RESUMEN
Worldwide, tuberculosis (TB) remains one of the most prevalent infectious diseases causing morbidity and death in >1.5 million patients annually. Mycobacterium tuberculosis (Mtb), the etiologic agent of TB, usually resides in the alveolar macrophages. Current tuberculosis treatment methods require more than six months, and low compliance often leads to therapeutic failure and multidrug resistant strain development. Critical to improving TB-therapy is shortening treatment duration and increasing therapeutic efficacy. In this study, we sought to determine if lung hemodynamics and pathological changes in Mtb infected cells can be used for the selective targeting of microparticles to infected tissue(s). Thioaptamers (TA) with CD44 (CD44TA) targeting moiety were conjugated to discoidal silicon mesoporous microparticles (SMP) to enhance accumulation of these agents/carriers in the infected macrophages in the lungs. In vitro, CD44TA-SMP accumulated in macrophages infected with mycobacteria efficiently killing the infected cells and decreasing survival of mycobacteria. In vivo, increased accumulations of CD44TA-SMP were recorded in the lung of M. tuberculosis infected mice as compared to controls. TA-targeted carriers significantly diminished bacterial load in the lungs and caused recruitment of T lymphocytes. Proposed mechanism of action of the designed vector accounts for a combination of increased uptake of particles that leads to infected macrophage death, as well as, activation of cellular immunity by the TA, causing increased T-cell accumulation in the treated lungs. Based on our data with CD44TA-SMP, we anticipate that this drug carrier can open new avenues in TB management.
Asunto(s)
Aptámeros de Nucleótidos/administración & dosificación , Portadores de Fármacos/administración & dosificación , Receptores de Hialuranos/genética , Mycobacterium tuberculosis , Tuberculosis/tratamiento farmacológico , Animales , Células Cultivadas , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos BALB C , Silicio/administración & dosificación , Linfocitos T/inmunología , Tuberculosis/inmunología , Tuberculosis/metabolismoRESUMEN
BACKGROUND: Sequence variation in the human 12/15 lipoxygenase (ALOX15) has been associated with atherosclerotic disease. We functionally characterized an ALOX15 promoter polymorphism, rs2255888, previously associated with carotid plaque burden. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate specific in vitro and in vivo binding of the cytoskeletal protein, vimentin, to the ALOX15 promoter. We show that the two promoter haplotypes carrying alternate alleles at rs2255888 exhibit significant differences in promoter activity by luciferase reporter assay in two cell lines. Differences in in-vitro vimentin-binding to and formation of DNA secondary structures in the polymorphic promoter sequence are also detected by electrophoretic mobility shift assay and biophysical analysis, respectively. We show regulation of ALOX15 protein by vimentin. CONCLUSIONS/SIGNIFICANCE: This study suggests that vimentin binds the ALOX15 promoter and regulates its promoter activity and protein expression. Sequence variation that results in changes in DNA conformation and vimentin binding to the promoter may be relevant to ALOX15 gene regulation.
Asunto(s)
Araquidonato 15-Lipooxigenasa/genética , Aterosclerosis/enzimología , Aterosclerosis/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas , Vimentina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Haplotipos/genética , Humanos , Luciferasas/metabolismo , Células MCF-7 , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Conformación de Ácido Nucleico , Oligonucleótidos/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Sales (Química)/farmacología , TransfecciónRESUMEN
Thioaptamers offer advantages over normal phosphate ester backbone aptamers due to their enhanced affinity, specificity, and higher stability, largely due to the properties of the sulfur backbone modifications. Over the past several years, in vitro thioaptamer selection and bead-based thioaptamer selection techniques have been developed in our laboratory. Furthermore, several thioaptamers targeting specific proteins such as transcription factor NF-kappaB and AP-1 proteins have been identified. Selected thioaptamers have been shown diagnostic promise in proteome screens. Moreover, some promising thioaptamers have been shown in preliminary animal therapeutic dosing to increase survival in animal models of infection with West Nile virus.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Animales , Humanos , Ratones , Análisis por Matrices de Proteínas , Proteómica , Tionucleótidos/uso terapéutico , Factores de Transcripción/antagonistas & inhibidores , Fiebre del Nilo Occidental/tratamiento farmacológico , Fiebre del Nilo Occidental/mortalidadRESUMEN
Nuclear factor-kappaB (NF-kappaB) transcription factors are important in regulating the immune response and play critical roles in the pathogenesis of chronic inflammatory diseases and a variety of human cancers. Agents that target specific NF-kappaB dimers may serve as therapeutic agents for the prevention of pathogenic immune responses. We have selected monothiophosphate-modified aptamers, or "thioaptamers", to the NF-kappaB p50/RelA heterodimer using combinatorial selection techniques. We also utilized a "double sieve" or editing approach for the generation of thioaptamers with enhanced selectivity to the RelA/RelA homodimer. The thioaptamers from these selections and our previous selections on the p50/p50 and RelA/RelA homodimers all had unique sequences and bound tightly to the recombinant NF-kappaB dimers against which they were selected. The selected thioaptamers also appear to maintain their selectivity and specificity among other cellular proteins, because they have the ability to bind NF-kappaB proteins within nuclear extracts from lipopolysaccharide (LPS)-induced macrophages and B cells.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , FN-kappa B/metabolismo , Oligorribonucleótidos/farmacología , Fosfatos , Linfocitos B/química , Secuencia de Bases , Núcleo Celular/química , Dimerización , Humanos , Lipopolisacáridos , Macrófagos/química , FN-kappa B/efectos de los fármacos , Subunidad p50 de NF-kappa B , Oligorribonucleótidos/química , Factor de Transcripción ReIA , Factores de TranscripciónAsunto(s)
Proteínas de Unión al Calcio/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas de Neoplasias/química , Biomarcadores de Tumor , Calcio/química , Clonación Molecular , ADN Complementario/metabolismo , Dimerización , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Neoplasias/metabolismo , Factores de Crecimiento Nervioso , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/química , Transducción de Señal , TemperaturaRESUMEN
As part of a study on chemical constituents of Camptotheca species, one new natural camptothecin analogue (2), two new alkaloids (3, 4), one new ellagic acid analogue (5), and 19 known compounds (1, 6-23) have been isolated from the root bark, stem bark, fruits, and leaves of Camptotheca acuminata Decaisne. The structures of 2-5 were determined from spectral data to be 10-methoxy-20-O-acetylcamptothecin (2), 20-O-beta-glucopyranosyl 18-hydroxycamptothecin (3), 20-formylbenz indolizino [1,2-b]quino-line-11(13H)-one (4), and 3,4-methylenedioxy-3'-O-methyl-5'-hydroxyellagic acid (5).