RESUMEN
Emotional distress (ED), commonly characterized by symptoms of depression and/or anxiety, is prevalent in patients with cancer. Preclinical studies suggest that ED can impair antitumor immune responses, but few clinical studies have explored its relationship with response to immune checkpoint inhibitors (ICIs). Here we report results from cohort 1 of the prospective observational STRESS-LUNG study, which investigated the association between ED and clinical efficacy of first-line treatment of ICIs in patients with advanced non-small-cell lung cancer. ED was assessed by Patient Health Questionnaire-9 and Generalized Anxiety Disorder 7-item scale. The study included 227 patients with 111 (48.9%) exhibiting ED who presented depression (Patient Health Questionnaire-9 score ≥5) and/or anxiety (Generalized Anxiety Disorder 7-item score ≥5) symptoms at baseline. On the primary endpoint analysis, patients with baseline ED exhibited a significantly shorter median progression-free survival compared with those without ED (7.9 months versus 15.5 months, hazard ratio 1.73, 95% confidence interval 1.23 to 2.43, P = 0.002). On the secondary endpoint analysis, ED was associated with lower objective response rate (46.8% versus 62.1%, odds ratio 0.54, P = 0.022), reduced 2-year overall survival rate of 46.5% versus 64.9% (hazard ratio for death 1.82, 95% confidence interval 1.12 to 2.97, P = 0.016) and detriments in quality of life. The exploratory analysis indicated that the ED group showed elevated blood cortisol levels, which was associated with adverse survival outcomes. This study suggests that there is an association between ED and worse clinical outcomes in patients with advanced non-small-cell lung cancer treated with ICIs, highlighting the potential significance of addressing ED in cancer management. ClinicalTrials.gov registration: NCT05477979 .
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Inhibidores de Puntos de Control Inmunológico , Neoplasias Pulmonares , Distrés Psicológico , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Femenino , Masculino , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Persona de Mediana Edad , Anciano , Estudios Prospectivos , Depresión/tratamiento farmacológico , Ansiedad/tratamiento farmacológico , Resultado del Tratamiento , Supervivencia sin Progresión , Adulto , Anciano de 80 o más AñosRESUMEN
The aim of this study is to explore the tissue distribution of PEGylated puerarin in acute myocardial ischemia model rats. Healthy male SD rats were randomly divided into two groups (30 each). Both were given PEGylated puerarin at a dose of 488 mg x kg(-1). After 5 min of medication, one group was normal rats, another group with acute myocardial ischemia was established by peritoneal injection of 50 mg x kg(-1) isoprenaline. After administration, the animals were executed at 30, 60, 90, 120, 150 and 180 min, then heart, liver, spleen, lung, kidney were extracted. The content of puerarin in organ tissue was determined by HPLC. The results showed that the AUC of tissue distribution of PEGylated puerarin in normal rats was liver > kidney > heart ≈ spleen > lung > brain. While the AUC of tissue distribution of PEGylated puerarin in acute myocardial ischemia model rats was liver ≈ heart > kidney > lung ≈ spleen > brain. AUC(heart) of PEGylated puerarin in acute myocardial ischemia model rats was 1.7 times than that of the normal rats, and there was significant difference (P < 0.05). Thus, PEGylated puerarin had a good heart-targeting property in early myocardial infarction area, drugs could accumulate in the ischemic myocardium. It provided important information for further study and clinic use of PEGylated puerarin.
Asunto(s)
Isoflavonas/farmacocinética , Isquemia Miocárdica/metabolismo , Animales , Encéfalo/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Bazo/metabolismo , Distribución TisularRESUMEN
PURPOSE: We identified and validated [(18)F]-CP18, a DEVD (the caspase 3 substrate recognition motif) containing substrate-based compound as an imaging tracer for caspase-3 activity in apoptotic cells. PROCEDURES: CP18 was radiolabeled with fluorine-18 using click chemistry. The affinity and selectivity of CP18 for caspase-3 were evaluated in vitro. The biodistribution and metabolism pattern of [(18)F]-CP18 were assessed in vivo. [(18)F]-CP18 positron emission tomography (PET) scans were performed in a dexamethasone-induced thymic apoptosis mouse model. After imaging, the mice were sacrificed, and individual organs were collected, measured in a gamma counter, and tested for caspase-3 activity. RESULTS: In vitro enzymatic caspase-3 assay demonstrated specific cleavage of CP18. In vivo, [(18)F]-CP18 is predominantly cleared through the kidneys and urine, and is rapidly eliminated from the bloodstream. There was a sixfold increase in caspase activity and a fourfold increase of [(18)F]-CP18 retention in the dexamethasone-induced thymus of treated versus control mice. CONCLUSIONS: We report the use [(18)F]-CP18 as a PET tracer for imaging apoptosis. Our data support further development of this tracer for clinical PET applications.
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Apoptosis , Glicopéptidos/farmacocinética , Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Análisis de Varianza , Animales , Caspasa 3/metabolismo , Línea Celular Tumoral , Dexametasona/efectos adversos , Glicopéptidos/química , Humanos , Modelos Lineales , Ratones , Radiofármacos/química , Radiofármacos/farmacocinética , Timo/química , Timo/efectos de los fármacos , Distribución TisularRESUMEN
PURPOSE: A novel caspase-3 substrate-based probe [(18)F]-CP18 was evaluated as an in vivo positron emission tomography (PET) imaging agent for monitoring apoptosis in tumors. METHODS: Uptake of [(18)F]-CP18 in cell assays and tumors was measured. Caspase-3/7 activities in cell lysates and tumor homogenates were determined. Autoradiography,Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and cleaved caspase-3 immunostaining were performed on adjacent tumor sections to identify areas of apoptosis. RESULTS: The in vitro cell assays showed caspase-3-dependent uptake of [(18)F]-CP18 in tumor cells when treated with an apoptosis inducer. The in vivo microPET imaging signal of [(18)F]-CP18 in xenograft tumors correlated with the ex vivo caspase-3/7 activities in these tumors. Furthermore, tumor autoradiographies of [(18)F]-CP18 in tumor sections matched adjacent sections stained by TUNEL and caspase-3 immunohistochemistry (IHC). CONCLUSIONS: [(18)F]-CP18 demonstrated high affinity and selectivity for activated caspase-3 both in vitro and in vivo, and the results support [(18)F]-CP18 as a promising new PET imaging agent for apoptosis.
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Apoptosis , Glicopéptidos/farmacocinética , Imagen Molecular/métodos , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Animales , Caspasa 3/análisis , Caspasa 3/metabolismo , Caspasa 7/análisis , Caspasa 7/metabolismo , Línea Celular Tumoral , Glicopéptidos/química , Humanos , Modelos Lineales , Ratones , Radiofármacos/química , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: We wished to develop a highly selective positron emission tomography (PET) imaging agent targeting PHF-tau in human Alzheimer's disease (AD) brains. METHODS: To screen potential tau binders, human AD brain sections were used as a source of native paired helical filament (PHF)-tau and Aß rather than synthetic tau aggregates or Aß fibrils generated in vitro to measure the affinity and selectivity of [(18)F]T807 to tau and Aß. Brain uptake and biodistribution of [(18)F]T807 in mice were also tested. RESULTS: In vitro autoradiography results show that [(18)F]T807 exhibits strong binding to PHF-tau-positive human brain sections. A dissociation constant (Kd) of [(18)F]T807 (14.6 nM) was measured using brain sections from the frontal lobe of AD patients. A comparison of autoradiography and double immunohistochemical staining of PHF-tau and Aß on adjacent sections demonstrated that [(18)F]T807 binding colocalized with immunoreactive PHF-tau pathology, but did not highlight Aß plaques. In vivo studies in mice demonstrated that [(18)F]T807 was able to cross the blood-brain barrier and washed out quickly. CONCLUSIONS: [(18)F]T807 demonstrates high affinity and selectivity to PHF-tau as well as favorable in vivo properties, making this a promising candidate as an imaging agent for AD.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Encéfalo/diagnóstico por imagen , Radioisótopos de Flúor , Proteínas tau/química , Proteínas tau/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/metabolismo , Animales , Autorradiografía , Encéfalo/patología , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Tomografía de Emisión de Positrones , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Distribución Tisular , Proteínas tau/genéticaRESUMEN
Tissue kallikrein (KLK1) processes low-molecular weight kininogen to produce vasoactive kinins, which exert biological functions via kinin receptor signaling. Using various delivery approaches, we have demonstrated that tissue kallikrein through kinin B2 receptor signaling exhibits a wide spectrum of beneficial effects by reducing cardiac and renal injuries, restenosis and ischemic stroke, and by promoting angiogenesis and skin wound healing, independent of blood pressure reduction. Protection by tissue kallikrein in oxidative organ damage is attributed to the inhibition of apoptosis, inflammation, hypertrophy and fibrosis. Tissue kallikrein also enhances neovascularization in ischemic heart and limb. Moreover, tissue kallikrein/kinin infusion not only prevents but also reverses kidney injury, inflammation and fibrosis in salt-induced hypertensive rats. Furthermore, there is a wide time window for kallikrein administration in protection against ischemic brain infarction, as delayed kallikrein infusion for 24 h after cerebral ischemia in rats is effective in reducing neurological deficits, infarct size, apoptosis and inflammation. Importantly, in the clinical setting, human tissue kallikrein has been proven to be effective in the treatment of patients with acute brain infarction when injected within 48 h after stroke onset. Finally, kallikrein promotes skin wound healing and keratinocyte migration by direct activation of protease-activated receptor 1.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Trastornos Cerebrovasculares/metabolismo , Enfermedades Renales/metabolismo , Piel/lesiones , Piel/metabolismo , Calicreínas de Tejido/metabolismo , Cicatrización de Heridas , Animales , Enfermedades Cardiovasculares/fisiopatología , Enfermedades Cardiovasculares/prevención & control , Trastornos Cerebrovasculares/fisiopatología , Trastornos Cerebrovasculares/prevención & control , Humanos , Enfermedades Renales/prevención & control , Piel/patología , Calicreínas de Tejido/antagonistas & inhibidores , Calicreínas de Tejido/química , Calicreínas de Tejido/uso terapéuticoRESUMEN
Delivery of short interfering RNA (siRNA) to cells in culture, and in vivo, is possible with combined use of a receptor-specific monoclonal antibody (mAb) and avidin-biotin technology. In the present studies, the luciferase gene is transiently expressed in human 293 epithelial cells. The siRNA delivery system is composed of the siRNA, monobiotinylated on the 3'-terminus of the sense strand, and a conjugate of streptavidin (SA) and a mAb to the human insulin receptor (HIR). Exposure of cells to 3'-biotinyl-siRNA bound to the HIRMAb/SA conjugate, but not to unconjugated SA, avidin, or the HIRMAb, causes a >90% reduction in luciferase gene expression. The receptor-targeted siRNA effect is maximal at 48 h after delivery of the siRNA to the cells, and the effect is lost by 7 days after a single application of the targeted siRNA in culture. The KI of the receptor-targeted siRNA inhibition of gene expression is 30.5 +/- 11.7 nM, and significant inhibition is observed with siRNA concentrations as low as 3 nM. In conclusion, the combination of a receptor-specific targeting ligand, such as the HIRMAb, and avidin-biotin technology allows for high affinity capture of the monobiotinylated siRNA by the targeting mAb. The siRNA is effectively delivered to the cytosol of cells, and knockdown of gene expression with the HIRMAb/SA delivery system is comparable to RNA interference effects obtained with cationic polyplexes. Whereas the use of cationic polyplexes in vivo is problematic, the bond between the targeting mAb and the siRNA is stable with avidin-biotin technology, and RNAi effects at distant sites such as brain are observed in vivo following an intravenous administration of the targeted siRNA.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD/inmunología , Avidina/química , Biotina/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/fisiología , Receptor de Insulina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Humanos , Luciferasas/genética , Ratones , Interferencia de ARN/fisiología , Estreptavidina/química , Factores de TiempoRESUMEN
The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.
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Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Biotina/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/farmacología , Receptores de Droga/efectos de los fármacos , Animales , Anticuerpos Monoclonales/biosíntesis , Avidina/química , Biotina/farmacocinética , Western Blotting , Células CHO , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , ADN Complementario/biosíntesis , ADN Complementario/genética , Diálisis , Sistemas de Liberación de Medicamentos , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Ingeniería Genética , Humanos , Microscopía Confocal , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Albúmina Sérica Bovina/química , UltrafiltraciónRESUMEN
Mucopolysaccharidosis Type I, Hurler's Syndrome, is a lysosomal storage disorder that affects the brain. The missing enzyme, alpha-L-iduronidase (IDUA), does not cross the blood-brain barrier (BBB). To enable BBB transport of the enzyme, human IDUA was fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb crosses the BBB on the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry into brain the IDUA. Transfection of COS cells resulted in high levels of IDUA enzyme activity both in the medium and in the intracellular space. The size of the fusion heavy chain, as measured with Western blotting and antibodies to either human IDUA or human IgG, was increased about 80 kDa, relative to the size of the heavy chain of the parent HIRMAb. The IDUA enzyme specific activity of the affinity purified HIRMAb-IDUA fusion protein was 363 +/- 37 U/microg protein, which is comparable to specific activity of recombinant IDUA. The accumulation of glycosoaminoglycans in Hurler fibroblasts was decreased 70% by treatment with the HIRMAb-IDUA fusion protein. Confocal microscopy showed targeting of the fusion protein to the lysosome. The HIRMAb-IDUA fusion protein bound with high affinity to the HIR, and was rapidly transported into the brain of the adult Rhesus monkey following intravenous administration. The HIRMAb-IDUA fusion protein is a new treatment for Hurler's syndrome, which has been specifically engineered to cross the human BBB.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Barrera Hematoencefálica/fisiología , Iduronidasa/farmacocinética , Mucopolisacaridosis I/terapia , Ingeniería de Proteínas/métodos , Receptor de Insulina/química , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Anticuerpos Monoclonales/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Células COS , Chlorocebus aethiops , Modelos Animales de Enfermedad , Fibroblastos , Humanos , Iduronidasa/administración & dosificación , Iduronidasa/química , Mucopolisacaridosis I/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Transfección/métodosRESUMEN
BACKGROUND: Rats with experimental Parkinson's disease (PD) are treated with intravenous glial-derived neurotrophic factor (GDNF) plasmid DNA, non-viral gene therapy using Trojan horse liposomes (THLs) targeted with a monoclonal antibody (MAb) to the rat transferrin receptor (TfR). Expression of the transgene is confined to catecholaminergic cells by placement of the GDNF gene under the influence of the rat tyrosine hydroxylase (TH) promoter. METHODS: A 13-kb eukaryotic expression plasmid, designated pTHpro-GDNF, is engineered in which the human prepro GDNF cDNA is driven by 8 kb of the 5'-flanking sequence of the rat TH promoter (pro), and is 3'-flanked by the bovine growth hormone transcription termination sequence. The pTHpro-GDNF plasmid DNA is encapsulated in THLs targeted with a TfRMAb, and a single intravenous injection is given to rats at 2 weeks after experimental PD is induced by intra-cerebral 6-hydroxydopamine. RESULTS: Expression of the GDNF gene, under the influence of the TH promoter, is restricted compared to GDNF expression under the influence of the cytomegalovirus promoter. GDNF is elevated only in organs of the rat where TH gene expression is observed, including the substantia nigra, liver and adrenal gland. The single, delayed intravenous administration of the GDNF gene therapy causes a lasting reduction in apormorphine-induced rotation, which is correlated with a 19-fold increase in striatal TH enzyme activity. Both dose-response and time-responses are observed. CONCLUSIONS: Sustained therapeutic effects are achieved in experimental PD with a delayed single intravenous dosing of GDNF plasmid DNA gene therapy, using receptor-targeted THLs and a region-specific promoter.
Asunto(s)
Terapia Genética/métodos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Liposomas/administración & dosificación , Trastornos Parkinsonianos/terapia , Regiones Promotoras Genéticas , Tirosina 3-Monooxigenasa/genética , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Factor Neurotrófico Derivado de la Línea Celular Glial/administración & dosificación , Humanos , Inmunohistoquímica , Liposomas/metabolismo , Modelos Biológicos , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , RatasRESUMEN
PURPOSE: The effective delivery of short interfering RNA (siRNA) to brain following intravenous administration requires the development of a delivery system for transport of the siRNA across the brain capillary endothelial wall, which forms the blood-brain barrier in vivo. METHODS: siRNA was delivered to brain in vivo with the combined use of a receptor-specific monoclonal antibody delivery system, and avidin-biotin technology. The siRNA was mono-biotinylated on either terminus of the sense strand, in parallel with the production of a conjugate of the targeting MAb and streptavidin. RESULTS: Rat glial cells (C6 or RG-2) were permanently transfected with the luciferase gene, and implanted in the brain of adult rats. Following the formation of intra-cranial tumors, the rats were treated with a single intravenous injection of 270 microg/kg of biotinylated siRNA attached to a transferrin receptor antibody via a biotin-streptavidin linker. The intravenous administration of the siRNA caused a 69-81% decrease in luciferase gene expression in the intracranial brain cancer in vivo. CONCLUSIONS: Brain delivery of siRNA following intravenous administration is possible with siRNAs that are targeted to brain with the combined use of receptor specific antibody delivery systems and avidin-biotin technology.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Biotina/metabolismo , Neoplasias Encefálicas/terapia , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Transferrina/inmunología , Estreptavidina/metabolismo , Animales , Biotinilación , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Genes Reporteros , Humanos , Luciferasas/genética , Masculino , ARN Interferente Pequeño/administración & dosificación , Ratas , Ratas Endogámicas F344 , Receptor de Insulina/inmunología , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: The present study examines whether chromosomal derived forms of therapeutic genes can be delivered to brain following intravenous administration. The brain expression of a rat tyrosine hydroxylase (TH) cDNA is compared to the brain expression of a plasmid DNA encoding the 18 kb rat TH gene. METHODS: TH gene expression is measured in cell culture and in vivo in brain in experimental Parkinson's disease (PD). A total of four eukaryotic expression plasmids encoding rat TH were engineered wherein the size of the TH expression cassette ranged from 1.5 kb, in the case of the cDNA form of the gene, to 17.5 kb, in the case of the largest size genomic construct. The TH expression plasmids were delivered to either cultured cells or to rat brain in vivo with Trojan horse liposomes (THLs), which target the non-viral plasmid DNA to cells via cell membrane receptors. RESULTS: The pattern of TH gene expression in cell culture and in vivo was similar: the cDNA form of the TH gene was fast-acting with short duration of action, and the genomic form of the TH gene was slow-acting with longer duration of action. The most sustained replacement of striatal TH enzyme activity in experimental PD was produced by combination gene therapy where both the cDNA and the genomic forms of the TH gene were administered simultaneously. CONCLUSIONS: Eukaryotic expression plasmids encoding genomic forms of therapeutic genes, as large as 18 kb, can be successfully incorporated in THLs and delivered to brain following intravenous administration.
Asunto(s)
Encéfalo/enzimología , ADN Complementario/genética , Terapia Genética/métodos , Genoma/genética , Enfermedad de Parkinson/terapia , Tirosina 3-Monooxigenasa/genética , Animales , Encéfalo/metabolismo , Células Cultivadas , Cuerpo Estriado/enzimología , Humanos , Liposomas , Neuroglía/enzimología , Plásmidos/metabolismo , Ratas , Mapeo Restrictivo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Delivery of monoclonal antibody therapeutics across the blood-brain barrier is an obstacle to the diagnosis or therapy of CNS disease with antibody drugs. The immune therapy of Alzheimer's disease attempts to disaggregate the amyloid plaque of Alzheimer's disease with an anti-Abeta monoclonal antibody. The present work is based on a three-step model of immune therapy of Alzheimer's disease: (1) influx of the anti-Abeta monoclonal antibody across the blood-brain barrier in the blood to brain direction, (2) binding and disaggregation of Abeta fibrils in brain, and (3) efflux of the anti-Abeta monoclonal antibody across the blood-brain barrier in the brain to blood direction. This is accomplished with the genetic engineering of a trifunctional fusion antibody that binds (1) the human insulin receptor, which mediates the influx from blood to brain across the blood-brain barrier, (2) the Abeta fibril to disaggregate amyloid plaque, and (3) the Fc receptor, which mediates the efflux from brain to blood across the blood-brain barrier. This fusion protein is a new antibody-based therapeutic for Alzheimer's disease that is specifically engineered to cross the human blood-brain barrier in both directions.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/farmacología , Anticuerpos Monoclonales/uso terapéutico , Barrera Hematoencefálica/metabolismo , Placa Amiloide/metabolismo , Receptores Fc/uso terapéutico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Transporte Biológico , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacologíaRESUMEN
Kallikrein cleaves low molecular weight kininogen to generate vasoactive kinins, which bind to the kinin B2 receptor, triggering a host of biological effects. Kallikrein gene delivery has been shown previously to reduce ischemia/reperfusion-induced cerebral infarction. In this study, we tested the hypothesis that the kinin B2 receptor plays a protective role in ischemic brain injury using kinin B2 receptor gene knockout (B2R-KO) mice subjected to middle cerebral artery occlusion (MCAO). The mortality rate and neurological deficit scores of B2R-KO mice (n=48) after MCAO were significantly increased compared with wild-type (WT) mice (n=40) when examined over a 14-day period. In addition, the infarct volume in B2R-KO mice was significantly larger than in WT mice at days 1 and 3 after MCAO. Similarly, apoptotic cells, detected by TUNEL labeling counterstained with propidium iodide, and caspase-3 activity in the ischemic brain increased significantly in B2R-KO mice at days 1 and 3 after MCAO. Furthermore, the accumulation of neutrophils in the ischemic brain of B2R-KO mice after MCAO increased when compared with WT mice and was associated with elevated tumor necrosis factor alpha expression. These alterations in B2R-KO mice correlated with decreased NO levels, Akt, and glycogen synthase kinase-3beta phosphorylation and increased nicotinamide-adenine dinucleotide oxidase activity. These results indicate that the kinin B2 receptor promotes survival and protects against brain injury by suppression of apoptosis and inflammation induced by ischemic stroke.
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Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Receptor de Bradiquinina B2/deficiencia , Animales , Apoptosis , Encéfalo/metabolismo , Encéfalo/fisiopatología , Isquemia Encefálica/complicaciones , Isquemia Encefálica/mortalidad , Infarto Cerebral/metabolismo , Infarto Cerebral/patología , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Enfermedades del Sistema Nervioso/etiología , Fármacos Neuroprotectores/metabolismo , Infiltración Neutrófila , Nitratos/metabolismo , Nitritos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Bradiquinina B2/metabolismo , Accidente Cerebrovascular/mortalidad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Stroke-induced neurological deficits and mortality are often associated with timing of treatment after the onset of stroke. We showed that local delivery of the human tissue kallikrein gene into rat brain immediately after middle cerebral artery occlusion (MCAO) exerts neuroprotection. In this study, we investigated the effect of systemic delivery of the kallikrein gene 8 hr after MCAO. Expression of recombinant human tissue kallikrein after gene transfer was identified in the ischemic brain region and blood vessels. Intravenous injection of adenovirus encoding the kallikrein gene significantly reduced neurological deficit scores 2 and 7 days after gene transfer. Kallikrein gene transfer also reduced ischemia-reperfusion (I/R)-induced cerebral infarction and promoted the survival and migration of glial cells from penumbra to the ischemic core from 3 to 14 days after gene delivery. Kallikrein reduced I/R-induced apoptosis of neuronal cells and inhibited inflammatory cell accumulation in the ischemic brain. These effects were blocked by the kinin B2 receptor antagonist icatibant. In addition, kallikrein enhanced angiogenesis and promoted neurogenesis after I/R and the stimulatory effect of kinin on neuronal cell proliferation was confirmed in primary cultured neuronal cells. The protective effects of kallikrein, through the kinin B2 receptor, were accompanied by increased cerebral nitric oxide and Bcl-2 levels, Akt phosphorylation, and reduced NAD(P)H oxidase activity, superoxide production, Bax levels, and caspase-3 activity. These results indicate that delayed systemic administration of the kallikrein gene after onset of stroke protects against ischemic brain injury by inhibiting apoptosis and inflammation and by promoting angiogenesis and neurogenesis.
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Apoptosis/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Calicreínas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Accidente Cerebrovascular/prevención & control , Animales , Apoptosis/genética , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Infarto Encefálico/tratamiento farmacológico , Infarto Encefálico/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Células Cultivadas , Técnicas de Transferencia de Gen , Humanos , Calicreínas/genética , Masculino , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/metabolismo , Proteína Oncogénica v-akt/efectos de los fármacos , Proteína Oncogénica v-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Daño por Reperfusión/terapiaRESUMEN
BACKGROUND: Our previous study showed that kallikrein gene transfer protects against gentamicin-induced nephrotoxicity and enhances renal function. In this study, we investigated the effects and potential mechanisms of kallikrein/kinin on inflammation and apoptosis induced by gentamicin. METHODS: Rats were injected subcutaneously with gentamicin daily for 10 days and received an intravenous injection of adenovirus carrying the human tissue kallikrein gene or control virus on the first day of gentamicin administration. RESULTS: After 10 days of gentamicin treatment, kallikrein gene transfer significantly attenuated gentamicin-induced tubular dilatation and lumenal protein casts. Moreover, kallikrein gene transfer reduced monocyte/macrophage infiltration, monocyte chemoattractant peptide-1 expression and renal cell apoptosis. Kallikrein's protective effects were accompanied by increased nitric oxide formation, and reduced NADH oxidase activity and superoxide production. Suppression of oxidative stress was associated with diminished c-jun N-terminal kinase activation and intercellular adhesion molecule-1 and transforming growth factor-beta protein levels. These biochemical effects were blocked by icatibant, indicating a kinin B2 receptor-mediated signalling event. CONCLUSIONS: This study indicates that kallikrein/kinin protects against gentamicin-induced nephrotoxicity by inhibiting inflammatory cell recruitment and apoptosis through suppression of oxidative stress-mediated signalling pathways. These findings raise the potential of applying kallikrein therapy approaches in treating aminoglycoside-induced renal damage.
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Lesión Renal Aguda/prevención & control , Apoptosis/efectos de los fármacos , Riñón/patología , Cininas/uso terapéutico , Calicreínas de Tejido/uso terapéutico , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Adenoviridae/genética , Animales , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Técnicas de Transferencia de Gen , Gentamicinas/toxicidad , Riñón/efectos de los fármacos , Riñón/metabolismo , Cininas/farmacocinética , Masculino , Ratas , Ratas Sprague-Dawley , Calicreínas de Tejido/farmacocinéticaRESUMEN
Adrenomedullin (AM) is a peptide hormone widely distributed in the central nervous system. Our previous study showed that AM gene delivery immediately after middle cerebral artery occlusion (MCAO) protected against cerebral ischemia/reperfusion (I/R) injury by promoting glial cell survival and migration. In the present study, we investigated the effect of delayed AM peptide infusion on ischemic brain injury at 24 h after MCAO. AM infusion significantly reduced neurological deficit scores at days 2, 4, and 8 after cerebral I/R. AM reduced cerebral infarct size at 8 and 15 days after surgery as determined by quantitative analysis. Double staining showed that AM infusion reduced TUNEL-positive apoptotic cells in both neurons and glial cells, as well as reduced caspase-3 activity in the ischemic area of the brain. In addition, AM treatment increased capillary density in the ischemic region at 15 days after I/R injury. Parallel studies revealed that AM treatment enhanced the proliferation of cultured endothelial cells as measured by both (3)H-thymidine incorporation and in situ BrdU labeling. Both in vitro and in vivo AM effects were blocked by calcitonin gene-related peptide (8-37), an AM receptor antagonist. Moreover, AM's effects were associated with increased cerebral nitric oxide (NO) levels, as well as decreased NAD(P)H oxidase activities and superoxide anion production. These results indicate that a continuous supply of exogenous AM peptide protects against I/R injury by improving the survival of neuronal and glial cells, and promoting angiogenesis through elevated NO formation and suppression of oxidative stress.
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Antiinflamatorios no Esteroideos/administración & dosificación , Apoptosis/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Péptidos/administración & dosificación , Accidente Cerebrovascular/prevención & control , Adrenomedulina , Análisis de Varianza , Animales , Infarto Encefálico/etiología , Infarto Encefálico/patología , Infarto Encefálico/prevención & control , Isquemia Encefálica/fisiopatología , Péptido Relacionado con Gen de Calcitonina/farmacología , Caspasa 3 , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Células Endoteliales/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Etiquetado Corte-Fin in Situ/métodos , Masculino , NADPH Oxidasas/metabolismo , Examen Neurológico , Nitratos/metabolismo , Nitritos/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
In DOCA-salt hypertension, renal kallikrein levels are increased and may play a protective role in renal injury. We investigated the effect of enhanced kallikrein levels on kidney remodeling of DOCA-salt hypertensive rats by systemic delivery of adenovirus containing human tissue kallikrein gene. Recombinant human kallikrein was detected in the urine and serum of rats after gene delivery. Kallikrein gene transfer significantly decreased DOCA- and salt-induced proteinuria, glomerular sclerosis, tubular dilatation, and luminal protein casts. Sirius red staining showed that kallikrein gene transfer reduced renal fibrosis, which was confirmed by decreased collagen I and fibronectin levels. Furthermore, kallikrein gene delivery diminished myofibroblast accumulation in the interstitium of the cortex and medulla, as well as transforming growth factor (TGF)-beta1 immunostaining in glomeruli. Western blot analysis and ELISA verified the decrease in immunoreactive TGF-beta1 levels. Kallikrein gene transfer also significantly reduced kidney weight, glomerular size, proliferating tubular epithelial cells, and macrophages/monocytes. Reduction of proliferation and hypertrophy was associated with reduced levels of the cyclin-dependent kinase inhibitor p27(Kip1), and the phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). The protective effects of kallikrein were accompanied by increased urinary nitrate/nitrite and cGMP levels, and suppression of superoxide formation. These results indicate that kallikrein protects against mineralocorticoid-induced renal fibrosis glomerular hypertrophy, and renal cell proliferation via inhibition of oxidative stress, JNK/ERK activation, and p27(Kip1) and TGF-beta1 expression.
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Terapia Genética/métodos , Hipertensión Renal/terapia , Calicreínas/genética , Proteinuria/terapia , Animales , Proteínas de Ciclo Celular/metabolismo , División Celular , GMP Cíclico/orina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Desoxicorticosterona/farmacología , Modelos Animales de Enfermedad , Matriz Extracelular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibrosis , Técnicas de Transferencia de Gen , Hipertensión Renal/metabolismo , Hipertensión Renal/patología , Hipertrofia , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Nitratos/orina , Estrés Oxidativo , Proteinuria/metabolismo , Proteinuria/patología , Ratas , Ratas Sprague-Dawley , Cloruro de Sodio Dietético/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Nitric oxide (NO) has been shown to play a key role in the regulation of cardiac hypertrophy and fibrosis in response to myocardial ischemia in part by antagonizing the action of angiotensin II (Ang II). In this study, we investigated the potential protective role of human endothelial nitric oxide synthase (eNOS) in left ventricular (LV) remodeling after myocardial infarction (MI) by a somatic gene transfer approach. Male Wistar rats underwent coronary artery ligation to induce MI. One week after surgery, adenovirus encoding the human eNOS or luciferase gene under the control of the CMV promoter/enhancer was injected into rats via the tail vein, and animals were sacrificed at 1 and 5 weeks after gene transfer. Successful gene transfer was evaluated based on increased levels of NO and cGMP in the heart, measured at one week after eNOS gene delivery. Six weeks after MI, the LV end-diastolic pressure, heart weight, LV axis length and cardiomyocyte size were markedly increased compared to the Sham group, while eNOS gene delivery significantly reduced these parameters. Rats receiving control virus developed considerably more fibrotic lesions identified by Sirius Red staining and collagen I immunostaining compared to Sham rats, and eNOS gene delivery significantly reduced collagen accumulation. eNOS gene transfer also reduced TUNEL-positive apoptotic cells. The cardioprotective effect of NO was accompanied by reduced NADH and NADPH oxidase activities and superoxide formation, TGF-beta1 and p27 levels, JNK activation, NF-kappa B nuclear translocation, and caspase-3 activity. This study shows that NO may play an important role in attenuating cardiac remodeling and apoptosis after myocardial infarction via suppression of oxidative stress-mediated signaling pathways.
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Terapia Genética/métodos , Infarto del Miocardio/terapia , Óxido Nítrico Sintasa/genética , Estrés Oxidativo/genética , Remodelación Ventricular/genética , Adenoviridae , Animales , Apoptosis/fisiología , Compuestos Azo , Caspasa 3 , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Colágeno , Vasos Coronarios/cirugía , GMP Cíclico/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Vectores Genéticos , Ventrículos Cardíacos/patología , Humanos , Immunoblotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ligadura , Luciferasas/genética , MAP Quinasa Quinasa 4 , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , NAD/metabolismo , NADP/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III , Tamaño de los Órganos , Ratas , Ratas Wistar , Superóxidos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1 , Proteínas Supresoras de Tumor/metabolismo , Función VentricularRESUMEN
Kallikrein/kinin has been shown to protect against ischemia/reperfusion-induced myocardial infarction and apoptosis. In the present study, we examined the potential neuroprotective action of kallikrein gene transfer in cerebral ischemia. Adult, male Sprague-Dawley rats were subjected to a 1-hour occlusion of the middle cerebral artery followed by intracerebroventricular injection of adenovirus harboring either the human tissue kallikrein gene or the luciferase gene. Kallikrein gene transfer significantly reduced ischemia-induced locomotor deficit scores and cerebral infarction after cerebral ischemia injury. Expression of recombinant human tissue kallikrein was identified and localized in monocytes/macrophages of rat ischemic brain by double immunostaining. Morphological analyses showed that kallikrein gene transfer enhanced the survival and migration of glial cells into the ischemic penumbra and core, as identified by immunostaining with glial fibrillary acidic protein. Cerebral ischemia markedly increased apoptotic cells, and kallikrein gene delivery reduced apoptosis to near-normal levels as seen in sham control rats. In primary cultured glial cells, kinin stimulated cell migration but inhibited hypoxia/reoxygenation-induced apoptosis in a dose-dependent manner. The effects of kinin on both migration and apoptosis were abolished by icatibant, a bradykinin B2 receptor antagonist. Enhanced cell survival after kallikrein gene transfer occurred in conjunction with markedly increased cerebral nitric oxide levels and phospho-Akt and Bcl-2 levels but reduced caspase-3 activation, NAD(P)H oxidase activity, and superoxide production. These results indicate that kallikrein gene transfer provides neuroprotection against cerebral ischemia injury by enhancing glial cell survival and migration and inhibiting apoptosis through suppression of oxidative stress and activation of the Akt-Bcl-2 signaling pathway.