponding with inhibition of HIV-1 and HIV-2 replication. Ex vivo HIV-1 reactivation using purified peripheral CD4+ T cells from HIV-infected ART-suppressed donors was also blocked by FX1 treatment (P < 0.01). Our results indicate that BCL6 function contributes to Tfh/non-Tfh CD4+ T cell activation and cellular susceptibility to HIV infection. BCL6 inhibition represents a novel therapeutic strategy to potentiate HIV suppression in Tfh/non-Tfh CD4+ T cells without reactivation of latent virus.IMPORTANCE The expansion and accumulation of HIV-infected BCL6+ Tfh CD4+ T cells are thought to contribute to the persistence of viral reservoirs in infected subjects undergoing ART. Two mechanisms have been raised for the preferential retention of HIV within Tfh CD4+ T cells: (i) antiretroviral drugs have limited tissue distribution, resulting in insufficient tissue concentration and lower efficacy in controlling HIV replication in lymphoid tissues, and (ii) cytotoxic CD8+ T cells within lymphoid tissues express low levels of chemokine receptor (CXCR5), thus limiting their ability to enter the GCs to control/eliminate HIV-infected Tfh cells. Our results indicate that the BCL6 inhibitor FX1 can not only repress HIV infection of tonsillar Tfh ex vivo but also suppress HIV infection and reactivation in primary, non-Tfh CD4+ T cells. Our study provides a rationale for targeting BCL6 protein to extend ART-mediated reduction of persistent HIV and/or support strategies toward HIV remission beyond ART cessation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/metabolismo , Indóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Tiazolidinedionas/farmacologia , Adulto , Regulação para Baixo , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Pessoa de Meia-Idade , Fosforilação , Replicação Viral/efeitos dos fármacos , Adulto JovemRESUMO
A novel series of ethyl ketone based HDACs 1, 2, and 3 selective inhibitors have been identified with good enzymatic and cellular activity and high selectivity over HDACs 6 and 8. These inhibitors contain a spirobicyclic group in the amide region. Compound 13 stands out as a lead due to its good potency, high selectivity, and reasonable rat and dog PK. Compounds 33 and 34 show good potency and rat PK profiles as well.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Cetonas/farmacologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacocinética , Linhagem Celular Tumoral , Cães , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacocinética , Humanos , Cetonas/síntese química , Cetonas/farmacocinética , Testes de Sensibilidade Microbiana , Ratos , Compostos de Espiro/síntese química , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologiaRESUMO
The synthesis and SAR development of a trisubstituted imidazole HDAC inhibitor is described. The compounds were synthesized with high diastereocontrol by leveraging Ellman sulfinyl imine chemistry. Structural elucidation provided insight into binding mode and supported design rational. Pharmacokinetic properties of lead compounds were determined.
Assuntos
Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Animais , Linfócitos T CD4-Positivos/virologia , Cristalografia por Raios X , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disease with pathological hallmarks including the formation of extracellular aggregates of amyloid-beta (Aß) known as plaques and intracellular tau tangles. Coincident with the formation of Aß plaques is recruitment and activation of glial cells to the plaque forming a plaque niche. In addition to histological data showing the formation of the niche, AD genetic studies have added to the growing appreciation of how dysfunctional glia pathways drive neuropathology, with emphasis on microglia pathways. Genomic approaches enable comparisons of human disease profiles between different mouse models informing on their utility to evaluate secondary changes to triggers such as Aß deposition. METHODS: In this study, we utilized two animal models of AD to examine and characterize the AD-associated pathology: the Tg2576 Swedish APP (KM670/671NL) and TgCRND8 Swedish plus Indiana APP (KM670/671NL + V717F) lines. We used laser capture microscopy (LCM) to isolate samples surrounding Thio-S positive plaques from distal non-plaque tissue. These samples were then analyzed using RNA sequencing. RESULTS: We determined age-associated transcriptomic differences between two similar yet distinct APP transgenic mouse models, known to differ in proportional amyloidogenic species and plaque deposition rates. In Tg2576, human AD gene signatures were not observed despite profiling mice out to 15 months of age. TgCRND8 mice however showed progressive and robust induction of lysomal, neuroimmune, and ITIM/ITAM-associated gene signatures overlapping with prior human AD brain transcriptomic studies. Notably, RNAseq analyses highlighted the vast majority of transcriptional changes observed in aging TgCRND8 cortical brain homogenates were in fact specifically enriched within the plaque niche samples. Data uncovered plaque-associated enrichment of microglia-related genes such as ITIM/ITAM-associated genes and pathway markers of phagocytosis. CONCLUSION: This work may help guide improved translational value of APP mouse models of AD, particularly for strategies aimed at targeting neuroimmune and neurodegenerative pathways, by demonstrating that TgCRND8 more closely recapitulates specific human AD-associated transcriptional responses.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Córtex Cerebral/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/genética , Fatores Etários , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebral/patologia , Correlação de Dados , Modelos Animais de Doenças , Humanos , Microdissecção e Captura a Laser , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Mutação/genética , Placa Amiloide/patologia , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
OBJECTIVES: Pre-exposure prophylaxis (PrEP) using antiretroviral drugs (ARVs) has been shown to reduce HIV transmission in people at high risk of HIV infection. Adherence to PrEP strongly correlates with the level of HIV protection. Long-acting injectable ARVs provide sustained systemic drug exposures over many weeks and can improve adherence due to infrequent parenteral administration. Here, we evaluated a new long-acting formulation of raltegravir for prevention of vaginal HIV transmission. METHODS: Long-acting raltegravir was administered subcutaneously to BALB/c, NSG (NOD-scid-gamma) and humanized BLT (bone marrow-liver-thymus) mice and rhesus macaques. Raltegravir concentration in peripheral blood and tissue was analysed. Suppression of HIV replication was assessed in infected BLT mice. Two high-dose HIV vaginal challenges were used to evaluate protection from HIV transmission in BLT mice. RESULTS: Two weeks after a single subcutaneous injection of long-acting raltegravir in BLT mice (7.5 mg) and rhesus macaques (160 mg), the plasma concentration of raltegravir was comparable to 400 mg orally, twice daily in humans. Serum collected from mice 3 weeks post-administration of long-acting raltegravir efficiently blocked HIV infection of TZM-bl indicator cells in vitro. Administration of long-acting raltegravir suppressed viral RNA in plasma and cervico-vaginal fluids of infected BLT mice, demonstrating penetration of active raltegravir into the female reproductive tract. Using transmitted/founder HIV we observed that BLT mice administered a single subcutaneous dose of long-acting raltegravir were protected from two high-dose HIV vaginal challenges 1 week and 4 weeks after drug administration. CONCLUSIONS: These preclinical results demonstrated the efficacy of long-acting raltegravir in preventing vaginal HIV transmission.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Transmissão de Doença Infecciosa/prevenção & controle , Infecções por HIV/prevenção & controle , Profilaxia Pré-Exposição/métodos , Raltegravir Potássico/administração & dosagem , Vagina/virologia , Animais , Quimioprevenção/métodos , Feminino , Infecções por HIV/transmissão , Injeções Subcutâneas , Macaca mulatta , Camundongos Endogâmicos BALB C , Camundongos SCID , Resultado do TratamentoRESUMO
In recent years, an increasing amount of literature is emerging on candidate urine and blood-based biomarkers associated with incidence and severity of preeclampsia (PE) in pregnant women. While enthusiasm on the usefulness of several of these markers in predicting PE is evolving, essentially all work so far has focused on the needs of high-resource settings and high-income countries, resulting primarily in multi-parameter laboratory assays based on proteomic and metabolomics analysis techniques. These highly complex methods, however, require laboratory capabilities that are rarely available or affordable in low-resource settings (LRS). The importance of quantifying maternal and perinatal risks and identifying which pregnancies can be safely prolonged is also much greater in LRS, where intensive care facilities that can rapidly respond to PE-related health threats for women and infants are limited. For these reasons, simple, low cost, sensitive, and specific point-of-care (POC) tests are needed that can be performed by antenatal health care providers in LRS and that can facilitate decisions about detection and management of PE. Our study aims to provide a comprehensive systematic review of current and emerging blood and urine biomarkers for PE, not only on the basis of their clinical performance, but also of their suitability to be used in LRS-compatible test formats, such as lateral flow and other variants of POC rapid assays.
Assuntos
Biomarcadores/sangue , Biomarcadores/urina , Recursos em Saúde , Testes Imediatos , Pré-Eclâmpsia/prevenção & controle , Pré-Eclâmpsia/terapia , Feminino , Humanos , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/metabolismo , GravidezRESUMO
A hallmark of Alzheimer's disease (AD) brain is the amyloid ß (Aß) plaque, which is comprised of Aß peptides. Multiple lines of evidence suggest that Aß oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was >2500× selective for Aß oligomers over Aß monomers with a limit of detection â¼ 0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized Aß multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3- to 5-fold increase in Aß oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. Aß oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the Aß oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.
Assuntos
Envelhecimento/líquido cefalorraquidiano , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/imunologia , Anticorpos/imunologia , Especificidade de Anticorpos , Biomarcadores/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática , Reações Falso-Positivas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Curva ROC , Reprodutibilidade dos Testes , Espalhamento de RadiaçãoRESUMO
BACKGROUND: Cerebrospinal fluid (CSF) tau is a common biomarker for Alzheimer disease (AD). Measurements of tau have historically been performed using immunoassays. Given the molecular diversity of tau in CSF, the selectivity of these immunoassays has often been questioned. Therefore, we aimed to develop an analytically sensitive and selective immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) (IA-MS) assay. METHODS: IA-MS sample analysis involved the addition of an internal standard, immunoaffinity purification of tau using a tau monoclonal antibody coupled to magnetic beads, trypsin digestion, and quantification of a surrogate tau peptide by LC-MS/MS using a Waters Trizaic nanoTile ultraperformance LC microfluidic device. Further characterization of tau peptides was performed by full-scan MS using a Thermo Orbitrap LC-MS. CSF samples from a cohort of age-matched controls and patients with AD were analyzed by the IA-MS method as well as a commercially available immunoassay. RESULTS: The IA-MS assay had intra- and interassay imprecision values of 3.2% to 8.1% CV and 7.8% to 18.9% C, respectively, a mean recovery of 106%, and a limit of quantification of 0.25 pmol/L and was able to quantify tau concentrations in all human specimens tested. The IA-MS assay showed a correlation of R(2) = 0.950 against a total-tau immunoassay. In patients with AD, tau was increased approximately 2-fold. CONCLUSIONS: Combining immunoaffinity enrichment with microflow LC-MS/MS analysis is an effective approach for the development of a highly selective assay to measure total tau and, potentially, other posttranslationally modified forms of tau in CSF.
Assuntos
Proteínas tau/líquido cefalorraquidiano , Anticorpos , Estudos de Casos e Controles , Cromatografia Líquida/métodos , Humanos , Isoformas de Proteínas/líquido cefalorraquidiano , Isoformas de Proteínas/imunologia , Espectrometria de Massas em Tandem/métodos , Proteínas tau/imunologiaRESUMO
Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches. Additionally, we determine the therapeutic index of our polymer conjugates in vivo and demonstrate that the incorporation of pH responsive elements dramatically expands the therapeutic index to 10-15, beyond that of the therapeutic index (less than 3), for polymer conjugates previously reported.
Assuntos
Concentração de Íons de Hidrogênio , Polímeros/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Animais , Polímeros/química , Polímeros/farmacocinética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética , RatosRESUMO
Efficient siRNA delivery is dependent not only on the ability of the delivery vehicle to target a specific organ but also on its ability to enable siRNA entry into the cytoplasm of the target cells. Polymers with endosomolytic properties are increasingly being used as siRNA delivery vehicles due to their potential to facilitate endosomal escape and intracellular delivery. Addition of disulfide bonds in the backbone of these polymers was expected to provide degradability through reduction by glutathione in cytosol. This paper describes the synthesis of new endosomolytic bioreducible poly(amido amine disulfide) polymers whose lytic potential can be masked at physiological pH, but can be restored at acidic endosomal pH. These polymer conjugates gave good in vitro knockdown (KD) and did not demonstrate cytotoxicity in a MTS assay. Efficient mRNA KD for apolipoprotein B in mouse liver was observed with these polyconjugates following intravenous dosing.
Assuntos
Dissulfetos/química , Sistemas de Liberação de Medicamentos , Endossomos/metabolismo , Poliaminas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Apolipoproteínas B/deficiência , Apolipoproteínas B/genética , Eritrócitos/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Estrutura Molecular , Oxirredução , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologiaRESUMO
Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (µVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DµVS), that integrates a µVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DµVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DµVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.
RESUMO
Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Infecções por HIV/tratamento farmacológico , Antivirais/uso terapêutico , Apoptose , Morte Celular , Linfócitos T CD4-Positivos , Replicação ViralRESUMO
Productively infected cells are generally thought to arise from HIV infection of activated CD4+ T cells, and these infected activated cells are thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate from direct infection of resting CD4+ T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting CD4+ T cells was correlated with the availability of susceptible target cells in lymphoid tissues largely restricted to resting CD4+ T cells in which expression of pTEFb enabled productive infection, and we documented persistence of HIV-producing resting T cells during antiretroviral therapy (ART). Thus, we provide evidence of a mechanism by which direct infection of resting T cells in lymphoid tissues to generate productively and latently infected cells creates a mechanism by which the productively infected cells can replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection.