Tc-99m Radiolabeled Peptide p5 + 14 is an Effective Probe for SPECT Imaging of Systemic Amyloidosis

Purpose

Systemic peripheral amyloidosis is a rare disease in which misfolded proteins deposit in various organs. We have previously developed I-124 labeled peptide p5?+?14 as a tracer for positron emission tomography imaging of amyloid in patients. In this report, we now document the labeling efficiency, bioactivity, and stability of Tc-99m labeled p5?+?14 for single-photon emission computed tomography (SPECT) imaging of amyloidosis, validated in a mouse model of systemic amyloidosis.

Procedures

Radiochemical yield, purity, and biological activity of [^99mTc]p5?+?14 were documented by instant thin-layer chromatography (ITLC), SDS-PAGE and a quantitative amyloid fibril pulldown assay. The efficacy and stability were documented in serum amyloid protein A (AA) amyloid-bearing or wild-type (WT) control mice imaged with SPECT/X-ray computed tomography (CT) at two time points. The uptake and retention of [^99mTc]p5?+?14 in hepatosplenic amyloid was evaluated using region of interest (ROI) and tissue counting measurements.

Results

Tc-99m p5?+?14 was produced with a radiochemical yield of 75 % with greater than 90 % purity and biological activity comparable to that of radioiodinated peptide. AA amyloid was visualized by SPECT/CT imaging with specific uptake seen in amyloid-laden organs at levels ～5 folds higher than in healthy mice. ROI analyses of decay-corrected SPECT/CT images showed <20 % loss of radiolabel from the 1 to 4 h imaging time points. Biodistribution data confirmed the specificity of the probe accumulation by amyloid-laden organs as compared to non-diseased tissues.

Conclusion

[^99mTc]p5?+?14 is a specific and stable radiotracer for systemic amyloid in mice and may provide a convenient and inexpensive alternative to imaging of peripheral amyloidosis in patients.

     

<Emphasis Type="Italic">In vivo</Emphasis> Biodistribution of Radiolabeled Acoustic Protein Nanostructures

Purpose

Contrast-enhanced ultrasound plays an expanding role in oncology, but its applicability to molecular imaging is hindered by a lack of nanoscale contrast agents that can reach targets outside the vasculature. Gas vesicles (GVs)—a unique class of gas-filled protein nanostructures—have recently been introduced as a promising new class of ultrasound contrast agents that can potentially access the extravascular space and be modified for molecular targeting. The purpose of the present study is to determine the quantitative biodistribution of GVs, which is critical for their development as imaging agents.

Procedures

We use a novel bioorthogonal radiolabeling strategy to prepare technetium-99m-radiolabeled ([^99mTc])GVs in high radiochemical purity. We use single photon emission computed tomography (SPECT) and tissue counting to quantitatively assess GV biodistribution in mice.

Results

Twenty minutes following administration to mice, the SPECT biodistribution shows that 84 % of [^99mTc]GVs are taken up by the reticuloendothelial system (RES) and 13 % are found in the gall bladder and duodenum. Quantitative tissue counting shows that the uptake (mean ± SEM % of injected dose/organ) is 0.6 ± 0.2 for the gall bladder, 46.2 ± 3.1 for the liver, 1.91 ± 0.16 for the lungs, and 1.3 ± 0.3 for the spleen. Fluorescence imaging confirmed the presence of GVs in RES.

Conclusions

These results provide essential information for the development of GVs as targeted nanoscale imaging agents for ultrasound.

     

pH-Sensitive,Long-Circulating Liposomes as an Alternative Tool to Deliver Doxorubicin into Tumors: a Feasibility Animal Study

Purpose

Therapeutic agents used in chemotherapy have low specificity leading to undesired severe side effects. Hence, the development of drug delivery systems that improve drug specificity, such as liposome moieties, is an alternative to overcome chemotherapy limitations and increase antitumor efficacy. In this study, the biodistribution profile evaluation of pH-sensitive long-circulating liposomes (SpHL) containing [^99mTc]DOX in 4T1 tumor-bearing BALB/c mice is described.

Procedures

[^99mTc]DOX was radiolabeled by direct method. Liposomes were prepared and characterized. [^99mTc]DOX was encapsulated into liposomes by freezing and thawing. Circulation time for SpHL-[^99mTc]DOX was determined by measuring the blood activity from healthy animals. Biodistribution studies were carried out in tumor-bearing mice at 1, 4, and 24 h after injection.

Results

Blood levels of the SpHL-[^99mTc]DOX declined in a biphasic manner, with an α half-life of 14.1 min and β half-life of 129.0 min. High uptake was achieved in the liver and spleen, due to the macrophages captured. Moreover, tumor uptake was higher than control tissue, resulting in high tumor-to-muscle ratios, indicating higher specificity for the tumor area.

Conclusion

[^99mTc]DOX was successfully encapsulated in liposomes. Biodistribution indicated high tumor-to-muscle ratios in breast tumor-bearing BALB/c mice. In summary, these results showed the higher accumulation of SpHL-[^99mTc]DOX in the tumor area, suggesting selective delivery of doxorubicin into tumor.

     

Selective Imaging of Vascular Endothelial Growth Factor Receptor-1 and Receptor-2 in Atherosclerotic Lesions in Diabetic and Non-diabetic ApoE<Superscript>−/−</Superscript> Mice

Purpose

Plaque vulnerability is associated with inflammation and angiogenesis, processes that rely on vascular endothelial growth factor (VEGF) signaling via two receptors, VEGFR-1 and VEGFR-2. We have recently reported that enhanced uptake of scVEGF-PEG-DOTA/Tc-99m (scV/Tc) single photon emission computed tomography (SPECT) tracer that targets both VEGFR-1 and VEGFR-2, identifies accelerated atherosclerosis in diabetic relative to non-diabetic ApoE^?/? mice. Since VEGFR-1 and VEGFR-2 may play different roles in atherosclerotic plaques, we reasoned that selective imaging of each receptor can provide more detailed information on plaque biology.

Procedures

Recently described VEGFR-1 and VEGFR-2 selective mutants of scVEGF, named scVR1 and scVR2, were site-specifically derivatized with Tc-99m chelator DOTA via 3.4 kDa PEG linker, and their selectivity to the cognate receptors was confirmed in vitro. scVR1 and scVR2 conjugates were radiolabeled with Tc-99m to specific activity of 110 ± 11 MBq/nmol, yielding tracers named scVR1/Tc and scVR2/Tc. 34–40 week old diabetic and age-matched non-diabetic ApoE^?/? mice were injected with tracers, 2–3 h later injected with x-ray computed tomography (CT) contrast agent and underwent hybrid SPECT/CT imaging. Tracer uptake, localized to proximal aorta and brachiocephalic vessels, was quantified as %ID from. Tracer uptake was also quantified as %ID/g from gamma counting of harvested plaques. Harvested atherosclerotic arterial tissue was used for immunofluorescent analyses of VEGFR-1 and VEGFR-2 and various lineage-specific markers.

Results

Focal, receptor-mediated uptake in proximal aorta and brachiocephalic vessels was detected for both scVR1/Tc and scVR2/Tc tracers. Uptake of scVR1/Tc and scVR2/Tc was efficiently inhibited only by “cold” proteins of the same receptor selectivity. Tracer uptake in this area, expressed as %ID, was higher in diabetic vs. non- diabetic mice for scVR1/Tc (p = 0.01) but not for scVR2/Tc. Immunofluorescent analysis revealed enhanced VEGFR-1 prevalence in and around plaque area in diabetic mice.

Conclusions

Selective VEGFR-1 and VEGFR-2 imaging of atherosclerotic lesions may be useful to explore plaque biology and identify vulnerability.

     

Multimodal <Emphasis Type="Italic">In Vivo</Emphasis> Imaging of Tumorigenesis and Response to Chemotherapy in a Transgenic Mouse Model of Mammary Cancer

Purpose

Transgenic mice expressing the polyoma middle T oncoprotein (PyMT) in the mammary epithelium were explored by multimodal imaging to monitor longitudinally spontaneous tumor growth and response to chemotherapy.

Procedures

Positron emission tomography (PET) with 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) and 3'-deoxy-3'-[¹⁸F]fluorothymidine ([¹⁸F]FLT), single photon emission tomography (SPECT) with [^99mTc]TcO₄ ([^99mTc]TEC), X-ray computed tomography, and fluorescent confocal endomicroscopy (FCE) images were acquired during tumor progression in female PyMT mice. Imaging with [¹⁸F]FDG and [^99mTc]TEC was also performed in untreated, doxorubicin-treated, and docetaxel-treated PyMT mice. Total tumor volumes were quantified. Tumors were collected and macroscopic and histological examinations were performed.

Results

All PyMT mice developed multifocal tumors of the mammary epithelium that became palpable at 8 weeks of age (W8). Computed tomography (CT) detected tumors at W14, while a clear tumoral uptake of [^99mTc]TEC and [¹⁸F]FDG was present as early as W6 and W8, respectively. No contrast between mammary tumors and surrounding tissue was observed at any stage with [¹⁸F]FLT. FCE detected an angiogenic switch at W10. Lung metastases were not clearly evidenced by imaging. Doxorubicin and docetaxel treatments delayed tumor growth, as shown by [¹⁸F]FDG and [^99mTc]TEC, but tumor growth resumed upon treatment discontinuation. Tumor growth fitted an exponential model with time constant rates of 0.315, 0.145, and 0.212 week^?1 in untreated, doxorubicin, and docetaxel groups, respectively.

Conclusions

Molecular imaging of mammary tumors in PyMT is precocious, precise, and predictive. [¹⁸F]FDG-PET and [^99mTc]TEC SPECT monitor tumor response to chemotherapy.

     

A Comparison of [<Superscript>99m</Superscript>Tc]Duramycin and [<Superscript>99m</Superscript>Tc]Annexin V in SPECT/CT Imaging Atherosclerotic Plaques

Purpose

Apoptosis is a key factor in unstable plaques. The aim of this study is to evaluate the utility of visualizing atherosclerotic plaques with radiolabeled duramycin and Annexin V.

Procedures

ApoE^?/? mice were fed with a high-fat diet to develop atherosclerosis, C57 mice as a control. Using a routine conjugation protocol, highly pure [^99mTc]duramycin and [^99mTc]Annexin V were obtained, which were applied for in vitro cell assays of apoptosis and in vivo imaging of atherosclerotic plaques in the animal model. Oil Red O staining, TUNEL, hematoxylin-eosin (HE), and CD68 immunostaining were used to evaluate the deposition of lipids and presence of apoptotic macrophages in the lesions where focal intensity positively correlated with the uptake of both tracers.

Results

[^99mTc]duramycin and [^99mTc]Annexin V with a high radiochemical purity (97.13 ± 1.52 and 94.94 ± 0.65 %, respectively) and a well stability at room temperature were used. Apoptotic cells binding activity to [^99mTc]duramycin (Kd, 6.92 nM and Bmax, 56.04 mol/10¹⁹ cells) was significantly greater than [^99mTc]Annexin V (Kd, 12.63 nM and Bmax, 31.55 mol/10¹⁹ cells). Compared with [^99mTc]Annexin V, [^99mTc]duramycin bound avidly to atherosclerotic lesions with a higher plaque-to-background ratio (P/B was 8.23 ± 0.91 and 5.45 ± 0.48 at 20 weeks, 15.02 ± 0.23 and 12.14 ± 0.22 at 30 weeks). No plaques were found in C57 control mice. Furthermore, Oil Red O staining showed lipid deposition areas were significantly increased in ApoE^?/? mice at 20 and 30 weeks, and TUNEL and CD68 staining confirmed that the focal uptake of both tracers contained abundant apoptotic macrophages.

Conclusions

This stable, fast clearing, and highly specific [^99mTc]duramycin, therefore, can be useful for the quantification of vulnerable atherosclerotic plaques.

     

Specific Targeting of Atherosclerotic Plaques in ApoE<Superscript>−/−</Superscript> Mice Using a New Camelid sdAb Binding the Vulnerable Plaque Marker LOX-1

Purpose

Molecular imaging has the potential to provide quantitative information about specific biological aspects of developing atherosclerotic lesions. This requires the generation of reliable, highly specific plaque tracers. This study reports a new camelid single-domain antibody fragment (sdAb) targeting the Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a biomarker for the detection and molecular phenotyping of vulnerable atherosclerotic plaques.

Procedures

A camelid sdAb was generated and selected for high affinity binding to LOX-1. Ex vivo biodistribution and in vivo single photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies were performed in wild-type mice and in fat-fed atherosclerotic apolipoprotein E-deficient mice with ^99mTc-labeled sdAbs. Gamma-counting and autoradiography analyses were performed on dissected aorta segments with different degrees of plaque burden. The specificity of the LOX-1-targeting sdAb was evaluated by blocking with unlabeled sdAb or by comparison with a nontargeting ^99mTc-labeled control sdAb.

Results

We generated a sdAb binding LOX-1 with a K_D of 280 pM?±?62 pM affinity. After ^99mTc-labeling, the tracer had radiochemical purity higher then 99 % and retained specificity in in vitro binding studies. Tracer blood clearance was fast with concomitant high kidney retention. At 3 h after injection, uptake in tissues other than plaques was low and not different than background, suggesting a restricted expression pattern of LOX-1. Conversely, uptake in aortic segments increased with plaque content and was due to specific LOX-1 binding. In vivo SPECT/CT imaging 160 min after injection in atherosclerotic mice confirmed specific targeting of LOX-1-expressing aortic plaques.

Conclusions

The LOX-sdAb specifically targets LOX-1-expressing atherosclerotic plaques within hours after injection. The possibility to image LOX-1 rapidly after administration combined with the favourable biodistribution of a sdAb are beneficial for molecular phenotyping of atherosclerotic plaques and the generation of a future prognostic tracer.

     

SPECT Imaging with <Superscript>99m</Superscript>Tc-Labeled EGFR-Specific Nanobody for <Emphasis Type="Italic">In Vivo</Emphasis> Monitoring of EGFR Expression

Purpose

Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnostics and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody®, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice.

Methods

Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The Nanobody was then labeled with ^99mTc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of ^99mTc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU145) overexpression of EGFR.

Results

FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of ^99mTc-8B6 Nanobody is relatively fast (half-life, 1.5 h) and mainly via the kidneys. At 3 h postinjection, total kidney accumulation is high (46.6?±?0.9%IA) compared to total liver uptake (18.9?±?0.6%IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2?±?0.5%IA/cm³) of ^99mTc-8B6 Nanobody compared to DU145 xenografts (1.8?±?0.3%IA/cm³, p?

Conclusion

The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with ^99mTc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

     

Immunoglobulin G (IgG)-Based Imaging Probe Accumulates in M1 Macrophage-Infiltrated Atherosclerotic Plaques Independent of IgG Target Molecule Expression

Purpose

Vulnerable plaques are key factors for ischemic diseases. Thus, their precise detection is necessary for the diagnosis of such diseases. Immunoglobulin G (IgG)-based imaging probes have been developed for imaging biomolecules related to plaque formation for the diagnosis of atherosclerosis. However, IgG accumulates nonspecifically in atherosclerotic regions, and its accumulation mechanisms have not yet been clarified in detail. Therefore, we explored IgG accumulation mechanisms in atherosclerotic lesions and examined images of radiolabeled IgG for the diagnosis of atherosclerosis.

Procedures

Mouse IgG without specificity to biomolecules was labeled with technetium-99m via 6-hydrazinonicotinate to yield [^99mTc]IgG. ApoE^?/? or C57BL/6J mice were injected intravenously with [^99mTc]IgG, and their aortas were excised 24 h after injection. After radioactivity measurement, serial aortic sections were autoradiographically and histopathologically examined. RAW264.7 macrophages were polarized into M1 or M2 and then treated with [^99mTc]IgG. The radioactivities in the cells were measured after 1 h of incubation. [^99mTc]IgG uptake in M1 macrophages was also evaluated after the pretreatment with an anti-Fcγ receptor (FcγR) antibody. The expression levels of FcγRs in the cells were measured by western blot analysis.

Results

[^99mTc]IgG accumulation levels in the aortas were significantly higher in apoE^?/? mice than in C57BL/6J mice (5.1 ± 1.4 vs 2.8 ± 0.5 %ID/g, p < 0.05). Autoradiographic images showed that the accumulation areas highly correlated with the macrophage-infiltrated areas. M1 macrophages showed significantly higher levels of [^99mTc]IgG than M2 or M0 (nonpolarized) macrophages [2.2 ± 0.3 (M1) vs 0.5 ± 0.1 (M2), 0.4 ± 0.1 (M0) %dose/mg protein, p < 0.01] and higher expression levels of FcγRI and FcγRII. [^99mTc]IgG accumulation in M1 macrophages was suppressed by pretreatment with the anti-FcγR antibody [2.2 ± 0.3 (nonpretreatment) vs 1.2 ± 0.2 (pretreatment) %ID/mg protein, p < 0.01].

Conclusions

IgG accumulated in pro-inflammatory M1 macrophages via FcγRs in atherosclerotic lesions. Thus, the target biomolecule-independent imaging of active inflammation should be taken into account in the diagnosis of atherosclerosis using IgG-based probes.

     

Imaging CXCR4 Expression with <Superscript>99m</Superscript>Tc-Radiolabeled Small-Interference RNA in Experimental Human Breast Cancer Xenografts

Purpose

Noninvasive quantification of chemokine receptor 4 (CXCR4) expression could serve as a prognostic indicator and may be of value for the design of personalized therapies and posttreatment monitoring. The objective of the present study was to assess the use of ^99mTc-radiolabeled small-interference RNA (siRNA) targeting CXCR4 to detect CXCR4 expression in vivo.

Procedures

CXCR4 siRNAs were radiolabeled with ^99mTc using the bifunctional chelator hydrazinonicotinamide (HYNIC), and the labeling efficiency, specific activity and radiochemical purity were determined. The stability of the probe in serum was assessed by measuring its radiochemical purity and inhibitory activity by RT-PCR and western blotting. Biodistribution studies and static imaging were performed in MDA-MB-231 tumor-bearing mice.

Results

Radiochemical purity remained highly stable in PBS and fresh human serum at room temperature and at 37 °C. Radiolabeled siRNA1 showed strong inhibitory effects similar to those of unlabeled siRNA1 on both CXCR4 messenger RNA (mRNA) and protein in vitro. The excretion of the probe occurred mainly through the liver and kidneys. Tumors were clearly visualized at 1–10 h after injection of the probe, but not after injection of the control probe.

Conclusions

^99mTc-labeled CXCR4 siRNA1 shows tumor-specific accumulation and could be a promising strategy for the visualization of CXCR4 expression in human breast cancer.

     

700-nm Zwitterionic Near-Infrared Fluorophores for Dual-Channel Image-Guided Surgery

Purpose

The purpose of this study was to develop a family of 700-nm zwitterionic pentamethine indocyanine near-infrared fluorophores that would permit dual-channel image-guided surgery.

Procedures

Three complementary synthetic schemes were used to produce novel zwitterionic chemical structures. Physicochemical, optical, biodistribution, and clearance properties were compared to Cy5.5, a conventional pentamethine indocyanine now used for biomedical imaging.

Results

ZW700-1a, ZW700-1b, and ZW700-1c were synthesized, purified, and analyzed extensively in vitro and in vivo. All molecules had extinction coefficients ≥199,000 M^?1 cm^?1, emission ≥660 nm, and stability ≥99 % after 24 h in warm serum. In mice, rats, and pigs, ≥80 % of the injected dose was completely eliminated from the body via renal clearance within 4 h. Either alone or conjugated to a tumor targeting ligand, ZW700-1a permitted dual-channel, high SBR, and simultaneous imaging with 800-nm NIR fluorophores using the FLARE® imaging system.

Conclusions

Novel 700-nm zwitterionic NIR fluorophores enable dual-NIR image-guided surgery.

     

Evaluation of Brain Nuclear Medicine Imaging Tracers in a Murine Model of Sepsis-Associated Encephalopathy

Purpose

The purpose of this study was to evaluate a set of widely used nuclear medicine imaging agents as possible methods to study the early effects of systemic inflammation on the living brain in a mouse model of sepsis-associated encephalopathy (SAE). The lipopolysaccharide (LPS)-induced murine systemic inflammation model was selected as a model of SAE.

Procedures

C57BL/6 mice were used. A multimodal imaging protocol was carried out on each animal 4 h following the intravenous administration of LPS using the following tracers: [^99mTc][2,2-dimethyl-3-[(3E)-3-oxidoiminobutan-2-yl]azanidylpropyl]-[(3E)-3-hydroxyiminobutan-2-yl]azanide ([^99mTc]HMPAO) and ethyl-7-[¹²⁵I]iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([¹²⁵I]iomazenil) to measure brain perfusion and neuronal damage, respectively; 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) to measure cerebral glucose uptake. We assessed microglia activity on another group of mice using 2-[6-chloro-2-(4-[¹²⁵I]iodophenyl)-imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-methyl-acetamide ([¹²⁵I]CLINME). Radiotracer uptakes were measured in different brain regions and correlated. Microglia activity was also assessed using immunohistochemistry. Brain glutathione levels were measured to investigate oxidative stress.

Results

Significantly reduced perfusion values and significantly enhanced [¹⁸F]FDG and [¹²⁵I]CLINME uptake was measured in the LPS-treated group. Following perfusion compensation, enhanced [¹²⁵I]iomazenil uptake was measured in the LPS-treated group’s hippocampus and cerebellum. In this group, both [¹⁸F]FDG and [¹²⁵I]iomazenil uptake showed highly negative correlation to perfusion measured with ([^99mTc]HMPAO uptake in all brain regions. No significant differences were detected in brain glutathione levels between the groups. The CD45 and P2Y12 double-labeling immunohistochemistry showed widespread microglia activation in the LPS-treated group.

Conclusions

Our results suggest that [¹²⁵I]CLINME and [^99mTc]HMPAO SPECT can be used to detect microglia activation and brain hypoperfusion, respectively, in the early phase (4 h post injection) of systemic inflammation. We suspect that the enhancement of [¹⁸F]FDG and [¹²⁵I]iomazenil uptake in the LPS-treated group does not necessarily reflect neural hypermetabolism and the lack of neuronal damage. They are most likely caused by processes emerging during neuroinflammation, e.g., microglia activation and/or immune cell infiltration.

     

Low mechanical index contrast mode versus high mechanical index contrast mode: which is a more sensitive method for detecting Sonazoid microbubbles in the liver of normal subjects?

Purpose

Using the low mechanical index (MI) contrast mode and the high MI contrast mode of contrast-enhanced ultrasonography, we evaluated which method is more sensitive for detecting Sonazoid microbubbles in the liver of normal subjects.

Methods

Thirteen normal subjects received an intravenous bolus injection of 0.2 mL of Sonazoid. We defined the intensity difference as the intensity post-injection minus the intensity pre-injection. We evaluated the intensity difference at the portal vein using both the low MI (0.21–0.23) and the high MI (0.7–1.2) at 1 min, at every 10 min between 10 to 60 min, and at every 30 min between 60 to 300 min post-injection. The intensity difference at the liver parenchyma was also evaluated at eight points (1, 10, 30, 60, 120, 180, 240, and 300 min) using the low MI and at three points (1, 10, and 300 min) using the high MI.

Results

The intensity differences at the portal vein measured using high MI were significantly higher than those measured using the low MI at each point between 1 and 240 min (P < 0.01) and at 270 min post-injection (P < 0.05). The intensity differences at the liver parenchyma measured using the high MI were also significantly higher than those measured using the low MI at each time point (P < 0.01).

Conclusion

Compared with the low MI, the high MI is more sensitive for detecting Sonazoid microbubbles in the liver of normal subjects.

     

Optimization of a Labeling and Kit Preparation Method for Ga-68 Labeled DOTATATE,Using Cation Exchange Resin Purified Ga-68 Eluates Obtained from a Tin Dioxide <Superscript>68</Superscript>Ge/<Superscript>68</Superscript>Ga Generator

Purpose

The aim of this study was to optimize a radiolabeling method using cationic processed Ga-68 eluates from a SnO₂-based ⁶⁸Ge/⁶⁸Ga generator, followed by the development of DOTA-Tyr³-Thre⁸-octreotide (DOTATATE) kits.

Procedures

Diluted generator eluates were adsorbed on a SCX resin and desorbed with acidified 5 M NaCl solution. Optimized labeling conditions were determined by variation of pH, using 35 μg DOTATATE and sodium acetate buffer. DOTATATE kits were developed based on optimized radiolabeling conditions, were labeled, and evaluated.

Results

Optimized labeling conditions resulted in a radiolabeling efficiency of around 99 % and radiochemical yield of almost 85 %. Different kit preparation methods did not significantly influence the radiolabeling results. Kits were found to be stable over 3 months.

Conclusion

A labeling method using SCX-processed Ga-68 eluates was optimized. DOTATATE kits specifically for these SCX-processed Ga-68 eluates were successfully formulated. A post-labeling Sep-Pak C18 purification should be optional.

     

Normal Databases for the Relative Quantification of Myocardial Perfusion

Purpose of Review

Myocardial perfusion imaging (MPI) with SPECT is performed clinically worldwide to detect and monitor coronary artery disease (CAD). MPI allows an objective quantification of myocardial perfusion at stress and rest. This established technique relies on normal databases to compare patient scans against reference normal limits. In this review, we aim to introduce the process of MPI quantification with normal databases and describe the associated perfusion quantitative measures that are used.

Recent Findings

New equipment and new software reconstruction algorithms have been introduced, which require the development of new normal limits. The appearance and regional count variations of normal MPI scans may differ between these new scanners and standard Anger cameras. Therefore, these new systems may require the determination of new normal limits to achieve optimal accuracy in relative myocardial perfusion quantification. Accurate diagnostic and prognostic results rivaling those obtained by expert readers can be obtained by this widely used technique.

Summary

Throughout this review, we emphasize the importance of the normal databases and the need for specific databases relative to distinct imaging procedures. Use of appropriate normal limits allows optimal quantification of MPI by taking into account subtle image differences due to the hardware and software used, and the population studied.

     

Terbutaline lessens protein fluxes across the alveolo-capillary barrier during high-volume ventilation

Objective

To evaluate whether a β₂-adrenergic agonist may reduce acute alveolo-capillary barrier alterations during high-volume ventilation.

Design

Experimental study.

Setting

Animal research laboratory.

Subjects

A total of 48 male Wistar rats.

Interventions

A zone of alveolar flooding was produced by liquid instillation in a distal airway. Proteins in the instilled solution were traced with ^99mTc-albumin. ¹¹¹In, which binds to transferrin, was injected into the systemic circulation. Terbutaline was administered in the instilled solution or intra-peritoneally. Conventional ventilation was applied for 30?min followed by different ventilation strategies for 90?min: conventional ventilation, high-volume ventilation with or without 6?cmH₂O PEEP.

Measurements and main results

Protein fluxes across the alveolar and microvascular barriers were evaluated by scintigraphy. High-volume ventilation resulted in immediate leakage of ^99mTc-albumin from alveolar spaces and increased pulmonary uptake of systemic ¹¹¹In-transferrin. Terbutaline in the instilled solution and PEEP lessened alveolar ^99mTc-albumin leakage and pulmonary ¹¹¹In-transferrin uptake due to high-volume ventilation, whereas terbutaline given intra-peritoneally only lessened ¹¹¹In-transferrin uptake. Terbutaline in the instilled solution also lessened the increase in lung wet-to-dry weight ratio due to high-volume ventilation.

Conclusions

Terbutaline reduces protein fluxes across the alveolar epithelial and pulmonary microvascular barriers during high-volume ventilation in vivo. The route of administration may be important.

     

Effect of synbiotic therapy on the incidence of ventilator associated pneumonia in critically ill patients: a randomised,double-blind,placebo-controlled trial

Objective

To investigate the effect of enteral Synbiotic 2000 FORTE^® (a mixture of lactic acid bacteria and fibre) on the incidence of ventilator associated pneumonia (VAP) in critically ill patients.

Design

Prospective, randomised, double blind, placebo controlled trial.

Setting

Tertiary referral centre, general Adult Intensive Care Unit (ICU).

Patients and participants

259 enterally fed patients requiring mechanical ventilation for 48 h or more were enrolled.

Intervention

All patients were enterally fed as per a standard protocol and randomly assigned to receive either synbiotic 2000 FORTE^® (twice a day) or a cellulose-based placebo for a maximum of 28 days.

Measurements and results

Treatment group (n = 130) was well matched with placebo group (n = 129) for age (mean 49.5 and 50 years, respectively) and APACHE II score (median 17 for both). Oropharyngeal microbial flora and colonisation rates were unaffected by synbiotics. The overall incidence of VAP was lower than anticipated (11.2%) and no statistical difference was demonstrated between groups receiving synbiotic and placebo in the incidence of VAP (9 and 13%, P = 0.42), VAP rate per 1,000 ventilator days (13 and 14.6, P = 0.91) or hospital mortality (27 and 33%, P = 0.39), respectively.

Conclusions

Enteral administration of Synbiotic 2000 FORTE^® has no statistically significant impact on the incidence of VAP in critically ill patients.

     

Imaging VEGF Receptors and α<Subscript>v</Subscript>β<Subscript>3</Subscript> Integrins in a Mouse Hindlimb Ischemia Model of Peripheral Arterial Disease

Purpose

To compare targeted imaging of vascular endothelial growth factor (VEGF) receptors vs. α_vβ₃ integrins in a mouse hindlimb ischemia model of peripheral artery disease.

Procedures

Male wild-type (WT) C57BL/6 mice (8- to 10-week old) (n?=?24) underwent left femoral artery ligation. The right leg served as control. Five days later, mice were injected with either VEGF receptor targeting [^99mTc]DOTA-PEG-scVEGF ([^99mTc]scV) (n?=?8) or with α_vβ₃-targeting tracer [^99mTc]HYNIC-cycloRGD ([^99mTc]RGD) (n?=?8) and underwent single photon emission computed tomography (SPECT) x-ray computed tomography imaging. To assess non-specific [^99mTc]scV uptake, six additional mice received a mixture of [^99mTc]scV and 30-fold excess of targeting protein, scVEGF. Tracer uptake as %ID was measured using volumetric regions encompassing the hindlimb muscles and as %ID/g from harvested limb muscles. Double and triple immunofluorescent analysis on tissue sections established localization of α_vβ₃, VEGFR-1, VEGFR-2, as well as certain cell lineage markers.

Results

Tracer uptake, as %ID/g, was higher in ligated limbs of mice injected with [^99mTc]scV compared to ligated hindlimbs in mice injected with [^99mTc]RGD (p?=?0.02). The ratio of tracer uptake for ligated/control hindlimb was borderline higher for [^99mTc]scV than for [^99mTc]RGD (p?=?0.06). Immunofluorescent analysis showed higher prevalence of VEGFR-1, VEGFR-2, and α_vβ₃, in damaged vs. undamaged hindlimb tissue, but with little co-localization of these markers. Double immunofluorescent staining showed partial co-localization of VEGFR-1, VEGFR-2, and α_vβ_3, with endothelial cell marker FVIII, but not with CD31. Immunostaining for VEGFR-1 and VEGFR-2 additionally co-localized with lineage markers for endothelial progenitor cell and monocytes/macrophages, with a more diverse pattern of co-localization for VEGFR-2.

Conclusion

In a mouse hindlimb ischemia model of peripheral artery disease, [^99mTc]scV SPECT tracer-targeting VEGF receptors showed a more robust signal than [^99mTc]RGD tracer-targeting α_vβ₃. Immunofluorescent analysis suggests that uptake of [^99mTc]scV and [^99mTc]RGD in damaged tissue is due to non-overlapping cell populations and reflects different dynamic processes and that enhanced uptake of [^99mTc]scV may be due to the presence of VEGF receptors on additional cell types.

     

Examining the Impact of Rehospitalization on Healthcare Cost of Myocardial Infarction Patients in Beijing: A Retrospective Observational Study

Introduction

To examine the impact of rehospitalization on the healthcare expenditure of myocardial infarction (MI) patients in Beijing.

Methods

Retrospective data of MI patients were retrieved from the Beijing Medical Insurance Database, an administrative database of social medical reimbursement activities for the urban population in Beijing, China. Ten percent of patients diagnosed with MI from January 1 to December 31, 2012 were randomly selected and their first hospitalization was considered as the index event. Their hospital utilization after the index event was extracted till September 30, 2013. Rehospitalization was defined as an event of hospital admission due to the same diagnosis and with a time interval of at least 14 days from the most recent admission. The healthcare cost of patients was analyzed, including inpatient cost and outpatient cost. Patients’ demographic characteristics, co-morbidities, and length of hospital stay were also collected from the database.

Results

Of the 1235 MI patients identified, 335 (mean age of 66.14 ± 15.04 years; 84.18% males) had rehospitalization. The rate of MI recurrence was 27.13%. The annual healthcare expense was significantly higher for MI patients with rehospitalization compared to MI patients without rehospitalization (99,920.43 ± 84,113.52 CNY vs. 58,877.89 ± 93,942.90 CNY; P < 0.001). The significant positive predictors of incurring healthcare expenditure were male gender, age 45 years old or more, length of stay at first hospital admission, whether having rehospitalization (yes/no), co-morbidity (heart failure and diabetes), and admission to a tertiary hospital at first hospital admission.

Conclusion

There was a high risk of MI recurrence for patients in Beijing. The annual healthcare cost of MI patients with rehospitalization was significantly higher than MI patients without rehospitalization. Male patients of 45 years old or more with heart failure and diabetes are likely to incur higher healthcare expenditure.