Toward Ultrasound Molecular Imaging With Phase-Change Contrast Agents: An In Vitro Proof of Principle

Phase-change contrast agents (PCCAs), which normally consist of nanoscale or microscale droplets of liquid perfluorocarbons in an encapsulating shell, can be triggered to undergo a phase transition to the highly echogenic gaseous state upon the input of sufficient acoustic energy. As a result of the subsequent volumetric expansion, a number of unique applications have emerged that are not possible with traditional ultrasound microbubble contrast agents. Although many studies have explored the therapeutic aspects of the PCCA platform, few have examined the potential of PCCAs for molecular imaging purposes. In this study, we demonstrate a PCCA-based platform for molecular imaging using α_vβ₃-targeted nanoscale PCCAs composed of low-boiling-point perfluorocarbons. In vitro, nanoscale PCCAs adhered to target cells, could be activated and imaged with a clinical ultrasound system and produced a six-fold increase in image contrast compared with non-targeted control PCCAs and a greater than fifty-fold increase over baseline. Data suggest that low-boiling-point nanoscale PCCAs could enable future ultrasound-based molecular imaging techniques in both the vascular and extravascular spaces.     

Mast Cell Granule-Mediated Uptake of Low Density Lipoproteins by Macrophages: A Novel Carrier Mechanism Leading to the Formation of Foam Cells

Mast cells are present in the arterial intima, the site of atherogenesis. To gain insight into the possible role of mast cells in the formation of the cholesterol-loaded macrophage foam cells typical of both early and late atherosclerotic lesions, a model system was developed in which isolated rat serosal mast cells were incubated with mouse peritoneal macrophages in medium to which low-density lipoproteins (LDL) had been added. Stimulation of the mast cells was found to induce a 50-fold enhancement of LDL uptake by the macrophages, which concomitantly accumulated LDL-derived cholesterol. This process, called the “granule-mediated uptake of LDL”, involves the following steps: (i) exocytosis of the cytoplasmic granules of the mast cells, (ii) escape of soluble granule components, such as histamine and a fraction of the granule heparin proteoglycans into the medium, leaving granule remnants consisting of neutral proteases embedded in a heparin proteoglycan matrix, (iii) binding of LDL to binding sites on the glycosaminoglycan side chains of the heparin proteoglycan component of the granule remnants, (iv) proteolytic degradation of the bound LDL by the neutral proteases of the granule remnants, (v) fusion of degraded LDL particles on the surfaces of the granule remnants, and (vi) phagocytosis of the LDL-laden granule remnants by the macrophages. Simultaneously, the soluble heparin proteoglycans, to which no proteolytic enzymes are bound, interact with LDL with formation of insoluble complexes which are also phagocytosed by the macrophages. Finally, cholesterol derived from the granule remnant-bound LDL and the LDL-heparin proteoglycan complexes becomes esterified by the macrophages, with formation of macrophage foam cells. Experiments in vivo showed that granule remnants may also carry LDL into macrophages in the peritoneal cavity of the rat, where mast cells and macrophages coexist. The results suggest that mast cells play an active role in the intracellular cholesteryl ester deposition characteristic of human atherosclerotic lesions.     

Comparison Between Color Doppler Twinkling Artifact and Acoustic Shadowing for Renal Calculus Detection: An In Vitro Study

To assess the ability of the color Doppler twinkling artifact to detect renal stones relative to acoustic shadowing, we scanned seven uric acid calculi embedded in a tissue mimicking phantom and in sheep kidneys using a high frequency linear array and a standard curved linear array ultrasound scanheads (L12–5 and C5–2; Philips Ultrasound, Bothel, WA, USA). The stones were scanned in and out of focus. The scans were optimized for shadow formation in gray-scale imaging and for color twinkling in color Doppler imaging. The images were analyzed using Image J (http://rsb.info.nih.gov/ij/). We calculated the contrast to noise ratios (C/N) for the acoustic shadows and the color twinkling artifact compared with background. These measurements were then evaluated using a single factor analysis of variance (ANOVA) and paired two-tailed t tests. With these comparisons, the C/Ns for twinkling were significantly higher than for acoustic shadowing. On average, twinkling produced 19.2 dB greater C/Ns for stones in the phantom and 17.6 dB more for the stones in the kidneys. In addition, ANOVA showed that twinkling is resistant to focusing and scanning frequency differences. The results suggest that the twinkling artifact is a robust method for detecting the presence of renal calculi. The color signature is easier to detect than is acoustic shadowing. Twinkling may be relatively resistant to many of the problems that plague ultrasound examinations for renal stones, i.e., out-of-focus scans that might be caused by beam aberration effects due to patient body habitus. (E-mail: jrubin@med.umich.edu)     

制备多模态成像对比剂并用于联合治疗葡萄膜黑色素瘤:体外实验

目的 制备肿瘤微环境响应性超声/MRI/光学成像纳米粒(Ce6@TA-Fe),观察其性能、体外多模态成像及光热联合氧气增强光动力治疗体外葡萄膜黑色素瘤(UM)的效果.方法 以Ce6、单宁酸(TA)及FeCl3·6H2O制备Ce6@TA-Fe纳米粒,检测其理化性质,观察其体外多模态显像效果,评估光热联合光动力治疗对UM细...     

Arthroscopic Ultrasound Technique for Simultaneous Quantitative Assessment of Articular Cartilage and Subchondral Bone: An In Vitro and In Vivo Feasibility Study

Traditional arthroscopic examination is subjective and poorly reproducible. Recently, we introduced an arthroscopic ultrasound method for quantitative diagnostics of cartilage lesions. Here we describe our investigation of the feasibility of ultrasound arthroscopy for simultaneous measurements of articular cartilage and subchondral bone. Human osteochondral samples (n = 13) were imaged using a clinical 9-MHz ultrasound system. Ultrasound reflection coefficients (R, IRC), the ultrasound roughness index (URI) and the apparent integrated backscattering coefficient (AIB) were determined for both tissues. Mechanical testing, histologic analyses and micro-scale computed tomography imaging were the reference methods. Ultrasound arthroscopies were conducted on two patients. The ultrasound reflection coefficient correlated with the Mankin score and Young's modulus of cartilage (|r| > 0.56, p < 0.05). Ultrasound parameters (R, IRC, AIB) for subchondral bone correlated with the bone surface/volume ratio (|r| > 0.70, p < 0.05) and trabecular thickness (|r| > 0.59, p < 0.05). Furthermore, R and subchondral bone mineral density were significantly correlated (|r| > 0.65, p < 0.05). Arthroscopic ultrasound examination provided diagnostically valuable information on cartilage and subchondral bone in vivo.     

Long-Term Results of Nervous Tissue Alterations Caused by Epineurial Electrode Application: An Experimental Study in Rat Sciatic Nerve

In order to evaluate the long-term effects of epineurial electrode application for functional electrical stimulation (FES) the left sciatic nerve of seven rats was exposed. Four ring-shaped stainless steel wire electrodes were sutured to the epineurium of each nerve in the same manner as performed clinically for carrousel stimulation in man. The nerves were reexposed 1 year after implantation and the stimulation threshold to obtain a tetanic contraction in the lower limb was determined for each electrode. Afterwards the animals were sacrificed. The electrodes were excised and cross sections of the sciatic nerve directly at site of the electrodes, 2-mm proximal and 2-mm distal to them were harvested for histologic and planimetric assessment of nerve lesions. The area of damaged neural tissue was expressed as a percentage of the total cross-sectional area within the perineural sheath. The sciatic nerves of the right side served as controls. The values for the stimulation thresholds ranged between 0.1 and 1.0 mA (mean 0.43 mA). By morphometric examination five of seven nerves were seen altered, the altered areas captured between 1% and 4.8% of the total cross-sectional area of the nerves within the perineural sheath. Besides two specimens, all altered nerve segments exhibited distinct signs of nerve fiber regeneration. The clinical implications of the results for long-term electrical stimulation, such as phrenic pacing, are discussed.     

Vascularity Index of Laryngeal Cancer Derived from 3-D Ultrasound: A Predicting Factor for the in vivo Assessment of Cervical Lymph Node Status

To demonstrate whether a calculated vascularity index (VI) can predict metastases of cervical lymph nodes, the VI values of the primary tumors were obtained by using 3-D sonography in 87 subjects with laryngeal cancer confirmed by laryngoscope and biopsy. N-staging of the subjects was determined by pathological nodal harvesting. The relationship between the VI and pathological N-staging was evaluated by correlation coefficient. To test the accuracy of the VI for predicting cervical lymph node involvement, a receiver operating characteristic (ROC) curve was constructed, and the best operating point was determined by Youden's index. For comparison, 2-D sonography was applied to detect metastatic cervical lymph nodes. The accuracy, sensitivity and specificity of the VI, 2-D sonography and a combination of the two methods for diagnosis of metastatic cervical lymph nodes were compared. There was a positive linear correlation between the VI and pN-staging (r = 0.740, p < 0.001). The area under the ROC curve for the VI was 0.919. The best operating point of the VI was 4.4565, which derived higher sensitivity than that of 2-D sonography (95% vs. 81%, p = 0.031), but lower specificity (75% vs. 95%, p = 0.012). The combination of the two methods yielded a higher accuracy (97% vs. 85% and 89%, p = 0.002 and 0.016), a higher sensitivity to 2-D sonography (95% vs. 81%, p = 0.031) and a higher specificity to VI (98% vs. 75%, p = 0.002). The VI of laryngeal cancer can be a useful factor for predicting metastases of cervical lymph nodes. (E-mail: zjsts8@163.com)     

Ag85B/IL-2融合蛋白在体外对PBMC增殖及Th1型细胞因子分泌的影响

[目的]研究Ag85B/IL-2融合蛋白对人外周血单个核细胞（PBMC）增殖及Th1型细胞因子分泌的影响.[方法]MTT法测定不同浓度的Ag85B/IL-2融合蛋白在不同时相对人PBMC的增殖活性,ELISA法检测Ag85B/IL-2融合蛋白作用人PBMC不同时间后IL-12和IFN-γ的分泌水平,并与Ag85B和BCG作对比.[结果]以0.8 μg/ml Ag85B/IL-2融合蛋白刺激组的人PBMC增殖活性为最强,此组在各时段均显著高于空白对照组（P〈0.01,P〈0.05）;各浓度Ag85B/IL-2融合蛋白刺激48 h后的IL-12水平均显著高于空白对照组（P〈0.01,P〈0.05）,以0.8 μg/ml的浓度刺激PBMC 72 h时,IL-12的分泌达最高;0.8 μg/ml和1.6 μg/ml刺激组的IFN-γ浓度在各个时相点均显著高于空白对照组（P〈0.01,P〈0.05）.Ag85B/IL-2融合蛋白刺激PBMC 分泌IL-12和IFN-γ的作用,均显著强于Ag85B和BCG（P〈0.01）.[结论]Ag85B/IL-2融合蛋白具有促人PBMC增殖和Th1型细胞因子分泌的作用.     

Magnetic Resonance Imaging for the Exploitation of Bubble-Enhanced Heating by High-Intensity Focused Ultrasound: A Feasibility Study in ex Vivo Liver

Bubble-enhanced heating (BEH) may be exploited to improve the heating efficiency of high-intensity focused ultrasound in liver and to protect tissues located beyond the focal point. The objectives of this study, performed in ex vivo pig liver, were (i) to develop a method to determine the acoustic power threshold for induction of BEH from displacement images measured by magnetic resonance acoustic radiation force imaging (MR-ARFI), and (ii) to compare temperature distribution with MR thermometry for HIFU protocols with and without BEH. The acoustic threshold for generation of BEH was determined in ex vivo pig liver from MR-ARFI calibration curves of local tissue displacement resulting from sonication at different powers. Temperature distributions (MR thermometry) resulting from “conventional” sonications (20 W, 30 s) were compared with those from “composite” sonications performed at identical parameters, but after a HIFU burst pulse (0.5 s, acoustic power over the threshold for induction of BEH). Displacement images (MR-ARFI) were acquired between sonications to measure potential modifications of local tissue displacement associated with modifications of tissue acoustic characteristics induced by the burst HIFU pulse. The acoustic threshold for induction of BEH corresponded to a displacement amplitude of approximately 50 μm in ex vivo liver. The displacement and temperature images of the composite group exhibited a nearly spherical pattern, shifted approximately 4 mm toward the transducer, in contrast to elliptical shapes centered on the natural focal position for the conventional group. The gains in maximum temperature and displacement values were 1.5 and 2, and the full widths at half-maximum of the displacement data were 1.7 and 2.2 times larger than in the conventional group in directions perpendicular to ultrasound propagation axes. Combination of MR-ARFI and MR thermometry for calibration and exploitation of BEH appears to increase the efficiency and safety of HIFU treatment.     

Evidence for an Additional Metastatic Route: <Emphasis Type=Italic>In Vivo</Emphasis> Imaging of Cancer Cells in the Primo-Vascular System Around Tumors and Organs

Purpose  

Researchers have been studying the mechanisms by which metastasis can be prevented via blocking the hematogenous and the lymphatic routes for a long time now. However, metastasis is still the single most challenging obstacle for successful cancer management. In a new twist that may require some retooling of this established approach, we investigated the hypothesis that tumor metastases can occur via an independent fluid-conducting system called the primo-vascular system.     

Observational Study of Intravenous versus Oral Corticosteroids for Acute Asthma: An Example of Confounding by Severity

Objectives: To demonstrate the problem of "confounding by severity" using the example of intravenous (IV) versus oral corticosteroids for the treatment of acute asthma. Double-blind, randomized trials have clearly demonstrated that IV and oral corticosteroids have comparable efficacy. Methods: Using a standardized protocol, 64 emergency departments enrolled 1,847 patients, aged 18–54 years, with acute asthma. Because route of corticosteroid therapy was not randomized, potential confounders of the association between corticosteroid route and hospital admission were controlled for by multivariate logistic regression and stratification. Results: Among the 1,193 patients, 383 (32%) received IV corticosteroids and 810 (68%) received oral corticosteroids. The two groups differed markedly at baseline, with patients receiving IV corticosteroids having more severe asthma. Overall, patients receiving IV corticosteroids were more likely to be admitted or experience a relapse event within 48 hours (51% vs. 19%; p < 0.001). On multivariate analysis, patients receiving IV corticosteroids remained more likely to be admitted or experience a relapse event within 48 hours (odds ratio = 2.6; 95% confidence interval = 1.2 to 6.0). Conclusions: In this observational study, patients with worse asthma exacerbations were more likely to receive IV corticosteroids as compared with oral corticosteroids. Although we controlled for many markers of asthma severity, we were unable to completely control for baseline differences between the IV and oral corticosteroid groups. Observational research continues to serve as an important tool for describing problems and for understanding many exposure–disease associations. For examining the impact of treatments on adverse outcomes, randomized trials are often required to avoid intractable confounding by severity.     

Adhesion of Activated Platelets to Endothelial Cells: Evidence for a GPIIbIIIa-dependent Bridging Mechanism and Novel Roles for Endothelial Intercellular Adhesion Molecule 1 (ICAM-1), αvβ3 Integrin, and GPIbα

Although it has been reported that activated platelets can adhere to intact endothelium, the receptors involved have not been fully characterized. Also, it is not clear whether activated platelets bind primarily to matrix proteins at sites of endothelial cell denudation or directly to endothelial cells. Thus, this study was designed to further clarify the mechanisms of activated platelet adhesion to endothelium. Unstimulated human umbilical vein endothelial cell (HUVEC) monolayers were incubated with washed, stained, and thrombin-activated human platelets. To exclude matrix involvement, HUVEC were harvested mechanically and platelet binding was measured by flow cytometry. Before the adhesion assay, platelets or HUVEC were treated with different receptor antagonists. Whereas blockade of platelet β₁ integrins, GPIbα, GPIV, P-selectin, and platelet-endothelial cell adhesion molecule (PECAM)-1 did not reduce platelet adhesion to HUVEC, blockade of platelet GPIIbIIIa by antibodies or Arg-Gly-Asp (RGD) peptides markedly decreased adhesion. Moreover, when platelets were treated with blocking antibodies to GPIIbIIIa-binding adhesive proteins, including fibrinogen and fibronectin, and von Willebrand factor (vWF), platelet binding was also reduced markedly. Addition of fibrinogen, fibronectin, or vWF further increased platelet adhesion, indicating that both endogenous platelet-exposed and exogenous adhesive proteins can participate in the binding process. Evaluation of the HUVEC receptors revealed predominant involvement of intercellular adhesion molecule (ICAM)-1 and α_vβ₃ integrin. Blockade of these two receptors by antibodies decreased platelet binding significantly. Also, there was evidence that a component of platelet adhesion was mediated by endothelial GPIbα. Blockade of β₁ integrins, E-selectin, P-selectin, PECAM-1, vascular cell adhesion molecule (VCAM)-1 and different matrix proteins on HUVEC did not affect platelet adhesion. In conclusion, we show that activated platelet binding to HUVEC monolayers is mediated by a GPIIbIIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1, α_vβ₃ integrin, and, to a lesser extent, GPIbα.     

Training the Trainer: An Educational Course for Training Pain Nursing Specialists Supported by the International Association for the Study of Pain (IASP)

To train pain nursing specialists through a pain education program, 20 nurses from six hospitals in Shanghai Province and seven in six provinces of China received the training of 2-month pain education and 4-month clinical practice. This nonrandomized pilot study examined the results of tests before and after the program, case report evaluations, future plan evaluations, clinical practice, and satisfaction questionnaire. After the program, the score of the test increased significantly compared with that before the program (44.1 ± 3.19; paired-sample t = 10.363; p < .0001). All of the participants thought that the program had broadened their vision, 19 (95%) thought that the program had raised the level of their theoretical knowledge in pain management, 17 (85%) thought that the program had improved their skills in clinical practice, and 15 (75%) thought that the program had played a role in enhancing their research abilities. Considering the whole program, most students (n = 17; 85%) were quite satisfied, and 3 (15%) were simply satisfied. By content analysis of the opening questions, we found that the participants had deeper and broader ideas about nurses' role and pain nursing specialists' responsibilities in pain management. The program improved nurses' attitudes, knowledge, and skills in pain management. The participants recognized pain nursing specialists' responsibilities in pain management more clearly.