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PURPOSE: The need to detect and quantify brain lactate accurately by MRS has stimulated the development of editing sequences based on J coupling effects. In J-difference editing of lactate, threonine can be co-edited and it contaminates lactate estimates due to the spectral proximity of the coupling partners of their methyl protons. We therefore implemented narrow-band editing 180° pulses (E180) in MEGA-PRESS acquisitions to resolve separately the 1.3-ppm resonances of lactate and threonine. METHODS: Two 45.3-ms rectangular E180 pulses, which had negligible effects 0.15-ppm away from the carrier frequency, were implemented in a MEGA-PRESS sequence with TE 139 ms. Three acquisitions were designed to selectively edit lactate and threonine, in which the E180 pulses were tuned to 4.1 ppm, 4.25 ppm, and a frequency far off resonance. Editing performance was validated with numerical analyses and acquisitions from phantoms. The narrow-band E180 MEGA and another MEGA-PRESS sequence with broad-band E180 pulses were evaluated in six healthy subjects. RESULTS: The 45.3-ms E180 MEGA offered a difference-edited lactate signal with lower intensity and reduced contamination from threonine compared to the broad-band E180 MEGA. The 45.3 ms E180 pulse had MEGA editing effects over a frequency range larger than seen in the singlet-resonance inversion profile. Lactate and threonine in healthy brain were both estimated to be 0.4 ± 0.1 mM, with reference to N-acetylaspartate at 12 mM. CONCLUSION: Narrow-band E180 MEGA editing minimizes threonine contamination of lactate spectra and may improve the ability to detect modest changes in lactate levels.
Assuntos
Encéfalo , Ácido Láctico , Humanos , Ácido Láctico/análise , Espectroscopia de Ressonância Magnética , Encéfalo/diagnóstico por imagem , Imagens de Fantasmas , TreoninaRESUMO
Promethazine, an antihistamine drug used in the clinical treatment of nausea, has been demonstrated the ability to bind Abeta in a transgenic mouse model of Alzheimer's disease. However, so far, all of the studies were performed in vitro using extracted tissues. In this work, we report the design and synthesis of a novel [11C]promethazine PET radioligand for future in vivo studies. The [11C]promethazine was isolated by RP-HPLC with radiochemical purity >95% and molar activity of 48 TBq/mmol. The specificity of the probe was demonstrated using human hippocampal tissues via autoradiography.
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Doença de Alzheimer/diagnóstico , Encéfalo/diagnóstico por imagem , Prometazina/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Animais , Autorradiografia , Encéfalo/efeitos dos fármacos , Humanos , Camundongos , Placa Amiloide/diagnóstico , Placa Amiloide/diagnóstico por imagem , Placa Amiloide/patologia , Tomografia por Emissão de Pósitrons , Prometazina/síntese química , Prometazina/química , Radioquímica , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/químicaRESUMO
We present an integrated delivery technology herein employing the aerosolized method to repurpose thioflavin S for imaging amyloid beta (Abeta) deposits in the retina as a surrogate of Abeta in the brain for early detection of Alzheimer's disease. The data showed that wild type (WT) mice also have Abeta deposits in the retinae, albeit much less than 5XFAD mice. Further, only in 5XFAD mice, significant Abeta deposits were found associated with retinal ganglion cells (RGCs) in whole-mount and cross-section data. Furthermore, the fluorescent signal depicted from thioflavin S corroborates with Abeta immunohistochemistry staining information. Overall, this probe delivery via inhalation method is also applicable to other Abeta-binding molecules, such as Congo red, curcumin, and thioflavin T. The advantage of imaging retinal amyloid deposits compared to the brain counterparts is that the eye is easily accessible by in vivo imaging and it reduces the effort to design a probe that must cross the formidable blood-brain barrier.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Benzotiazóis/metabolismo , Inalação , Retina/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Camundongos , Imagem MolecularRESUMO
The NMR hyperpolarization of uniformly 15 N-labeled [15 N3 ]metronidazole is demonstrated by using SABRE-SHEATH. In this antibiotic, the 15 NO2 group is hyperpolarized through spin relays created by 15 N spins in [15 N3 ]metronidazole, and the polarization is transferred from parahydrogen-derived hydrides over six chemical bonds. In less than a minute of parahydrogen bubbling at approximately 0.4â µT, a high level of nuclear spin polarization (P15N ) of around 16 % is achieved on all three 15 N sites. This product of 15 N polarization and concentration of 15 N spins is around six-fold better than any previous value determined for 15 N SABRE-derived hyperpolarization. At 1.4â T, the hyperpolarized state persists for tens of minutes (relaxation time, T1 ≈10â min). A novel synthesis of uniformly 15 N-enriched metronidazole is reported with a yield of 15 %. This approach can potentially be used for synthesis of a wide variety of in vivo metabolic probes with potential uses ranging from hypoxia sensing to theranostic imaging.
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In vivo evaluation of [18F]BMS-754807 binding in mice and rats using microPET and biodistribution methods is described herein. The radioligand shows consistent binding characteristics, in vivo, in both species. Early time frames of the microPET images and time activity curves of brain indicate poor penetration of the tracer across the blood brain barrier (BBB) in both species. However, microPET experiments in mice and rats show high binding of the radioligand outside the brain to heart, pancreas and muscle, the organs known for higher expression of IGF1R/1R. Biodistribution analysis 2h after injection of [18F]BMS-754807 in rats show negligible [18F]defluorination as reflected by the low bone uptake and clearance from blood. Overall, the data indicate that [18F]BMS-754807 can potentially be a radiotracer for the quantification of IGF1R/IR outside the brain using PET.
Assuntos
Tomografia por Emissão de Pósitrons/métodos , Pirazóis/farmacocinética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Triazinas/farmacocinética , Animais , Radioisótopos de Flúor/metabolismo , Xenoenxertos , Humanos , Camundongos , Ensaio Radioligante , RatosRESUMO
An open-source hyperpolarizer producing (13)C hyperpolarized contrast agents using parahydrogen induced polarization (PHIP) for biomedical and other applications is presented. This PHIP hyperpolarizer utilizes an Arduino microcontroller in conjunction with a readily modified graphical user interface written in the open-source processing software environment to completely control the PHIP hyperpolarization process including remotely triggering an NMR spectrometer for efficient production of payloads of hyperpolarized contrast agent and in situ quality assurance of the produced hyperpolarization. Key advantages of this hyperpolarizer include: (i) use of open-source software and hardware seamlessly allowing for replication and further improvement as well as readily customizable integration with other NMR spectrometers or MRI scanners (i.e., this is a multiplatform design), (ii) relatively low cost and robustness, and (iii) in situ detection capability and complete automation. The device performance is demonstrated by production of a dose (â¼2-3 mL) of hyperpolarized (13)C-succinate with %P13C â¼ 28% and 30 mM concentration and (13)C-phospholactate at %P13C â¼ 15% and 25 mM concentration in aqueous medium. These contrast agents are used for ultrafast molecular imaging and spectroscopy at 4.7 and 0.0475 T. In particular, the conversion of hyperpolarized (13)C-phospholactate to (13)C-lactate in vivo is used here to demonstrate the feasibility of ultrafast multislice (13)C MRI after tail vein injection of hyperpolarized (13)C-phospholactate in mice.
Assuntos
Meios de Contraste/metabolismo , Hidrogênio/química , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Animais , Automação , Isótopos de Carbono/química , Catálise , Meios de Contraste/química , Ácido Láctico/metabolismo , Camundongos , Camundongos Nus , Software , Succinatos/química , Succinatos/metabolismo , Água/químicaRESUMO
Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level.
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Adenocarcinoma/diagnóstico , Antígenos Glicosídicos Associados a Tumores/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/diagnóstico , Diagnóstico por Imagem/métodos , Adenocarcinoma/metabolismo , Animais , Colonoscopia , Neoplasias Colorretais/metabolismo , Detecção Precoce de Câncer , Feminino , Fluorescência , Corantes Fluorescentes , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Nanosferas , Ratos , Ratos Nus , Células Tumorais CultivadasRESUMO
We are investigating an imaging agent that detects early-stage primary colorectal cancer on the mucosal surface in real time under colonoscopic observation. The imaging agent, which is named the nanobeacon, is fluorescent nanospheres conjugated with peanut agglutinin and poly(N-vinylacetamide). Its potential use as an imaging tool for colorectal cancer has been thoroughly validated in numerous studies. Here, toxicities of the nanobeacon were assessed in rats. The nanobeacon was prepared according to the synthetic manner which is being established as the Good Manufacturing Practice-guided production. The rat study was performed in accordance with Good Laboratory Practice regulations. No nanobeacon treatment-related toxicity was observed. The no observable adverse effect levels (NOAEL) of the nanobeacon in 7-day consecutive oral administration and single intrarectal administration were estimated to be more than 1000mg/kg/day and 50mg/kg/day, respectively. We concluded that the nanobeacon could be developed as a safe diagnostic agent for colonoscopy applications. FROM THE CLINICAL EDITOR: Colon cancer remains a major cause of death. Early detection can result in early treatment and thus survival. In this article, the authors tested potential systemic toxicity of coumarin 6-encapsulated polystyrene nanospheres conjugated with peanut agglutinin (PNA) and poly(N-vinylacetamide) (PNVA), which had been shown to bind specifically to colonic cancer cells and thus very promising in colonoscopic detection of cancer cells.
Assuntos
Acetamidas/toxicidade , Colonoscopia , Cumarínicos/toxicidade , Corantes Fluorescentes/toxicidade , Nanosferas/toxicidade , Aglutinina de Amendoim/toxicidade , Poliestirenos/toxicidade , Polivinil/toxicidade , Tiazóis/toxicidade , Acetamidas/administração & dosagem , Acetamidas/química , Animais , Peso Corporal/efeitos dos fármacos , Células CHO , Células CACO-2 , Colo/efeitos dos fármacos , Colo/patologia , Neoplasias Colorretais/diagnóstico , Cumarínicos/administração & dosagem , Cumarínicos/química , Cricetulus , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/química , Humanos , Masculino , Nanosferas/administração & dosagem , Nanosferas/química , Aglutinina de Amendoim/administração & dosagem , Aglutinina de Amendoim/química , Poliestirenos/administração & dosagem , Poliestirenos/química , Polivinil/administração & dosagem , Polivinil/química , Ratos , Reto/efeitos dos fármacos , Reto/patologia , Tiazóis/administração & dosagem , Tiazóis/químicaRESUMO
With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Inativação Gênica/fisiologia , Imageamento por Ressonância Magnética , RNA Interferente Pequeno/fisiologia , Espectroscopia de Luz Próxima ao Infravermelho , Adenocarcinoma/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Humanos , Camundongos , Sondas MolecularesRESUMO
Here, we present a new approach for the delivery of a metabolic contrast agent for in vivo molecular imaging. The use of a phosphate-protecting group that facilitates parahydrogen-induced polarization of 1-(13)C-phospholactate potentially enables the in vivo administration of a hydrogenated hyperpolarized adduct. When injected, nonhyperpolarized 1-(13)C-phospholactate is retained in the vasculature during its metabolic conversion to 1-(13)C-lactate by blood phosphatases as demonstrated here using a mucin 1 mouse model of breast cancer and ex vivo high-resolution (13)C NMR. This multisecond process is a suitable mechanism for the delivery of relatively short-lived (13)C and potentially (15)N hyperpolarized contrast agents using -OH phosphorylated small molecules, which is demonstrated here for the first time as an example of 1-(13)C-phospholactate. Through this approach, DL-1-(13)C-lactate is taken up by tissues and organs including the liver, kidneys, brain, heart, and tumors according to a timescale amenable to hyperpolarized magnetic resonance imaging.
Assuntos
Meios de Contraste/farmacocinética , Lactatos/farmacocinética , Ácido Láctico/análogos & derivados , Compostos Radiofarmacêuticos/farmacocinética , Animais , Isótopos de Carbono/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Tumoral , Meios de Contraste/síntese química , Feminino , Lactatos/síntese química , Neoplasias Mamárias Experimentais/diagnóstico , Camundongos , Fosforilação , Compostos Radiofarmacêuticos/síntese química , Distribuição TecidualRESUMO
Several new lines of research have demonstrated that a significant number of amyloid-ß peptides found in Alzheimer's disease (AD) are truncated and undergo post-translational modification by glutaminyl cyclase (QC) at the N-terminal. Notably, QC's products of Abeta-pE3 and Abeta-pE11 have been active targets for investigational drug development. This work describes the design, synthesis, characterization, and in vivo validation of a novel PET radioligand, [18F]PB0822, for targeted imaging of QC. We report herein a simplified and robust chemistry for the synthesis of the standard compound, [19F]PB0822, and the corresponding [18F]PB0822 radioligand. The PET probe was developed with 99.9% radiochemical purity, a molar activity of 965 Ci.mmol-1, and an IC50 of 56.3 nM, comparable to those of the parent PQ912 inhibitor (62.5 nM). Noninvasive PET imaging showed that the probe is distributed in the brain 5 min after intravenous injection. Further, in vivo PET imaging with [18F]PB0822 revealed that AD 5XFAD mice harbor significantly higher QC activity than WT counterparts. The data also suggested that QC activity is found across different brain regions of the tested animals.
Assuntos
Doença de Alzheimer , Aminoaciltransferases , Tomografia por Emissão de Pósitrons , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Aminoaciltransferases/metabolismo , Aminoaciltransferases/antagonistas & inibidores , Animais , Camundongos , Radioisótopos de Flúor/química , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/enzimologia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/síntese química , Biomarcadores/metabolismo , Humanos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/análise , LigantesRESUMO
The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.
RESUMO
The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Placa Amiloide , Anticorpos de Domínio Único , Animais , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Camundongos , Placa Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Humanos , Encéfalo/metabolismo , Encéfalo/patologia , Barreira Hematoencefálica/metabolismo , Camundongos Transgênicos , Camelídeos Americanos , Modelos Animais de DoençasRESUMO
This research aimed to validate the specificity of the newly developed nanobeacon for imaging the Thomsen-Friedenreich (TF) antigen, a potential biomarker of colorectal cancer. The imaging agent is comprised of a submicron-sized polystyrene nanosphere encapsulated with a Coumarin 6 dye. The surface of the nanosphere was modified with peanut agglutinin (PNA) and poly(N-vinylacetamide (PNVA) moieties. The former binds to Gal-ß(1-3)GalNAc with high affinity while the latter enhances the specificity of PNA for the carbohydrates. The specificity of the nanobeacon was evaluated in human colorectal cancer cells and specimens, and the data were compared with immunohistochemical staining and flow cytometric analysis. Additionally, distribution of the nanobeacon in vivo was assessed using an "intestinal loop" mouse model. Quantitative analysis of the data indicated that approximately 2 µg of PNA were detected for each milligram of the nanobeacon. The nanobeacon specifically reported colorectal tumors by recognizing the tumor-specific antigen through the surface-immobilized PNA. Removal of TF from human colorectal cancer cells and tissues resulted in a loss of fluorescence signal, which suggests the specificity of the probe. Most importantly, the probe was not absorbed systematically in the large intestine upon topical application. As a result, no registered toxicity was associated with the probe. These data demonstrate the potential use of this novel nanobeacon for imaging the TF antigen as a biomarker for the early detection and prediction of the progression of colorectal cancer at the molecular level.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Neoplasias Colorretais/diagnóstico , Cumarínicos , Diagnóstico por Imagem/métodos , Nanosferas , Aglutinina de Amendoim , Tiazóis , Animais , Antígenos Glicosídicos Associados a Tumores/genética , Western Blotting , Estudos de Casos e Controles , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Cumarínicos/farmacocinética , Corantes Fluorescentes , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Transgênicos , Aglutinina de Amendoim/farmacocinética , Poliestirenos/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reto/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Propriedades de Superfície , Tiazóis/farmacocinética , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Apoptosis is irreversible programmed cell death, characterized by a cellular cascade activation of caspase 3, which subsequently degrades proteins and other components of cells with a motif sequence. Here we report a novel reporter system to detect apoptosis, growth arrest, and cell death based on controlled and self-amplified protein degradation. The key element of the reporter system is an apoptotic sensor chimerical protein which consists of three components: procaspase 3, ubiquitin (Ub), and a strong consensus sequence of N-degron. Between each of these units is a DEVD (Asp-Glu-Val-Asp) sequence, which acts as the cleavage target of caspase 3. This non-conventional signal loss approach is much more sensitive than other native methods that are based on signal gain. The superior sensitivity is demonstrated by its effective application in 386-well high-throughput screening (HTS) with low drug concentrations and a short incubation time. The HTS selection process using this reporter system is very simple and economic. The simplicity eliminates potential errors introduced by multiple steps; there is no need for any substrate. Furthermore, the cells in the assay need not be disrupted, and the morphology of the cells can provide additional information on mechanisms. After HTS, the intact cells can also be used for other analytic analysis. This system thus has a potentially important role in the discovery and development of new anticancer drugs. It also appears to be very versatile, can be used both in vitro and in vivo with different linked reporter genes, and can be used for a variety of imaging applications.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Genes Reporter/efeitos dos fármacos , Apoptose , Bioensaio , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Microscopia Confocal , Proteínas Recombinantes/genética , Reprodutibilidade dos TestesRESUMO
Polylysine (PL) has been used to facilitate dendritic cell (DC) uptake of super paramagnetic iron oxide (SPIO) nanoparticles for use in magnetic resonance imaging (MRI). In this work, we examined the effect of PL on cell toxicity and induction of cell maturation as manifested by the up-regulation of surface molecules. We found that PL became toxic to bone marrow-derived DCs (BMDCs) at the 10 µg/ml threshold. Incubation of BMDCs with 20 µg/ml of PL for 1h resulted in approximately 90% cell death. However, addition of SPIO nanoparticles rescued DCs from PL-induced death as the combination of SPIO with PL did not cause cytotoxicity until the PL concentration was 1000 µg/ml. Prolonged exposure to PL induced BMDC maturation as noted by the expression of surface molecules such as MHC class II, CD40, CCR7 and CD86. However, the combination of SPIO and PL did not induce BMDC maturation at 1h. However prolonged exposure to SPIO nanoparticles induced CD40 expression and protein expression of TNFα and KC. The data suggest that the use of PL to enhance the labeling of DCs with SPIO nanoparticles is a dedicated work. Appropriate calibration of the incubation time and concentrations of PL and SPIO nanoparticles is crucial to the development of MRI technology for noninvasive imaging of DCs in vivo. FROM THE CLINICAL EDITOR: The authors of this study present detailed data on toxicity and efficiency of polylysine-facilitated uptake of USPIO-s by dendritic cells for cell-specific MR imaging.
Assuntos
Meios de Contraste/administração & dosagem , Células Dendríticas/citologia , Portadores de Fármacos/metabolismo , Compostos Férricos/administração & dosagem , Nanopartículas de Magnetita/administração & dosagem , Polilisina/metabolismo , Animais , Células da Medula Óssea/citologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/análise , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/ultraestrutura , Portadores de Fármacos/toxicidade , Humanos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Polilisina/toxicidadeRESUMO
Extracellular circulating microRNAs (miRNAs) have been discussed as potential biomarkers for Alzheimer's disease (AD) diagnosis. As the retina is a part of the CNS, we hypothesize that miRNAs expression levels in the brain, particularly neocortex-hippocampus, eye tissues, and tear fluids are similar at different stages of AD progression. Ten miRNA candidates were systematically investigated in transgenic APP-PS1 mice, noncarrier siblings, and C57BL/6J wild-type controls at young and old ages. Relative expression levels of tested miRNAs revealed a similar pattern in both APP-PS1 mice and noncarrier siblings when compared with age- and sex-matched wild-type controls. However, the differences seen in expression levels between APP-PS1 mice and noncarrier siblings could possibly have resulted from underlying molecular etiology of AD. Importantly, miRNAs associated with amyloid beta (Aß) production (-101a, -15a, and -342) and proinflammation (-125b, -146a, and -34a) showed significant up-regulations in the tear fluids with disease progression, as tracked by cortical Aß load and reactive astrogliosis. Overall, for the first time, the translational potential of up-regulated tear fluid miRNAs associated with AD pathogenesis was comprehensively demonstrated.
Assuntos
Doença de Alzheimer , MicroRNAs , Camundongos , Animais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , MicroRNAs/genética , Precursor de Proteína beta-Amiloide/genética , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Introduction: Ergothioneine (Ergo) is a naturally occurring dietary antioxidant. Ergo uptake is dependent on the transporter, organic cation transporter novel-type 1 (OCTN1) distribution. OCTN1 is highly expressed in blood cells (myeloid lineage cells), brain and ocular tissues that are likely predisposed to oxidative stress. Ergo may protect the brain and eye against oxidative damage and inflammation, however, the underlying mechanism remains unclear. Amyloid beta (Aß) clearance is a complex process mediated by various systems and cell types including vascular transport across the blood-brain barrier, glymphatic drainage, and engulfment and degradation by resident microglia and infiltrating innate immune cells. Impaired Aß clearance is a major cause for Alzheimer's disease (AD). Here we investigated neuroretinas to explore the neuroprotective effect of Ergo in a transgenic AD mouse model. Methods: Age-matched groups of Ergo-treated 5XFAD, non-treated 5XFAD, and C57BL/6J wildtype (WT controls) were used to assess Ergo transporter OCTN1 expression and Aß load along with microglia/macrophage (IBA1) and astrocyte (GFAP) markers in wholemount neuroretinas (n = 26) and eye cross-sections (n = 18). Immunoreactivity was quantified by fluorescence or by semi-quantitative assessments. Results and discussion: OCTN1 immunoreactivity was significantly low in the eye cross-sections of Ergo-treated and non-treated 5XFAD vs. WT controls. Strong Aß labeling, detected in the superficial layers in the wholemounts of Ergo-treated 5XFAD vs. non-treated 5XFAD reflects the existence of an effective Aß clearance system. This was supported by imaging of cross-sections where Aß immunoreactivity was significantly low in the neuroretina of Ergo-treated 5XFAD vs. non-treated 5XFAD. Moreover, semi-quantitative analysis in wholemounts identified a significantly reduced number of large Aß deposits or plaques, and a significantly increased number of IBA1(+)ve blood-derived phagocytic macrophages in Ergo-treated 5XFAD vs. non-treated 5XFAD. In sum, enhanced Aß clearance in Ergo-treated 5XFAD suggests that Ergo uptake may promote Aß clearance possibly by blood-derived phagocytic macrophages and via perivascular drainage.
RESUMO
L-ergothioneine (ERGO) is a potent antioxidant with cytoprotective effects. To study ERGO biodistribution and detect oxidative stress in vivo, we report an efficient and reproducible preparation of [11 C]-labeled ERGO PET radioligand based on protecting the histidine carboxylic group with a methyl ester. Overall, this new protection approach using methyl ester improved the chemical yield of a 4-step reaction from 14% to 24% compared to the previous report using t-butyl ester. The [11 C]CH3 methylation of the precursor provided the desired product with 55 ± 10% radiochemical purity and a molar activity of 450 ± 200 TBq·mmol-1 . The [11 C]ERGO radioligand was able to detect threshold levels of oxidative stress in a preclinical animal model of Alzheimer's disease.
Assuntos
Doença de Alzheimer , Ergotioneína , Doença de Alzheimer/diagnóstico por imagem , Animais , Ésteres , Estresse Oxidativo , Tomografia por Emissão de Pósitrons/métodos , Distribuição TecidualRESUMO
Amyloid beta (Aß) deposits in the retina of the Alzheimer's disease (AD) eye may provide a useful diagnostic biomarker for AD. This study focused on the relationship of Aß with macroglia and microglia, as these glial cells are hypothesized to play important roles in homeostasis and clearance of Aß in the AD retina. Significantly higher Aß load was found in AD compared to controls, and specifically in the mid-peripheral region. AD retina showed significantly less immunoreactivity against glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) compared to control eyes. Immunoreactivity against ionized calcium binding adapter molecule-1 (IBA-1), a microglial marker, demonstrated a higher level of microgliosis in AD compared to control retina. Within AD retina, more IBA-1 immunoreactivity was present in the mid-peripheral retina, which contained more Aß than the central AD retina. GFAP co-localized rarely with Aß, while IBA-1 co-localized with Aß in more layers of control than AD donor retina. These results suggest that dysfunction of the Müller and microglial cells may be key features of the AD retina.