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BACKGROUND: Apoptosis in atherosclerotic lesions contributes to plaque vulnerability by lipid core enlargement and fibrous cap attenuation. Apoptosis is associated with exteriorization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell membrane. Although PS-avid radiolabeled annexin-V has been employed for molecular imaging of high-risk plaques, PE-targeted imaging in atherosclerosis has not been studied. OBJECTIVES: This study sought to evaluate the feasibility of molecular imaging with PE-avid radiolabeled duramycin in experimental atherosclerotic lesions in a rabbit model and compare duramycin targeting with radiolabeled annexin-V. METHODS: Of the 27 rabbits, 21 were fed high-cholesterol, high-fat diet for 16 weeks. Nine of the 21 rabbits received 99mTc-duramycin (test group), 6 received 99mTc-linear duramycin (duramycin without PE-binding capability, negative radiotracer control group), and 6 received 99mTc-annexin-V for radionuclide imaging. The remaining normal chow-fed 6 animals (disease control group) received 99mTc-duramycin. In vivo microSPECT/microCT imaging was performed, and the aortas were explanted for ex vivo imaging and for histological characterization of atherosclerosis. RESULTS: A significantly higher duramycin uptake was observed in the test group compared with that of disease control and negative radiotracer control animals; duramycin uptake was also significantly higher than the annexin-V uptake. Quantitative duramycin uptake, represented as the square root of percent injected dose per cm (âID/cm) of abdominal aorta was >2-fold higher in atherosclerotic lesions in test group (0.08 ± 0.01%) than in comparable regions of disease control animals (0.039 ± 0.0061%, p = 3.70·10-8). Mean annexin uptake (0.060 ± 0.010%) was significantly lower than duramycin (p = 0.001). Duramycin uptake corresponded to the lesion severity and macrophage burden. The radiation burden to the kidneys was substantially lower with duramycin (0.49% ID/g) than annexin (5.48% ID/g; p = 4.00·10-4). CONCLUSIONS: Radiolabeled duramycin localizes in lipid-rich areas with high concentration of apoptotic macrophages in the experimental atherosclerosis model. Duramycin uptake in atherosclerotic lesions was significantly greater than annexin-V uptake and produced significantly lower radiation burden to nontarget organs.
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Apoptosis/fisiología , Aterosclerosis/metabolismo , Membrana Celular/metabolismo , Imagen Molecular/métodos , Fosfolípidos/metabolismo , Animales , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/etiología , Bacteriocinas/metabolismo , Membrana Celular/patología , Dieta Alta en Grasa/efectos adversos , Humanos , Masculino , Péptidos/metabolismo , Conejos , Cintigrafía/métodosAsunto(s)
Apoptosis , Bacteriocinas/administración & dosificación , Doxorrubicina , Imagen Molecular/métodos , Miocardio/patología , Compuestos de Organotecnecio/administración & dosificación , Radiofármacos/administración & dosificación , Volumen Sistólico , Tomografía Computarizada de Emisión de Fotón Único , Disfunción Ventricular Izquierda/diagnóstico por imagen , Función Ventricular Izquierda , Animales , Modelos Animales de Enfermedad , Detección Precoz del Cáncer , Masculino , Valor Predictivo de las Pruebas , Ratas Sprague-Dawley , Factores de Tiempo , Disfunción Ventricular Izquierda/inducido químicamente , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatología , Microtomografía por Rayos XRESUMEN
Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus-based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of ß-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.
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INTRODUCTION: Monitoring the effectiveness of therapy early and accurately continues to be challenging. We hypothesize that determination of Human Epidermal Growth Factor Receptor 2 (HER2) mRNA in malignant breast cancer (BC) cells by positron emission tomography (PET) imaging, before and after treatment, would reflect therapeutic efficacy. METHOD: WT4340, a peptide nucleic acid (PNA) 12-mer complementary to HER2 mRNA was synthesized together with -CSKC, a cyclic peptide, which facilitated internalization of the PNA via IGFR expressed on BC cells, and DOTA that chelated Cu-64. Mice (n = 8) with BT474 ER+/HER2+ human BC received doxorubicin (DOX, 1.5mg/kg) i.p. once a week for six weeks. Mice (n = 8) without DOX served as controls. All mice were PET imaged with F-18-FDG and 48 h later with Cu-64-WT4340. PET imaging were performed before and 72 h after each treatment. Standardized uptake values (SUVs) were determined and percent change calculated. Animal body weight (BW) and tumor volume (TV) were measured. RESULTS: SUVs for Cu-64-WT4340 after DOX treatment declined by 54% ± 17% after the second dose, 41% ± 15% after the fourth dose, and 29% ± 7% after the sixth dose, compared with 42% ± 22%, 31% ± 18%, and 13% ± 9% (p<0.05) for F-18-FDG. In untreated mice, the corresponding percent SUVs for Cu-64-WT4340 were 145% ± 82%, 165% ± 39%, and 212% ± 105% of pretreatment SUV, compared with 108% ± 28%, 151% ± 8%, and 152% ± 35.5%, (p<0.08) for F-18-FDG. TV in mice after second dose was 114.15% ± 61.83%, compared with 144.7% ± 64.4% for control mice. BW of DOX-treated mice was 103.4% ± 7.6% of pretreatment, vs. 100.1% ± 4.3% for control mice. CONCLUSION: Therapeutic efficacy was apparent sooner by molecular PET imaging than by determination of reduction in TV.
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Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/tratamiento farmacológico , Ácidos Nucleicos de Péptidos , Tomografía de Emisión de Positrones , Receptor ErbB-2/genética , Animales , Neoplasias de la Mama/patología , Transformación Celular Neoplásica , Radioisótopos de Cobre , Doxorrubicina/uso terapéutico , Femenino , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Factor I del Crecimiento Similar a la Insulina/química , Ratones , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
A fluorescent zinc 2,2'-dipicolylamine coordination complex PSVue®794 (probe 1) is known to selectively bind to phosphatidylserine exposed on the surface of apoptotic and necrotic cells. In this study, we investigated the cell death targeting properties of probe 1 in myocardial ischemia-reperfusion injury. A rat heart model of ischemia-reperfusion was used. Probe 1, control dye, or 99mTc glucarate was intravenously injected in rats subjected to 30-minute and 5-minute myocardial ischemia followed by 2-hour reperfusion. At 90 minutes or 20 hours postinjection, myocardial uptake was evaluated ex vivo by fluorescence imaging and autoradiography. Hematoxylin-eosin and cleaved caspase-3 staining was performed on myocardial sections to demonstrate the presence of ischemia-reperfusion injury and apoptosis. Selective accumulation of probe 1 could be detected in the area at risk up to 20 hours postinjection. Similar topography and extent of uptake of probe 1 and 99mTc glucarate were observed at 90 minutes postinjection. Histologic analysis demonstrated the presence of necrosis, but only a few apoptotic cells could be detected. Probe 1 selectively accumulates in myocardial ischemia-reperfusion injury and is a promising cell death imaging tool.
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Aminas/química , Colorantes Fluorescentes , Ácido Glucárico/análogos & derivados , Daño por Reperfusión Miocárdica/diagnóstico , Compuestos de Organotecnecio , Ácidos Picolínicos/química , Radiofármacos , Zinc/química , Animales , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
We previously showed that rabies virus (RABV) virions are excellent vehicles for antigen presentation. Here, a reverse genetic approach was applied to generate recombinant RABV that express a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin type A (BoNT/A) and RABV glycoprotein (G). To promote surface expression and incorporation of HC50/A into RABV virions, the RABV glycoprotein (G) ER translocation sequence, various fragments of RABV ectodomain (ED) and cytoplasmic domain were fused to HC50/A. The HC50/A chimeric proteins were expressed on the surface of cells infected with all of the recombinant RABVs, however, the highest level of surface expression was detected by utilizing 30 amino acids of the RABV G ED (HV50/A-E30). Our results also indicated that this chimeric protein was effectively incorporated into RABV virions. Immunization of mice with inactivated RABV-HC50/A-E30 virions induced a robust anti-HC50/A IgG antibody response that efficiently neutralized circulating BoNT/A in vivo, and protected mice against 1000 fold the lethal dose of BoNT/A.
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Anticuerpos Antibacterianos/sangre , Antígenos Virales/inmunología , Vacunas Bacterianas/inmunología , Toxinas Botulínicas Tipo A/química , Botulismo/prevención & control , Glicoproteínas/inmunología , Virus de la Rabia/genética , Proteínas del Envoltorio Viral/inmunología , Virión/genética , Animales , Anticuerpos Neutralizantes/sangre , Antígenos Virales/genética , Antígenos Virales/metabolismo , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/inmunología , Toxinas Botulínicas Tipo A/metabolismo , Botulismo/inmunología , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Inmunización , Inmunoglobulina G/sangre , Ratones , Virus de la Rabia/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismoRESUMEN
Three live rabies virus (RV) recombinant vaccine candidates, SPBNGA, SPBNGA-Cyto c (+), and SPBNGA-GA, were examined for their production levels and stability. Maximum production levels up to 10(10) infectious particles/mL were achieved using bioreactor technology. All virus lots exhibited thermostability profiles typical for RV vaccines and were non-pathogenic for intracranially inoculated immunocompetent mice. Moreover, sequence analysis indicated high genetic stability in all three RVs during 10 consecutive passages in newborn mice. This analysis revealed no change in the extra RV G gene in the SPBNGA-GA vaccine or in the cytochrome c gene in the SPBNGA-Cyto c (+) vaccine. Moreover, no changes were detected in the G gene codon for Glu333, which renders the virus non-pathogenic. However, after the fifth passage, a mutation resulting in an Asn194 --> Lys194 exchange emerged in the G genes of all three RVs. This mutation was associated with a modest increase in pathogenicity in SPBNGA and SPBNGA-Cyto c (+), but not in SPBNGA-GA, which contained the mutation in only one of its two G genes and which remained non-pathogenic. These results demonstrate the feasibility of producing RV vaccines that remain highly stable even after multiple passages.