The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo.

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is great interest in using lytic bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of intravenously administered bacteriophage in uninfected mice. A single dose of LPS-5, an antipseudomonal bacteriophage recently used in human clinical trials, was administered intravenously to both wild-type BALB/c and neutropenic ICR mice. Phage concentrations were assessed in peripheral blood and spleen at 0.5, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance is only minimally affected by neutropenia. Indeed, the half-life of phages in blood in BALB/c and ICR mice is 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that substantial functional inactivation of circulating phages occurs over time. These data indicate that circulating factors, but not neutrophils, inactivate intravenously administered phages.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings.

Immunomodulation Therapy Using Tolerogenic Macrophages in a Rodent Model of Pulmonary Hypertension.

Inflammation plays a major role in the pathogenesis of pulmonary hypertension (PH). We sought to investigate the effects of a cell-based immunomodulation in a dysimmune model of PH. PH was induced in athymic nude rats using semaxinib (Su group, n = 6). Tolerogenic macrophages (toM) were generated from monocyte isolation and then injected either the day before semaxinib injection (Prevention group, n = 6) or 3 weeks after (Reversion group, n = 6). Six athymic nude rats were used as controls. In vivo trafficking of toM was investigated with bioluminescence imaging showing that toM were mainly located into the lungs until 48 h after injection. Right ventricular (RV) end-systolic pressure and RV systolic function were assessed at 4 weeks using echocardiography. Morphometric analysis and RNA sequencing of the lungs were realized at 4 weeks. Rats treated with toM (Prevention and Reversion groups) had a significantly lower RV end-systolic pressure at 4 weeks (respectively, 25 ± 8 and 30 ± 6 mmHg vs. 67 ± 9 mmHg, P < 0.001), while RV systolic dysfunction was observed in Su and Reversion groups. Mean medial wall thickness of small arterioles was lower in Prevention and Reversion groups compared with the Su group (respectively, 10.9% ± 0.8% and 16.4% ± 1.3% vs. 28.2% ± 2.1%, P < 0.001). Similarly, cardiomyocyte area was decreased in rats treated with toM (150 ± 18 and 160 ± 86 µm2 vs. 279 ± 50 µm2, P < 0.001). A trend toward upregulation of genes involved in pulmonary arterial hypertension pathobiology was found in Su rats, while KCNK3 was significantly downregulated (fold-change = 9.8, P < 0.001). Injection of toM was associated with a less severe phenotype of PH in rats exposed to angioproliferative stress. Preserved expression of KCNK3 may explain the protective effect of toM.

A novel roadmap connecting the 1H-MRS total choline resonance to all hallmarks of cancer following targeted therapy.

This review describes a cellular adaptive stress signalling roadmap connecting the 1H magnetic resonance spectroscopy (MRS) total choline peak at 3.2 ppm (tCho) to cancer response after targeted therapy (TT). Recent research on cell signalling, tCho metabolism, and TT of cancer has been retrospectively re-examined. Signalling research describes how the unfolded protein response (UPR), a major stress signalling network, transduces, regulates, and rewires the total membrane turnover in different cancer hallmarks after a TT stress. In particular, the UPR signalling maintains or increases total membrane turnover in all pro-survival hallmarks, whilst dramatically decreases turnover during apoptosis, a pro-death hallmark. Recent research depicts the TT-induced stress as a crucial event responsible for interrupting UPR pro-survival pathways, leading to an UPR-mediated cell death. The 1H-MRS tCho resonance represents the total mobile precursors and products during the enzymatic modification of phosphatidylcholine membrane abundance. The tCho profile represents a biomarker that noninvasively monitors TT-induced enzymatic changes in total membrane turnover in a wide variety of existing and new anticancer treatments targeting specific layers of the UPR signalling network. Our overview strongly suggests further evaluating and validating the 1H-MRS tCho peak as a powerful noninvasive imaging biomarker of cancer response in TT clinical trials.

Molecular Imaging of Apoptosis in Atherosclerosis by Targeting Cell Membrane Phospholipid Asymmetry.

BACKGROUND: Apoptosis in atherosclerotic lesions contributes to plaque vulnerability by lipid core enlargement and fibrous cap attenuation. Apoptosis is associated with exteriorization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell membrane. Although PS-avid radiolabeled annexin-V has been employed for molecular imaging of high-risk plaques, PE-targeted imaging in atherosclerosis has not been studied. OBJECTIVES: This study sought to evaluate the feasibility of molecular imaging with PE-avid radiolabeled duramycin in experimental atherosclerotic lesions in a rabbit model and compare duramycin targeting with radiolabeled annexin-V. METHODS: Of the 27 rabbits, 21 were fed high-cholesterol, high-fat diet for 16 weeks. Nine of the 21 rabbits received 99mTc-duramycin (test group), 6 received 99mTc-linear duramycin (duramycin without PE-binding capability, negative radiotracer control group), and 6 received 99mTc-annexin-V for radionuclide imaging. The remaining normal chow-fed 6 animals (disease control group) received 99mTc-duramycin. In vivo microSPECT/microCT imaging was performed, and the aortas were explanted for ex vivo imaging and for histological characterization of atherosclerosis. RESULTS: A significantly higher duramycin uptake was observed in the test group compared with that of disease control and negative radiotracer control animals; duramycin uptake was also significantly higher than the annexin-V uptake. Quantitative duramycin uptake, represented as the square root of percent injected dose per cm (√ID/cm) of abdominal aorta was >2-fold higher in atherosclerotic lesions in test group (0.08 ± 0.01%) than in comparable regions of disease control animals (0.039 ± 0.0061%, p = 3.70·10-8). Mean annexin uptake (0.060 ± 0.010%) was significantly lower than duramycin (p = 0.001). Duramycin uptake corresponded to the lesion severity and macrophage burden. The radiation burden to the kidneys was substantially lower with duramycin (0.49% ID/g) than annexin (5.48% ID/g; p = 4.00·10-4). CONCLUSIONS: Radiolabeled duramycin localizes in lipid-rich areas with high concentration of apoptotic macrophages in the experimental atherosclerosis model. Duramycin uptake in atherosclerotic lesions was significantly greater than annexin-V uptake and produced significantly lower radiation burden to nontarget organs.

Deep-learning-assisted diagnosis for knee magnetic resonance imaging: Development and retrospective validation of MRNet.

BACKGROUND: Magnetic resonance imaging (MRI) of the knee is the preferred method for diagnosing knee injuries. However, interpretation of knee MRI is time-intensive and subject to diagnostic error and variability. An automated system for interpreting knee MRI could prioritize high-risk patients and assist clinicians in making diagnoses. Deep learning methods, in being able to automatically learn layers of features, are well suited for modeling the complex relationships between medical images and their interpretations. In this study we developed a deep learning model for detecting general abnormalities and specific diagnoses (anterior cruciate ligament [ACL] tears and meniscal tears) on knee MRI exams. We then measured the effect of providing the model's predictions to clinical experts during interpretation. METHODS AND FINDINGS: Our dataset consisted of 1,370 knee MRI exams performed at Stanford University Medical Center between January 1, 2001, and December 31, 2012 (mean age 38.0 years; 569 [41.5%] female patients). The majority vote of 3 musculoskeletal radiologists established reference standard labels on an internal validation set of 120 exams. We developed MRNet, a convolutional neural network for classifying MRI series and combined predictions from 3 series per exam using logistic regression. In detecting abnormalities, ACL tears, and meniscal tears, this model achieved area under the receiver operating characteristic curve (AUC) values of 0.937 (95% CI 0.895, 0.980), 0.965 (95% CI 0.938, 0.993), and 0.847 (95% CI 0.780, 0.914), respectively, on the internal validation set. We also obtained a public dataset of 917 exams with sagittal T1-weighted series and labels for ACL injury from Clinical Hospital Centre Rijeka, Croatia. On the external validation set of 183 exams, the MRNet trained on Stanford sagittal T2-weighted series achieved an AUC of 0.824 (95% CI 0.757, 0.892) in the detection of ACL injuries with no additional training, while an MRNet trained on the rest of the external data achieved an AUC of 0.911 (95% CI 0.864, 0.958). We additionally measured the specificity, sensitivity, and accuracy of 9 clinical experts (7 board-certified general radiologists and 2 orthopedic surgeons) on the internal validation set both with and without model assistance. Using a 2-sided Pearson's chi-squared test with adjustment for multiple comparisons, we found no significant differences between the performance of the model and that of unassisted general radiologists in detecting abnormalities. General radiologists achieved significantly higher sensitivity in detecting ACL tears (p-value = 0.002; q-value = 0.019) and significantly higher specificity in detecting meniscal tears (p-value = 0.003; q-value = 0.019). Using a 1-tailed t test on the change in performance metrics, we found that providing model predictions significantly increased clinical experts' specificity in identifying ACL tears (p-value < 0.001; q-value = 0.006). The primary limitations of our study include lack of surgical ground truth and the small size of the panel of clinical experts. CONCLUSIONS: Our deep learning model can rapidly generate accurate clinical pathology classifications of knee MRI exams from both internal and external datasets. Moreover, our results support the assertion that deep learning models can improve the performance of clinical experts during medical imaging interpretation. Further research is needed to validate the model prospectively and to determine its utility in the clinical setting.

Deep learning for chest radiograph diagnosis: A retrospective comparison of the CheXNeXt algorithm to practicing radiologists.

BACKGROUND: Chest radiograph interpretation is critical for the detection of thoracic diseases, including tuberculosis and lung cancer, which affect millions of people worldwide each year. This time-consuming task typically requires expert radiologists to read the images, leading to fatigue-based diagnostic error and lack of diagnostic expertise in areas of the world where radiologists are not available. Recently, deep learning approaches have been able to achieve expert-level performance in medical image interpretation tasks, powered by large network architectures and fueled by the emergence of large labeled datasets. The purpose of this study is to investigate the performance of a deep learning algorithm on the detection of pathologies in chest radiographs compared with practicing radiologists. METHODS AND FINDINGS: We developed CheXNeXt, a convolutional neural network to concurrently detect the presence of 14 different pathologies, including pneumonia, pleural effusion, pulmonary masses, and nodules in frontal-view chest radiographs. CheXNeXt was trained and internally validated on the ChestX-ray8 dataset, with a held-out validation set consisting of 420 images, sampled to contain at least 50 cases of each of the original pathology labels. On this validation set, the majority vote of a panel of 3 board-certified cardiothoracic specialist radiologists served as reference standard. We compared CheXNeXt's discriminative performance on the validation set to the performance of 9 radiologists using the area under the receiver operating characteristic curve (AUC). The radiologists included 6 board-certified radiologists (average experience 12 years, range 4-28 years) and 3 senior radiology residents, from 3 academic institutions. We found that CheXNeXt achieved radiologist-level performance on 11 pathologies and did not achieve radiologist-level performance on 3 pathologies. The radiologists achieved statistically significantly higher AUC performance on cardiomegaly, emphysema, and hiatal hernia, with AUCs of 0.888 (95% confidence interval [CI] 0.863-0.910), 0.911 (95% CI 0.866-0.947), and 0.985 (95% CI 0.974-0.991), respectively, whereas CheXNeXt's AUCs were 0.831 (95% CI 0.790-0.870), 0.704 (95% CI 0.567-0.833), and 0.851 (95% CI 0.785-0.909), respectively. CheXNeXt performed better than radiologists in detecting atelectasis, with an AUC of 0.862 (95% CI 0.825-0.895), statistically significantly higher than radiologists' AUC of 0.808 (95% CI 0.777-0.838); there were no statistically significant differences in AUCs for the other 10 pathologies. The average time to interpret the 420 images in the validation set was substantially longer for the radiologists (240 minutes) than for CheXNeXt (1.5 minutes). The main limitations of our study are that neither CheXNeXt nor the radiologists were permitted to use patient history or review prior examinations and that evaluation was limited to a dataset from a single institution. CONCLUSIONS: In this study, we developed and validated a deep learning algorithm that classified clinically important abnormalities in chest radiographs at a performance level comparable to practicing radiologists. Once tested prospectively in clinical settings, the algorithm could have the potential to expand patient access to chest radiograph diagnostics.

Molecular Imaging of Apoptosis in Ischemia Reperfusion Injury With Radiolabeled Duramycin Targeting Phosphatidylethanolamine: Effective Target Uptake and Reduced Nontarget Organ Radiation Burden.

OBJECTIVES: The purpose of this study was to evaluate the feasibility of imaging apoptosis in experimental ischemia-reperfusion model by technetium-99m (99mTc)-labeled Duramycin, and compare it to an established tracer, 99mTc-labeled Annexin-V, which has a relative disadvantage of high radiation burden to nontarget organs. BACKGROUND: During apoptosis, the cell membrane phospholipids-phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed and can be targeted by Annexin-V and Duramycin, respectively, for in vivo imaging. Identification of a reversible cell death process should permit therapeutic intervention to help reduce myocyte loss and left ventricle dysfunction. METHODS: In a 40-min left coronary artery ischemia-reperfusion model in 17 rabbits, 7 mCi of 99mTc-labeled Duramycin (n = 10), 99mTc-linear Duramycin (a negative tracer control; n = 3), or 99mTc-Annexin-V (a positive tracer-control; n = 4) were intravenously administered 30 min after reperfusion. Of the 10 Duramycin group animals, 4 animals were treated with an antiapoptotic agent, minocycline at the time of reperfusion. In vivo and ex vivo micro-single-photon emission computed tomography (µSPECT) and micro-computed tomography (µCT) imaging was performed 3 h after reperfusion, followed by quantitative assessment of tracer uptake and pathological characterization. Fluorescent Duramycin and Annexin-V were injected in 4 rats to visualize colocalization in infarct areas in a 40-min left coronary artery occlusion and 30-min reperfusion model. RESULTS: Intense uptake of Duramycin and Annexin-V was observed in the apical (infarcted) areas. The percent injected dose per gram uptake of Duramycin in apical region (0.751 ± 0.262%) was significantly higher than remote area in same animals (0.045 ± 0.029%; p < 0.01). Duramycin uptake was insignificantly lower than Annexin-V uptake (1.23 ± 0.304%; p > 0.01) but demonstrated substantially lower radiation burden to kidneys (0.358 ± 0.210% vs. 1.58 ± 0.316%, respectively; p < 0.001). Fluorescence studies with Duramycin and Annexin V showed colocalization in infarct areas. Minocycline treatment substantially resolved Duramycin uptake (0.354% ± 0.0624%; p < 0.01). CONCLUSIONS: Duramycin is similarly effective in imaging apoptotic cell death as Annexin-V with lower nontarget organ radiation. Clinical feasibility of apoptosis imaging with a PE-seeking tracer should be tested.

Cardioprotective Effects of HSP72 Administration on Ischemia-Reperfusion Injury.

BACKGROUND: Although early reperfusion is the most desirable intervention after ischemic myocardial insult, it may add to damage through oxidative stress. OBJECTIVES: This study investigated the cardioprotective effects of a single intravenous dose of heat shock protein-72 (HSP72) coupled to a single-chain variable fragment (Fv) of monoclonal antibody 3E10 (3E10Fv) in a rabbit ischemia-reperfusion model. The Fv facilitates rapid transport of HSP72 into cells, even with intact membranes. METHODS: A left coronary artery occlusion (40 min) reperfusion (3 h) model was used in 31 rabbits. Of these, 12 rabbits received the fusion protein (Fv-HSP72) intravenously. The remaining 19 control rabbits received a molar equivalent of 3E10Fv alone (n = 6), HSP72 alone (n = 6), or phosphate-buffered saline (n = 7). Serial echocardiographic examinations were performed to assess left ventricular function before and after reperfusion. Micro-single-photon emission computed tomography imaging of 99mTc-labeled annexin-V was performed with micro-computed tomography scanning to characterize apoptotic damage in vivo, followed by gamma counting of the excised myocardial specimens to quantify cell death. Histopathological characterization of the myocardial tissue and sequential cardiac troponin I measurements were also undertaken. RESULTS: Myocardial annexin-V uptake was 43% lower in the area at risk (p = 0.0003) in Fv-HSP72-treated rabbits compared with control animals receiving HSP72 or 3E10Fv alone. During reperfusion, troponin I release was 42% lower and the echocardiographic left ventricular ejection fraction 27% higher in the Fv-HSP72-treated group compared with control animals. Histopathological analyses confirmed penetration of 3E10Fv-containing molecules into cardiomyocytes in vivo, and treatment with Fv-HSP72 showed fewer apoptotic nuclei compared with control rabbits. CONCLUSIONS: Single-dose administration of Fv-HSP72 fusion protein at the time of reperfusion reduced myocardial apoptosis by almost one-half and improved left ventricular functional recovery after myocardial ischemia-reperfusion injury in rabbits. It might have potential to serve as an adjunct to early reperfusion in the management of myocardial infarction.

Targeted scVEGF/(177)Lu radiopharmaceutical inhibits growth of metastases and can be effectively combined with chemotherapy.

BACKGROUND: scVEGF/(177)Lu is a novel radiopharmaceutical targeted by recombinant single-chain (sc) derivative of vascular endothelial growth factor (VEGF) that binds to and is internalized by vascular endothelial growth factor receptors (VEGFR). scVEGF/(177)Lu potential as adjuvant and neoadjuvant anti-angiogenic therapy was assessed in metastatic and orthotopic mouse models of triple-negative breast cancer. METHODS: Metastatic lesions in Balb/c mice were established by intracardiac injection of luciferase-expressing 4T1luc mouse breast carcinoma cells. Mice with metastatic lesions received single intravenous (i.v.) injection of well-tolerated dose of scVEGF/(177)Lu (7.4 MBq/mouse) at day 8 after 4T1luc cell injection. Primary orthotopic breast tumors in immunodeficient mice were established by injecting luciferase-expressing MDA231luc human breast carcinoma cells into mammary fat pad. Tumor-bearing mice were treated with single injections of scVEGF/(177)Lu (7.4 MBq/mouse, i.v), or liposomal doxorubicin (Doxil, 1 mg doxorubicin per kg, i.v.), or with a combination of Doxil and scVEGF/(177)Lu given at the same doses, but two hours apart. "Cold" scVEGF-targeting conjugate was included in controls and in Doxil alone group. The effects of treatments were defined by bioluminescent imaging (BLI), computed tomography (CT), computed microtomography (microCT), measurements of primary tumor growth, and immunohistochemical analysis. RESULTS: In metastatic model, adjuvant treatment with scVEGF/(177)Lu decreased overall metastatic burden and improved survival. In orthotopic primary tumor model, a combination of Doxil and scVEGF/(177)Lu was more efficient in tumor growth inhibition than each treatment alone. scVEGF/(177)Lu treatment decreased immunostaining for VEGFR-1, VEGFR-2, and pro-tumorigenic M2-type macrophage marker CD206. CONCLUSIONS: Selective targeting of VEGFR with well-tolerated doses of scVEGF/(177)Lu is effective in metastatic and primary breast cancer models and can be combined with chemotherapy. As high level of VEGFR expression is a common feature in a variety of cancers, targeted delivery of (177)Lu for specific receptor-mediated uptake warrants further exploration.

(99m)Tc-Annexin A5 quantification of apoptotic tumor response: a systematic review and meta-analysis of clinical imaging trials.

PURPOSE: (99m)Tc-Annexin A5 has been used as a molecular imaging probe for the visualization, characterization and measurement of apoptosis. In an effort to define the quantitative (99m)Tc-annexin A5 uptake criteria that best predict tumor response to treatment, we performed a systematic review and meta-analysis of the results of all clinical imaging trials found in the literature or publicly available databases. METHODS: Included in this review were 17 clinical trials investigating quantitative (99m)Tc-annexin A5 (qAnx5) imaging using different parameters in cancer patients before and after the first course of chemotherapy and/or radiation therapy. Qualitative assessment of the clinical studies for diagnostic accuracy was performed using the QUADAS-2 criteria. Of these studies, five prospective single-center clinical trials (92 patients in total) were included in the meta-analysis after exclusion of one multicenter clinical trial due to heterogeneity. Pooled positive predictive values (PPV) and pooled negative predictive values (NPV) (with 95% CI) were calculated using Meta-Disc software version 1.4. RESULTS: Absolute quantification and/or relative quantification of (99m)Tc-annexin A5 uptake were performed at baseline and after the start of treatment. Various quantitative parameters have been used for the calculation of (99m)Tc-annexin A5 tumor uptake and delta (Δ) tumor changes post-treatment compared to baseline including: tumor-to-background ratio (TBR), ΔTBR, tumor-to-noise ratio, relative tumor ratio (TR), ΔTR, standardized tumor uptake ratio (STU), ΔSTU, maximum count per pixel within the tumor volume (Cmax), Cmax%, absolute ΔU and percentage (ΔU%), maximum ΔU counts, semiquantitative visual scoring, percent injected dose (%ID) and %ID/cm(3). Clinical trials investigating qAnx5 imaging have included patients with lung cancer, lymphoma, breast cancer, head and neck cancer and other less common tumor types. In two phase I/II single-center clinical trials, an increase of ≥25% in uptake following treatment was considered a significant threshold for an apoptotic tumor response (partial response, complete response). In three other phase I/II clinical trials, increases of ≥28%, ≥42% and ≥47% in uptake following treatment were found to be the mean cut-off levels in responders. In a phase II/III multicenter clinical trial, an increase of ≥23% in uptake following treatment was found to be the minimum cut-off level for a tumor response. In one clinical trial, no significant difference in (99m)Tc-annexin A5 uptake in terms of %ID was found in healthy tissues after chemotherapy compared to baseline. In two other clinical trials, intraobserver and interobserver measurements of (99m)Tc-annexin A5 tumor uptake were found to be reproducible (mean difference <5%, kappa = 0.90 and 0.82, respectively) and to be highly correlated with treatment outcome (Spearman r = 0.99, p < 0.0001). The meta-analysis demonstrated a pooled positive PPV of 100% (95% CI 92 - 100%) and a pooled NPV of 70% (95% CI 55 - 82%) for prediction of a tumor response after the first course of chemotherapy and/or radiotherapy in terms of ΔU%. In a symmetric sROC analysis, the AUC was 0.919 and the Q* index was 85.21 %. CONCLUSION: Quantitative (99m)Tc-annexin A5 imaging has been investigated in clinical trials for the assessment of apoptotic tumor responses. This meta-analysis showed a high pooled PPV and a moderate pooled NPV with ΔU cut-off values ranging between 20% and 30%. Standardization of quantification and harmonization of results are required for high-quality clinical research. A standardized uptake value score (SUV, ΔSUV) using quantitative SPECT/CT imaging may be a promising approach to the simple, reproducible and semiquantitative assessment of apoptotic tumor changes.

Enhanced caspase activity contributes to aortic wall remodeling and early aneurysm development in a murine model of Marfan syndrome.

OBJECTIVE: Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome. APPROACH AND RESULTS: Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs. CONCLUSIONS: Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome.

[99mTc]Annexin V-128 SPECT Monitoring of Splenic and Disseminated Listeriosis in Mice: a Model of Imaging Sepsis.

PURPOSE: Here, we evaluate [(99m)Tc]annexin V-128, an in vivo marker of apoptosis, for single photon emission computed tomography (SPECT) imaging of localization and antibiotic treatment of disseminated bacterial infection, using a well-described mouse model that employs bioluminescent Listeria monocytogenes. PROCEDURES: Sixteen groups of five mice in six separate experiments were infected with bioluminescent Listeria, and in vivo bioluminescence imaging (BLI) was performed each day, to assess the location and extent of infection and response to antibiotics. [(99m)Tc]annexin V-128 was then injected for SPECT imaging, and the two sets of images were correlated and validated. RESULTS: Signals from BLI and [(99m)Tc]annexin V-128 SPECT co-localized within the spleen and other organs including bone marrow, intestine, nasopharynx, and brain. Decreases in [(99m)Tc]annexin V-128 uptake and BLI signal within the spleen directly reflected the reduction of bacterial infection by ampicillin treatment. CONCLUSIONS: Tc-99m-Annexin V-128 uptake as observed by SPECT allowed for the detection of systemic listeriosis and ampicillin treatment in mice. [(99m)Tc]annexin V-128 should be further explored for the assessment of bacterial spread and antibiotic efficacy in patients with disseminated bacterial infection.

Attenuation of apoptosis by telmisartan in atherosclerotic plaques of apolipoprotein E-/- mice: evaluation using technetium 99m-annexin A5.

Technetium 99m (99mTc)-annexin A5, a marker of ongoing apoptosis, is supposed to be useful in the detection of metabolically active atheroma. The aim of this study was to determine the potential of 99mTc-annexin A5 for evaluating the therapeutic effects of an angiotensin II receptor type 1 blocker (ARB) (telmisartan) on atherosclerosis. Male apolipoprotein E-/- mice were divided into telmisartan-treated (3 mg/kg/d, n  =  10) and control (n  =  10) groups. After 16 to 21 weeks of treatment, 99mTc-annexin A5 was injected and cryostat sections of aortic tissues (n  =  10-12/aorta) were prepared. The 99mTc-annexin A5 accumulation level in the plaques was evaluated by autoradiography. Serial sections of the plaques were histologically examined to identify the lesion phenotypes (normal vessels, early lesions, atheromatous lesions, and fibrotic lesions), plaque size, macrophage infiltration levels, and lipid deposition levels. Telmisartan treatment significantly decreased the plaque size (0.05 ± 0.05 vs 0.11 ± 0.08, mm2), macrophage infiltration level (0.02 ± 0.02 vs 0.03 ± 0.02, mm2), lipid deposition level (0.01 ± 0.01 vs 0.02 ± 0.02, mm2), and 99mTc-annexin A5 accumulation level (1.30 ± 1.09 vs 2.15 ± 1.91, × 10-6/g). 99mTc-annexin A5 accumulation levels in the plaques positively correlated with macrophage infiltration (r  =  .69, p < .05) and lipid deposition (r  =  .66, p < .05) levels. Apoptosis imaging with 99mTc-annexin A5 may be useful for evaluating the therapeutic effects of ARBs on atherosclerosis.

Synchrotron infrared imaging of advanced glycation endproducts (AGEs) in cardiac tissue from mice fed high glycemic diets.

Recent research findings correlate an increased risk for dieases such as diabetes, macular degeneration and cardiovascular disease (CVD) with diets that rapidly raise the blood sugar levels; these diets are known as high glycemic index (GI) diets which include white breads, sodas and sweet deserts. Lower glycemia diets are usually rich in fruits, non-starchy vegetables and whole grain products. The goal of our study was to compare and contrast the effects of a low vs. high glycemic diet using the biochemical composition and microstructure of the heart. The improved spatial resolution and signal-to-noise for SR-FTIR obtained through the coupling of the bright synchrotron infrared photon source to an infrared spectral microscope enabled the molecular-level observation of diet-related changes within unfixed fresh frozen histologic sections of mouse cardiac tissue. High and low glycemic index (GI) diets were started at the age of five-months and continued for one year, with the diets only differing in their starch distribution (high GI diet = 100% amylopectin versus low GI diet = 30% amylopectin/70% amylose). Serial cryosections of cardiac tissue for SR-FTIR imaging alternated with adjacent hematoxylin and eosin (H&E) stained sections allowed not only fine-scale chemical analyses of glycogen and glycolipid accumulation along a vein as well as protein glycation hotspots co-localizing with collagen cold spots but also the tracking of morphological differences occurring in tandem with these chemical changes. As a result of the bright synchrotron infrared photon source coupling, we were able to provide significant molecular evidence for a positive correlation between protein glycation and collagen degradation in our mouse model. Our results bring a new insight not only to the effects of long-term GI dietary practices of the public but also to the molecular and chemical foundation behind the cardiovascular disease pathogenesis commonly seen in diabetic patients.

Recent advances in the molecular imaging of programmed cell death: Part II--non-probe-based MRI, ultrasound, and optical clinical imaging techniques.

There is much that can be done to detect apoptosis and other forms of cell death with existing clinical modalities including ultrasound, MRI, and optical imaging without the need for current or new intravenous contrast agents. We will discuss how these widely available imaging technologies can readily be applied to the imaging of apoptosis in patients undergoing chemotherapy or radiation treatment. The limiting factor of course is the lack of knowledge of the optimal times after the start of treatment for the most accurate assessment of apoptosis and necrosis with each modality and specific technique. It is hoped that imaging studies that systematically look at treatment response can soon be performed to address these issues.

Recent advances in the molecular imaging of programmed cell death: part I--pathophysiology and radiotracers.

In humans, apoptosis (programmed cell death) is the most common form of cell death after necrosis. Apoptosis is a series of genetically preprogrammed biochemical and morphologic energy-requiring events that, after a specific external or internal stimulus, results in the physiologic disappearance of a cell via its self-disintegration and packaging of its contents into membrane vesicles called apoptotic bodies. Apoptotic bodies can readily be ingested, with their nutrients and even organelles recycled by neighboring cells or phagocytes without local inflammation. In contrast, necrosis is characterized by the primary loss of plasma membrane integrity and the uncontrolled release of a cell's contents, often causing local inflammation, tissue damage, and scarring. Alternate forms of cell death also exist, associated with specific molecular mechanisms involving enzymes, organelles, genes, external stimuli, or blockade of normal cell proliferation. In this review we will briefly outline the molecular mechanisms of apoptosis that can be imaged with radiotracers now under development.

Brain uptake of Tc99m-HMPAO correlates with clinical response to the novel redox modulating agent EPI-743 in patients with mitochondrial disease.

While decreased ATP production and redox imbalance are central to mitochondrial disease pathogenesis, efforts to develop effective treatments have been hampered by the lack of imaging markers of oxidative stress. In this study we wished to determine if Tc99m-HMPAO, a SPECT imaging marker of cerebral blood flow and glutathione/protein thiol content, could be used to monitor the effect(s) of EPI-743, an oral redox modulating, para-benzoquinone based therapeutic for mitochondrial disease. We hypothesized that treatment changes in HMPAO uptake would be inversely proportional to changes in oxidative stress within the brain and directly correlate to clinical response to EPI-743 therapy. Twenty-two patients with mitochondrial disease were treated with EPI-743. Each underwent baseline and 3-month Tc99m-HMPAO SPECT scanning along with clinical/neurologic evaluations. Diseases treated were: Leigh syndrome (n=7), polymerase γ deficiency (n=5), MELAS (n=5), Friedreich ataxia (n=2), Kearns-Sayre syndrome, Pearson syndrome, and mtDNA depletion syndrome. Neuro-anatomic uptake analyses of HMPAO were performed with NeuroGam™ (Segami Corp.) statistical software and clinical response was assessed by the Newcastle Paediatric Mitochondrial Disease Scale or Newcastle Mitochondrial Disease Adult Scale depending on patient age. For all 22 patients there was a significant linear correlation between the change in cerebellar uptake of HMPAO and the improvement in Newcastle score (r=0.623, **p=0.00161). The MELAS subgroup showed a significant relationship of whole brain uptake (n=5, r=0.917, *p=0.028) to improvement in Newcastle score. We conclude that Tc99m-HMPAO SPECT scanning has promise as a general marker of the oxidative state of the brain and its response to redox modulating therapies. Further studies will be needed to confirm these findings in a more homogenous study population.

Quantitative determination of apoptosis of pancreatic ß-cells in a murine model of type 1 diabetes mellitus.

UNLABELLED: Type 1 diabetes mellitus is characterized by a significant deficit in pancreatic ß-cell mass, presumably caused by ß-cell apoptosis. We investigated the incidence of ß-cell apoptosis in streptozotocin-treated mice and nonobese diabetic (NOD) mice with (99m)Tc-annexin A5. METHODS: Vehicle-treated mice, streptozotocin-treated mice, and NOD mice at the ages of 5, 9, 16, and 20 wk (5-8 mice per group) were injected with (99m)Tc-annexin A5 and sacrificed 6 h later for autoradiography, and the regional (99m)Tc-annexin A5 level in the pancreas was evaluated. Pancreatic islets were identified by insulin immunohistochemical staining, and apoptotic cells were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The (99m)Tc-annexin A5 level in pancreatic islets was expressed as the percentage injected dose per area of pancreatic islets and normalized by animal body weight (%ID × 10(6)/mm(2)/kg). The level of apoptotic cells in pancreatic islets was expressed as the number of TUNEL-positive cells per area of pancreatic islets (cells/mm(2)). RESULTS: The (99m)Tc-annexin A5 accumulation level was significantly higher (2.5 ± 0.7 vs. 0.7 ± 0.1 %ID × 10(6)/mm(2)/kg, P < 0.05) and the number of TUNEL-positive cells was significantly higher (1,170 ± 535 vs. 5 ± 6 cells/mm(2), P < 0.05) in the pancreatic islets of the streptozotocin-treated mice than in those of the vehicle-treated mice. The (99m)Tc-annexin A5 accumulation level was significantly higher (1.1 ± 0.4 vs. 0.5 ± 0.1 %ID × 10(6)/mm(2)/kg, P < 0.05) and the number of TUNEL-positive cells was significantly higher (152 ± 82 vs. 4 ± 9 cells/mm(2), P < 0.05) in the pancreatic islets of 16-wk-old NOD mice than in those of 5-wk-old NOD mice. In addition, the level of (99m)Tc-annexin A5 correlated with the number of TUNEL-positive cells in the pancreatic islets of the streptozotocin-treated mice (r = 0.821, P < 0.001) and NOD mice (r = 0.721, P < 0.001). CONCLUSION: There is significant islet cell apoptosis with (99m)Tc-annexin A5 accumulation in the pancreas of both streptozotocin and NOD mice.