RESUMEN
Blockade of immune-checkpoint programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 can enhance effector T-cell responses. However, the lack of response in many patients to checkpoint-inhibitor therapies emphasizes the need for combination immunotherapies to pursue maximal antitumor efficacy. We have previously demonstrated that antagonism of C-X-C chemokine receptor type 4 (CXCR4) by plerixafor (AMD3100) can decrease regulatory T (Treg)-cell intratumoral infiltration. Therefore, a combination of these 2 therapies might increase antitumor effects. Here, we evaluated the antitumor efficacy of AMD3100 and anti-PD-1 (αPD-1) antibody alone or in combination in an immunocompetent syngeneic mouse model of ovarian cancer. We found that AMD3100, a highly specific CXCR4 antagonist, directly down-regulated the expression of both C-X-C motif chemokine 12 (CXCL12) and CXCR4 in vitro and in vivo in tumor cells. AMD3100 and αPD-1 significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice when given as monotherapy. Combination of these 2 agents significantly enhanced antitumor effects compared with single-agent administration. Benefits of tumor control and animal survival were associated with immunomodulation mediated by these 2 agents, which were characterized by increased effector T-cell infiltration, increased effector T-cell function, and increased memory T cells in tumor microenvironment. Intratumoral Treg cells were decreased, and conversion of Treg cells into T helper cells was increased by AMD3100 treatment. Intratumoral myeloid-derived suppressor cells were decreased by the combined treatment, which was associated with decreased IL-10 and IL-6 in the ascites. Also, the combination therapy decreased suppressive leukocytes and facilitated M2-to-M1 macrophage polarization in the tumor. These results suggest that AMD3100 could be used to target the CXCR4-CXCL12 axis to inhibit tumor growth and prevent multifaceted immunosuppression alone or in combination with αPD-1 in ovarian cancer, which could be clinically relevant to patients with this disease.-Zeng, Y., Li, B., Liang, Y., Reeves, P. M., Qu, X., Ran, C., Liu, Q., Callahan, M. V., Sluder, A. E., Gelfand, J. A., Chen, H., Poznansky, M. C. Dual blockade of CXCL12-CXCR4 and PD-1-PD-L1 pathways prolongs survival of ovarian tumor-bearing mice by prevention of immunosuppression in the tumor microenvironment.
Asunto(s)
Antígeno B7-H1 , Quimiocina CXCL12 , Compuestos Heterocíclicos/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Proteínas de Neoplasias , Neoplasias Ováricas , Receptor de Muerte Celular Programada 1 , Receptores CXCR4 , Transducción de Señal , Microambiente Tumoral , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Bencilaminas , Línea Celular Tumoral , Quimiocina CXCL12/antagonistas & inhibidores , Quimiocina CXCL12/inmunología , Ciclamas , Femenino , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen. Finally, in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs/saCas9 AAV-DJ/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials.
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Endonucleasas/genética , Terapia Genética/métodos , Genoma Viral , Infecciones por VIH/terapia , VIH-1/genética , Provirus/genética , ARN Guía de Kinetoplastida/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Endonucleasas/metabolismo , Edición Génica/métodos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Infecciones por VIH/patología , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , VIH-1/metabolismo , Humanos , Ratones , Ratones Transgénicos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Provirus/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/enzimología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 escapes antiretroviral agents by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. Transcriptional activation is prerequisite for reactivation and the eradication of latent HIV-1 proviruses. dCas9-SunTag-VP64 transcriptional system has been reported that it can robustly activate the expression of an endogenous gene using a single guide RNA (sgRNA). Here, we systematically investigated the potential of dCas9-SunTag-VP64 with the designed sgRNAs for reactivating latent HIV-1. We found dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 targeted from -164 to -146 or -124 to -106 bp upstream of the transcription start sites of HIV-1 could induce high expression of luciferase reporter gene after screening of sgRNAs targeting different regions of the HIV-1 promoter. Further, we confirmed that dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 can effectively reactivate latent HIV-1 transcription in several latently infected human T-cell lines. Moreover, we confirmed that the reactivation of latent HIV-1 by dCas9-SunTag-VP64 with the designed sgRNA occurred through specific binding to the HIV-1 LTR promoter without genotoxicity and global T-cell activation. Taken together, our data demonstrated dCas9-SunTag-VP64 system can effectively and specifically reactivate latent HIV-1 transcription, suggesting that this strategy could offer a novel approach to anti-HIV-1 latency.
Asunto(s)
Sistemas CRISPR-Cas , VIH-1/fisiología , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/genética , Proteínas Recombinantes de Fusión , Activación Transcripcional , Activación Viral/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , Expresión Génica , Regulación Viral de la Expresión Génica , Orden Génico , Marcación de Gen , Genes Reporteros , Vectores Genéticos/genética , Duplicado del Terminal Largo de VIH , Humanos , Unión ProteicaRESUMEN
HIV-infected individuals currently cannot be completely cured because existing antiviral therapy regimens do not address HIV provirus DNA, flanked by long terminal repeats (LTRs), already integrated into host genome. Here, we present a possible alternative therapeutic approach to specifically and directly mediate deletion of the integrated full-length HIV provirus from infected and latently infected human T cell genomes by using specially designed zinc-finger nucleases (ZFNs) to target a sequence within the LTR that is well conserved across all clades. We designed and screened one pair of ZFN to target the highly conserved HIV-1 5'-LTR and 3'-LTR DNA sequences, named ZFN-LTR. We found that ZFN-LTR can specifically target and cleave the full-length HIV-1 proviral DNA in several infected and latently infected cell types and also HIV-1 infected human primary cells in vitro. We observed that the frequency of excision was 45.9% in infected human cell lines after treatment with ZFN-LTR, without significant host-cell genotoxicity. Taken together, our data demonstrate that a single ZFN-LTR pair can specifically and effectively cleave integrated full-length HIV-1 proviral DNA and mediate antiretroviral activity in infected and latently infected cells, suggesting that this strategy could offer a novel approach to eradicate the HIV-1 virus from the infected host in the future.
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ADN Viral/metabolismo , Endodesoxirribonucleasas/metabolismo , Duplicado del Terminal Largo de VIH , VIH-1/genética , Provirus/genética , Linfocitos T/virología , Células Cultivadas , VIH-1/fisiología , Humanos , Células Jurkat , Eliminación de Secuencia , Integración Viral , Latencia del Virus , Dedos de ZincRESUMEN
Reactivation of latent HIV-1 is a promising strategy for the clearance of the viral reservoirs. Because of the limitations of current agents, identification of new latency activators is urgently required. Using an established model of HIV-1 latency, we examined the effect of Oxaliplatin on latent HIV-1 reactivation. We showed that Oxaliplatin, alone or in combination with valproic acid (VPA), was able to reactivate HIV-1 without inducing global T cell activation. We also provided evidence that Oxaliplatin reactivated HIV-1 expression by inducing nuclear factor kappa B (NF-κB) nuclear translocation. Our results indicated that Oxaliplatin could be a potential drug candidate for anti-latency therapies.
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VIH-1/inmunología , Activación de Linfocitos/inmunología , FN-kappa B/inmunología , Compuestos Organoplatinos/farmacología , Linfocitos T/inmunología , Linfocitos T/virología , Activación Viral/inmunología , Latencia del Virus/inmunología , Células Cultivadas , VIH-1/efectos de los fármacos , Humanos , Activación de Linfocitos/efectos de los fármacos , Oxaliplatino , Linfocitos T/efectos de los fármacos , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacosRESUMEN
The long-lived latently infected cells persist in spite of prolonged highly active anti-retroviral therapy and present a major barrier to a cure of human immunodeficiency virus type 1 (HIV-1) infection. Elimination of this reservoir requires reactivation of the latent virus. None of the current agents can safely and effectively reactivate latent HIV-1 reservoirs. Dilazep, a nucleoside transport inhibitor, is used to treat ischemic dysfunction. However, little is known about the effect of dilazep in inducing HIV expression in latently infected cells. Using the Jurkat T cell model of HIV-1 latency, we found that dilazep effectively reactivates latent HIV-1 gene expression in a dose manner. We observed that dilazep synergistically reactivated latent HIV-1 transcription with valproic acid. We also found that dilazep activates viral latency without inducing cell surface activation markers CD25 and CD69 activation. In summary, dilazep, alone or in combination with VPA, could be useful in future eradication strategies.
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Dilazep/farmacología , Reservorios de Enfermedades/virología , VIH-1/efectos de los fármacos , Activación Viral/efectos de los fármacos , Latencia del Virus/fisiología , Sinergismo Farmacológico , Citometría de Flujo , Proteínas Fluorescentes Verdes , Humanos , Células Jurkat , Ácido Valproico/farmacologíaRESUMEN
A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can efficiently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.
Asunto(s)
ADN Viral/aislamiento & purificación , Endonucleasas/metabolismo , Genes Virales , VIH-1/genética , Provirus/genética , Dedos de Zinc , Síndrome de Inmunodeficiencia Adquirida/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proliferación Celular , Biología Computacional , ADN Viral/genética , Eliminación de Gen , Marcación de Gen , Genoma Humano , Células HEK293 , VIH-1/fisiología , Humanos , Células Jurkat , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Secuencias Repetidas Terminales , Replicación ViralRESUMEN
Omega-6 (n-6) arachidonic acid (AA) and its pro-inflammatory metabolites, including prostaglandin E2 (PGE(2)), are known to promote tumorigenesis. Delta-6 desaturase (D6D) is the rate-limiting enzyme for converting n-6 linoleic acid (LA) to AA. Our objective was to determine if AA synthesis, specifically D6D activity, and PGE(2) levels are increased in cancerous breast tissue, and whether these variables differ between estrogen receptor positive (ER+) and negative (ER-) breast cancers. Gas chromatography was performed on surgical breast tissue samples collected from 69 women with breast cancer. Fifty-four had ER+ breast cancer, and 15 had ER- breast cancer. Liquid chromatography-mass spectrometry was used to determine PGE(2) levels. Lipid analysis revealed higher levels of LA metabolites (C18:3 n-6, C20:3 n-6, and AA) in cancerous tissue than in adjacent noncancerous tissue (P < 0.01). The ratio of LA metabolites to LA, a measure of D6D activity, was increased in cancerous tissue, suggesting greater conversion of LA to AA (P < 0.001), and was higher in ER- than in ER+ patients, indicating genotype-related trends. Similarly, PGE(2) levels were increased in cancerous tissue, particularly in ER- patients. The results showed that the endogenous AA synthetic pathway, D6D activity, and PGE(2) levels are increased in breast tumors, particularly those of the ER- genotype. These findings suggest that the AA synthetic pathway and the D6D enzyme in particular may be involved in the pathogenesis of breast cancer. The development of drugs and nutritional interventions to alter this pathway may provide new strategies for breast cancer prevention and treatment.
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Ácido Araquidónico/metabolismo , Neoplasias de la Mama/metabolismo , Linoleoil-CoA Desaturasa/metabolismo , Cromatografía de Gases , Cromatografía Liquida , Femenino , Humanos , Espectrometría de Masas , Receptores de Estrógenos/metabolismoRESUMEN
Metabotropic glutamate receptor 2 (mGluR2) has been extensively studied for the treatment of various neurological and psychiatric disorders. Understanding of the mGluR2 function is pivotal in supporting the drug discovery targeting mGluR2. Herein, the positive allosteric modulation of mGluR2 was investigated via the in vivo positron emission tomography (PET) imaging using 2-((4-(2-[11C]methoxy-4-(trifluoromethyl)phenyl)piperidin-1-yl)methyl)-1-methyl-1H-imidazo[4,5-b]pyridine ([11C]mG2P001). Distinct from the orthosteric compounds, pretreatment with the unlabeled mG2P001, a potent mGluR2 positive allosteric modulator (PAM), resulted in a significant increase instead of decrease of the [11C]mG2P001 accumulation in rat brain detected by PET imaging. Subsequent in vitro studies with [3H]mG2P001 revealed the cooperative binding mechanism of mG2P001 with glutamate and its pharmacological effect that contributed to the enhanced binding of [3H]mG2P001 in transfected CHO cells expressing mGluR2. The in vivo PET imaging and quantitative analysis of [11C]mG2P001 in non-human primates (NHPs) further validated the characteristics of [11C]mG2P001 as an imaging ligand for mGluR2. Self-blocking studies in primates enhanced accumulation of [11C]mG2P001. Altogether, these studies show that [11C]mG2P001 is a sensitive biomarker for mGluR2 expression and the binding is affected by the tissue glutamate concentration.
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Receptores de Glutamato Metabotrópico , Ratas , Cricetinae , Animales , Ratas Sprague-Dawley , Cricetulus , Tomografía de Emisión de PositronesRESUMEN
Docosahexaenoic acid (DHA), an n-3 long chain polyunsaturated fatty acid (LC-PUFA), highly enriched in the central nervous system, is critical for brain development and function. It has been shown that DHA deficiency impairs cognitive performance whereas DHA supplementation improves the condition. However, the mechanisms underlying the role of DHA in brain development and function remain to be elucidated. By using transgenic fat-1 mice rich in endogenous n-3 PUFA, we show that increased brain DHA significantly enhances hippocampal neurogenesis shown by an increased number of proliferating neurons and neuritogenesis, evidenced by increased density of dendritic spines of CA1 pyramidal neurons in the hippocampus. Concurrently, fat-1 mice exhibit a better spatial learning performance in the Morris water maze compared with control WT littermates. In vitro experiments further demonstrate that DHA promotes differentiation and neurite outgrowth of neuronal cells derived from mouse ES cells and increases the proliferation of cells undergoing differentiation into neuronal lineages from the ES cells. These results together provide direct evidence for a promoting effect of DHA on neurogenesis and neuritogenesis and suggest that this effect may be a mechanism underlying its beneficial effect on behavioral performance.
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Ácidos Docosahexaenoicos/farmacología , Aprendizaje/efectos de los fármacos , Neuritas/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Conducta Espacial/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Suplementos Dietéticos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Ácidos Grasos/metabolismo , Hipocampo/citología , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Mutantes , Ratones Transgénicos , Neuritas/metabolismoRESUMEN
Human transmission of SARS-CoV-2 and emergent variants of concern continue to occur globally, despite mass vaccination campaigns. Public health strategies to reduce virus spread should therefore rely, in part, on frequent screening with rapid, inexpensive, and sensitive tests. We evaluated two digitally integrated rapid tests and assessed their performance using stored nasal swab specimens collected from individuals with or without COVID-19. An isothermal amplification assay combined with a lateral flow test had a limit of detection of 10 RNA copies per reaction, and a positive percent agreement (PPA)/negative percent agreement (NPA) during the asymptomatic and symptomatic phases of 100%/100% and 95.83/100%, respectively. Comparatively, an antigen-based lateral flow test had a limit of detection of 30,000 copies and a PPA/NPA during the asymptomatic and symptomatic phases of 82.86%/98.68% and 91.67/100%, respectively. Both the isothermal amplification and antigen-based lateral flow tests had optimized detection of SARS-CoV-2 during the peak period of transmission; however, the antigen-based test had reduced sensitivity in clinical samples with qPCR Ct values greater than 29.8. Low-cost, high-throughput screening enabled by isothermal amplification or antigen-based techniques have value for outbreak control.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Humanos , Pruebas Inmunológicas , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/genética , Sensibilidad y EspecificidadRESUMEN
Fragile X syndrome (FXS) is a monogenic disorder characterized by intellectual disability and behavioral challenges. It is caused by aberrant methylation of the fragile X mental retardation 1 (FMR1) gene. Given the failure of clinical trials in FXS and growing evidence of a role of metabotropic glutamate subtype 5 receptors (mGluR5) in the pathophysiology of the disorder, we investigated mGluR5 function in FMR1 Knockout (FMR1-KO) mice and age- and sex-matched control mice using longitudinal positron emission tomography (PET) imaging to better understand the disorder. The studies were repeated at four time points to examine age- and disease-induced changes in mGluR5 availability using 3-fluoro-[18F]5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB). We found that the binding potential (BP) of [18F]FPEB was significantly lower in the KO mice in mGluR5-implicated brain areas including striatum, cortex, hippocampus, thalamus, and olfactory bulb. The BP also changed with age, regardless of disorder status, increasing in early adulthood in male but not in female mice before decreasing later in both sexes. The difference in mGluR5 availability between the FMR1-KO and control mice and the change in BP in the KO mice as a function of age and sex illustrate the nature of the disorder and its progression, providing mechanistic insights for treatment design.
RESUMEN
PURPOSE: Metabotropic glutamate receptor 2 (mGluR2) has been implicated in various psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. We have previously developed [11C]7 as a PET radioligand for imaging mGluR2. Herein, [18F]JNJ-46356479 ([18F]8) was synthesized and characterized as the first 18F-labeled mGluR2 imaging ligand to enhance diagnostic approaches for mGluR2-related disorders. PROCEDURES: JNJ-46356479 (8) was radiolabeled via the copper (I)-mediated radiofluorination of organoborane 9. In vivo PET imaging experiments with [18F]8 were conducted first in C57BL/6 J mice and Sprague-Dawley rats to obtain whole body biodistribution and brain uptake profile. Subsequent PET studies were done in a cynomolgus monkey (Macaca fascicularis) to investigate the uptake of [18F]8 in the brain, its metabolic stability, as well as pharmacokinetic properties. RESULTS: JNJ-46356479 (8) exhibited excellent selectivity against other mGluRs. In vivo PET imaging studies showed reversible and specific binding characteristic of [18F]8 in rodents. In the non-human primate, [18F]8 displayed good in vivo metabolic stability, excellent brain permeability, fast and reversible kinetics with moderate heterogeneity across brain regions. Pre-treatment studies with compound 7 revealed time-dependent decrease of [18F]8 accumulation in mGluR2 rich regions based on SUV values with the highest decrease in the nucleus accumbens (18.7 ± 5.9%) followed by the cerebellum (18.0 ± 7.9%), the parietal cortex (16.9 ± 7.8%), and the hippocampus (16.8 ± 6.9%), similar to results obtained in the rat studies. However, the volume of distribution (VT) results derived from 2T4k model showed enhanced VT from a blocking study with compound 7. This is probably because of the potentiating effect of compound 7 as an mGluR2 PAM as well as related non-specific binding in the tissue data. CONCLUSIONS: [18F]8 readily crosses the blood-brain barrier and demonstrates fast and reversible kinetics both in rodents and in a non-human primate. Further investigation of [18F]8 on its binding specificity would warrant translational study in human.
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Encéfalo/metabolismo , Fluorodesoxiglucosa F18/química , Radiofármacos/síntesis química , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Fluorodesoxiglucosa F18/farmacocinética , Ligandos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Radiofármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Myocilin (MYOC) gene is expressed in many ocular tissues, including the trabecular meshwork, a specialized eye tissue essential in regulating intraocular pressure. Many mutations in MYOC have been detected in primary open-angle glaucoma (POAG). We investigated whether MYOC mutations contributed to the susceptibility to POAG in a Chinese family. In a four-generation family affected with POAG, ocular examinations were performed on all members of the pedigree to determine their disease status, and 200 healthy matched controls were recruited. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing were used to determine the mutations in MYOC. Biological software was used to analyze the corresponding proteins for missense mutations. The c.1084G>- was found, for the first time, in four of eight affected patients and in one of two patients with suspected POAG. The c.1006C>T mutation was found in two of eight patients and in one of 19 subjects who were asymptomatic. The frequencies of c.1084G>- and c.1006C>T were 12.82 and 7.69%, respectively, in patients but not in the controls. These data provide additional clues to the pathogenesis of POAG because no other mutation was detected in either group. Our results suggest that the MYOC c.1084G>- may contribute to a genetic predisposition to POAG.
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Pueblo Asiatico/genética , Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Predisposición Genética a la Enfermedad , Glaucoma de Ángulo Abierto/genética , Glicoproteínas/genética , Mutación/genética , Adolescente , Adulto , Anciano , Secuencia de Bases , Niño , Preescolar , China , Análisis Mutacional de ADN , Familia , Femenino , Glaucoma de Ángulo Abierto/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Disco Óptico/patología , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Campos Visuales/fisiología , Adulto JovenRESUMEN
Three benzimidazole derivatives (13-15) have been synthetized as potential positron emission tomography (PET) imaging ligands for mGluR2 in the brain. Of these compounds, 13 exhibits potent binding affinity (IC50 = 7.6 ± 0.9 nM), positive allosteric modulator (PAM) activity (EC50 = 51.2 nM), and excellent selectivity against other mGluR subtypes (>100-fold). [11C]13 was synthesized via O-[11C]methylation of its phenol precursor 25 with [11C]methyl iodide. The achieved radiochemical yield was 20 ± 2% (n = 10, decay-corrected) based on [11C]CO2 with a radiochemical purity of >98% and molar activity of 98 ± 30 GBq/µmol EOS. Ex vivo biodistribution studies revealed reversible accumulation of [11C]13 and hepatobiliary and urinary excretions. PET imaging studies in rats demonstrated that [11C]13 accumulated in the mGluR2-rich brain regions. Pre-administration of mGluR2-selective PAM, 17 reduced the brain uptake of [11C]13, indicating a selective binding. Therefore, [11C]13 is a potential PET imaging ligand for mGluR2 in different central nervous system-related conditions.
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Bencimidazoles/química , Encéfalo/diagnóstico por imagen , Diseño de Fármacos , Tomografía de Emisión de Positrones , Receptores AMPA/análisis , Animales , Bencimidazoles/síntesis química , Bencimidazoles/farmacocinética , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Ligandos , Ratones , Ratones Noqueados , Modelos Moleculares , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Receptores AMPA/deficiencia , Relación Estructura-Actividad , Distribución TisularRESUMEN
We have synthesized and characterized [18F]-N-(4-chloro-3-((fluoromethyl-d2)thio)phenyl)-picolinamide ([18F]15) as a potential ligand for the positron emission tomography (PET) imaging of mGluR4 in the brain. Radioligand [18F]15 displays central nervous system drug-like properties, including mGluR4 affinity, potent mGluR4 PAM activity, and selectivity against other mGluRs, as well as sufficient metabolic stability. Radiosynthesis was carried out in two steps. The radiochemical yield of [18F]15 was 11.6 ± 2.9% (n = 7, decay corrected) with a purity of 99% and a molar activity of 84.1 ± 11.8 GBq/µmol. Ex vivo biodistribution studies showed reversible binding of [18F]15 in all investigated tissues including the brain, liver, heart, lungs, and kidneys. PET imaging studies in male Sprague Dawley rats showed that [18F]15 accumulates in the brain regions known to express mGluR4. Pretreatment with the unlabeled mGluR4 PAM compounds 13 (methylthio analogue) and 15 showed significant dose-dependent blocking effects. These results suggest that [18F]15 is a promising radioligand for PET imaging mGluR4 in the brain.
Asunto(s)
Picolinas/farmacología , Radiofármacos/farmacología , Receptores de Glutamato Metabotrópico/análisis , Animales , Encéfalo/metabolismo , Estabilidad de Medicamentos , Radioisótopos de Flúor/química , Ligandos , Masculino , Microsomas Hepáticos/metabolismo , Picolinas/síntesis química , Picolinas/farmacocinética , Tomografía de Emisión de Positrones , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
The sigma-1 receptor (σ 1R) is a unique intracellular protein. σ 1R plays a major role in various pathological conditions in the central nervous system (CNS), implicated in several neuropsychiatric disorders. Imaging of σ 1R in the brain using positron emission tomography (PET) could serve as a noninvasively tool for enhancing the understanding of the disease's pathophysiology. Moreover, σ 1R PET tracers can be used for target validation and quantification in diagnosis. Herein, we describe the radiosynthesis, in vivo PET/CT imaging of novel σ 1R 11C-labeled radioligands based on 6-hydroxypyridazinone, [11C]HCC0923 and [11C]HCC0929. Two radioligands have high affinities to σ 1R, with good selectivity. In mice PET/CT imaging, both radioligands showed appropriate kinetics and distributions. Additionally, the specific interactions of two radioligands were reduced by compounds 13 and 15 (self-blocking). Of the two, [11C]HCC0929 was further investigated in positive ligands blocking studies, using classic σ 1R agonist SA 4503 and σ 1R antagonist PD 144418. Both σ 1R ligands could extensively decreased the uptake of [11C]HCC0929 in mice brain. Besides, the biodistribution of major brain regions and organs of mice were determined in vivo. These studies demonstrated that two radioligands, especially [11C]HCC0929, possessed ideal imaging properties and might be valuable tools for non-invasive quantification of σ 1R in brain.
RESUMEN
PURPOSE: To investigate the genetic linkage of primary open-angle glaucoma (POAG) in a Chinese family. METHODS: We have screened for myocilin (MYOC) gene mutations in a glaucoma family of five generations. There are fifty-six members of whom 11 were confirmed to have POAG , two with ocular hypertension were considered as POAG suspect, and the remaining 43 were asymptomatic. We also recruited 200 unrelated healthy Chinese subjects as normal control. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing were used to identify mutations in the three exons of MYOC. Presymptomatic diagnoses were made for the family members seeking consultation based on the results of both clinical examination and genetic analysis. RESULTS: Among three allelic variants identified in this pedigree (Pro13Leu [38 C-->T], Arg76Lys [227G-->A], and Gln337Stop [1009C del]), Pro13Leu and Gln337Stop were reported to be novel mutations while Arg76Lys has been previously documented. Our results show that all 11 POAG patients carry the Gln337Stop mutation and that four POAG patients and one POAG suspect (V:2) were found to have the Pro13Leu mutation. In addition, Arg76Lys polymorphism was identified in two patients and a POAG suspect (V:5). CONCLUSIONS: Pro13Leu and Gln337Stop mutations of MYOC are likely responsible for the etiology of POAG in this pedigree, but the causative mechanism needs further research.
Asunto(s)
Pueblo Asiatico/genética , Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Glaucoma de Ángulo Abierto/genética , Glicoproteínas/genética , Mutación/efectos de los fármacos , Adolescente , Adulto , Anciano , Secuencia de Bases , China , Proteínas del Citoesqueleto/química , Análisis Mutacional de ADN , Proteínas del Ojo/química , Femenino , Glaucoma de Ángulo Abierto/fisiopatología , Glicoproteínas/química , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Linaje , Fenotipo , Estructura Secundaria de Proteína , Campos Visuales/fisiologíaRESUMEN
Highly active anti-retroviral therapy (HAART) cannot clear infected cells harboring HIV-1 proviral DNA from HIV-1-infected patients. We previously demonstrated that zinc-finger nucleases (ZFNs) can specifically and efficiently excise HIV-1 proviral DNA from latently infected human T cells by targeting long terminal repeats (LTRs), a novel and alternative antiretroviral strategy for eradicating HIV-1 infection. To prevent unwanted off-target effects from constantly expressed ZFNs, in this study, we engineered the expression of ZFNs under the control of HIV-1 LTR, by which ZFN expression can be activated by the HIV-1 (Trans-Activator of Transcription) Tat protein. Our results show that functional expression of ZFNs induced by Tat excise the integrated proviral DNA of HIV-NL4-3-eGFP in approximately 30% of the population of HIV-1-infected cells. The results from HIV-1-infected human primary T cells and latently infected T cells treated with the inducible ZFNs further validated that proviral DNA can be excised. Taken together, positively regulated expression of ZFNs in the presence of HIV-1 Tat may provide a safer and novel implementation of genome-editing technology for eradicating HIV-1 proviral DNA from infected host cells.
RESUMEN
Cigarette smoking is a well-known risk factor in the development and progression of malignant diseases. Nicotine, the major constituent in cigarette smoke, has also shown negative effects on stem cells. Mesenchymal stem cells (MSCs) have been widely demonstrated to migrate into tumors and play key roles in cancer progression. However, the mechanisms by which nicotine impacts MSCs and tumorigenesis of lung cancer are still undetermined. In this study we investigated the effects of nicotine on human umbilical cord mesenchymal stem cells (hUC-MSCs) and the impacts of nicotine-treated hUC-MSCs on tumor formation and progression. We found that nicotine has a toxic effect on hUC-MSCs and changes the morphology, inhibits proliferation and promotes apoptosis of hUC-MSCs in a dose-dependent manner. Nicotine-treated hUC-MSCs produce higher level of IL-6. Moreover, nicotine promotes migration, stemness and epithelial-mesenchymal transition (EMT) of hUC-MSCs by inhibiting E-cadherin expression and upregulating mesenchymal markers such as N-cadherin and Vimentin, leading to the induction of stem cell markers Sox2, Nanog, Sall4, Oct4 and CD44. Migration and proliferation of non-small cell lung cancer A549 cells and breast cancer MCF-7 cells are promoted after their coculture with nicotine-treated hUC-MSCs in a cell-cell contact-independent manner. Furthermore, nicotine-treated hUC-MSCs promote tumor formation and growth of A549 cells in nude mice. These studies demonstrated that the enhanced stemness and EMT of hUC-MSCs induced by nicotine are critical for the development of tobacco-related cancers.