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An oxygen sensor-mounted fine-needle biopsy tool was used for in vivo measurement of oxygen levels in tumor xenografts. The system provides a means of measuring the oxygen content in harvested tumor tissue from specific locations. Oxygen in human tumor xenografts in a murine model was observed for over 1 min. Tissues were mapped in relation to oxygen tension (pO2) readings and sampled for conventional cytological examination. Careful modeling of the pO2 readings over 60 seconds yielded a diffusion coefficient for oxygen at the sensor tip, providing additional diagnostic information about the tissue before sampling. Oxygen level measurement may provide a useful adjunct to the use of biomarkers in tumor diagnosis.
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For many cancer types, being undetectable from early symptoms or blood tests, or often detected at late stages, medical imaging emerges as the most efficient tool for cancer screening. MRI, ultrasound, X-rays (mammography), and X-ray CT (CT) are currently used in hospitals with variable costs. Diagnostic materials that can detect breast tumors through molecular recognition and amplify the signal at the targeting site in combination with state-of-the-art CT techniques, such as dual-energy CT, could lead to a more precise detection and assist significantly in image-guided intervention. Herein, we have developed a ligand-specific X-ray contrast agent that recognizes α5ß1 integrins overexpressed in MDA-MB-231 breast cancer cells for detection of triple (-) cancer, which proliferates very aggressively. In vitro studies show binding and internalization of our nanoprobes within those cells, towards uncoated nanoparticles (NPs) and saline. In vivo studies show high retention of ~3 nm ligand-PEG-S-AuNPs in breast tumors in mice (up to 21 days) and pronounced CT detection, with statistical significance from saline and iohexol, though only 0.5 mg of metal were utilized. In addition, accumulation of ligand-specific NPs is shown in tumors with minimal presence in other organs, relative to controls. The prolonged, low-metal, NP-enhanced spectral-CT detection of triple (-) breast cancer could lead to breakthrough advances in X-ray cancer diagnostics, nanotechnology, and medicine.
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Nanopartículas Metálicas , Neoplasias , Animais , Meios de Contraste/química , Ouro/química , Ligantes , Mamografia/métodos , Nanopartículas Metálicas/química , Camundongos , Tomografia Computadorizada por Raios X/métodosRESUMO
Despite the central role of IFN-γ in vitiligo pathogenesis, systemic IFN-γ neutralization is an impractical treatment option owing to strong immunosuppression. However, most patients with vitiligo present with <20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFN-γ signaling during vitiligo, we hypothesized that tethering an IFN-γâneutralizing antibody to keratinocytes would limit antiâIFN-γ effects on the treated skin for the localized treatment. To that end, we developed a bispecific antibody capable of blocking IFN-γ signaling while binding to desmoglein expressed by keratinocytes. We characterized the effect of the bispecific antibody in vitro, ex vivo, and in a mouse model of vitiligo. Single-photon emission computed tomography/computed tomography biodistribution and serum assays after local footpad injection revealed that the bispecific antibody had improved skin retention, faster elimination from the blood, and less systemic IFN-γ inhibition than the nontethered version. Furthermore, the bispecific antibody conferred localized protection almost exclusively to the treated footpad during vitiligo, which was not possible by local injection of the nontethered antiâIFN-γ antibody. Thus, keratinocyte tethering proved effective while significantly diminishing the off-tissue effects of IFN-γ blockade, offering a safer treatment strategy for localized skin diseases, including vitiligo.
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Produtos Biológicos , Vitiligo , Camundongos , Animais , Vitiligo/tratamento farmacológico , Distribuição Tecidual , Queratinócitos/metabolismo , Pele/patologia , Produtos Biológicos/uso terapêuticoRESUMO
OBJECTIVE: The objective of this study is to develop a novel method for monitoring the integrity of motor neurons in vivo by quantifying net retrograde axonal transport. METHODS: The method uses single photon emission computed tomography to quantify retrograde transport to spinal cord of tetanus toxin fragment C (125 I-TTC) following intramuscular injection. We characterized the transport profiles in 3 transgenic mouse models carrying amyotrophic lateral sclerosis (ALS)-associated genes, aging mice, and SOD1G93A transgenic mice following CRISPR/Cas9 gene editing. Lastly, we studied the effect of prior immunization of tetanus toxoid on the transport profile of TTC. RESULTS: This technique defines a quantitative profile of net retrograde axonal transport of TTC in living mice. The profile is distinctly abnormal in transgenic SOD1G93A mice as young as 65 days (presymptomatic) and worsens with disease progression. Moreover, this method detects a distinct therapeutic benefit of gene editing in transgenic SOD1G93A mice well before other clinical parameters (eg, grip strength) show improvement. Symptomatic transgenic PFN1C71G/C71G ALS mice display gross reductions in net retrograde axonal transport, which is also disturbed in asymptomatic mice harboring a human C9ORF72 transgene with an expanded GGGGCC repeat motif. In wild-type mice, net retrograde axonal transport declines with aging. Lastly, prior immunization with tetanus toxoid does not preclude use of this assay. INTERPRETATION: This assay of net retrograde axonal transport has broad potential clinical applications and should be particularly valuable as a physiological biomarker that permits early detection of benefit from potential therapies for motor neuron diseases. ANN NEUROL 2022;91:716-729.
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Esclerose Lateral Amiotrófica , Transporte Axonal , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/genética , Animais , Transporte Axonal/genética , Biomarcadores , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Profilinas , Medula Espinal/diagnóstico por imagem , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Toxoide TetânicoRESUMO
PURPOSE: Previously, we demonstrated specific accumulation into bacteria of a 12-mer phosphorodiamidate morpholino (MORF) oligomer complementary to a ribosomal RNA (rRNA) segment found in all bacteria using the universal probe called Eub338 (Eub). Here, two MORF oligomers Eco and Kpn with sequences specific to the rRNA of Escherichia coli (Eco) and Klebsiella pneumoniae (Kpn) were investigated along with Eub and control (nonEub). PROCEDURES: To determine bacterial rRNA binding, oligomers were tagged with Alexa Fluor 633 (AF633) for fluorescence in situ hybridization (FISH) and fluorescence microscopy, and radiolabeled with technetium-99m (Tc-99m) for biodistribution and SPECT imaging in infected mice. RESULTS: By both FISH and fluorescence microscopy, Eub showed a positive signal in both E. coli and K. pneumoniae as expected, and Kpn showed significantly higher accumulation in K. pneumoniae with near background in E. coli (p < 0.01). Conversely, Eco was positive in both E. coli and K. pneumoniae, hence nonspecific. As determined by biodistribution, the accumulation of [(99m)Tc]Kpn was higher in the thigh infected with live K. pneumoniae than with live E. coli (p = 0.05), and significantly higher than with heat-killed K. pneumoniae (p = 0.02) in the target thigh. By SPECT imaging, the accumulation of [(99m)Tc]Kpn was obviously higher in its specific target of K. pneumoniae compared to an E. coli infected thigh. CONCLUSIONS: Kpn complementary to the rRNA of K. pneumoniae, labeled with Tc-99m or AF633, demonstrated specific binding to fixed and live K. pneumoniae in culture and in infected mice such that Tc-99m-labeled Kpn as the MORF oligomer may be useful for K. pneumoniae infection detection through imaging.
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Infecções por Klebsiella/diagnóstico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/isolamento & purificação , Oligonucleotídeos Antissenso/farmacologia , RNA Ribossômico/metabolismo , Animais , Hibridização in Situ Fluorescente , Infecções por Klebsiella/diagnóstico por imagem , Infecções por Klebsiella/patologia , Camundongos , Microscopia de Fluorescência , Morfolinos/farmacologia , Compostos Radiofarmacêuticos/metabolismo , Tecnécio/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: scVEGF/(177)Lu is a novel radiopharmaceutical targeted by recombinant single-chain (sc) derivative of vascular endothelial growth factor (VEGF) that binds to and is internalized by vascular endothelial growth factor receptors (VEGFR). scVEGF/(177)Lu potential as adjuvant and neoadjuvant anti-angiogenic therapy was assessed in metastatic and orthotopic mouse models of triple-negative breast cancer. METHODS: Metastatic lesions in Balb/c mice were established by intracardiac injection of luciferase-expressing 4T1luc mouse breast carcinoma cells. Mice with metastatic lesions received single intravenous (i.v.) injection of well-tolerated dose of scVEGF/(177)Lu (7.4 MBq/mouse) at day 8 after 4T1luc cell injection. Primary orthotopic breast tumors in immunodeficient mice were established by injecting luciferase-expressing MDA231luc human breast carcinoma cells into mammary fat pad. Tumor-bearing mice were treated with single injections of scVEGF/(177)Lu (7.4 MBq/mouse, i.v), or liposomal doxorubicin (Doxil, 1 mg doxorubicin per kg, i.v.), or with a combination of Doxil and scVEGF/(177)Lu given at the same doses, but two hours apart. "Cold" scVEGF-targeting conjugate was included in controls and in Doxil alone group. The effects of treatments were defined by bioluminescent imaging (BLI), computed tomography (CT), computed microtomography (microCT), measurements of primary tumor growth, and immunohistochemical analysis. RESULTS: In metastatic model, adjuvant treatment with scVEGF/(177)Lu decreased overall metastatic burden and improved survival. In orthotopic primary tumor model, a combination of Doxil and scVEGF/(177)Lu was more efficient in tumor growth inhibition than each treatment alone. scVEGF/(177)Lu treatment decreased immunostaining for VEGFR-1, VEGFR-2, and pro-tumorigenic M2-type macrophage marker CD206. CONCLUSIONS: Selective targeting of VEGFR with well-tolerated doses of scVEGF/(177)Lu is effective in metastatic and primary breast cancer models and can be combined with chemotherapy. As high level of VEGFR expression is a common feature in a variety of cancers, targeted delivery of (177)Lu for specific receptor-mediated uptake warrants further exploration.
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Cell death is involved in many pathological conditions, and there is a need for clinical and preclinical imaging agents that can target and report cell death. One of the best known biomarkers of cell death is exposure of the anionic phospholipid phosphatidylserine (PS) on the surface of dead and dying cells. Synthetic zinc(II)-bis(dipicolylamine) (Zn2BDPA) coordination complexes are known to selectively recognize PS-rich membranes and act as cell death molecular imaging agents. However, there is a need to improve in vivo imaging performance by selectively increasing target affinity and decreasing off-target accumulation. This present study compared the cell death targeting ability of two new deep-red fluorescent probes containing phenoxide-bridged Zn2BDPA complexes. One probe was a bivalent version of the other and associated more strongly with PS-rich liposome membranes. However, the bivalent probe exhibited self-quenching on the membrane surface, so the monovalent version produced brighter micrographs of dead and dying cells in cell culture and also better fluorescence imaging contrast in two living animal models of cell death (rat implanted tumor with necrotic core and mouse thymus atrophy). An (111)In-labeled radiotracer version of the monovalent probe also exhibited selective cell death targeting ability in the mouse thymus atrophy model, with relatively high amounts detected in dead and dying tissue and low off-target accumulation in nonclearance organs. The in vivo biodistribution profile is the most favorable yet reported for a Zn2BDPA complex; thus, the monovalent phenoxide-bridged Zn2BDPA scaffold is a promising candidate for further development as a cell death imaging agent in living subjects.
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Morte Celular , Corantes Fluorescentes/química , Imagem Molecular/métodos , Neoplasias/patologia , Compostos Organometálicos/química , Ácidos Picolínicos/química , Timo/patologia , Animais , Atrofia/diagnóstico , Atrofia/patologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Masculino , Camundongos , Microscopia de Fluorescência/métodos , Neoplasias/diagnóstico , Imagem Óptica/métodos , Fosfatidilserinas/análise , Ratos , Ratos Wistar , Timo/citologiaRESUMO
In this Letter, different from conventional pretargeting, an additional novel DNA polymer with multiple copies of a target was first designed to be administrated between the antitumor antibody, and the labeled effector served as an amplification pretargeting strategy. Two phosphorothioate DNA strands, a bridging and a target strand, were hybridized to form a polymer. Polymer size, as a function of molar ratios, was then monitored by size exclusion HPLC and electrophoretic mobility shift assay. Moreover, binding efficiency of polymers with the radiolabeled effector and polymer size after hybridization were measured by HPLC as well. As the polymer was expected to produce more binding sites that would be targeted by effectors, amplification pretargeting can greatly improve accumulation of effectors in tumor. This novel proof-of-concept was then well demonstrated by the in vitro test of signal amplification in antibody-binding protein L coated plate and LS174T cells. Compared to conventional pretargeting, significantly increasing radioactive signal was observed in this designed amplification pretargeting, which would serve as a useful paradigm of the potential of oligomer polymers to improve pretargeting and other related approaches.
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PURPOSE: This study prepared three structurally related zinc-dipicolylamine (ZnDPA) tracers with [(111)In] labels and conducted biodistribution and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging studies of a mouse leg infection model. PROCEDURES: Two monovalent tracers, ZnDPA-[(111)In]DTPA and ZnDPA-[(111)In]DOTA, each with a single zinc-dipicolylamine targeting unit, and a divalent tracer, Bis(ZnDPA)-[(111)In]DTPA, with two zinc-dipicolylamine units were prepared. Organ biodistribution and SPECT and CT imaging studies were performed on living mice with a leg infection created by injection of clinically relevant Gram positive Streptococcus pyogenes. Fluorescent and luminescent Eu(3+)-labeled versions of these tracers were also prepared and used to measure relative affinity for the exterior membrane surface of bacterial cells and mimics of healthy mammalian cells. RESULTS: All three (111)In-labeled radiotracers were prepared with a radiopurity of >90 %. The biodistribution studies showed that the two monovalent tracers were cleared from the body through the liver and kidney, with retained percentage injected dose for all organs of <8 % at 20 h and infected leg target to non-target ratio (T/NT) ratio of ≤3.0. Clearance of the divalent tracer from the bloodstream was slower and primarily through the liver, with a retained percentage injected dose for all organs <37 % at 20 h and T/NT ratio rising to 6.2 after 20 h. The SPECT/CT imaging indicated the same large difference in tracer pharmacokinetics and higher accumulation of the divalent tracer at the site of infection. CONCLUSIONS: All three [(111)In]-ZnDPA tracers selectively targeted the site of a clinically relevant mouse infection model that could not be discerned by visual external inspection of the living animal. The highest target selectivity, observed with a divalent tracer equipped with two zinc-dipicolylamine targeting units, compares quite favorably with the imaging selectivities previously reported for other nuclear tracers that target bacterial cell surfaces. The tracer pharmacokinetics depended heavily on tracer molecular structure suggesting that it may be possible to rapidly fine tune the structural properties for optimized in vivo imaging performance and clinical translation.
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Infecções Bacterianas/diagnóstico por imagem , Compostos Organometálicos , Ácidos Picolínicos , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Európio , Extremidades/diagnóstico por imagem , Extremidades/microbiologia , Radioisótopos de Índio , Camundongos Nus , Microscopia de Fluorescência , Compostos Organometálicos/sangue , Ácidos Picolínicos/sangue , Streptococcus pyogenes/citologia , Distribuição Tecidual , Tomografia Computadorizada por Raios X , Imagem Corporal TotalRESUMO
Although an increasing number of antibody conjugates are being used in the clinic, there remain many unmet needs in antibody targeting. Normal tissue background is one of the key issues that limits the therapeutic efficacy and the detection sensitivity. Background reduction coupled with dose increase may provide the required target accumulation of the label or toxin at an acceptable normal tissue background. However, the knowledge about the in vivo interaction between antibody and a clearing agent is currently inadequate for designing a rational clearance regimen or system. The current investigation focuses on the clearability of antibody for background reduction, an important topic to antibody targeting in general. The investigation employs pretargeting as a research tool and avidin as a model clearing agent. By comparing the effects of natural clearance at a longer post-injection time and avidin clearance, we demonstrated that avidin clearance is much more effective. By directly attaching avidin to a biotinylated antibody prior to injection, we found that the biotinylated antibody in blood, once bound to the clearing agent, can be removed from the circulation immediately and completely, while the real non-clearable antibody without biotin stays. The study of multiple avidin injections confirmed that the presence of clearable biotinylated antibodies after an avidin injection is due to their temporary inaccessibility and subsequent return from tissue compartments. The collective clearance efficiency of 91% by three avidin injections indicates a continuous IV infusion would be recommended to remove all of the biotinylated IgG molecules. In conclusion, the use of antibody pretargeting as a tool in this study has improved understanding of the incomplete clearance by avidin and can aid in overcoming this obstacle.
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INTRODUCTION: The differences between two agents often need to be accurately defined in vivo. Usually they are injected respectively into two groups of subjects. However, if the two agents do not interact with each other in vivo, a coinjection would serve the same purpose. We believe some individual differences in biodistribution may be circumvented through this approach by calculating organ level ratios. METHODS: A model system of MORF/cMORF pretargeting (MORF/cMORF is a complementary pair of DNA analogues) was employed in connection with an on-going tumor therapeutic project. Human LS174T cells were implanted into the flank of severely immuno-compromised NOD-scid IL2rg(null) mice. The tumor was confirmed to express TAG-72 antigens. At 16 days post tumor inoculation, mice received IV 60 µg of MORF-conjugated CC49 (an antiTAG-72 antibody), followed 2 days later by a low-mass-dose IV coinjection containing 2.5 µg of (90)Y-cMORF and 2.5 µg of (99m)Tc-cMORF. At 3 h post radioactivity injection, the distribution of (99m)Tc was imaged on a SPECT/CT camera and then organs were excised and counted for (90)Y and (99m)Tc. Because the two labeled cMORFs do not react or interact with each other in vivo, the two groups of (90)Y and (99m)Tc data enabled a conventional group comparison. In a new effort, (90)Y/(99m)Tc ratios were calculated. Student's t-test and retrospective power analysis were performed for both approaches. In the new approach, the ratios were set at 1 as the null hypothesis. RESULTS: The Student's t-test in the conventional group approach indicated that the two labeled cMORFs distributed similarly, but significant differences were observed in salivary gland and large intestines. The coinjection-ratio approach certainly did not subvert the results of the conventional approach but revealed subtler differences. The P values were reduced, the powers were increased in most organs, and more significant differences were observed. The increased sensitivity was due to the reduced CV%s (SD/average*100%) of the (90)Y/(99m)Tc ratios. Therefore, some individual differences were circumvented and notably the ratio approach differentiated individual differences into ratio-correctable and ratio-uncorrectable. CONCLUSIONS: Although the conventional approach is reliable, the coinjection-ratio approach using organ level ratios is more sensitive and therefore is recommended whenever possible. In addition, it differentiates individual differences into "coinjection correctable" and "coinjection uncorrectable".
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Morfolinos , Radioisótopos de Ítrio , Animais , Antígenos de Neoplasias/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Injeções , Camundongos , Morfolinos/genética , Morfolinos/farmacocinética , TecnécioRESUMO
PURPOSE: Radiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents. METHODS AND RESULTS: Identical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were (99m)Tc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of (99m)Tc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8pmol, respectively, per 10(10) cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90min for both (99m)Tc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of (99m)Tc-study MORF was significantly higher (p=0.009) than that of the control in the thigh with sterile inflammation. CONCLUSION: A (99m)Tc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.
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Morfolinos/química , Compostos de Organotecnécio/química , RNA Bacteriano/metabolismo , Compostos Radiofarmacêuticos/química , Animais , Infecções Bacterianas/diagnóstico , Escherichia coli/genética , Meia-Vida , Hibridização in Situ Fluorescente , Klebsiella pneumoniae/genética , Camundongos , Microscopia de Fluorescência , Morfolinos/farmacocinética , RNA Ribossômico 16S/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Staphylococcus aureus/genética , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios XRESUMO
Abstract Intraperitoneal (IP) injection is frequently reported to be as effective as intravenous (IV) injection. Because it allows administering a larger volume with more radioactivity, we have investigated this route and the possibility of using it to circumvent the volume constraint we earlier experienced with pretargeting radiotherapy. Using (99m)Tc as the label, the pharmacokinetics (PK) of the cMORF effector (a DNA analogue) was evaluated after IP or IV injection in normal mice by necropsy and SPECT/CT imaging. In another experiment, nude mice bearing tumors were used and they received MORF-CC49 pretargeting antibody IV 2 days earlier than labeled cMORF IV or IP. Tumor accumulations of cMORF were measured at 6 hours after its injections. The absorbed radiation doses for (188)Re or (90)Y pretargeting were estimated using the (99m)Tc data and a self-absorbed model. Although the absorbed radiation doses to other organs were comparable, the dose to intestines after IP injection was 30-fold higher than IV injection due to the slow entry into the circulation. It had reached such a level as high as the dose to the kidneys that cleared the radioactivity and usually were at the highest level. Nevertheless, the slow entry did not reduce the tumor accumulation. In conclusion, using IP in place of IV led to an unacceptably high absorbed radiation dose to the intestines although the tumor accumulation was not compromised. This effect may be applicable to other radiotherapeutic agents as well.
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Morfolinos/administração & dosagem , Morfolinos/farmacocinética , Neoplasias/metabolismo , Neoplasias/radioterapia , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/farmacocinética , Dosagem Radioterapêutica , Tecnécio/administração & dosagem , Tecnécio/farmacocinética , Adsorção , Animais , Anticorpos Antinucleares , Área Sob a Curva , Disponibilidade Biológica , Injeções Intraperitoneais , Injeções Intravenosas , Mucosa Intestinal/metabolismo , Rim/metabolismo , Camundongos , Camundongos Nus , Morfolinos/imunologia , Doses de Radiação , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios XRESUMO
PURPOSE: Invasive aspergillosis is a major cause of infectious morbidity and mortality in immunocompromised patients. The fungus Aspergillus fumigatus (A. fumigatus) is the primary causative agent of invasive aspergillosis. However, A. fumigatus infections remain difficult to diagnose particularly in the early stages due to the lack of a rapid, sensitive and specific diagnostic approach. In this study, we investigated (99m)Tc labeled MORF oligomers targeting fungal ribosomal RNA (rRNA) for the imaging detection of fungal infections. PROCEDURES: Three phosphorodiamidate morpholino (MORF) oligomer (a DNA analogue) probes were designed: AGEN, complementary to a sequence of the fungal 28S ribosomal RNA (rRNA) of Aspergillus, as a genus-specific probe; AFUM, complementary to the 28S rRNA sequence of A. fumigatus, as a fungus species-specific probe; and cMORF, irrelevant to all fungal species, as a control probe. The probes were conjugated with Alexa Fluor 633 carboxylic acid succinimidyl ester (AF633) for fluorescence imaging or with NHS-mercaptoacetyl triglycine (NHS-MAG3) for nuclear imaging with (99m)Tc and then evaluated in vitro and in vivo. RESULTS: The specific binding of AGEN and AFUM to fungal total RNA was confirmed by dot blot hybridization while specific binding of AGEN and AFUM in fixed and live A. fumigatus was demonstrated by both fluorescent in situ hybridization (FISH) analysis and accumulation in live cells. SPECT imaging of BALB/c mice with pulmonary A. fumigatus infections and administered (99m)Tc labeled AGEN and AFUM showed immediate and obvious accumulation in the infected lungs, while no significant accumulation of the control (99m)Tc-cMORF in the infected lung was observed. Compared to non-infected mice, with sacrifice at 1h, the accumulation of (99m)Tc-AGEN and (99m)Tc-AFUM in the lungs of mice infected with A. fumigatus was 2 and 2.7 fold higher respectively. CONCLUSIONS: In vivo targeting fungal ribosomal RNA with (99m)Tc labeled MORF probes AGEN and AFUM may be useful for A. fumigatus infection imaging and may provide a new strategy for the noninvasive diagnosis of invasive aspergillosis and other fungal infections.
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Aspergillus fumigatus/fisiologia , Pneumopatias Fúngicas/diagnóstico por imagem , Morfolinos/metabolismo , Compostos de Organotecnécio , RNA Fúngico/metabolismo , RNA Ribossômico 28S/metabolismo , Animais , Aspergillus fumigatus/genética , Sequência de Bases , Pulmão/diagnóstico por imagem , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Morfolinos/genética , Morfolinos/farmacocinética , Imagem Multimodal , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Sondas de Oligonucleotídeos/farmacocinética , Tomografia por Emissão de Pósitrons , Especificidade da Espécie , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologia , Tomografia Computadorizada por Raios XRESUMO
A three-component nanoparticle consisting of biotinylated Trastuzumab antiHer2 antibody, tat transferring peptide and radiolabeled antisense oligomer, linked together through streptavidin, have shown promise in the delivery to Her2+ tumor in mice following intravenous administration and with evidence of radiotherapeutic efficacy. These results have encouraged us to consider the nanoparticle as a delivery vehicle for RNA interference therapy in which the radiolabeled antisense oligomer is replaced with an unlabeled siRNA duplex. The siRNA stability within the nanoparticle was first confirmed by incubation with RNase A. The interferon responses, that indicate off-target cytotoxicity, were evaluated by quantitative real-time RT-PCR in BT-474 (Her2+) human breast cancer cells by measuring the mRNA expression of 2', 5'-oligoadenylate synthetase (OAS1) and Stat-1, two key interferon-responsive genes. Thereafter the cytotoxicity induced by the siRNA nanoparticle was evaluated by a clonogenic survival assay in BT-474 cells while the Her2 expression of these target cells was evaluated for evidence of specific gene silencing. The siRNA within the three-component anti- Her2/neu siRNA nanoparticle was largely protected from RNase-dependent degradation and did not activate an interferon response. The nanoparticle effectively and significantly inhibited colony formation of the target cells and silenced the Her2 gene expression at 5 nM compared with the identical nanoparticle with a scrambled siRNA. Our delivery nanoparticle, with tumor targeting provided by the antibody and its accumulation without entrapment, possibly due to the transfecting peptide, delivered an siRNA duplex to the proper subcellular localization for specific and effective gene silencing in culture by what appears to be an siRNA mechanism.
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Genes erbB-2 , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Estreptavidina/administração & dosagem , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Biotinilação/métodos , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Interferons/genética , Interferons/metabolismo , RNA Mensageiro/genética , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transfecção/métodos , TrastuzumabRESUMO
Molecules associated with the transforming growth factor ß (TGF-ß) superfamily, such as bone morphogenic proteins (BMPs) and TGF-ß, are key regulators of inflammation, apoptosis and cellular transitions. Here we show that the BMP receptor activin-like kinase 3 (Alk3) is elevated early in diseased kidneys after injury. We also found that its deletion in the tubular epithelium leads to enhanced TGF-ß1-Smad family member 3 (Smad3) signaling, epithelial damage and fibrosis, suggesting a protective role for Alk3-mediated signaling in the kidney. A structure-function analysis of the BMP-Alk3-BMP receptor, type 2 (BMPR2) ligand-receptor complex, along with synthetic organic chemistry, led us to construct a library of small peptide agonists of BMP signaling that function through the Alk3 receptor. One such peptide agonist, THR-123, suppressed inflammation, apoptosis and the epithelial-to-mesenchymal transition program and reversed established fibrosis in five mouse models of acute and chronic renal injury. THR-123 acts specifically through Alk3 signaling, as mice with a targeted deletion for Alk3 in their tubular epithelium did not respond to therapy with THR-123. Combining THR-123 and the angiotensin-converting enzyme inhibitor captopril had an additive therapeutic benefit in controlling renal fibrosis. Our studies show that BMP signaling agonists constitute a new line of therapeutic agents with potential utility in the clinic to induce regeneration, repair and reverse established fibrosis.
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Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/agonistas , Rim/lesões , Rim/metabolismo , Peptídeos/metabolismo , Regeneração/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Apoptose/genética , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Captopril/farmacologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Transição Epitelial-Mesenquimal , Fibrose/metabolismo , Inflamação/genética , Inflamação/metabolismo , Túbulos Renais/metabolismo , Camundongos , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/farmacocinética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteína Smad3/genética , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/genéticaRESUMO
INTRODUCTION: A zinc-dipicolylamine analog (Zn-DPA) conjugated with a fluorophore (PSVue®794) has been shown to image bacterial infections in mice. However, radiolabeled Zn-DPA has not previously been considered for nuclear imaging of infection. METHODS: Both 111In-labeled DOTA-biotin and Zn-DPA-biotin were combined using streptavidin (SA) as a noncovalent linker. Mice injected intramuscularly with Streptococcus pyogenes (infection model) or with lipopolysaccharide (LPS) (inflammation model) were coinjected intravenously with 6 µg of DPA as PSVue794 and as 111In-DOTA-biotin/SA/biotin-Zn-DPA. Periodic fluorescent and SPECT (single photon emission computed tomography)/CT (computed tomography) images were acquired, and biodistributions were obtained at 22 h. RESULTS: Histological examination confirmed the validity of both the infection and inflammation animal models. Both the whole-body optical and nuclear images showed obvious accumulations in the target thigh in both models at all time points. At 22 h, the average target thigh accumulation of 111In was 1.66%ID/g (S.D. 0.15) in the infection mice compared to 0.58%ID/g (S.D. 0.07) in the inflammation mice (P<.01), and the 111In target/normal thigh ratio was 2.8 fold higher in the infection animals compared to the inflammation animals. CONCLUSIONS: These preliminary results show that Zn-DPA within streptavidin targets S. pyogenes-infected mice similarly to its free fluorescent analogue. The significantly higher accumulation in the live bacterial infection thigh compared to that of the LPS-induced inflammation thigh suggests that Zn-DPA may be a promising imaging agent to distinguish between bacterial infections and sterile inflammations.
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Compostos Organometálicos , Ácidos Picolínicos , Infecções Estreptocócicas/diagnóstico por imagem , Streptococcus pyogenes/patogenicidade , Animais , Biotinilação , Quelantes/química , Corantes Fluorescentes/química , Compostos Heterocíclicos com 1 Anel/química , Inflamação/induzido quimicamente , Inflamação/diagnóstico por imagem , Inflamação/metabolismo , Inflamação/patologia , Marcação por Isótopo , Lipopolissacarídeos/efeitos adversos , Camundongos , Imagem Multimodal , Nanopartículas/química , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacocinética , Ácidos Picolínicos/química , Ácidos Picolínicos/metabolismo , Ácidos Picolínicos/farmacocinética , Tomografia por Emissão de Pósitrons , Estreptavidina/metabolismo , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/patologia , Tomografia Computadorizada por Raios XRESUMO
INTRODUCTION: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting. METHODS: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111In-labeled cMORF to direct targeting by 111In-labeled HPi1. RESULTS: HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting. CONCLUSIONS: Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ.
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Anticorpos/imunologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Morfolinos/metabolismo , Pele , Animais , Linhagem Celular , Humanos , Radioisótopos de Índio , Células Secretoras de Insulina/transplante , Marcação por Isótopo , Camundongos , Morfolinos/farmacocinéticaRESUMO
INTRODUCTION: Auger radiotherapy requires adequate tumor delivery and high nuclear accumulation and retention. We hypothesize that the noncovalent nature of a streptavidin/biotin three-component nanoparticle possessing these qualities may be required for dissociation of the radiolabeled oligomer and its accumulation into the cell nucleus. METHODS: As a test of our hypothesis, the intracellular fate of an antisense oligomer when incubated as the nanoparticle and when incubated while covalently conjugated to the antibody was compared. The three-component noncovalent nanoparticle consisted of streptavidin linking three biotinylated components: a Cy3-labeled anti-RIα antisense phosphorodiamidate morpholino (MORF) oligomer, a tat transfecting peptide and the anti-Her2 herceptin antibody. The covalent constructs included an anti-RIα antisense DNA conjugated to a radiolabeled herceptin and a fluorescent DNA conjugated to native herceptin. Fluorescence microscopy in SK-BR-3 (Her2+) cells was used to evaluate the fate of the fluorescent Cy5.5-DNA and Cy3-MORF, while the subcellular accumulation of the (111)In-labeled herceptin and herceptin-DNA in both SK-BR-3 and MDA-MB-231 (Her2) cells was determined by isolating and counting the nuclear fractions. RESULTS: Previously, we demonstrated that when incubated as the three-component nanoparticle consisting of herceptin and streptavidin and (99m)Tc-labeled antisense MORF, only the MORF accumulated in the nucleus of Her2+ cells. In this investigation, clear evidence was observed of nuclear accumulation of the antisense oligomer within the noncovalent nanoparticle as before, but when incubated as the covalent construct, by both fluorescence microscopy and nuclear counting, no evidence of nuclear accumulation was observed. CONCLUSION: The weaker noncovalent biotin-streptavidin bond may be essential for adequate delivery of the radiolabeled antisense oligomer to the nucleus of tumor cells.