Improved Detection of preclinical Colorectal Liver Metastases by High Resolution Ultrasound including Molecular Ultrasound Imaging using the targeted Contrast Agent BR55.

PURPOSE: Aim of the present study was to investigate the sensitivity of high resolution ultrasound (HRU), standard contrast-enhanced ultrasound (CEUS) and CEUS using a novel vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast agent for the detection of hepatic metastases in a mouse model of colorectal cancer using clinical standard technology. MATERIALS AND METHODS: The human colon cancer cell line HT29, transfected with luciferase cDNA for in vivo bioluminescence monitoring, was injected intrasplenically into CB17.SCID mice. Mice were monitored weekly by bioluminescence and after 2 and 4.5 weeks by HRU and CEUS. Contrast media (untargeted BR1, targeted BR55) was applied and digital cine loops from the arterial phase (15 - 45 sec), portal venous phase (50 - 120 s) and late phases (3 - 5 min, 1hour) of the whole liver were analyzed. Data were correlated with postmortem histopathology. RESULTS: Without contrast enhancement, lesions > 4 mm were reliably detected. After use of untargeted CEUS, lesions > 2 mm were reliably detected and enhanced rim vascularization and late-phase wash-out was shown. With BR55, lesions > 0.8 mm were reliably detected with excellent documentation of vascularization. A persistent contrast enhancement was seen > 30 min after injection. Contrast-enhancement patterns with BR55 significantly correlated with CD31 (R2 = 0.74) and VEGFR2-immunohistochemistry (R2 = 0.66). CONCLUSION: Detection of metastases by HRU and CEUS was earlier and more accurate than monitoring via bioluminescence. In vivo monitoring of hepatic micrometastases can thus be performed without prior modification of cancer cells using standard technology.

Efficacy of a therapeutic treatment using gas-filled microbubble-associated phospholipase A2 in a mouse model of honeybee venom allergy.

BACKGROUND: Venom immunotherapy is efficient to desensitize people suffering from insect sting allergies. However, the numerous injections required over several years and important risks of severe side reactions complicate the widespread use of immunotherapy. In the search for novel approaches to blunt the overwhelming pro-allergic Th2 response, we evaluated the therapeutic efficacy of a treatment based on a denatured form of the major allergen, phospholipase A2, associated with microbubbles (PLA2denat -MB) in a mouse model of honeybee venom allergy. METHODS: Antibodies measured by ELISA, T-cell responses assessed by CFSE-based proliferation assays and ELISA, and basophil degranulation were examined after PLA2denat -MB-based therapeutic treatment of sensitized mice. Mice were challenged with a lethal dose of PLA2 to evaluate protection against anaphylaxis. RESULTS: Therapeutic subcutaneous administration of two different PLA2denat -MB formulations, in contrast to PLA2denat alone, reduced allergic symptoms and protected all mice from anaphylaxis-mediated death after allergen challenge. At the functional level, the use of PLA2denat decreased IgE-mediated basophil degranulation as compared to the native form of the allergen. In comparison with PLA2denat alone, both PLA2denat -MB formulations decreased allergen-specific Th2 CD4 T-cell reactivity. At the mechanistic level, PLA2denat -MB containing 20% palmitic acid and PEG induced PLA2-specific IgA and increased Foxp3(+) Treg frequencies and TGF-ß production, whereas the formulation bearing 80% palmitic acid triggered the production of IFN-γ, IgG2a, and IgG3. CONCLUSIONS: In contrast to conventional PLA2 subcutaneous immunotherapy, the therapeutic administration of PLA2-MB treatment to mice that already had established allergy to PLA2 protects all subsequently challenged animals.

Prophylactic immunization of mice with phospholipase A2-loaded gas-filled microbubbles is protective against Th2-mediated honeybee venom allergy.

BACKGROUND: People suffering from honeybee venom allergy can be treated by venom immunotherapy, which consists in the subcutaneous injection of increasing doses of allergen extracts over a period of 3-5 years. Such a procedure is time-consuming, and the risks of severe side reactions are important. Approaches based on the use of novel adjuvants to blunt pro-allergic Th2-type immune responses represent a sound alternative. OBJECTIVES: In this study, we evaluated in a mouse model of honeybee venom allergy the protection induced by the prophylactic use of the major allergen phospholipase A2 (PLA2) associated with microbubbles (MB). METHODS: Antibody (Ab) and T cell responses, as detected by ELISA and CFSE-based proliferation assays, were first examined after prophylactic immunization of CBA/J mice with PLA2-MB, and second after sensitization with native PLA2. Mice were eventually challenged with a lethal dose of PLA2 to assess protection against anaphylaxis. RESULTS: Prophylactic immunization with PLA2-MB induced PLA2-specific IgG and IgA Ab, triggered the production of IFN-γ and IL-10 and the differentiation of PLA2-specific Foxp3(+) Treg. Immunized/sensitized mice displayed the following: (1) increased titres of potent blocking IgG1, IgG2a and IgG3 Ab, (2) both reduced allergen-specific T cell proliferation and Th2-type cytokine production and (3) elevated frequencies of specific Foxp3(+) Treg and increased production of TGF-ß, as compared to naïve/sensitized animals. Immunomodulation correlated with reduced signs of anaphylaxis after allergen challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Our data demonstrate the ability of PLA2-MB to prophylactically protect mice against subsequent sensitization and death-inducing PLA2 challenge for up to 4 months, revealing so far unravelled immunomodulatory properties of MB. These data, combined with the safe use of MB as contrast agents for in situ imaging in humans, render them an immunotherapeutic agent of great interest for further evaluation.

Sonothrombolysis: the contribution of stable and inertial cavitation to clot lysis.

Microbubble-mediated sonothrombolysis (STL) is a remarkable approach to vascular occlusion therapy. However, STL remains a complex process with multiple interactions between clot, ultrasound (US), microbubbles (MB) and thrombolytic drug. The aim of this study was to evaluate the ability of combining US and MB to degrade fibrin and, more specifically, to assess the roles of both stable (SC) and inertial (IC) cavitation. Human blood clots containing radiolabeled fibrin were exposed to different combinations of recombinant tissue plasminogen activator (rtPA), US (1 MHz) and phospholipid MB. Three acoustic pressures were tested: 200, 350 and 1,300 kPa (peak-negative pressure). Clot lysis was assessed by diameter loss and release of radioactive fibrin degradation products. The combination rtPA + US + MB clearly revealed that IC (1,300 kPa) was able to enhance fibrin degradation significantly (66.3 ± 1.8%) compared with rtPA alone (51.7 ± 2.0%, p < 0.001). However, SC failed to enhance fibrin degradation at an acoustic pressure of 200 kPa. At 350 kPa, a synergistic effect between rtPA and US + MB was observed with an absolute increase of 6% compared to rtPA alone (p < 0.001). Conversely, without rtPA, the combination of US + MB was unable to degrade the fibrin network (0.3 ± 0.1%, p > 0.05 vs. control), but induced a distinct loss of red blood cells throughout the entire thickness of the clot, implying that MB were able to penetrate and cavitate inside the clot.

Ultrasound contrast agents for ultrasound molecular imaging.

Ultrasound is a real-time imaging technique which is widely used in many clinical applications for its capacity to provide anatomic information with high spatial and temporal resolution. The advent of ultrasound contrast agents in combination with contrast-specific imaging modes has given access to perfusion assessments at an organ level, leading to an improved diagnostic accuracy. More recently, the development of biologically-targeted ultrasound contrast agents has expanded the role of ultrasound even further into molecular imaging applications. Ultrasound molecular imaging can be used to visualize the expression of intravascular markers, and to assess their local presence over time and/or during therapeutic treatment. Major applications are in the field of inflammation and neoangiogenesis due to the strictly intravascular presence of microbubbles. Various technologies have been investigated for attaching the targeting moiety to the shell from simple biotin-avidin constructs to more elaborated insertion within the shell through attachment to PEG residues. This important improvement has allowed a clinical translation of initial pre-clinical investigations, opening the way for an early detection and an accurate characterization of lesions in patients. The combination of anatomic, functional and molecular information/data provided by contrast ultrasound is a powerful tool which is still in its infancy due to the lack of agents suitable for clinical use. The advantages of ultrasound techniques combined with the molecular signature of lesions will represent a significant advance in imaging in the field of personalized medicine.

Perfusion quantification in contrast-enhanced ultrasound (CEUS)--ready for research projects and routine clinical use.

With contrast-enhanced ultrasound (CEUS) now established as a valuable imaging modality for many applications, a more specific demand has recently emerged for quantifying perfusion and using measured parameters as objective indicators for various disease states. However, CEUS perfusion quantification remains challenging and is not well integrated in daily clinical practice. The development of VueBox™ alleviates existing limitations and enables quantification in a standardized way. VueBox™ operates as an off-line software application, after dynamic contrast-enhanced ultrasound (DCE-US) is performed. It enables linearization of DICOM clips, assessment of perfusion using patented curve-fitting models, and generation of parametric images by synthesizing perfusion information at the pixel level using color coding. VueBox™ is compatible with most of the available ultrasound platforms (nonlinear contrast-enabled), has the ability to process both bolus and disruption-replenishment kinetics loops, allows analysis results and their context to be saved, and generates analysis reports automatically. Specific features have been added to VueBox™, such as fully automatic in-plane motion compensation and an easy-to-use clip editor. Processing time has been reduced as a result of parallel programming optimized for multi-core processors. A long list of perfusion parameters is available for each of the two administration modes to address all possible demands currently reported in the literature for diagnosis or treatment monitoring. In conclusion, VueBox™ is a valid and robust quantification tool to be used for standardizing perfusion quantification and to improve the reproducibility of results across centers.

A novel minimal invasive closed chest myocardial ischaemia reperfusion model in rhesus monkeys (Macaca mulatta): improved stability of cardiorespiratory parameters.

The aim of this study was to report the cardiorespiratory events observed during coronary artery occlusion and reperfusion in a minimally invasive closed chest myocardial occlusion-reperfusion model in rhesus monkeys. We hypothesized that a minimally invasive technique may lead to fewer cardiac arrhythmias and complications. Eight male rhesus macaques 10-15 kg and 10-15 years old were sedated with ketamine (2 mg/kg), midazolam (1.3 mg/kg), atropine (0.01 mg/kg) and buprenorphine 0.02 mg/kg intramuscularly. Etomidate 1-2 mg/kg was injected intravenously to allow tracheal intubation. Anaesthesia was maintained with isoflurane. Pulse oximetry, electrocardiogram (ECG), heart rate, mean arterial blood pressure (MAP), inspired isoflurane fractions (F(I)ISO) and core temperature were recorded every 10 min. The coronary artery occlusion was induced by a balloon-tipped catheter advanced via the femoral artery into the left anterior descending artery and inflated to completely occlude the vessel for 20-50 min (IT) before reperfusion. Sequences of elevated ST segment, QRS complex prolongation, ventricular premature complexes and ventricular fibrillation were observed with a lower incidence than previously described in the literature. IT was (min: 17; max: 50) min long. F(I)ISO was lower than the minimal alveolar concentration in these species. Hypotension (MAP < 70 mmHg) and hypothermia (T°C < 36°C) were observed in all macaques. This minimally invasive closed chest model was successful in providing better cardiorespiratory physiological parameters than reported in previous models. The benefit (achieving ischaemia) versus risk (lethal arrhythmia) of the duration of the coronary occlusion should be considered.

[Value of contrast-enhanced ultrasound in the evaluation of indeterminate renal cysts on CT]. / Évaluation de l'apport de l'échographie de contraste dans l'analyse des kystes rénaux de nature indéterminée au scanner.

PURPOSE: To demonstrate the value of contrast-enhanced ultrasound (CEUS) in the management of Bosniak type 2F and 3 renal cysts on CT. PATIENTS AND METHODS: Retrospective study of 14 patients with 15 Bosniak type 2F and 3 renal cysts on contrast enhanced CT. All patients underwent CEUS of the kidneys after injection of SonoVue(®). Using predetermined criteria, the lesions were classified as benign, malignant or indeterminate. Patients either underwent surgery or follow-up CT at one to three years. RESULTS: From the 15 indeterminate cysts on CT, 12 were either benign (n=8) or malignant (n=4) on CEUS. From the eight cysts considered benign on CEUS, seven remained stable on follow-up CT after a minimum of one year interval and one was surgically resected and confirmed to be benign. All four cysts considered malignant on CEUS were surgically resected and three were confirmed to be malignant and one was confirmed to be benign. Three cysts were indeterminate: two lesions were surgically resected and one was benign while the other was malignant and one lesion was stable at one year follow-up CT. CONCLUSION: CEUS was able to characterize indeterminate lesions as benign or malignant in 80% of cases with 92% reliability. Twenty percent of cysts remained indeterminate on CEUS. CEUS is reliable as a complement to CT in the evaluation of Bosniak type 2F and 3 renal cysts.

Good practice guide for cervical ultrasound scan and echo-guided techniques in treating differentiated thyroid cancer of vesicular origin.

Good practice guide for cervical ultrasound scan and echo-guided techniques in treating differentiated thyroid cancer of vesicular origin. American, European and French Recommendations for the treatment of differentiated vesicular thyroid cancer were recently published. Cervical ultrasound scanning is now considered a key examination in the follow-up of these cancers. This examination is noninvasive, easy to perform and to obtain, is not costly, but remains operator-dependent. To date, there are no recommendations published that assemble all the technical aspects, results, indications and the limits of this examination in the initial medical report and the follow-up of these cancers. In order to standardise the procedure and validate the quality of the examination, a workgroup made up of a panel of experts particularly involved in carrying out ultrasound scans was set up. The aim was to draw up a good practice guide for performing cervical ultrasound scans and echo-guided techniques in treating patients with differentiated thyroid cancer of vesicular origin. The main objectives are to: (a) standardise the procedure and reports, (b) define the criteria for establishing whether lesions identified during a cervical ultrasound scan are malignant or benign, (c) standardise the indications for carrying out cytological tests and an in situ assay of markers, (d) help doctors to select the patients who ought to receive a cervical ultrasound scan and or cytological tests, (e) discuss how frequently the examinations should be carried out depending on the risk of recurrence.

[French first results using magnetic resonance-guided focused ultrasound for myoma treatment]. / Résultats préliminaires de la destruction des fibromes utérins par ultrasons focalisés contrôlée par résonance magnétique.

OBJECTIVE: Many women with myomas desire uterine conservation. Magnetic resonance-guided focused ultrasound surgery (MRgFUS) is a new non-invasive therapy. We describe our early results regarding efficacy and safety of MRgFUS for the treatment of uterine leiomyomas. PATIENTS AND METHODS: Fifty-two French women, over 18 years of age, who were candidates for surgical myomectomy, hysterectomy or uterine artery embolization due to symptomatic myomas were treated by MRgFUS (ExAblate 2000(®), InSightec) and followed up for at least 6 months. Intramural or subserous myomas with a size between 4-12 cm and a T2 low intensity MRI image were selected in women with no abdominal scar and no bowel interposition. The modified symptom severity score (SSS) was examined before and after the treatment at 6 months. Second treatment rate during the first follow-up year was reported. RESULTS: No serious complications were recorded during the treatments or follow-up period. Seven women had initial failure mostly because of bowel interposition, six of them had uterine artery embolisation. The mean modified SSS value for patients before MRgFUS was 41/100 and the values diminished significantly to a mean value of 22/100. A total of 65% of women had a reduction of at least 10 points (n=22) (initial score of 41 [29; 62] and 22 [16; 46] at 6 month follow-up). Good correlation was observed between myomas destruction and the symptoms score. During the follow-up period, ten patients (19%) required invasive interventions (six hysterectomies, two embolisations and two myomectomies). The average reduction in myoma volume determined by MR imaging at 6 months after treatment was 14%. This volume reduction is poorly correlated with the myoma's treated volume of 36.4% (6-74%). CONCLUSION: MRgFUS can safely be used for symptomatic treatment and avoid the need for surgical intervention in most patients. Additional follow-up is needed to determine the long-term durability of this promising non-invasive approach and to obtain reimbursement.

Intravenous contrast ultrasound examination using contrast-tuned imaging (CnTI) and the contrast medium SonoVue for discrimination between benign and malignant adnexal masses with solid components.

OBJECTIVE: To determine whether intravenous contrast ultrasound examination is superior to gray-scale or power Doppler ultrasound for discrimination between benign and malignant adnexal masses with complex ultrasound morphology. METHODS: In an international multicenter study, 134 patients with an ovarian mass with solid components or a multilocular cyst with more than 10 cyst locules, underwent a standardized transvaginal ultrasound examination followed by contrast examination using the contrast-tuned imaging technique and intravenous injection of the contrast medium SonoVue(R). Time intensity curves were constructed, and peak intensity, area under the intensity curve, time to peak, sharpness and half wash-out time were calculated. The sensitivity and specificity with regard to malignancy were calculated and receiver-operating characteristics (ROC) curves were drawn for gray-scale, power Doppler and contrast variables and for pattern recognition (subjective assignment of a certainly benign, probably benign, uncertain or malignant diagnosis, using gray-scale and power Doppler ultrasound findings). The gold standard was the histological diagnosis of the surgically removed tumors. RESULTS: After exclusions (surgical removal of the mass > 3 months after the ultrasound examination, technical problems), 72 adnexal masses with solid components were used in our statistical analyses. The values for peak contrast signal intensity and area under the contrast signal intensity curve in malignant tumors were significantly higher than those in borderline tumors and benign tumors, while those for the benign and borderline tumors were similar. The area under the ROC curve of the best contrast variable with regard to diagnosing borderline or invasive malignancy (0.84) was larger than that of the best gray-scale (0.75) and power Doppler ultrasound variable (0.79) but smaller than that of pattern recognition (0.93). CONCLUSION: Findings on ultrasound contrast examination differed between benign and malignant tumors but there was a substantial overlap in contrast findings between benign and borderline tumors. It appears that ultrasound contrast examination is not superior to conventional ultrasound techniques, which also have difficulty in distinguishing between benign and borderline tumors, but can easily differentiate invasive malignancies from other tumors.

Prognostic value of absent end-diastolic flow within the first week following renal transplantation.

BACKGROUND: Doppler sonogram of the graft is used as a routine assessment in renal transplantation. When the resistance index (RI) equals 1, absent end-diastolic flow (AEDF) is observed; the prognostic value of AEDF is presently unknown. PATIENTS AND METHODS: Between 1988 and 1996, 342 patients received a first cadaveric kidney transplant in our ward. AEDF was observed in 30 patients who were compared with 60 controls who showed an RI < 0.75 within the first 7 days after transplantation. They were matched for year of transplantation (+/-1 year); recipient age (+/-2 years); recipient sex; and HLA antibodies (3 classes: 0%, 1-75%, >75%). The follow-up was 4 years. RESULTS: AEDF was observed at day 1 in 64%, at day 3 in 96%, and at day 7 in 28%. Recipient age, donor age, recipient sex, cold and warm ischemia durations, HLA A, B, and DR mismatches, and cytomegalovirus (CMV) status were not different between the 2 groups. Immediate graft function and 3- to 24-month creatinine levels were better in the control than the AEDF group. However, there was no difference in serum creatinine at 3 and 4 years or in patient and graft survivals during follow-up. CONCLUSIONS: AEDF observed within the first week following transplantation is associated with impaired renal functional recovery. However, whether AEDF is a prognostic marker of poor long-term graft function or survival remains to be proven.

[Real-time contrast-enhanced ultrasound in the evaluation of focal liver lesions: diagnostic efficacy and economical issues from a French multicentric study]. / Echographie de contraste temps réel dans la prise en charge diagnostique des lésions nodulaires hépatiques: évaluation des performances diagnostiques et de l'impact économique sur une étude multicentrique française.

The recent introduction of high-end ultrasound equipment combined with recent contrast agents provides marked improvements in the characterization of focal liver lesions as previously reported by monocentric studies. The aim of the present study was to evaluate the diagnostic performance of Contrast-Enhanced Ultrasonography (CEUS) using SonoVue as well as its medico-economic value for characterization of focal liver lesions. These nodules were not characterized on previous CT or conventional sonography. This prospective multicentric study conducted in 15 French centres found diagnostic performances similar to those reported for CT and MRI, with a concordance rate of 84.5%, sensitivity greater than 80% and specificity greater than 90% for all types of lesions. Higher acceptance was found for CEUS compared to other imaging modalities. Economical assessment based on examination reimbursment and contrast agent cost showed a lower cost for contrast ultrasound versus CT and MRI. This French multicentric study confirmed the high diagnostic value of CEUS for focal liver lesion characterization and demonstrated a lower economical impact compared to other imaging modalities such as CT and MRI.

[Guidelines for contrast enhanced ultrasound (CEUS)--update 2008]. / Nouvelles recommandations pour l'utilisation des agents de contraste ultrasonores: mise à jour 2008.

These new recommendations are following the text published in 2004 by the group of experts of the EFSUMB that was augmented to reevaluate the indications of ultrasound contrast agents. Initially limited to focal liver lesions, new recommendations include the study of renal and pancreatic diseases, as well as vesico-ureteric reflux, blunt abdominal trauma and trans-cranial Doppler. Contrast-enhanced ultrasound improves the diagnosis of renal pseudo-tumors but does not allow characterization of solid focal masses. Its major contribution includes characterization of complex cystic masses, diagnosis of peripheral vascular disorders such as infarction and cortical necrosis, and radiofrequency ablation follow-up. It is also useful for the study of pancreatic masses visible at ultrasound in order to improve lesion visualization and characterization (adenocarcinoma and neuro-endocrine tumors), as well as for vascular staging.

[Elastosonography of thyroid lesions]. / Elastographie ultrasonore des lésions thyroïdiennes.

UNLABELLED: While ultrasound is the imaging modality of choice for diagnosis of thyroid lesions, characterization remains limited and tissue diagnosis frequently is required for management. The availability of additional tools such as elastography may improve lesion characterization and direct management. MATERIALS AND METHODS: A total of 96 patients (11 males and 85 females; 58+/-24 years) referred for fine needle aspiration (FNA) of mainly solid thyroid nodules 9-32 mm in diameter underwent conventional US and elastosonography. Results on elastography were correlated with histological results from FNA and classified as follows: suspected malignant lesion, suspected benign lesion, suspicious, indeterminate. RESULTS: The nodules were classified as follows: 95 nodules were soft (classes I and II) and 13 nodules were hard (classes III and IV). No cancers were detected in class and II lesions and 6 cancers were detected in class III and IV lesions. FNA provided insufficient cellular material for diagnosis in 5 class I-II nodules and 2 class III-IV nodules. CONCLUSION: Real-time elastosonography may be a useful adjunct to conventional US in the evaluation and characterization of thyroid nodules allowing identification of patients at high risk of malignancy for whom tissue diagnosis and/or close follow-up is required.

Acoustical bubble trapper applied to hemodialysis.

Gaseous microemboli can arise in extracorporeal lines and devices such as dialysis machines. They are associated with severe pulmonary side effects in patients undergoing chronic hemodialysis sessions. The goal of this study was to develop a gaseous emboli trapper using ultrasound waves to remove any air bubble from the tubing system before they reach the patient. A homemade bubble trapper, developed in the laboratory, consists of a Perspex block containing a main channel connected to the tubing of a hemodialysis machine and a second subchannel positioned perpendicularly to the main one, used to trap the air microemboli. The microemboli flowing in the main channel were insonified through an acoustic window with an ultrasound wave, at a frequency of 500 kHz and with a maximal acoustic pressure of 500 kPa, generated by a single-element transducer positioned 3 cm away from the main flow. The radiation force induced by the ultrasound beam acts directly on the flowing air emboli, by pushing them into the subchannel. Two Doppler probes operating both at 2 MHz, connected to a DWL Doppler machine were placed before and after the bubble trapper to count sequentially the number of embolic events. The flow of the machine was varied between 200 mL/min and 500 mL/min. Depending on the flow velocity, the number of microembolic signals (MES) detected by the Doppler probes before and after the trapping system was identical and ranged from 5 to 150 MES/min in absence of the ultrasound irradiation. When the air bubble trapper was activated, a reduction of the number of MES, up to 70%, was achieved. Doppler recordings suggest that the circulating bubbles were either fragmented into smaller bubble fragments or directly got pushed into the second subchannel where they were collected. This simple approach using an ultrasound-based trapping system was shown to operate adequately with the current settings and can be used to filter air microemboli.