Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase–mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.publishedVersio

Fluorescence-Based Molecular Imaging of Porcine Urinary Bladder Sentinel Lymph Nodes.

The primary objective was to test the ability of a laparoscopic camera system to detect the fluorescent signal emanating from sentinel lymph nodes (SLNs) approximately 2 d after injection and imaging of a positron-emitting molecular imaging agent into the submucosa of the porcine urinary bladder. Methods: Three female pigs underwent a submucosal injection of the bladder with fluorescent-tagged tilmanocept, radiolabeled with both 68Ga and 99mTc. One hour after injection, a pelvic PET/CT scan was acquired for preoperative SLN mapping. Approximately 36 h later, robotic SLN mapping was performed using a fluorescence-capable camera system. After identification of the fluorescent lymph nodes, a pelvic lymph node dissection was completed with robotic assistance. All excised nodal packets (n = 36) were assayed for 99mTc activity, which established a lymph node as an SLN. 99mTc activity was also used to calculate the amount of dye within each lymph node. Results: All of the SLNs defined by the ex vivo γ-well assay of 99mTc activity were detected by fluorescence mode imaging. The time between injection and robotic SLN mapping ranged from 32 to 38 h. A total of 5 fluorescent lymph nodes were detected; 2 pigs had 2 fluorescent lymph nodes and 1 pig exhibited a single lymph node. Four of the 5 SLNs exhibited increased SUVs of 12.4-139.0 obtained from PET/CT. The dye content of the injection sites ranged from 371 to 1,441 pmol, which represented 16.5%-64.1% of the injected dose; the amount of dye within the SLNs ranged from 8.5 to 88 pmol, which was equivalent to 0.38%-3.91% of the administered dose. Conclusion: Fluorescent-labeled 68Ga-tilmanocept allows for PET imaging and real-time intraoperative detection of SLNs during robotic surgery

Fluorescent Guided Sentinel Lymph Mapping of the Oral Cavity with Fluorescent‐Labeled Tilmanocept

ObjectiveWith 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.MethodsAn 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.ResultsBuccal 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.ConclusionFluorescent-labeled Tc-99 m-tilmanocept represents a highly accurate adjunct to enhance SLNB for oral cavity cancer.Level of evidenceN/A Laryngoscope, 134:1299-1307, 2024

Molecular Imaging of endometrial sentinel lymph nodes utilizing fluorescent-labeled Tilmanocept during robotic-assisted surgery in a porcine model.

Molecular imaging with a fluorescent version of Tilmanocept may permit an accurate and facile detection of sentinel nodes of endometrial cancer. Tilmanocept accumulates in sentinel lymph nodes (SLN) by binding to a cell surface receptor unique to macrophages and dendritic cells. Four female Yorkshire pigs underwent cervical stromal injection of IRDye800-Tilmanocept, a molecular imaging agent tagged with near-infrared fluorescent dye and radiolabeled with gallium-68 and technetium-99m. PET/CT scans 1.5 hours post-injection provided pre-operative SLN mapping. Robotic-assisted lymphadenectomy was performed two days after injection, using the FireFly imaging system to identify nodes demonstrating fluorescent signal. After removal of fluorescent nodes, pelvic and periaortic node dissections were performed. Nodes were assayed for technetium-99m activity, and SLNs were established using the "10%-rule", requiring that the radioactivity of additional SLNs be greater than 10% of the "hottest" SLN. Thirty-four nodal samples were assayed ex vivo for radioactivity. All the SLNs satisfying the "10%-rule" were detected using the FireFly system. Five fluorescent nodes were detected, corresponding with preoperative PET/CT scan. Three pigs had one SLN and one pig had two SLNs, with 100% concordance between fluorescence and radioactivity. Fluorescent-labeled Tilmanocept permits real-time intraoperative detection of SLNs during robotic-assisted lymphadenectomy for endometrial cancer in a porcine model. When radiolabeled with gallium-68, Tilmanocept allows for preoperative localization of SLNs using PET/CT, and shows specificity to SLNs with persistent fluorescent signal, detectable using the FireFly system, for two days post-injection. In conclusion, these findings suggest that a phase I trial in human subjects is warranted, and that a long-term goal of an intra-operative administration of non-radioactive fluorescent-labeled Tilmanocept is possible

Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by in Vivo Whole Animal Imaging and ex Vivo Super-Resolution Fluorescence Imaging

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls

Extended Lifetime In Vivo Pulse Stimulated Ultrasound Imaging.

An on-demand long-lived ultrasound contrast agent that can be activated with single pulse stimulated imaging (SPSI) has been developed using hard shell liquid perfluoropentane filled silica 500-nm nanoparticles for tumor ultrasound imaging. SPSI was tested on LnCAP prostate tumor models in mice; tumor localization was observed after intravenous (IV) injection of the contrast agent. Consistent with enhanced permeability and retention, the silica nanoparticles displayed an extended imaging lifetime of 3.3±1 days (mean±standard deviation). With added tumor specific folate functionalization, the useful lifetime was extended to 12 ± 2 days; in contrast to ligand-based tumor targeting, the effect of the ligands in this application is enhanced nanoparticle retention by the tumor. This paper demonstrates for the first time that IV injected functionalized silica contrast agents can be imaged with an in vivo lifetime ~500 times longer than current microbubble-based contrast agents. Such functionalized long-lived contrast agents may lead to new applications in tumor monitoring and therapy

Hollow iron-silica nanoshells for enhanced high intensity focused ultrasound.

BackgroundHigh intensity-focused ultrasound (HIFU) is an alterative ablative technique currently being investigated for local treatment of breast cancer and fibroadenomas. Current HIFU therapies require concurrent magnetic resonance imaging monitoring. Biodegradable 500 nm perfluoropentane-filled iron-silica nanoshells have been synthesized as a sensitizing agent for HIFU therapies, which aid both mechanical and thermal ablation of tissues. In low duty cycle high-intensity applications, rapid tissue damage occurs from mechanical rather than thermal effects, which can be monitored closely by ultrasound obviating the need for concurrent magnetic resonance imaging.Materials and methodsIron-silica nanoshells were synthesized by a sol-gel method on polystyrene templates and calcined to yield hollow nanoshells. The nanoshells were filled with perfluoropentane and injected directly into excised human breast tumor, and intravenously (IV) into healthy rabbits and Py8119 tumor-bearing athymic nude mice. HIFU was applied at 1.1 MHz and 3.5 MPa at a 2% duty cycle to achieve mechanical ablation.ResultsEx vivo in excised rabbit livers, the time to visually observable damage with HIFU was 20 s without nanoshells and only 2 s with nanoshells administered IV before sacrifice. Nanoshells administered IV into nude mice with xenograft tumors were activated in vivo by HIFU 24 h after administration. In this xenograft model, applied HIFU resulted in a 13.6 ± 6.1 mm(3) bubble cloud with the IV injected particles and no bubble cloud without particles.ConclusionsIron-silica nanoshells can reduce the power and time to perform HIFU ablative therapy and can be monitored by ultrasound during low duty cycle operation

Hollow iron-silica nanoshells for enhanced high intensity focused ultrasound.

BackgroundHigh intensity-focused ultrasound (HIFU) is an alterative ablative technique currently being investigated for local treatment of breast cancer and fibroadenomas. Current HIFU therapies require concurrent magnetic resonance imaging monitoring. Biodegradable 500 nm perfluoropentane-filled iron-silica nanoshells have been synthesized as a sensitizing agent for HIFU therapies, which aid both mechanical and thermal ablation of tissues. In low duty cycle high-intensity applications, rapid tissue damage occurs from mechanical rather than thermal effects, which can be monitored closely by ultrasound obviating the need for concurrent magnetic resonance imaging.Materials and methodsIron-silica nanoshells were synthesized by a sol-gel method on polystyrene templates and calcined to yield hollow nanoshells. The nanoshells were filled with perfluoropentane and injected directly into excised human breast tumor, and intravenously (IV) into healthy rabbits and Py8119 tumor-bearing athymic nude mice. HIFU was applied at 1.1 MHz and 3.5 MPa at a 2% duty cycle to achieve mechanical ablation.ResultsEx vivo in excised rabbit livers, the time to visually observable damage with HIFU was 20 s without nanoshells and only 2 s with nanoshells administered IV before sacrifice. Nanoshells administered IV into nude mice with xenograft tumors were activated in vivo by HIFU 24 h after administration. In this xenograft model, applied HIFU resulted in a 13.6 ± 6.1 mm(3) bubble cloud with the IV injected particles and no bubble cloud without particles.ConclusionsIron-silica nanoshells can reduce the power and time to perform HIFU ablative therapy and can be monitored by ultrasound during low duty cycle operation

Molecular Imaging of the Glomerulus via Mesangial Cell Uptake of Radiolabeled Tilmanocept

An unmet need for the clinical management of chronic kidney disease is a predictive tool of kidney function during the first decade of the disease, when there is silent loss of glomerular function. The objective of this study was to demonstrate receptor-mediated binding of tilmanocept to CD206 within the kidney and provide evidence of kinetic sensitivity of this binding to renal function. Methods: Rats were positioned in a PET scanner with the liver and kidneys within the field of view. After an intravenous injection of 68Ga-IRDye800-tilmanocept, using 1 of 2 scaled molar doses (0.02 nmol/g, n = 5; or 0.10 nmol/g, n = 5), or coinjection (n = 3) of 68Ga-IRDye800-tilmanocept (0.10 nmol/g) and unlabeled tilmanocept (5.0 nmol/g), or a negative control, 68Ga-IRDye800-DTPA-galactosyl-dextran (0.02 nmol/g, n = 5), each animal was imaged for 20 min followed by a whole-body scan. Frozen kidney sections were stained for podocytes and CD206 using immunofluorescence. Molecular imaging of diabetic db/db mice (4.9 wk, n = 6; 7.3 wk, n = 4; 13.3 wk, n = 6) and nondiabetic db/m mice (n = 6) was performed with fluorescence-labeled 99mTc-tilmanocept (18.5 MBq, 2.6 nmol). Thirty minutes after injection, blood, liver, kidneys, and urine were assayed for radioactivity. Renal time-activity curves were generated. Results: Rat PET whole-body images and time-activity curves of 68Ga-IRDye800-tilmanocept demonstrated receptor-mediated renal accumulation with evidence of glomerular uptake. Activity within the renal cortex persisted during the 40-min study. Histologic examination demonstrated colocalization of CD206 and IRDye800-tilmanocept within the glomerulus. The glomerular accumulation of the coinjection and the negative control studies were significantly less than the CD206-targeted agent. The db/db mice displayed a multiphasic renal time-activity curve with high urinary bladder accumulation; the nondiabetic mice exhibited renal uptake curves dominated by a single phase with low bladder accumulation. Conclusion: This study demonstrated receptor-mediated binding to the glomerular mesangial cells and kinetic sensitivity of tilmanocept to chronic renal disease. Given the role of mesangial cells during the progression of diabetic nephropathy, PET or SPECT renal imaging with radiolabeled tilmanocept may provide a noninvasive quantitative assessment of glomerular function

Imaging and SLN mapping parameters.

<p>Imaging and SLN mapping parameters.</p