ABSTRACT
RATIONALE: The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target. METHODS: Wild-type C57BL/6J or endothelial cell (EC)-cNAMPT -/- knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced ("one-hit") or a combined LPS/ventilator ("two-hit")-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo. RESULTS: Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT -/- mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models. CONCLUSIONS: These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.
Subject(s)
Acute Lung Injury , COVID-19 , Animals , Antibodies, Monoclonal , Humans , Mice , Mice, Inbred C57BL , SARS-CoV-2ABSTRACT
It has been reported that hyaluronic acid (HA) with a 35 kDa molecular weight (HA35) acts biologically to protect tissue from injury, but its biological properties are not yet fully characterized. This study aimed to evaluate the cellular effects and biodistribution of HA35 compared to HA with a 1600 kDa molecular weight (HA1600). We assessed the effects of HA35 and HA1600 on cell migration, NO and ROS generation, and gene expression in cultured macrophages, microglia, and lymphocytes. HA35 was separately radiolabeled with 99mTc and 125I and administered to C57BL/6J mice for in vivo biodistribution imaging. In vitro studies indicated that HA35 and HA1600 similarly enhanced cell migration through HA receptor binding mechanisms, reduced the generation of NO and ROS, and upregulated gene expression profiles related to cell signaling pathways in immune cells. HA35 showed a more pronounced effect in regulating a broader range of genes in macrophages and microglia than HA1600. Upon intradermal or intravenous administration, radiolabeled HA35 rapidly accumulated in the liver, spleen, and lymph nodes. In conclusion, HA35 not only exhibits effects on cellular bioactivity comparable to those of HA1600 but also exerts biological effects on a broader range of immune cell gene expression. The findings herein offer valuable insights for further research into the therapeutic potential of HA35 in inflammation-mediated tissue injury.
ABSTRACT
Myocardial infarction (MI) triggers adverse ventricular remodeling (VR), cardiac fibrosis, and subsequent heart failure. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is postulated to play a significant role in VR processing via activation of the TLR4 inflammatory pathway. We hypothesized that an eNAMPT specific monoclonal antibody (mAb) could target and neutralize overexpressed eNAMPT post-MI and attenuate chronic cardiac inflammation and fibrosis. We investigated humanized ALT-100 and ALT-300 mAb with high eNAMPT-neutralizing capacity in an infarct rat model to test our hypothesis. ALT-300 was 99mTc-labeled to generate 99mTc-ALT-300 for imaging myocardial eNAMPT expression at 2 hours, 1 week, and 4 weeks post-IRI. The eNAMPT-neutralizing ALT-100 mAb (0.4 mg/kg) or saline was administered intraperitoneally at 1 hour and 24 hours post-reperfusion and twice a week for 4 weeks. Cardiac function changes were determined by echocardiography at 3 days and 4 weeks post-IRI. 99mTc-ALT-300 uptake was initially localized to the ischemic area at risk (IAR) of the left ventricle (LV) and subsequently extended to adjacent non-ischemic areas 2 hours to 4 weeks post-IRI. Radioactive uptake (%ID/g) of 99mTc-ALT-300 in the IAR increased from 1 week to 4 weeks (0.54 ± 0.16 vs. 0.78 ± 0.13, P < 0.01). Rats receiving ALT-100 mAb exhibited significantly improved myocardial histopathology and cardiac function at 4 weeks, with a significant reduction in the collagen volume fraction (%LV) compared to controls (21.5 ± 6.1% vs. 29.5 ± 9.9%, P < 0.05). Neutralization of the eNAMPT/TLR4 inflammatory cascade is a promising therapeutic strategy for MI by reducing chronic inflammation, fibrosis, and preserving cardiac function.
Subject(s)
Cardiomyopathies , Myocardial Infarction , Ventricular Dysfunction, Left , Rats , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Toll-Like Receptor 4 , Myocardial Infarction/diagnostic imaging , Myocardial Infarction/drug therapy , Myocardial Infarction/pathology , Cardiomyopathies/diagnostic imaging , Cardiomyopathies/drug therapy , Cardiomyopathies/etiology , Ventricular Remodeling/physiology , Fibrosis , InflammationABSTRACT
PURPOSE: Previous studies indicate that 99mTc- and fluorescent-labeled c[Cys-Thr-Pro-Ser-Pro-Phe-Ser-His-Cys]OH (TCP-1) peptides were able to detect colorectal cancer (CRC) and tumor-associated vasculature. This study was designed to characterize the targeting properties of PEGylated and non-PEGylated TCP-1 peptides for CRC imaging. PROCEDURES: Cell uptake of cyanine 7 (Cy7)-labeled TCP-1 probes (Cy7-PEG4-TCP-1 and Cy7-TCP-1) was investigated in three CRC cell lines (human, HCT116 and HT29; mouse, CT26). Xenograft and orthotopic CRC tumor models with HCT116 and CT26 cells were used to characterize biodistribution and CRC tumor-targeting properties of TCP-1 fluorescence and radioligand with and without PEGylation, [99mTc]Tc-HYNIC-PEG4-TCP-1 vs. [99mTc]Tc-HYNIC-TCP-1. RESULTS: Fluorescence images showed that TCP-1 probes were distributed in the cytoplasm and nucleus of CRC cells. When CT26 cells were treated with unlabeled TCP-1 peptide prior to the cell incubation with Cy7-PEG4-TCP-1, cell fluorescent signals were significantly reduced relative to the cells without blockade. Relative to Cy7-TCP-1, superior brilliance and visibility of fluorescence was observed in the tumor with Cy7-PEG4-TCP-1 and maintained up to 18 h post-injection. [99mTc]Tc-HYNIC-PEG4-TCP-1 images in xenograft and orthotopic CRC models demonstrated that TCP-1 PEGylation preserved tumor-targeting capability of TCP-1, but its distribution (%ID/g) in the liver and intestine was higher than that of [99mTc]Tc-HYNIC-TCP-1 (1.51 ± 0.29 vs 0.53 ± 0.12, P < 0.01). Better tumor visualization by [99mTc]Tc-HYNIC-TCP-1 was observed in the orthotopic CRC model due to lower intestinal radioactivity. CONCLUSIONS: TCP-1-based probes undergo endocytosis and localize in the cytoplasm and nucleus of human and mouse CRC cells. Tumor detectability of fluorescent TCP-1 peptide with a PEG4 spacer is promising due to its enhanced tumor binding affinity and rapid clearance kinetics from nontumor tissues. Non-PEGylated [99mTc]Tc-HYNIC-TCP-1 exhibits lower nonspecific accumulation in the liver and gastrointestinal tract and might have better capability for detecting CRC lesions in clinical sites. TCP-1 may represent an innovative targeting molecule for detecting CRC noninvasively.
Subject(s)
Colorectal Neoplasms , Peptides , Humans , Animals , Mice , Tissue Distribution , Peptides/metabolism , Tomography, Emission-Computed, Single-Photon/methods , Colorectal Neoplasms/diagnostic imaging , Cell Line, Tumor , Organotechnetium Compounds/chemistryABSTRACT
A fluorescent zinc 2,2'-dipicolylamine coordination complex PSVue®794 (probe 1) is known to selectively bind to phosphatidylserine exposed on the surface of apoptotic and necrotic cells. In this study, we investigated the cell death targeting properties of probe 1 in myocardial ischemia-reperfusion injury. A rat heart model of ischemia-reperfusion was used. Probe 1, control dye, or 99mTc glucarate was intravenously injected in rats subjected to 30-minute and 5-minute myocardial ischemia followed by 2-hour reperfusion. At 90 minutes or 20 hours postinjection, myocardial uptake was evaluated ex vivo by fluorescence imaging and autoradiography. Hematoxylin-eosin and cleaved caspase-3 staining was performed on myocardial sections to demonstrate the presence of ischemia-reperfusion injury and apoptosis. Selective accumulation of probe 1 could be detected in the area at risk up to 20 hours postinjection. Similar topography and extent of uptake of probe 1 and 99mTc glucarate were observed at 90 minutes postinjection. Histologic analysis demonstrated the presence of necrosis, but only a few apoptotic cells could be detected. Probe 1 selectively accumulates in myocardial ischemia-reperfusion injury and is a promising cell death imaging tool.
Subject(s)
Amines/chemistry , Fluorescent Dyes , Glucaric Acid/analogs & derivatives , Myocardial Reperfusion Injury/diagnosis , Organotechnetium Compounds , Picolinic Acids/chemistry , Radiopharmaceuticals , Zinc/chemistry , Animals , Immunohistochemistry , Male , Rats , Rats, Sprague-DawleyABSTRACT
Acute respiratory distress syndrome (ARDS) is accompanied by a dramatic increase in lung hyaluronic acid (HA), leading to a dose-dependent reduction of pulmonary oxygenation. This pattern is associated with severe infections, such as COVID-19, and other important lung injury etiologies. HA actively participates in molecular pathways involved in the cytokine storm of COVID-19-induced ARDS. The objective of this study was to evaluate an imaging approach of radiolabeled HA for assessment of dysregulated HA deposition in mouse models with skin inflammation and lipopolysaccharide (LPS)-induced ARDS using a novel portable intensified Quantum Imaging Detector (iQID) gamma camera system. METHODS: HA of 10 kDa molecular weight (HA10) was radiolabeled with 125I and 99mTc respectively to produce [125I]I-HA10 and [99mTc]Tc-HA10, followed by comparative studies on stability, in vivo biodistribution, and uptake at inflammatory skin sites in mice with 12-O-tetradecanoylphorbol-13-acetate (TPA)-inflamed ears. [99mTc]Tc-HA10 was used for iQID in vivo dynamic imaging of mice with ARDS induced by intratracheal instillation of LPS. RESULTS: [99mTc]Tc-HA10 and [125I]I-HA10 had similar biodistribution and localization at inflammatory sites. [99mTc]Tc-HA10 was shown to be feasible in measuring skin injury and monitoring skin wound healing. [99mTc]Tc-HA10 dynamic pulmonary images yielded good visualization of radioactive uptake in the lungs. There was significantly increased lung uptake and slower lung washout in mice with LPS-induced ARDS than in control mice. Postmortem biodistribution measurement of [99mTc]TcHA10 (%ID/g) was 11.0 ± 3.9 vs. 1.3 ± 0.3 in the ARDS mice (n = 6) and controls (n = 6) (P < 0.001), consistent with upregulated HA expression as determined by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) staining. CONCLUSIONS: [99mTc]Tc-HA10 is promising as a biomarker for evaluating HA dysregulation that contributes to pulmonary injury in ARDS. Rapid iQID imaging of [99mTc]Tc-HA10 clearance from injured lungs may provide a functional template for timely assessment and quantitative monitoring of pulmonary pathophysiology and intervention in ARDS.
Subject(s)
COVID-19 , Respiratory Distress Syndrome , Animals , Mice , Hyaluronic Acid , Tissue Distribution , Lipopolysaccharides , Respiratory Distress Syndrome/diagnostic imagingABSTRACT
Therapeutic strategies to prevent or reduce the severity of radiation pneumonitis are a serious unmet need. We evaluated extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a damage-associated molecular pattern protein (DAMP) and Toll-Like Receptor 4 (TLR4) ligand, as a therapeutic target in murine radiation pneumonitis. Radiation-induced murine and human NAMPT expression was assessed in vitro, in tissues (IHC, biochemistry, imaging), and in plasma. Wild type C57Bl6 mice (WT) and Nampt+/- heterozygous mice were exposed to 20Gy whole thoracic lung irradiation (WTLI) with or without weekly IP injection of IgG1 (control) or an eNAMPT-neutralizing polyclonal (pAb) or monoclonal antibody (mAb). BAL protein/cells and H&E staining were used to generate a WTLI severity score. Differentially-expressed genes (DEGs)/pathways were identified by RNA sequencing and bioinformatic analyses. Radiation exposure increases in vitro NAMPT expression in lung epithelium (NAMPT promoter activity) and NAMPT lung tissue expression in WTLI-exposed mice. Nampt+/- mice and eNAMPT pAb/mAb-treated mice exhibited significant histologic attenuation of WTLI-mediated lung injury with reduced levels of BAL protein and cells, and plasma levels of eNAMPT, IL-6, and IL-1ß. Genomic and biochemical studies from WTLI-exposed lung tissues highlighted dysregulation of NFkB/cytokine and MAP kinase signaling pathways which were rectified by eNAMPT mAb treatment. The eNAMPT/TLR4 pathway is essentially involved in radiation pathobiology with eNAMPT neutralization an effective therapeutic strategy to reduce the severity of radiation pneumonitis.
Subject(s)
Antibodies, Neutralizing/pharmacology , Cytokines/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism , Radiation Pneumonitis/metabolism , Toll-Like Receptor 4/metabolism , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Cytokines/blood , Cytokines/genetics , Cytokines/immunology , Humans , Lung/metabolism , Lung/pathology , Lung/radiation effects , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/radiation effects , Male , Mice, Inbred C57BL , Mice, Mutant Strains , NF-kappa B/metabolism , Nicotinamide Phosphoribosyltransferase/blood , Nicotinamide Phosphoribosyltransferase/genetics , Nicotinamide Phosphoribosyltransferase/immunology , Radiation Pneumonitis/drug therapy , Signal Transduction/drug effectsABSTRACT
The aim of this study was the development of (99m)Tc labeled bis(zinc(II)-dipicolylamine) (Zn²âº-DPA) coordination complexes, and the in vivo evaluation of their usefulness as radiotracers for the detection of cell death. DPA ligand 1 was labeled with (99m)Tc via the (99m)Tc-tricarbonyl core ([(99m)Tc(CO)3-1]³âº) or via HYNIC ((99m)Tc-HYNIC-1) in good radiochemical yields. Highest in vitro stabilities were demonstrated for [(99m)Tc(CO)3-1]³âº. A mouse model of hepatic apoptosis (anti-Fas mAb) was used to demonstrate binding to apoptotic cells. (99m)Tc-HYNIC-1 showed the best targeting of apoptotic hepatic tissue with a 2.2 times higher liver uptake in anti-Fas treated mice as compared to healthy animals. A rat model of ischemia-reperfusion injury was used to further explore the ability of the (99m)Tc-labeled Zn²âº-DPA coordination complexes to target cell death. Selective accumulation could be detected for both tracers in the area at risk, correlating with histological proof of cell death. Area at risk to normal tissue uptake ratios were 3.82 for [(99m)Tc(CO)3-1]³âº and 5.45 for (99m)Tc-HYNIC-1.
Subject(s)
Apoptosis , Coordination Complexes/chemical synthesis , Necrosis , Organotechnetium Compounds/chemical synthesis , Pyridines/chemical synthesis , Radiopharmaceuticals/chemical synthesis , Zinc/chemistry , Animals , Coordination Complexes/pharmacokinetics , Liver/diagnostic imaging , Male , Mice , Mice, Inbred BALB C , Organotechnetium Compounds/pharmacokinetics , Pyridines/pharmacokinetics , Radionuclide Imaging , Radiopharmaceuticals/pharmacokinetics , Rats , Rats, Sprague-Dawley , Reperfusion Injury/diagnostic imaging , Reperfusion Injury/pathology , Technetium/chemistry , Tissue DistributionABSTRACT
BACKGROUND: Intense liver uptake of (99m)Tc-sestamibi (MIBI) often interferes with visualization of myocardial perfusion in the inferior wall of the left ventricle. To develop improved myocardial perfusion agents, crown ether-containing dithiocarbamates and bisphosphines have been introduced in recent years. This study was designed to investigate the myocardial imaging properties and in vivo kinetics of a cationic (99m)Tc(I)-tricarbonyl complex, (99m)Tc-15C5-PNP, in comparison with MIBI. METHODS: Dynamic cardiac images were acquired for 60 minutes after intravenous tracer injection using a small-animal SPECT system in healthy control rats and rats with myocardial infarcts. Myocardial and liver time-activity curves were generated for radiopharmaceutical kinetic analysis. RESULTS: Good visualization of the left ventricular wall and perfusion defects could be achieved 20 minutes after (99m)Tc-15C5-PNP administration. (99m)Tc-15C5-PNP images in all hearts with infarcts showed perfusion defects, which were comparable to MIBI images. The kinetic curves plotted from 1 to 60 minutes demonstrated that (99m)Tc-15C5-PNP has a shorter washout half-life (6.4 ± 3.2 vs 124 ± 30.5 minutes, P < .01) in the liver, lower residual liver activity (14.5 ± 10.2% vs 36.5 ± 28.9%, P < .01), and higher heart/liver ratio than MIBI. CONCLUSIONS: (99m)Tc-15C5-PNP has potential for rapid myocardial perfusion imaging with low liver uptake.
Subject(s)
Myocardial Perfusion Imaging/methods , Organotechnetium Compounds , Radiopharmaceuticals , Tomography, Emission-Computed, Single-Photon/methods , Animals , Liver/metabolism , Male , Myocardium/metabolism , Organotechnetium Compounds/pharmacokinetics , Radiopharmaceuticals/pharmacokinetics , Rats , Rats, Sprague-Dawley , Technetium Tc 99m Sestamibi , Tissue DistributionABSTRACT
INTRODUCTION: The objective of this study was to investigate the cardioprotective effects of a dodecafluoropentane (DDFP)-based perfluorocarbon emulsion (DDFPe) as an artificial carrier for oxygen delivery to ischemic myocardium, using 99mTc-duramycin SPECT imaging. METHODS: Rat hearts with Ischemia-reperfusion (I/R) was prepared by coronary ligation for 45-min followed by reperfusion. The feasibility of 99mTc-duramycin in detecting myocardial I/R injury and its kinetic profile were first verified in the ischemic hearts with 2-h reperfusion (nâ¯=â¯6). DDFPe (0.6â¯mL/kg) was intravenously administered at 10â¯min after coronary ligation in fifteen rats and saline was given in thirteen rats as controls. 99mTc-duramycin SPECT images were acquired in the DDFPe-treated hearts and saline controls at 2-h (DDFPe-2â¯h, nâ¯=â¯7 and Saline-2â¯h, nâ¯=â¯6) or 24-h (DDFPe-24â¯h, nâ¯=â¯8 and Saline-24â¯h, nâ¯=â¯7) of reperfusion. RESULTS: SPECT images, showing "hot-spot" 99mTc-duramycin uptake in the ischemic myocardium, exhibited significantly lower radioactive retention and smaller hot-spot size in the DDFPe-2â¯h and DDFPe-24â¯h hearts compared to controls. The infarcts in the Saline-24â¯h hearts extended significantly relative to measurements in the Saline-2â¯h. The extension of infarct size did not reach a statistical difference between the DDFPe-2â¯h and DDFPe-24â¯h hearts. Ex vivo measurement of 99mTc-duramycin activity (%ID/g) was lower in the ischemic area of DDFPe-2â¯h and DDFPe-24â¯h than that of the Saline-2â¯h and Saline-24â¯h hearts (Pâ¯<â¯0.05). The area of injured myocardium, delineated by the uptake of 99mTc-duramycin, extended more substantially outside the infarct zone in the controls. CONCLUSIONS: Significant reduction in myocardial I/R injury, as assessed by 99mTc-duramycin cell death imaging and histopathological analysis, was induced by DDFPe treatment after acute myocardial ischemia. 99mTc-duramycin imaging can reveal myocardial cell death in ischemic hearts and may provide a tool for the non-invasive assessment of cardioprotective interventions.
Subject(s)
Cardiotonic Agents/administration & dosage , Cardiotonic Agents/pharmacology , Fluorocarbons/administration & dosage , Fluorocarbons/pharmacology , Myocardial Infarction/diagnostic imaging , Myocardial Infarction/metabolism , Oxygen/metabolism , Tomography, Emission-Computed, Single-Photon , Animals , Bacteriocins , Humans , Kinetics , Myocardial Infarction/pathology , Myocardium/metabolism , Organotechnetium Compounds , Rats , Rats, Sprague-DawleyABSTRACT
UNLABELLED: Apoptosis of macrophages and smooth muscle cells is linked to atherosclerotic plaque destabilization. The apoptotic cascade leads to exposure of phosphatidylethanolamine (PE) on the outer leaflet of the cell membrane, thereby making apoptosis detectable using probes targeting PE. The objective of this study was to exploit capabilities of a PE-specific imaging probe, (99m)Tc-duramycin, in localizing atherosclerotic plaque and assessing plaque evolution in apolipoprotein-E knockout (ApoE(-/-)) mice. METHODS: Atherosclerosis was induced in ApoE(-/-) mice by feeding an atherogenic diet. (99m)Tc-duramycin images were acquired using a small-animal SPECT imager. Six ApoE(-/-) mice at 20weeks of age (Group I) were imaged and then sacrificed for ex vivo analyses. Six additional ApoE(-/-) mice (Group II) were imaged at 20 and 40weeks of age before sacrifice. Six ApoE wild-type (ApoE(+/+)) mice (Group III) were imaged at 40weeks as controls. Five additional ApoE(-/-) mice (40weeks of age) (Group IV) were imaged with a (99m)Tc-labeled inactive peptide, (99m)Tc-LinDUR, to assess (99m)Tc-duramycin targeting specificity. RESULTS: Focal (99m)Tc-duramycin uptake in the ascending aorta and aortic arch was detected at 20 and 40weeks in the ApoE(-/-) mice but not in ApoE(+/+) mice. (99m)Tc-duramycin uptake in the aortic lesions increased 2.2-fold on quantitative imaging in the ApoE(-/-) mice between 20 and 40weeks. Autoradiographic and histological data indicated significantly increased (99m)Tc-duramycin uptake in the ascending aorta and aortic arch associated with advanced plaques. Quantitative autoradiography showed that the ratio of activity in the aortic arch to descending thoracic aorta, which had no plaques or radioactive uptake, was 2.1 times higher at 40weeks than at 20weeks (6.62±0.89 vs. 3.18±0.29, P<0.01). There was barely detectable focal uptake of (99m)Tc-duramycin in the aortic arch of ApoE(+/+) mice. No detectable (99m)Tc-LinDUR uptake was observed in the aortas of ApoE(-/-) mice. CONCLUSIONS: PE-targeting properties of (99m)Tc-duramycin in the atherosclerotic mouse aortas were noninvasively characterized. (99m)Tc-duramycin is promising in localizing advanced atherosclerotic plaques.
Subject(s)
Apolipoproteins E/deficiency , Bacteriocins/chemistry , Peptides/chemistry , Plaque, Atherosclerotic/diagnostic imaging , Technetium/chemistry , Tomography, Emission-Computed, Single-Photon/methods , Animals , Aorta/metabolism , Bacteriocins/metabolism , Biological Transport , Kinetics , Mice , Peptides/metabolism , Plaque, Atherosclerotic/metabolismABSTRACT
Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m ((99m)Tc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). (99m)Tc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for (99m)Tc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with (99m)Tc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n=5) received no blockade; Group II (n=5) received a blocking dose of non-radiolabeled TCP-1. Group III (n=5) were imaged with (99m)Tc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n=5) were also imaged with (99m)Tc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of (18)F-fluorodeoxyglucose ((18)F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro(99m)Tc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04±0.52nM. In cancer xenografts, (99m)Tc-TCP-1 radioactivity (%ID/g) was 1.01±0.15 in the absence of blockade and was reduced to 0.26±0.04 (P<0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with (99m)Tc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by (18)F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions.
Subject(s)
Colonic Neoplasms/diagnostic imaging , Molecular Imaging/methods , Peptide Fragments/administration & dosage , Technetium/administration & dosage , Animals , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , HCT116 Cells , Humans , Mice , Mice, SCID , Peptide Fragments/metabolism , Protein Binding/physiology , Technetium/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced nonmelanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared with those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here, we explored the use of topical rapamycin as a chemopreventive agent in the context of solar-simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared with controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared with vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.
Subject(s)
Apoptosis/drug effects , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Signal Transduction/drug effects , Sirolimus/pharmacology , Skin Neoplasms/prevention & control , Administration, Topical , Animals , Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/pharmacology , Apoptosis/radiation effects , Blotting, Western , Female , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Keratinocytes/radiation effects , Mice , Mice, Hairless , Phosphorylation/drug effects , Phosphorylation/radiation effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/radiation effects , Sirolimus/administration & dosage , Skin Neoplasms/etiology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Sulfonamides/pharmacology , Sunlight/adverse effects , TOR Serine-Threonine Kinases/metabolism , Thiadiazoles/pharmacologyABSTRACT
Ultrasound current source density imaging (UCSDI), based on the acoustoelectric (AE) effect, is a noninvasive method for mapping electrical current in 4-D (space + time). This technique potentially overcomes limitations with conventional electrical mapping procedures typically used during treatment of sustained arrhythmias. However, the weak AE signal associated with the electrocardiogram is a major challenge for advancing this technology. In this study, we examined the effects of the electrode configuration and ultrasound frequency on the magnitude of the AE signal and quality of UCSDI using a rabbit Langendorff heart preparation. The AE signal was much stronger at 0.5 MHz (2.99 µV/MPa) than 1.0 MHz (0.42 µV/MPa). Also, a clinical lasso catheter placed on the epicardium exhibited excellent sensitivity without penetrating the tissue. We also present, for the first time, 3-D cardiac activation maps of the live rabbit heart using only one pair of recording electrodes. Activation maps were used to calculate the cardiac conduction velocity for atrial (1.31 m/s) and apical (0.67 m/s) pacing. This study demonstrated that UCSDI is potentially capable of real-time 3-D cardiac activation wave mapping, which would greatly facilitate ablation procedures for treatment of arrhythmias.
Subject(s)
Action Potentials/physiology , Cardiac Catheterization/methods , Heart Conduction System/physiology , Imaging, Three-Dimensional/methods , Ultrasonography/methods , Algorithms , Animals , Heart Conduction System/diagnostic imaging , Image Interpretation, Computer-Assisted/methods , In Vitro Techniques , Neural Conduction/physiology , Rabbits , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
UNLABELLED: Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) play a critical role in initiating and accelerating atherosclerosis. This study evaluated the imaging properties of (99m)Tc-TNFR2-Fc-IL-1RA ((99m)Tc-TFI), a dual-domain cytokine radioligand that targets TNF-α and IL-1ß pathways, in assessing atherosclerosis development in apolipoprotein-E-deficient (ApoE(-)(/)(-)) mice. METHODS: The feasibility and specificity of detecting atherosclerosis with (99m)Tc-TFI SPECT imaging were investigated in ApoE(-)(/)(-) and ApoE(+)(/)(+) mice. Fifty-four ApoE(-)(/)(-) mice were fed either an atherogenic diet (AGD) or a normal diet (ND) beginning at 5 weeks of age. Eighteen Apo-E wild-type (ApoE(+)(/)(+)) mice were fed an ND. Two groups of ApoE(-)(/)(-) mice (n=12 each group) on AGD and ND were imaged three times with (99m)Tc-TFI and a high-resolution SPECT system at 20-25, 30-40, and 48-52 weeks to study the evolution of atherosclerotic plaque. RESULTS: Focal radioactive accumulations in the aortic arch region were observed in the ApoE(-)(/)(-) mice (n=12) on AGD but not in the ApoE(+)(/)(+) mice on ND (n=10). Apo-E(-)(/)(-) mice on ND (n=11) exhibited lower radioactive uptake than ApoE(-)(/)(-) mice on AGD (P<0.05). Co-injection of an excess of cold ligand with (99m)Tc-TFI resulted in significant reduction of (99m)Tc-TFI uptake in the ApoE(-)(/)(-) mice on AGD. Longitudinal studies showed that (99m)Tc-TFI uptake in the aortas of ApoE(-)(/)(-) mice progressively increased from 20 to 48 weeks. Real-time PCR assays demonstrated that atherosclerotic aortas expressed significantly higher IL-1ß and TNF-α than the aortas from wild-type controls. CONCLUSIONS: Atherosclerotic plaques were detected by (99m)Tc-TFI imaging in ApoE(-)(/)(-) mice. (99m)Tc-TFI is promising for specific detection of inflammatory response in atherosclerotic plaques.
Subject(s)
Apolipoproteins E/physiology , Atherosclerosis/diagnosis , Inflammation/diagnosis , Molecular Imaging/methods , Organotechnetium Compounds , Animals , Atherosclerosis/complications , Atherosclerosis/metabolism , Female , Image Processing, Computer-Assisted , Inflammation/complications , Inflammation/metabolism , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-1beta/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Tumor Necrosis Factor, Type II/metabolism , Tomography, Emission-Computed, Single-Photon/methods , Tumor Necrosis Factor-alpha/metabolismABSTRACT
UNLABELLED: Soluble tumor necrosis factor (TNF) receptor-2 (TNFR2) and interleukin-1 receptor antagonist (IL-1ra) were fused to the Fc portion of IgG1 using recombinant DNA technology. The resulting dual-domain cytokine ligand, TNFR2-Fc-IL-1ra, specifically binds to TNF and to the type I IL-1 receptor (IL-1RI). This study was designed to characterize the kinetic profile of (99m)Tc-labeled TNFR2-Fc-IL-1ra (TFI) for imaging inflammatory response in an ischemic-reperfused (IR) rat heart model. METHODS: The IR model was created by ligating the left coronary artery for 45 min, followed by 2-h reperfusion. Cardiac SPECT images of TFI in the IR model (n = 6) were dynamically acquired for 3 h. Correlative data of myocardial TFI distribution versus microsphere-determined tissue blood flow were acquired in 3 extra IR hearts. Inflammation targeting affinity of TFI was compared with 2 individual cytokine radioligands, (99m)Tc-IL-1ra-Fc (IF) and (99m)Tc-TNFR2-Fc (TF) (n = 6 each group). Myocardial cytokine expression was evaluated by immunochemical assay. RESULTS: Increased TFI uptake was found in the ischemic area and correlated with the severity of ischemia. At 3 h after injection, the ratio of hot-spot accumulation in the ischemic area to a remote viable zone was 5.39 ± 1.11 for TFI, which was greater than that for IF (3.28 ± 0.81) and TF (3.29 ± 0.75) (P < 0.05). The in vivo uptake profiles of TFI, TF, and IF were consistent with ex vivo radioactive measurements and correlated with upregulated IL-1 and TNF expression. CONCLUSION: The dual-domain TFI is promising for noninvasive detection of inflammatory reactions in IR myocardium because of its more potent affinity to the inflammatory sites compared with TF and IF.
Subject(s)
Cytokines/metabolism , Heart/diagnostic imaging , Interleukin 1 Receptor Antagonist Protein , Organotechnetium Compounds , Receptors, Tumor Necrosis Factor, Type II , Reperfusion Injury/diagnostic imaging , Tomography, Emission-Computed, Single-Photon , Animals , Biological Transport , Coronary Circulation , Gene Expression Regulation , Heart/physiopathology , Inflammation/diagnostic imaging , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-1beta/metabolism , Isotope Labeling , Ligands , Male , Rats , Rats, Sprague-Dawley , Receptors, Tumor Necrosis Factor, Type II/metabolism , Recombinant Fusion Proteins/metabolism , Reperfusion Injury/metabolism , Reperfusion Injury/physiopathology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVES: Prompt identification of necrosis and apoptosis in the infarct core and penumbra region is critical in acute stroke for delineating the underlying ischemic/reperfusion molecular pathologic events and defining therapeutic alternatives. The objective of this study was to investigate the capability of (99m)Tc-labeled duramycin in detecting ischemia-reperfusion injury in rat brain after middle cerebral artery (MCA) occlusion. METHODS: Ischemic cerebral injury was induced in ten rats by vascular insertion of a nylon suture in the left MCA for 3 hr followed by 21-24hr reperfusion. After i.v. injection of (99m)Tc-duramycin (1.0-3.5 mCi), dynamic cerebral images were acquired for 1 hr in six rats using a small-animal SPECT imager. Four other rats were imaged at 2 hr post-injection. Ex vivo images were obtained by autoradiography after sacrifice. Histologic analyses were performed to assess cerebral infarction and apoptosis. RESULTS: SPECT images showed that (99m)Tc-duramycin uptake in the left cerebral hemisphere was significantly higher than that in the right at 1 and 2 hr post-injection. The level of radioactive uptake in the ischemic brain varied based on ischemic severity. The average ratio of left cerebral hot-spot uptake to right hemisphere radioactivity, as determined by computerized ROI analysis, was 4.92±0.79. Fractional washout at 1 hr was 38.2±4.5% of peak activity for left cerebral hot-spot areas and 80.9±2.0% for remote control areas (P<0.001). Based on triphenyltetrazolium chloride staining and autoradiograph image data, the hotspot uptake may be associated primarily with the ischemic penumbra, in which high apoptotic activity was observed by cleaved caspase-3 immunocytochemical staining. CONCLUSIONS: (99m)Tc-duramycin SPECT imaging may be useful for detecting and quantifying ongoing apoptotic neuronal cell loss induced by ischemia-reperfusion injury.
Subject(s)
Bacteriocins , Infarction, Middle Cerebral Artery/complications , Organotechnetium Compounds , Peptides , Reperfusion Injury/complications , Reperfusion Injury/diagnostic imaging , Tomography, Emission-Computed, Single-Photon/methods , Animals , Brain/diagnostic imaging , Brain/pathology , Isotope Labeling , Kinetics , Male , Rats , Rats, Sprague-Dawley , Reperfusion Injury/pathologyABSTRACT
INTRODUCTION: TNFR2-Fc and IL-1ra-Fc are recombinant cytokine ligands that target TNF and IL-1. TNFR2-Fc-IL-1ra, a dual-domain agent that incorporates both ligands, allows bifunctional binding of IL-1 receptors and TNF. This study was designed to characterize (99m)Tc-labeled forms of these ligands, (99m)Tc-IL-1ra-Fc (IF), (99m)Tc-TNFR2-Fc (TF), and (99m)Tc-TNFR2-Fc-IL-1ra (TFI), for inflammation imaging. METHODS: The cytokine ligands were labeled with (99m)Tc by a direct approach via 2-iminothiolane (2-IT) reduction at various 2-IT/protein molar ratios. In vivo inflammation targeting studies were carried out in a mouse ear edema model created by topical application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the right ear of ICR mice. RESULTS: Radiolabeling yields increased with increasing amounts of 2-IT. When the 2-IT/protein ratio reached 1000, the radiolabeling yield was greater than 90% without significant colloid production. TPA-treated ears showed high radioligand uptake, which was clearly detected by SPECT and autoradiographic imaging. The activities (%ID/g) in the inflamed and control ears at 3h after injection were 2.76 ± 0.20 vs. 0.69 ± 0.12 for IF, 5.86 ± 0.40 vs. 2.86 ± 0.61 for TF, and 7.61 ± 0.86 vs. 1.99 ± 0.31 for TFI (P<0.05 vs. controls). TFI showed significantly higher uptake in the inflamed ears compared to TF and IF (P<0.05). Blocking study results indicated specificity of radioligand binding with decreased radioactive uptake in the inflamed ears. Western blotting and ELISA analysis further confirmed a high expression of IL-1ß and TNF-α in the inflamed ears. CONCLUSIONS: (99m)Tc-labeled cytokine ligands are a promising approach for detecting and understanding the inflammatory process. TFI may be more useful than the single-domain ligands for noninvasive detection of inflammatory sites.
Subject(s)
Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-1/metabolism , Molecular Imaging/methods , Organotechnetium Compounds , Receptors, Tumor Necrosis Factor, Type II/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Drug Stability , Edema/chemically induced , Edema/diagnosis , Edema/metabolism , Gene Expression Regulation , Inflammation/diagnosis , Inflammation/metabolism , Inflammation/pathology , Interleukin 1 Receptor Antagonist Protein/pharmacokinetics , Interleukin-1beta/metabolism , Isotope Labeling , Ligands , Male , Mice , Protein Transport , Rats , Receptors, Tumor Necrosis Factor, Type II/pharmacokinetics , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
INTRODUCTION: Interleukin (IL)-1 and IL-18 are potent proinflammatory cytokines in inflammation-related diseases. Their actions are regulated by IL-1 receptor antagonist (IL-1ra) and IL-18 binding protein (IL-18bp). This study was designed to (99m)Tc-radiolabel an IL-1ra and IL-18bp dual-domain cytokine ligand, IL-18bp-Fc-IL-1ra, for specific inflammation targeting. METHODS: The (99m)Tc-IL-18bp-Fc-IL-1ra was obtained by direct labeling via 2-iminothiolane reduction. Competitive binding of (99m)Tc-labeled and unlabeled IL-18bp-Fc-IL-1ra to rat polymorphonuclear leukocytes was assessed in vitro. A mouse ear edema model was used to evaluate specific targeting properties of (99m)Tc-IL-18bp-Fc-IL1ra in vivo. The correlation between (99m)Tc-IL-18bp-Fc-IL-1ra uptake and (111)In-labeled polymorphonuclear neutrophil infiltration was studied using ischemic-reperfused rat hearts. RESULTS: Direct (99m)Tc-labeling yielded a stable dual-domain cytokine radioligand with radiochemical purity greater than 95% after gel filtration. Competitive binding studies showed specific targeting of (99m)Tc-IL-18bp-Fc-IL-1ra to inflammatory cells. The (99m)Tc-IL-18bp-Fc-IL-1ra uptake was 1.80±0.17 % injected dose per gram (%ID/g) in the inflamed ear without blocking, whereas uptake in the presence of IL-18bp-Fc-IL-1ra was 1.09±0.08 %ID/g (P<.05). The amounts of IL-1ß and IL-18 were significantly increased in the inflamed ears compared to the vehicle controls. A significant correlation of (99m)Tc-IL-18bp-Fc-IL-1ra with (111)In-labeled neutrophil distribution was observed in the ischemic-reperfused hearts (P<.001). CONCLUSION: Targeting proinflammatory cytokines with (99m)Tc-IL-18bp-Fc-IL-1ra may provide a suitable approach for specific detection of inflammatory sites.