Dong-Wei Gao

Pinhole single-photon emission computed tomography for myocardial perfusion imaging of mice

OBJECTIVES Although transgenic mice have emerged as powerful experimental models of cardiovascular disease, methods for in vivo phenotypic assessment and characterization remain limited, motivating the development of new instruments for biologic measurement. BACKGROUND We have developed a single-photon emission computed tomography system with a pinhole collimator (pinhole SPECT) for high-resolution cardiovascular imaging of mice. In this study, we describe a protocol for myocardial perfusion imaging of mice using technetium-99m ((99m)Tc)-sestamibi and demonstrate the feasibility for measurement of perfusion defect size from pinhole SPECT images. METHODS Mice were anesthetized and injected with 370 MBq (10 mCi) of (99m)Tc-sestamibi. Tomographic projection images were acquired by rotating each mouse in a vertical axis in front of a stationary clinical scintillation camera equipped with a pinhole collimator. BALB/c mice (n = 15) were imaged after the permanent ligation of the left anterior descending coronary artery. The resulting defect size was measured from circumferential profiles of short-axis images. After imaging, the hearts were excised and sectioned to obtain ultra-high resolution digital autoradiographs of (99m)Tc-sestamibi, from which the actual infarct size was determined. RESULTS Reconstructed image quality was equivalent to that obtained for clinical myocardial perfusion imaging. Linear regression analysis produced a correlation coefficient of 0.83 (p < 0.001) between the measured and actual values of the defect size. CONCLUSIONS These results demonstrate that myocardial perfusion can be characterized qualitatively and quantitatively in mice using pinhole SPECT.

Identification of MCAM/CD146 as the target antigen of a human monoclonal antibody that recognizes both epithelioid and sarcomatoid types of mesothelioma.

The prognosis for patients diagnosed with mesothelioma is generally poor, and currently available treatments are usually ineffective. Therapies that specifically target tumor cells hold much promise for the treatment of cancers that are resistant to current approaches. We have previously selected phage antibody display libraries on mesothelioma cell lines to identify a panel of internalizing human single chain (scFv) antibodies that target mesothelioma-associated, clinically represented cell surface antigens and further exploited the internalizing function of these scFvs to specifically deliver lethal doses of liposome-encapsulated small molecule drugs to both epithelioid and sarcomatous subtypes of mesothelioma cells. Here, we report the identification of MCAM/MUC18/CD146 as the surface antigen bound by one of the mesothelioma-targeting scFvs using a novel cloning strategy based on yeast surface human proteome display. Immunohistochemical analysis of mesothelioma tissue microarrays confirmed that MCAM is widely expressed by both epithelioid and sarcomatous types of mesothelioma tumor cells in situ but not by normal mesothelial cells. In addition, quantum dot-labeled anti-MCAM scFv targets primary meosthelioma cells in tumor fragment spheroids cultured ex vivo. As the first step in evaluating the therapeutic potential of MCAM-targeting antibodies, we performed single-photon emission computed tomography studies using the anti-MCAM scFv and found that it recognizes mesothelioma organotypic xenografts in vivo. The combination of phage antibody library selection on tumor cells and rapid target antigen identification by screening the yeast surface-displayed human proteome could be a powerful method for mapping the targetable tumor cell surface epitope space.

Heterogeneous Sympathetic Innervation Influences Local Myocardial Repolarization in Normally Perfused Rabbit Hearts

BACKGROUND Heterogeneity of sympathetic innervation is thought to contribute to the potential for fatal arrhythmia. However, little is known about the effects of heterogeneous innervation on repolarization. METHODS AND RESULTS To assess this relationship, we measured activation recovery intervals (ARIs) from 64 epicardial sites in 11 rabbits studied 2 weeks after regional denervation produced by phenol and 4 sham-operated rabbits. ARI results were compared with the distribution of sympathetic innervation measured from 3D reconstructions of serial autoradiographs of [(125)I]metaiodobenzylguanidine and (99m)Tc-sestamibi. ARIs were recorded during baseline sinus rhythm, norepinephrine (NE) infusion (0.1 microg. kg(-1). min(-1)), and left stellate ganglion stimulation (SS). NE shortened ARI in 98% of electrodes in the denervated region. The degree of ARI shortening and dispersion increased (P<0.001 and P<0.01, respectively) as denervation became more severe. SS shortened ARI in 30% of electrodes in the denervated area, with increased shortening and dispersion related to increased severity of denervation (P<0.01). SS prolonged ARI in 70% of electrodes in the denervated area, with no correlation with severity of denervation. CONCLUSIONS The magnitude and dispersion of local repolarization responses are related to the severity of denervation, as well as the type of stimulation: neural (SS) versus humoral (NE). The differences may relate to the concentration of NE released.

Hepatic Fibrosis: Evaluation with Semiquantitative Contrast-enhanced CT

PURPOSE To evaluate the feasibility of using contrast material-enhanced computed tomographic (CT) measurements of hepatic fractional extracellular space (fECS) and macromolecular contrast material (MMCM) uptake to measure severity of liver fibrosis. MATERIALS AND METHODS All procedures were approved by and executed in accordance with University of California, San Francisco, institutional animal care and use committee regulations. Twenty-one rats that received intragastric CCl(4) for 0-12 weeks were imaged with respiratory-gated micro-CT by using both a conventional contrast material and a novel iodinated MMCM. Histopathologic hepatic fibrosis was graded qualitatively by using the Ishak fibrosis score and quantitatively by using morphometry of the fibrosis area. Hepatic fECS and MMCM uptake were calculated for each examination and correlated with histopathologic findings by using uni- and multivariate linear regressions. RESULTS Ishak fibrosis scores ranged from a baseline of 0 in untreated animals to a maximum of 5. Histopathologic liver fibrosis area increased from 0.46% to 3.5% over the same interval. Strong correlations were seen between conventional contrast-enhanced CT measurements of fECS and both the Ishak fibrosis scores (R(2) = 0.751, P < .001) and the fibrosis area (R(2) = 0.801, P < .001). Strong negative correlations were observed between uptake of MMCM in the liver and Ishak fibrosis scores (R(2) = 0.827, P < .001), as well as between uptake of MMCM in the liver and fibrosis area (R(2) = 0.643, P = .001). Multivariate linear regression analysis showed a trend toward independence for fECS and MMCM uptake in the prediction of Ishak fibrosis scores, with an R(2) value of 0.86 (P = .081 and P = .033, respectively). CONCLUSION Contrast-enhanced CT measurements of fECS and MMCM uptake are individually capable of being used to estimate the degree of early hepatic fibrosis in a rat model. SUPPLEMENTAL MATERIAL http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112452/-/DC1.

Telomeric transgenes are silenced in adult mouse tissues and embryo fibroblasts but are expressed in embryonic stem cells.

In addition to their role in protecting the ends of chromosomes, telomeres also influence the expression of adjacent genes, a process called telomere‐position effect. We previously reported that the neo and HSV‐tk transgenes located adjacent to telomeres in mouse embryonic stem cells are initially expressed at low levels and then become gradually silenced upon passage in culture through a process involving DNA methylation. We also reported extensive DNA methylation in these telomeric transgenes in three different tissues isolated from mice generated from one of these embryonic stem cell clones. In the present study, we demonstrate that embryo fibroblasts isolated from two different mouse strains show extensive DNA methylation and silencing of the telomeric transgenes. Consistent with this observation, we also demonstrate little or no detectable expression of the HSV‐tk telomeric transgene in somatic tissues using whole body imaging. In contrast, both telomeric transgenes are expressed at low levels and have little DNA methylation in embryonic stem cell lines isolated from these same mouse strains. Our results demonstrate that telomere‐position effect in mammalian cells can be observed either as a low level of expression in embryonic stem cells in the preimplantation embryo or as complete silencing and DNA methylation in differentiated cells and somatic tissues. This pattern of expression of the telomeric transgenes demonstrates that subtelomeric regions, like much of the genome, are epigenetically reprogrammed in the preimplantation embryo, a process that has been proposed to be important in early embryonic development.

The effect of internalizing human single chain antibody fragment on liposome targeting to epithelioid and sarcomatoid mesothelioma

Immunoliposomes (ILs) anchored with internalizing human antibodies capable of targeting all subtypes of mesothelioma can be useful for targeted imaging and therapy of this malignant disease. The objectives of this study were to evaluate both the in vitro and in vivo tumor targeted internalization of novel internalizing human single chain antibody (scFv) anchored ILs on both epithelioid (M28) and sarcomatoid (VAMT-1) subtypes of human mesothelioma. ILs were prepared by post-insertion of mesothelioma-targeting human scFv (M1) onto preformed liposomes and radiolabeled with (111)In ((111)In-IL-M1), along with control non-targeted liposomes ((111)In-CL). Incubation of (111)In-IL-M1 with M28, VAMT-1, and a control non-tumorigenic cell line (BPH-1) at 37 °C for 24 h revealed efficient binding and rapid internalization of ILs into both subtypes of tumor cells but not into the BPH-1 cells; internalization accounted for approximately 81-94% of total cell accumulation in mesothelioma cells compared to 37-55% in control cells. In tumor-bearing mice intravenous (i.v.) injection of (111)In-IL-M1 led to remarkable tumor accumulation: 4% and 4.7% injected dose per gram (% ID/g) for M28 and VAMT-1 tumors, respectively, 48 h after injection. Furthermore, tumor uptake of (111)In-IL-M1 in live xenograft animal models was verified by single photon emission computed tomography (SPECT/CT). In contrast, i.v. injection of (111)In-CL in tumor-bearing mice revealed very low uptake in both subtypes of mesothelioma, 48 h after injection. In conclusion, M1 scFv-anchored ILs showed selective tumor targeting and rapid internalization into both epithelioid and sarcomatoid subtypes of human mesothelioma, demonstrating its potential as a promising vector for enhanced tumor drug targeting.

Extravasated Contrast Material in Penetrating Abdominopelvic Trauma: Dual-Contrast Dual-Energy CT for Improved Diagnosis—Preliminary Results in an Animal Model

PURPOSE To compare the diagnostic performance of dual-energy (DE) computed tomography (CT) with two simultaneously administered contrast agents (hereafter, dual contrast) with that of conventional CT in the evaluation of the presence and source of extravasation in penetrating abdominopelvic trauma. MATERIALS AND METHODS Institutional animal care and use committee approval was obtained, and the study was performed in accordance with National Institutes of Health guidelines for the care and use of laboratory animals. Five rabbits with bowel trauma, vascular penetrating trauma, or both were imaged with simultaneous iodinated intravenous and bismuth subsalicylate enteric contrast material at DE CT. Four attending radiologists and six radiology residents without prior DE CT experience each evaluated 10 extraluminal collections to identify the vascular and/or enteric origin of extravasation and assess their level of diagnostic confidence, first with virtual monochromatic images simulating conventional CT and then with DE CT material decomposition attenuation maps. RESULTS Overall accuracy of identification of source of extravasation increased from 78% with conventional CT to 92% with DE CT (157 of 200 diagnoses vs 184 of 200 diagnoses, respectively; P < .001). Nine radiologists were more accurate with DE CT; one had no change. Mean confidence increased from 67% to 81% with DE CT (P < .001). CONCLUSION In a rabbit abdominopelvic trauma model, dual-contrast DE CT significantly increased accuracy and confidence in the diagnosis of vascular versus enteric extravasated contrast material.

Targeted In Vivo Extracellular Matrix Formation Promotes Neovascularization in a Rodent Model of Myocardial Infarction

Background The extracellular matrix plays an important role in tissue regeneration. We investigated whether extracellular matrix protein fragments could be targeted with antibodies to ischemically injured myocardium to promote angiogenesis and myocardial repair. Methodology/Principal Findings Four peptides, 2 derived from fibronectin and 2 derived from Type IV Collagen, were assessed for in vitro and in vivo tendencies for angiogenesis. Three of the four peptides—Hep I, Hep III, RGD—were identified and shown to increase endothelial cell attachment, proliferation, migration and cell activation in vitro. By chemically conjugating these peptides to an anti-myosin heavy chain antibody, the peptides could be administered intravenously and specifically targeted to the site of the myocardial infarction. When administered into Sprague-Dawley rats that underwent ischemia-reperfusion myocardial infarction, these peptides produced statistically significantly higher levels of angiogenesis and arteriogenesis 6 weeks post treatment. Conclusions/Significance We demonstrated that antibody-targeted ECM-derived peptides alone can be used to sufficiently alter the extracellular matrix microenvironment to induce a dramatic angiogenic response in the myocardial infarct area. Our results indicate a potentially new non-invasive strategy for repairing damaged tissue, as well as a novel tool for investigating in vivo cell biology.

Novel Human Single Chain Antibody Fragments That Are Rapidly Internalizing Effectively Target Epithelioid and Sarcomatoid Mesotheliomas

Human antibodies targeting all subtypes of mesothelioma could be useful to image and treat this deadly disease. Here we report tumor targeting of a novel internalizing human single chain antibody fragment (scFv) labeled with (⁹⁹m)Tc ((⁹⁹m)Tc-M40) in murine models of mesothelioma of both epithelioid (M28) and sarcomatoid (VAMT-1) origins. (⁹⁹m)Tc-M40 was taken up rapidly and specifically by both subtype tumor cells in vitro, with 68% to 92% internalized within 1 hour. The specificity of binding was evidenced by blocking (up to 95%) with 10-fold excess of unlabeled M40. In animal studies, tumors of both subtypes were clearly visualized by SPECT/CT as early as 1 hour postinjection of (⁹⁹m)Tc-M40. Tumor uptake measured as percent of injected dose per gram tissue (%ID/g) at 3 hours was 4.38 and 5.84 for M28 and VAMT-1 tumors, respectively, significantly greater than all organs or tissues studied (liver, 2.62%ID/g; other organs or tissues <1.7%ID/g), except the kidneys (130.7%ID/g), giving tumor-to-blood ratios of 5:1 and 7:1 and tumor-to-muscle ratios of 45:1 and 60:1, for M28 and VAMT-1, respectively. The target-mediated uptake was confirmed by a nearly 70% reduction in tumor activity following administration of 10-fold excess of unlabeled scFv. Taken together, these results indicate that M40 can rapidly and specifically target epithelioid and sarcomatoid tumor cells, demonstrating the potential of this agent as a versatile targeting ligand for imaging and therapy of all subtypes of mesothelioma.

In vivo comparison of tantalum, tungsten, and bismuth enteric contrast agents to complement intravenous iodine for double‐contrast dual‐energy CT of the bowel

To assess the ability of dual-energy CT (DECT) to separate intravenous contrast of bowel wall from intraluminal contrast, we scanned 16 rabbits on a clinical DECT scanner: n = 3 using only iodinated intravenous contrast, and n = 13 double-contrast enhanced scans using iodinated intravenous contrast and experimental enteric non-iodinated contrast agents in the bowel lumen (five bismuth, four tungsten, and four tantalum based). Representative image pairs from conventional CT images and DECT iodine density maps of small bowel (116 pairs from 232 images) were viewed by four abdominal imaging attending radiologists to independently score each comparison pair on a visual analog scale (-100 to +100%) for (1) preference in small bowel wall visualization and (2) preference in completeness of intraluminal enteric contrast subtraction. Median small bowel wall visualization was scored 39 and 42 percentage points (95% CI 30-44% and 36-45%, both p < 0.001) higher for double-contrast DECT than for conventional CT with enteric tungsten and tantalum contrast, respectively. Median small bowel wall visualization for double-contrast DECT was scored 29 and 35 percentage points (95% CI 20-35% and 33-39%, both p < 0.001) higher with enteric tungsten and tantalum, respectively, than with bismuth contrast. Median completeness of intraluminal enteric contrast subtraction in double-contrast DECT iodine density maps was scored 28 and 29 percentage points (95% CI 15-31% and 28-33%, both p < 0.001) higher with enteric tungsten and tantalum, respectively, than with bismuth contrast. Results suggest that in vivo double-contrast DECT with iodinated intravenous and either tantalum- or tungsten-based enteric contrast provides better visualization of small bowel than conventional CT. Copyright