Yared Tekabe

Development of Receptor for Advanced Glycation End Products-Directed Imaging of Atherosclerotic Plaque in a Murine Model of Spontaneous Atherosclerosis

Background—The receptor for advanced glycation end products (RAGE) is implicated in the development and progression of atherosclerosis. We tested the hypothesis that 99mTc-labeled anti-RAGE F(ab′)2 can be used as a noninvasive tool to image atherosclerotic lesions in apolipoprotein E–deficient (apoE−/−) mice. Methods and Results—A sequence in the V-type Ig extracellular domain of RAGE was used to develop a peptide injected into rabbits; serum was retrieved, IgG prepared and affinity-purified, and pepsin-digested into F(ab′)2. Thirteen 6-week apoE−/− mice were fed a Western diet. At 20 weeks, 6 were injected with 15.2±1.9 MBq (350 to 411 &mgr;Ci) 99mTc-labeled anti-RAGE F(ab′)2, 6 were injected with 99mTc-labeled control nonspecific IgG F(ab′)2, and 1 was injected with dual-labeled 99mTc and rhodamine anti-RAGE F(ab′)2. Four 20-week C57BL/6 mice were injected with 99mTc-labeled anti-RAGE F(ab′)2. All mice were imaged on a high resolution mini-&ggr; camera 4 hours after injection and euthanized. The aortic tree was dissected and photographed, and the proximal aorta was sectioned for staining after &ggr; scintillation counting. All 6 apoE−/− mice injected with 99mTc-labeled anti-RAGE F(ab′)2 fragments showed focal tracer uptake in the proximal aorta (mean %ID/g, 1.98%). Disease and antibody controls showed no focal tracer uptake in the thorax (%ID/g, <1.0%). Histological sections of the proximal aorta showed American Heart Association class III lesions with lipid laden macrophages, smooth muscle cells, and positive staining for RAGE. On immunofluorescence, RAGE colocalized with macrophages. Conclusion—These data show the feasibility of noninvasively imaging RAGE in atherosclerotic lesions in a murine model and confirm levels of RAGE expression sufficient to allow detection on in vivo imaging.

Targeting very small model lesions pretargeted with bispecific antibody with 99mTc-labeled high-specific radioactivity polymers.

BackgroundTwo-step targeting with bispecific antibody and 99mTc-labeled high-specific radioactivity polymers was used for molecular imaging of two very small model lesions in rats. Methods and resultsSprague–Dawley rats (group I) were injected with surrogate antigen-coated beads (SA beads) in the right hind leg or unmodified beads in the contralateral hind leg. In group II, femoral artery de-endothelialization was induced in the left hind leg and sham operation was performed in the contralateral hind leg. Bispecific antibody Z2D3 F(ab′)2-anti-DTPA F(ab′)2 was injected intravenously 24 h after SAB injection or 1 week after endothelial denudation. 99mTc-labeled polymers were injected intravenously 24 h later and &ggr;-images obtained at 2 and 24 h in group I or approximately 2.5 h in group II. Lesions were visualized by 2 h. In group I, SA beads-specific uptake in muscles was significantly greater than with unmodified beads (P<0.015). In group II, lesions were visualized by 2.5 h after radiopolymer injection with uptake activity 2.1±0.6 times greater than in the contralateral side from in-vivo images (P<0.004) and 1.8±0.7 times by &ggr;-scintillation counting (P<0.04). ConclusionPretargeting with Z2D3 bispecific antibody for the localization of radiolabeled polymers enabled successful in-vivo &ggr;-imaging of very small lesions in two rat models of extravascular and intravascular targets. Biodistribution data confirmed that pretargeting with bispecific antibody enabled targeted visualization of two different very small model lesions by in-vivo &ggr;-imaging.

Pretargeted gamma imaging of murine metastatic melanoma lung lesions with bispecific antibody and radiolabeled polymer drug conjugates.

IntroductionBispecific monoclonal antibodies (bsMAbs) have been developed as a pretargeting tool to reduce background activity, thereby increasing target to background (T : B) ratios. To enhance visualization of small lesions in vivo, we have used the pretargeting approach of bsMAb and negatively charged polymers radiolabeled with high-specific radioactivity. MethodsImaging of metastatic melanoma lesions localized in lung tissues pretargeted with bsMAb and targeted with high-specific radioactivity polymers was carried out. The bsMAb was prepared by covalent conjugation of an antinucleosomal antibody (2C5) recognizing a nucleosomal pan cancer antigen and an anti-diethylene triaminepentaacetic acid antibody (6C31H3) by means of thioether linkage. BsMAb was injected intravenously 10 days after the initiation of the induction of murine melanoma metastasized to the lungs. The next day, 37 MBq 99mTc-diethylene triaminepentaacetic acid–succinylated polylysine were injected intravenously and in-vivo imaging was carried out after the injection. In-vivo and ex-vivo target (T) to background (B) activity ratios were assessed by computer planimetry and biodistribution studies. ResultsLesions were visualized unequivocally in 3 h by gamma scintigraphy. Ex-vivo gamma-scintillation counting corrected for the lesion mass showed that the mean lesion activity was 24.85±13.53 percent injected dose per gram when pretargeted with bsMAb, whereas it was 0.977±0.465 percent injected dose per gram (P<0.01) in the control group injected only with radioactive polymers also corrected similarly. ConclusionThe use of bsMAb complexes and 99mTc-diethylene triaminepentaacetic acid–succinylated polylysine enabled early in-vivo visualization of small metastatic melanoma lesions in the lungs.

A Novel Monoclonal Antibody for RAGE-Directed Imaging Identifies Accelerated Atherosclerosis in Diabetes

Receptor for advanced glycation end products (RAGE) binds advanced glycation end products and other inflammatory ligands and is expressed in atherosclerotic plaques in diabetic and nondiabetic subjects. The higher expression in diabetes mellitus corresponds to the accelerated course of the atherosclerosis. This study was designed to test the hypothesis that the level of RAGE expression in atherosclerosis can be detected by quantitative in vivo SPECT and that counts in the target will correlate with the strength of the biologic signal. Methods: A monoclonal murine antibody was developed against the V-domain of RAGE, fragmented into F(ab′)2 and labeled with 99mTc, and injected at a dose of 15.14 ± 1.23 MBq into 24-wk-old male apolipoprotein E null (ApoE−/−) mice (n = 22), including mice with streptozotocin-induced diabetes mellitus (n = 8), nondiabetic mice (n = 8), and control ApoE−/−/RAGE−/− double-knock-out mice (n = 6). Four hours later (allowing for blood-pool clearance), the mice were imaged and sacrificed, and the proximal aorta was removed and counted to calculate the percentage injected dose of RAGE per gram of tissue, followed by histologic and immunohistochemical characterization. Results: Radiotracer uptake in the aortic lesions was clearly visualized noninvasively by SPECT. RAGE uptake as percentage injected dose in diabetic ApoE−/− mice (1.39 ± 0.16 × 10−2) was significantly higher than that in nondiabetic ApoE−/− mice (0.48 ± 0.27 × 10−2) (P < 0.0001). The radiotracer uptake was highly correlated with RAGE expression by quantitative immunohistomorphometry (r = 0.82, P = 0.002) and with percentage of macrophages (r = 0.86, P < 0.0001). Conclusion: In this study, 99mTc-labeled anti-RAGE F(ab′)2 SPECT successfully identified early accelerated disease in diabetes mellitus for age-matched ApoE−/− mice and quantified RAGE expression over a range of lesion severities.

Hyperspectral fluorescence tomography of quantum dots using the simplified spherical harmonics equations

The proposed image reconstruction method exploits the spectrally dependent absorption properties of biological tissue and quantum dots for recovering the three-dimensional reporter distribution. Only a single light source with macro-illumination needs to be used for the purpose of light emission stimulation and image reconstruction. The light propagation in strongly absorbing tissue is modeled with the simplified spherical harmonics (SPN) equations.

Beneficial Effect of Glucose Control on Atherosclerosis Progression in Diabetic ApoE−/− Mice: Shown by Rage Directed Imaging

Objective. Receptor for advanced glycated endproducts (RAGE) plays an important role in atherogenesis in diabetes. We imaged RAGE to investigate the effect of glucose control to suppress RAGE and reduce atherosclerosis in apolipoprotein E null (apoE−/−) diabetic mice. Methods and Results. Thirty-three apoE−/− mice received streptozotocin and 6 weeks later 15 began treatment with insulin implants. Blood glucose measurements during study averaged: 140 ± 23 mg/dL (treated) and 354 ± 14 mg/dL (untreated). After 15 wk 30 mice were injected with 99mTc-anti-RAGE F(ab′)2, 3 with 99mTc-nonimmune IgG F(ab′)2, and all with CT contrast agent and underwent SPECT/CT imaging. At necropsy, the proximal aorta was weighed, counted, and sectioned and the % injected dose per gram (%ID/g) was calculated. From the merged SPECT/CT scans, tracer uptake localized to arteries was lower in the treated mice: 3.15 ± 1.82 × 10−3 versus 8.69 ± 4.58 × 10−3%ID (P = 0.001). Percent cross-sectional lesion area was smaller in the treated (14.3 ± 7.8% versus 29.5 ± 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Lesion size as percent cross-sectional area was smaller in the treated (14.3 ± 7.8% versus 29.5 ± 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Conclusions. These results support the importance of suppressing RAGE to reduce atherosclerotic complications of diabetes and value of molecular imaging to assess treatment effect.

Cerenkov Light Tomography of 131I-labeled Ligands in Small Animal Tumor Models

Cerenkov light tomography calculates the distribution of 131I-labeled ligands by taking optical images at the tissue surface. We demonstrate the feasibility on a SK-NEP-1 tumor model and validate the results with SPECT.