RESUMO
Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a abundant extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase) from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of neuronal Ndst1- deficient mice, neuronal, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance.
Assuntos
Neurônios , Doenças Priônicas , Príons , Sulfotransferases , Animais , Camundongos , Heparitina Sulfato/metabolismo , Camundongos Knockout , Neurônios/enzimologia , Doenças Priônicas/metabolismo , Proteínas Priônicas/genética , Príons/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
γ-Tilmanocept ((99m)Tc-labeled-tilmanocept or [(99m)Tc]-tilmanocept) is the first mannose-containing, receptor-directed, radiolabeled tracer for the highly sensitive imaging of sentinel lymph nodes in solid tumor staging. To elucidate the mannose-binding receptor that retains tilmanocept in this microenvironment, human macrophages were used that have high expression of the C-type lectin mannose receptor (MR; CD206). Cy3-labeled tilmanocept exhibited high specificity binding to macrophages that was nearly abolished in competitive inhibition experiments. Furthermore, Cy3-tilmanocept binding was markedly reduced on macrophages deficient in the MR by small interfering RNA treatment and was increased on MR-transfected HEK 293 cells. Finally, confocal microscopy revealed colocalization of Cy3-tilmanocept with the macrophage membrane MR and binding of labeled tilmanocept to MR(+) cells (macrophages and/or dendritic cells) in human sentinel lymph node tissues. Together these data provide strong evidence that CD206 is a major binding receptor for γ-tilmanocept. Identification of CD206 as the γ-tilmanocept-binding receptor enables opportunities for designing receptor-targeted advanced imaging agents and therapeutics for cancer and other diseases.
Assuntos
Dextranos/metabolismo , Lectinas Tipo C/metabolismo , Mananas/metabolismo , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Biópsia de Linfonodo Sentinela/métodos , Pentetato de Tecnécio Tc 99m/análogos & derivados , Carbocianinas/química , Células Cultivadas , Dextranos/química , Citometria de Fluxo , Células HEK293 , Humanos , Imuno-Histoquímica , Lectinas Tipo C/genética , Linfonodos/metabolismo , Macrófagos/metabolismo , Mananas/química , Receptor de Manose , Lectinas de Ligação a Manose/genética , Microscopia Confocal , Estrutura Molecular , Neoplasias/diagnóstico , Neoplasias/metabolismo , Ligação Proteica , Interferência de RNA , Compostos Radiofarmacêuticos/metabolismo , Receptores de Superfície Celular/genética , Pentetato de Tecnécio Tc 99m/química , Pentetato de Tecnécio Tc 99m/metabolismoRESUMO
New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [(18)F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [(18)F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases.
Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Radioisótopos de Flúor , Tomografia por Emissão de Pósitrons/métodos , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Cetuximab/metabolismo , Humanos , Camundongos , Imagem Óptica , Radioquímica , Estreptavidina/metabolismoRESUMO
BACKGROUND: No prior studies have examined injection pain associated with Technetium-99m Tilmanocept (TcTM). METHODS: This was a randomized, double-blinded study comparing postinjection site pain between filtered Technetium Sulfur Colloid (fTcSC) and TcTM in breast cancer lymphoscintigraphy. Pain was evaluated with a visual analogue scale (VAS) (0-100 mm) and the short-form McGill Pain Questionnaire (SF-MPQ). The primary endpoint was mean difference in VAS scores at 1-min postinjection between fTcSC and TcTM. Secondary endpoints included a comparison of SF-MPQ scores between the groups at 5 min postinjection and construction of a linear mixed effects model to evaluate the changes in pain during the 5-min postinjection period. RESULTS: Fifty-two patients underwent injection (27-fTcSC, 25-TcTM). At 1-min postinjection, patients who received fTcSC experienced a mean change in pain of 16.8 mm (standard deviation (SD) 19.5) compared with 0.2 mm (SD 7.3) in TcTM (p = 0.0002). At 5 min postinjection, the mean total score on the SF-MPQ was 2.8 (SD 3.0) for fTcSC versus 2.1 (SD 2.5) for TcTM (p = 0.36). In the mixed effects model, injection agent (p < 0.001), time (p < 0.001) and their interaction (p < 0.001) were associated with change in pain during the 5-min postinjection period. The model found fTcSC resulted in significantly more pain of 15.2 mm (p < 0.001), 11.3 mm (p = 0.001), and 7.5 mm (p = 0.013) at 1, 2, and 3 min postinjection, respectively. CONCLUSIONS: Injection with fTcSC causes significantly more pain during the first 3 min postinjection compared with TcTM in women undergoing lymphoscintigraphy for breast cancer.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Dextranos/efeitos adversos , Linfocintigrafia/efeitos adversos , Mananas/efeitos adversos , Dor/diagnóstico , Biópsia de Linfonodo Sentinela/efeitos adversos , Pentetato de Tecnécio Tc 99m/análogos & derivados , Coloide de Enxofre Marcado com Tecnécio Tc 99m/efeitos adversos , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Linfonodos/diagnóstico por imagem , Linfonodos/patologia , Linfonodos/cirurgia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Dor/etiologia , Prognóstico , Compostos Radiofarmacêuticos/efeitos adversos , Pentetato de Tecnécio Tc 99m/efeitos adversosRESUMO
BACKGROUND: The efficacy of sentinel lymph node (SLN) surgery requires targeted removal of first-draining nodes; however, frequently more nodes are removed than necessary. [(99m)Tc]tilmanocept (TcTM) is a molecular-targeted radiopharmaceutical specifically designed for SLN mapping. We evaluated technical outcomes of SLN biopsy in breast cancer patients mapped with TcTM + vital blue dye (VBD) versus filtered [(99m)Tc]sulfur colloid (fTcSC) + VBD. METHODS: There were 84 versus 115 patients in the TcTM versus fTcSC cohorts, respectively. Main measures were the number of SLNs removed per patient and factors influencing number of nodes removed. We also evaluated whether the radiotracer injected affected the proportion of positive nodes removed in node-positive patients. RESULTS: Fewer nodes were removed among patients mapped with TcTM compared to fTcSC (mean TcTM: 1.85 vs. fTcSC: 3.24, p < 0.001). Logistic regression analysis adjusted for tumor characteristics showed that injection of fTcSC (p < 0.001) independently predicted removal of greater than 3 nodes. A similar proportion of patients was identified as node-positive, whether mapped with TcTM or with fTcSC (TcTM: 24 % vs. fTcSC: 17 %, p = 0.3); however, TcTM detected a greater proportion of positive nodes among node-positive patients compared with fTcSC (0.73 vs. 0.43, p = 0.001). CONCLUSIONS: Patients undergoing SLN biopsy with TcTM required fewer SLNs to identify the same rate of node-positive patients compared with fTcSC in breast cancer patients with similar risk of axillary metastatic disease. These data suggest that a molecularly targeted mechanism of SLN identification may reduce the total number of nodes necessary for accurate axillary staging.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/diagnóstico por imagem , Carcinoma Lobular/diagnóstico por imagem , Dextranos , Linfonodos/diagnóstico por imagem , Mananas , Compostos Radiofarmacêuticos , Pentetato de Tecnécio Tc 99m/análogos & derivados , Coloide de Enxofre Marcado com Tecnécio Tc 99m , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/cirurgia , Carcinoma Lobular/patologia , Carcinoma Lobular/cirurgia , Feminino , Seguimentos , Humanos , Linfonodos/patologia , Linfonodos/cirurgia , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Cintilografia , Estudos Retrospectivos , Biópsia de Linfonodo SentinelaRESUMO
A sentinel lymph node (SLN) is the first lymph node to drain a solid tumor and likely the first place metastasis will travel. SLN biopsy has been well established as a staging tool for melanoma and breast cancer to guide lymph node dissection (LND); its utility in bladder cancer is debated. We performed a systematic search of PubMed for both human and animal studies that looked at SLN detection in cases of urothelial carcinoma of the bladder. We identified a total of nine studies that assessed a variety of imaging techniques to identify SLNs in patients with urothelial carcinoma of the bladder. Eight studies investigated human patients while one looked at animal (dog) models. Seven studies representing 156 patients noted the negative predictive value of the SLN to predict a metastasis free state was 92% (92/100). The SLN biopsy was less accurate in metastatic patients with a positive predictive value of only 77% (43/56) with a false negative range of in individual studies of 0-19%. Clinically, positive nodes routinely do not take up the pharmaceutical agent for SLN. Therefore, SLN biopsy is a promising concept with a 92% negative predictive value; however, the false negative rates are high which may be improved by standardizing populations and indications. Novel technologies are improving the detection of SLN and may provide the surgeon with an improved ability to detect micrometastasis, guide surgery, and reduce patient morbidity.
RESUMO
BACKGROUND: Dendritic cells (DC) are localized in close proximity to cancer cells in many well-known tumors, and thus maybe a useful target for tumor margin assessment. MATERIALS AND METHODS: [(99m)Tc]- cyanine 7 (Cy7)-tilmanocept was synthesized and in vitro binding assays to bone marrow-derived DC were performed. Fifteen mice, implanted with either 4T1 mouse mammary or K1735 mouse melanoma tumors, were administered 1.0 nmol of [(99m)Tc]-Cy7-tilmanocept via tail vein injection. After fluorescence imaging 1 or 2 h after injection, the tumor, muscle, and blood were assayed for radioactivity to calculate percent-injected dose. Digital images of the tumors after immunohistochemical staining for DC were analyzed to determine DC density. RESULTS: In vitro binding demonstrated subnanomolar affinity of [(99m)Tc]-Cy7-tilmanocept to DC (KA = 0.31 ± 0.11 nM). After administration of [(99m)Tc]-Cy7-tilmanocept, fluorescence imaging showed a 5.5-fold increase in tumor signal as compared with preinjection images and a 3.3-fold difference in fluorescence activity when comparing the tumor with the surgical bed after tumor excision. Immunohistochemical staining analysis demonstrated that DC density positively correlated with tumor percent of injected dose per gram (r = 0.672, P = 0.03), and higher DC density was observed at the periphery versus center of the tumor (186 ± 54 K versus 64 ± 16 K arbitrary units, P = 0.001). CONCLUSIONS: [(99m)Tc]-Cy7-tilmanocept exhibits in vitro and in vivo tumor-specific binding to DC and maybe useful as a tumor margin targeting agent.
Assuntos
Benzotiazóis , Carbocianinas , Células Dendríticas/patologia , Dextranos , Neoplasias Mamárias Experimentais/patologia , Mananas , Melanoma Experimental/patologia , Pentetato de Tecnécio Tc 99m/análogos & derivados , Animais , Benzotiazóis/química , Antígeno CD11c/análise , Antígeno CD11c/química , Carbocianinas/química , Linhagem Celular , Linhagem Celular Tumoral , Células Dendríticas/química , Dextranos/química , Feminino , Neoplasias Mamárias Experimentais/química , Mananas/química , Melanoma Experimental/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Microscopia de Fluorescência , Pentetato de Tecnécio Tc 99m/química , Raios UltravioletaRESUMO
OBJECTIVE: With the shift toward utilization of sentinel lymph node biopsy (SLNB) in oral cavity cancer, improved techniques for intraoperative sentinel node identification are needed. This study investigates the feasibility of fluorescently labeled tilmanoscept in SLNB in an oral cancer rabbit model. METHODS: An animal study was designed using 21 healthy male New Zealand rabbits. Gallium-68-labeled tilmanocept labeled with IRDye800CW was injected submucosally into the buccal mucosa (n = 6) or lateral tongue (n = 7) followed by PET imaging. One hour after injection, SLNB was performed using fluorescence imaging followed by a bilateral neck dissection and sampling of non-nodal surrounding tissue. All tissues were measured for radioactivity and fluorescence. In addition, eight rabbits were injected with delayed SLNB performed 48 h after injection. RESULTS: Buccal injections all had ipsilateral SLN drainage and tongue injections exhibited 18.2% contralateral drainage. An average of 1.9 ± 1.0 SLN (range 1-5) were identified. In addition, an average of 16.9 ± 3.3 non-sentinel lymph nodes were removed per animal. SLNs had an average of 0.69 ± 0.60 percent-of-injected dose (%ID) compared with non-sentinel nodes with 0.012 ± 0.025 %ID and surrounding tissue with 0.0067 ± 0.015 %ID. There was 98.0% agreement between sentinel lymph nodes identified using fluorescence compared to radioactivity with Cohen's kappa coefficient of 0.879. In 48-h delayed SLNB, results were consistent with 97.8% agreement with radioactivity and Cohen's Kappa coefficient of 0.884. Fluorescence identified additional lymph nodes that were not identified by radioactivity, and with one false negative. CONCLUSION: Fluorescent-labeled Tc-99 m-tilmanocept represents a highly accurate adjunct to enhance SLNB for oral cavity cancer. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1299-1307, 2024.
Assuntos
Neoplasias Bucais , Linfonodo Sentinela , Masculino , Animais , Coelhos , Biópsia de Linfonodo Sentinela/métodos , Linfonodos/patologia , Linfonodo Sentinela/diagnóstico por imagem , Linfonodo Sentinela/cirurgia , Linfonodo Sentinela/patologia , Neoplasias Bucais/diagnóstico por imagem , Neoplasias Bucais/cirurgia , Neoplasias Bucais/patologiaRESUMO
Decompression sickness (DCS) is caused by gaseous nitrogen dissolved in tissues forming bubbles during decompression. To date, no method exists to identify nitrogen within tissues, but with advances in positron-emission tomography (PET) technology, it may be possible to track gaseous radionuclides into tissues. We aimed to develop a method to track nitrogen movement in vivo and under hyperbaric pressure that could then be used to further our understanding of DCS using nitrogen-13 (13N2). A single anesthetized female Sprague-Dawley rat was exposed to 625 kPa, composed of air, isoflurane, and 13N2 for 10 min. The PET scanner recorded 13N2 during the hyperbaric exposure with energy windows of 250-750 keV. The PET showed an increase in 13N2 concentration in the lung, heart, and abdominal regions, which all reached a plateau after â¼4 min. This showed that it is possible to gain noninvasive in vivo measurements of nitrogen kinetics through the body while at hyperbaric pressures. Tissue samples showed radioactivity above background levels in the blood, brain, liver, femur, and thigh muscle when assessed using a γ counter. The method can be used to evaluate an array of challenges to our understanding of decompression physiology by quantifying nitrogen load through γ counts of 13N2, and signal intensity of the PET. Further development of the method will improve the specificity of the measured outcomes, and enable it to be used with larger mammals, including humans.NEW & NOTEWORTHY This article describes a method for the in vivo quantification and tracking of nitrogen through the mammalian body whilst exposed to hyperbaric pressure. The method has the potential to further our understanding of decompression sickness, and quantitatively evaluate the effectiveness of both the treatment and prevention of decompression sickness.
Assuntos
Doença da Descompressão , Mergulho , Oxigenoterapia Hiperbárica , Radioisótopos de Nitrogênio , Humanos , Ratos , Animais , Feminino , Nitrogênio , Doença da Descompressão/diagnóstico por imagem , Mergulho/fisiologia , Ratos Sprague-Dawley , Descompressão/efeitos adversos , Gases , Oxigenoterapia Hiperbárica/métodos , Tomografia por Emissão de Pósitrons , MamíferosRESUMO
BACKGROUND: Sentinel lymph node (SLN) surgery is used worldwide for staging breast cancer patients and helps limit axillary lymph node dissection. [(99m)Tc]Tilmanocept is a novel receptor-targeted radiopharmaceutical evaluated in 2 open-label, nonrandomized, within-patient, phase 3 trials designed to assess the lymphatic mapping performance. METHODS: A total of 13 centers contributed 148 patients with breast cancer. Each patient received [(99m)Tc]tilmanocept and vital blue dye (VBD). Lymph nodes identified intraoperatively as radioactive and/or blue stained were excised and histologically examined. The primary endpoint, concordance (lower boundary set point at 90 %), was the proportion of nodes detected by VBD and [(99m)Tc]tilmanocept. RESULTS: A total of 13 centers contributed 148 patients who were injected with both agents. Intraoperatively, 207 of 209 nodes detected by VBD were also detected by [(99m)Tc]tilmanocept for a concordance rate of 99.04 % (p < 0.0001). [(99m)Tc]tilmanocept detected a total of 320 nodes, of which 207 (64.7 %) were detected by VBD. [(99m)Tc]Tilmanocept detected at least 1 SLN in more patients (146) than did VBD (131, p < 0.0001). In 129 of 131 patients with ≥1 blue node, all blue nodes were radioactive. Of 33 pathology-positive nodes (18.2 % patient pathology rate), [(99m)Tc]tilmanocept detected 31 of 33, whereas VBD detected only 25 of 33 (p = 0.0312). No pathology-positive SLNs were detected exclusively by VBD. No serious adverse events were attributed to [(99m)Tc]tilmanocept. CONCLUSION: [(99m)Tc]Tilmanocept demonstrated success in detecting a SLN while meeting the primary endpoint. Interestingly, [(99m)Tc]tilmanocept was additionally noted to identify more SLNs in more patients. This localization represented a higher number of metastatic breast cancer lymph nodes than that of VBD.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Dextranos , Linfonodos/diagnóstico por imagem , Mananas , Compostos Radiofarmacêuticos , Biópsia de Linfonodo Sentinela/métodos , Pentetato de Tecnécio Tc 99m/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Axila , Corantes , Dextranos/efeitos adversos , Feminino , Humanos , Linfonodos/patologia , Linfonodos/cirurgia , Metástase Linfática , Mananas/efeitos adversos , Pessoa de Meia-Idade , Cintilografia , Compostos Radiofarmacêuticos/efeitos adversos , Pentetato de Tecnécio Tc 99m/efeitos adversosRESUMO
PURPOSE: The hepatic asialoglycoprotein receptor is responsible for degradation of desialylated glycoproteins through receptor-mediated endocytosis. It has been shown that imaging of the receptor density using [(99m)Tc]diethylenetriamine pentaacetic acid (DTPA) galactosyl human serum albumin ([(99m)Tc]GSA) allows non-invasive determination of functional hepatocellular mass. Here we present the synthesis and evaluation of [(68)Ga]GSA for the potential use with positron emission tomography (PET). METHODS: Labelling of GSA with (68)Ga was carried out using a fractionated elution protocol. For quality control thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) techniques were evaluated. Stability of [(68)Ga]GSA was studied in phosphate-buffered saline (PBS) and human serum. For in vivo evaluation [(68)Ga]GSA distribution in Lewis rats was compared with [(99m)Tc]GSA by using a dual isotope protocol. PET and planar imaging studies were performed using the same scaled molar dose of [(68)Ga]GSA and [(99m)Tc]GSA. Time-activity curves (TAC) for heart and liver were generated and corresponding parameters calculated (t50, t90). RESULTS: [(68)Ga]GSA can be produced with high radiochemical purity. The best TLC methods for determining potential free (68)Ga include 0.1 M sodium citrate as eluent. None of the TLC methods tested were able to determine potential colloids. This can be achieved by SEC. HPLC confirmed high radiochemical purity (>98%). Stability after 120 min incubation at 37 °C was high in PBS (>95% intact tracer) and low in human serum (â¼27% intact tracer). Biodistribution studies simultaneously injecting both tracers showed comparable liver uptake, whereas activity concentration in blood was higher for [(68)Ga]GSA compared to [(99m)Tc]GSA. The [(99m)Tc]GSA TACs exhibited a small degree of hepatic metabolism compared to the [(68)Ga]GSA curves. The mean [(68)Ga]GSA t90 was higher than the mean t90 for [(99m)Tc]GSA. The mean [(68)Ga]GSA t50 was not significantly different from the mean t50 for [(99m)Tc]GSA. CONCLUSION: This study provides a promising new (68)Ga-labelled compound based on a commercially used kit for imaging the functional hepatocellular mass.
Assuntos
Albuminas/síntese química , Radioisótopos de Gálio/química , Fígado/diagnóstico por imagem , Compostos Organometálicos/síntese química , Compostos Radiofarmacêuticos/síntese química , Albuminas/química , Albuminas/farmacocinética , Animais , Radioisótopos de Gálio/farmacocinética , Humanos , Compostos Organometálicos/química , Compostos Organometálicos/farmacocinética , Ácido Pentético/química , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Agregado de Albumina Marcado com Tecnécio Tc 99m/farmacocinética , Pentetato de Tecnécio Tc 99m/farmacocinética , Distribuição TecidualRESUMO
Organic anion transporter 1 (OAT1/SLC22A6, NKT) is a multispecific drug transporter in the kidney with numerous substrates, including pharmaceuticals, endogenous metabolites, natural products, and uremic toxins. Here, we show that OAT1 regulates levels of gut microbiome-derived metabolites. We depleted the gut microbiome of Oat1-KO and WT mice and performed metabolomics to analyze the effects of genotype (KO versus WT) and microbiome depletion. OAT1 is an in vivo intermediary between the host and the microbes, with 40 of the 162 metabolites dependent on the gut microbiome also impacted by loss of Oat1. Chemoinformatic analysis revealed that the altered metabolites (e.g., indoxyl sulfate, p-cresol sulfate, deoxycholate) had more ring structures and sulfate groups. This indicates a pathway from gut microbes to liver phase II metabolism, to renal OAT1-mediated transport. The idea that multiple gut-derived metabolites directly interact with OAT1 was confirmed by in vitro transport and magnetic bead binding assays. We show that gut microbiome-derived metabolites dependent on OAT1 are impacted in a chronic kidney disease (CKD) model and human drug-metabolite interactions. Consistent with the Remote Sensing and Signaling Theory, our results support the view that drug transporters (e.g., OAT1, OAT3, OATP1B1, OATP1B3, MRP2, MRP4, ABCG2) play a central role in regulating gut microbe-dependent metabolism, as well as interorganismal communication between the host and microbiome.
Assuntos
Microbioma Gastrointestinal , Animais , Humanos , Camundongos , Transporte Biológico/genética , Rim/metabolismo , Proteínas de Membrana Transportadoras , MetabolômicaRESUMO
Sentinel lymph node biopsy (SLNB) has been used across oncological specialties for prognostication, staging, and identification of occult nodal metastasis. Recent studies demonstrated the potential clinical utility of SLNB in oral cavity squamous cell carcinoma (OCSCC). Elective neck dissection is the current standard of care in early management of OCSCC with depth of invasion greater than 2-4 mm; however, majority of patients ultimately do not have nodal disease on final pathology. SLNB is an alternative procedure widely adopted in early cancer management in many oncological subspecialities. Several considerations such as depth of invasion, nodal mapping, histopathology methods, operator variability, postoperative complications, and advancement in preoperative and intraoperative imaging technology can guide the appropriate application to SLNB in OCSCC. The aim of this review is to discuss the current evidence for SLNB in the treatment of early stage OCSCC, imaging technologies that support SLNB procedures, and studies that are currently underway.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Neoplasias Bucais/cirurgia , Neoplasias Bucais/patologia , Biópsia de Linfonodo Sentinela , Carcinoma de Células Escamosas/cirurgia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Metástase Linfática/patologia , Neoplasias de Cabeça e Pescoço/patologia , Estadiamento de Neoplasias , Linfonodos/patologiaRESUMO
PURPOSE: To determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection. MATERIALS AND METHODS: The animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m (99mTc)-labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of 99mTc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of 99mTc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test. RESULTS: In vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L±0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6×10(3) counts·sec(-1)·µW(-1) per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P=.005 to P=.042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation. CONCLUSION: The multireporter probe 99mTc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging.
Assuntos
Corantes , Dextranos , Linfonodos/diagnóstico por imagem , Mananas , Compostos de Organotecnécio , Ácido Pentético , Compostos Radiofarmacêuticos , Animais , Corantes/química , Dextranos/química , Linfonodos/patologia , Metástase Linfática , Mananas/química , Camundongos , Imagem Óptica , Compostos de Organotecnécio/química , Ácido Pentético/química , Cintilografia , Compostos Radiofarmacêuticos/química , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Pentetato de Tecnécio Tc 99m/análogos & derivadosRESUMO
Peutz-Jeghers syndrome (PJS) is a familial cancer disorder due to inherited loss of function mutations in the LKB1/ STK11 serine/threonine kinase. PJS patients develop gastrointestinal hamartomas with 100% penetrance often in the second decade of life, and demonstrate an increased predisposition toward the development of a number of additional malignancies. Among mitogenic signaling pathways, the mammalian-target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in tissues and tumors derived from LKB1-deficient mice. Consistent with a central role for mTORC1 in these tumors, rapamycin as a single agent results in a dramatic suppression of preexisting GI polyps in LKB1+/- mice. However, the key targets of mTORC1 in LKB1-deficient tumors remain unknown. We demonstrate here that these polyps, and LKB1- and AMPK-deficient mouse embryonic fibroblasts, show dramatic up-regulation of the HIF-1alpha transcription factor and its downstream transcriptional targets in an rapamycin-suppressible manner. The HIF-1alpha targets hexokinase II and Glut1 are up-regulated in these polyps, and using FDG-PET, we demonstrate that LKB1+/- mice show increased glucose utilization in focal regions of their GI tract corresponding to these gastrointestinal hamartomas. Importantly, we demonstrate that polyps from human Peutz-Jeghers patients similarly exhibit up-regulated mTORC1 signaling, HIF-1alpha, and GLUT1 levels. Furthermore, like HIF-1alpha and its target genes, the FDG-PET signal in the GI tract of these mice is abolished by rapamycin treatment. These findings suggest a number of therapeutic modalities for the treatment and detection of hamartomas in PJS patients, and potential for the screening and treatment of the 30% of sporadic human lung cancers bearing LKB1 mutations.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Síndrome de Peutz-Jeghers/enzimologia , Síndrome de Peutz-Jeghers/patologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Glucose/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos , Tomografia por Emissão de Pósitrons , Proteínas , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Fatores de Transcrição/metabolismo , Carga Tumoral/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Despite increased survivability for people living with HIV (PLWH), HIV-related cognitive deficits persist. Determining biological mechanism(s) underlying abnormalities is critical to minimize the long-term impact of HIV. Positron emission tomography (PET) studies reveal that PLWH exhibit elevated neuroinflammation, potentially contributing to these problems. PLWH are hypersensitive to environmental insults that drive elevated inflammatory profiles. Gp120 is an envelope glycoprotein exposed on the surface of the HIV envelope which enables HIV entry into a cell contributing to HIV-related neurotoxicity. In vivo evidence for mice overexpressing gp120 (transgenic) mice exhibiting neuroinflammation remains unclear. Here, we conducted microPET imaging in gp120 transgenic and wildtype mice, using the radiotracer [(18)F]FEPPA (binds to the translocator protein expressed by activated microglial serving as a neuroinflammatory marker). Imaging was performed at baseline and 24 h after lipopolysaccharide (LPS; 5 mg/kg) treatment (endotoxin that triggers an immune response). Gp120 transgenic mice exhibited elevated [(18F)]FEPPA in response to LPS vs. wildtype mice throughout the brain including dorsal and ventral striata, hypothalamus, and hippocampus. Gp120 transgenic mice are hypersensitive to environmental inflammatory insults, consistent with PLWH, measurable in vivo. It remains to-be-determined whether this heightened sensitivity is connected to the behavioral abnormalities of these mice or sensitive to any treatments.
Assuntos
Infecções por HIV , Receptores de GABA , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Infecções por HIV/complicações , Infecções por HIV/diagnóstico por imagem , Infecções por HIV/metabolismo , Humanos , Inflamação/diagnóstico por imagem , Inflamação/metabolismo , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Receptores de GABA/metabolismoRESUMO
A symposium at George Washington University on Receptor-Binding Radiotracers in 1980 and three follow-up meetings held at University of California, San Diego provided a forum for debating the critical concepts involved in the new field of designing and evaluating radiotracers for imaging receptors and transporters. This review is intended to educate young investigators who may be relatively new to receptor radiopharmaceutical development. Our anticipated audience includes researchers in basic pharmacology, radiochemistry, imaging technology and kinetic data analysis and how these disciplines have worked together to build our understanding of the human biology of transporters and receptor signaling in health and disease. We have chosen to focus on radiochemical design of a useful imaging agent and how design is coupled to analysis of data collected from dynamic imaging with that agent. Some pharmacology may be required for designing the imaging agent and some imaging physics may be important in optimizing the quality of data that is collected. However, the key to a successful imaging agent is matching the radiotracer to the target receptor and to analysis of the time-course data that is used to parse delivery from specific binding and subsequent metabolism or degradation. Properly designed imaging agents are providing critical information about human biology in health and disease as well as pharmacodynamic response to drug interventions. The review emphasizes some of the ideas that were controversial at the 1980 conference and chronicles with literature examples how they have resolved over the four decades of using radiotracers to study transporters and receptors in human subjects. These examples show that there are situations where a very small KD, i.e. high affinity, has the potential to yield an image that reflects blood flow more than receptor density. The examples also show that by combining two studies, one with high specific activity and a second with low specific activity injections one can unravel the pseudo-first order rate B'max into the true second-order rate constant, k3, and the unoccupied receptor density. The final section describes how mathematical methods first presented to the receptor-imaging community in 1980 are now being used to provide confidence in the analysis of kinetic biodistribution studies. Our hope is that by bringing these concepts together in a single review, the next generation of scientists developing receptor imaging agents can be much more efficient than their pioneers in developing useful imaging methods.
Assuntos
Desenho de Fármacos , Compostos Radiofarmacêuticos/metabolismo , Animais , Humanos , Traçadores RadioativosRESUMO
INTRODUCTION: Blood-brain barrier (BBB) disruption and subsequent neuro-inflammation occur following traumatic brain injury (TBI), resulting in a spectrum of human nervous system disorders. [99mTc]Tc-tilmanocept is a receptor-binding radiopharmaceutical FDA-approved for sentinel lymph node mapping. We hypothesize that after an intravenous (i.v.) injection, [99mTc]Tc-tilmanocept, will traverse a disrupted BBB and bind to CD206-bearing microglial cells. METHODS: Age-matched mice were divided into three groups: 5-days post TBI (nâ¯=â¯4), and 5-days post sham (nâ¯=â¯4), and naïve controls (nâ¯=â¯4). IRDye800CW-labeled [99mTc]Tc-tilmanocept (0.15â¯nmol per gram body weight) and FITC-labeled bovine serum albumin (FITC-BSA) were injected (i.v.) into each mouse. Mice were imaged with a high-resolution gamma camera for 45â¯min. Immediately after imaging, the brains were perfused with fixative, excised, imaged with a fluorescence scanner, assayed for radioactivity, and prepared for histology. RESULTS: In vivo nuclear imaging, ex vivo fluorescence imaging, ex vivo gamma well counting, and histo-microscopy demonstrated enhanced tilmanocept uptake in the TBI region. The normalized [99mTc]Tc-tilmanocept uptake value from nuclear imaging and the maximum pixel intensity from fluorescence imaging of the TBI group (1.12⯱â¯0.12 and 2288⯱â¯278â¯a.u., respectively) were significantly (Pâ¯<â¯0.04) higher than the sham group (0.64⯱â¯0.28 and 1708⯱â¯101â¯a.u., respectively) and the naive group (0.76⯱â¯0.24 and 1643⯱â¯391â¯a.u., respectively). The mean [99mTc]Tc-tilmanocept scaled uptake in the TBI brains (0.058⯱â¯0.013%/g) was significantly (Pâ¯<â¯0.010) higher than the scaled brain uptake of the sham group (0.031⯱â¯0.011%/g) and higher (Pâ¯=â¯0.04) than the uptake of the naïve group (0.020⯱â¯0.002%/g). Fluorescence microscopy demonstrated increased uptake of the IRDye800CW-tilmanocept and FITC-BSA in the TBI brain regions. CONCLUSION: [99mTc]Tc-tilmanocept traverses disrupted blood-brain barrier and localizes within the injured region. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: [99mTc]Tc-tilmanocept could serve as an imaging biomarker for TBI-associated neuroinflammation and any disease process that involves a disruption of the blood-brain barrier.
Assuntos
Lesões Encefálicas Traumáticas/diagnóstico por imagem , Lesões Encefálicas Traumáticas/metabolismo , Dextranos/metabolismo , Mananas/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Pentetato de Tecnécio Tc 99m/análogos & derivados , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Pentetato de Tecnécio Tc 99m/metabolismoRESUMO
We combine a novel boronate trap for F(-) with a near-infrared fluorophore into a single molecule. Attachment to targeting ligands enables localization by positron emission tomography (PET) and near-infrared fluorescence (NIRF). Our first application of this generic tag is to label Lymphoseek (tilmanocept), an agent designed for receptor-specific sentinel lymph node (SLN) mapping. The new conjugate incorporates (18)F(-) in a single, aqueous step, targets mouse SLN rapidly (1 h) with reduced distal lymph node accumulation, permits PET or scintigraphic imaging of SLN, and enables NIRF-guided excision and histological verification even after (18)F decay. This embodiment is superior to current SLN mapping agents such as nontargeted [(99m)Tc]sulfur colloids and Isosulfan Blue, as well as the phase III targeted ligand [(99m)Tc]SPECT Lymphoseek counterpart, species that are visible by SPECT or visible absorbance separately. Facile incorporation of (18)F into a NIRF probe should promote many synergistic PET and NIRF combinations.
Assuntos
Corantes Fluorescentes , Raios Infravermelhos , Marcação por Isótopo/métodos , Linfonodos/metabolismo , Imagem Molecular/métodos , Fenômenos Ópticos , Tomografia por Emissão de Pósitrons/métodos , Animais , Ácidos Borônicos/química , Ácidos Borônicos/farmacocinética , Dextranos/química , Dextranos/farmacocinética , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Radioisótopos de Flúor , Cinética , Linfonodos/diagnóstico por imagem , Linfonodos/cirurgia , Mananas/química , Mananas/farmacocinética , Camundongos , Compostos de Organotecnécio/química , Compostos de Organotecnécio/farmacocinética , Ácido Pentético/química , Ácido Pentético/farmacocinética , Radioquímica , Biópsia de Linfonodo Sentinela , Pentetato de Tecnécio Tc 99m/análogos & derivados , Tomografia Computadorizada por Raios XRESUMO
Deletion of integrin-beta1 (Itgb1) in the kidney collecting system led to progressive renal dysfunction and polyuria. The defect in the concentrating ability of the kidney was concomitant with decreased medullary collecting duct expression of aquaporin-2 and arginine vasopressin receptor 2, while histological examination revealed hypoplastic renal medullary collecting ducts characterized by increased apoptosis, ectasia and cyst formation. In addition, a range of defects from small kidneys with cysts and dilated tubules to bilateral renal agenesis was observed. This was likely due to altered growth and branching morphogenesis of the ureteric bud (the progenitor tissue of the renal collecting system), despite the apparent ability of the ureteric bud-derived cells to induce differentiation of the metanephric mesenchyme. These data not only support a role for Itgb1 in the development of the renal collecting system but also raise the possibility that Itgb1 links morphogenesis to terminal differentiation and ultimately collecting duct function and/or maintenance.