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The radiological surveillance of cirrhosis to detect the development of hepatocellular carcinoma (HCC) is problematic because no highly sensitive and specific imaging investigation is available. Ultrasound is typically the first modality used but is less accurate than other imaging modalities. We report the first case of a patient with cirrhosis in whom US imaging with liver-specific microbubbles detected an HCC prior to its detection by MR. The use of liver-specific microbubble US contrast agents is an exciting development in the detection of HCC in chronic liver disease and may help to rectify some of the shortcomings of US. [ABSTRACT FROM AUTHOR]

Background: Therapy with naked oligodeoxynucleotides (ODNs, molecular weight: 3000–7500) provides an elegant means of modulating gene expression without the problems associated withconventional gene therapy, but the relatively low transfer efficiency on intravascular administration is a limitation to clinical application. Ultrasound, which can be potentiated by microbubbles, shows promise as a method of delivering macromolecules such as plasmid DNA and other transgenes into cells. Since uptake of molecules into cells depends on their molecular weight, it might be expected that the delivery of ODNs, which are relatively small, will be facilitated by ultrasound and microbubbles.Methods & Results: In the present study, we delivered ODNs into veins using ultrasound and microbubbles. First, we quantified the uptake of fluorescent-labeled ODNs into intact ex vivo human saphenous veins, and isolated smooth muscle cells from the veins, evaluating the effect of ultrasound and microbubbles on uptake. Ultrasound potentiated the delivery of ODN in cells except at high concentrations. When intact veins were studied, we achieved nuclear localization of fluorescent-labeled ODNs in cells. This increased with increasing concentration and incubation time and was not potentiated by ultrasound even when microbubbles were used. We then applied a therapeutic ODN (antisense to intercellular adhesion molecule 1, ICAM-1) to vein samples and documented a functional inhibition of gene expression in a sequence-specific manner at the protein level with immunohistochemistry and Western Blot analysis. Again, no significant difference was seen with adjunct ultrasound.Conclusion: These observations suggest high diffusion of ODNs into human saphenous veins in this ex vivo model, indicating potential applications to inhibition of vascular bypass graft occlusion and other vasculopathies. Although microbubble-ultrasound was of value with cells in culture it was not beneficial with intact veins.Molecular Therapy (2006) 13, S212–S212; doi: 10.1016/j.ymthe.2006.08.622 [ABSTRACT FROM AUTHOR]

The combination of ultrasound and ultrasound contrast agents (UCAs) is able to induce transient membrane permeability leading to direct delivery of exogenous molecules into cells. Cavitation bubbles are believed to be involved in the membrane permeability; however, the detailed mechanism is still unknown. In the present study, the effects of ultrasound and the UCAs, Optison on transfection in vitro for different medium heights and the related dynamic behaviors of cavitation bubbles were investigated. Cultured CHO-E cells mixed with reporter genes (luciferase or beta-gal plasmid DNA) and UCAs were exposed to 1 MHz ultrasound in 24-well plates. Ultrasound was applied from the bottom of the well and reflected at the free surface of the medium, resulting in the superposition of ultrasound waves within the well. Cells cultured on the bottom of 24-well plates were located near the first node (displacement node) of the incident ultrasound downstream. Transfection activity was a function determined with the height of the medium (wave traveling distance), as well as the concentration of UCAs and the exposure time was also determined with the concentration of UCAs and the exposure duration. Survival fraction was determined by MTT assay, also changes with these values in the reverse pattern compared with luciferase activity. With shallow medium height, high transfection efficacy and high survival fraction were obtained at a low concentration of UCAs. In addition, capillary waves and subsequent atomized particles became significant as the medium height decreased. These phenomena suggested cavitation bubbles were being generated in the medium. To determine the effect of UCAs on bubble generation, we repeated the experiments using crushed heat-treated Optison solution instead of the standard microbubble preparation. The transfection ratio and survival fraction showed no additional benefit when ultrasound was used. These results suggested that cavitation bubbles created by the collapse of UCAs were a key factor for transfection, and their intensities were enhanced by the interaction of the superpose ultrasound with the decreasing the height of the medium. Hypothesizing that free cavitation bubbles were generated from cavitation nuclei created by fragmented UCA shells, we carried out numerical analysis of a free spherical bubble motion in the field of ultrasound. Analyzing the interaction of the shock wave generated by a cavitation bubble and a cell membrane, we estimated the shock wave propagation distance that would induce cell membrane damage from the center of the cavitation bubble.

Pluronics have been investigated as vectors for drug and gene delivery in vitro and in vivo and were demonstrated to have high efficiency for gene transfer in vivo. However, they alone do not enhance gene transfer in vitro. We examined three pluronics, F127, L61 and P85, for their effects on ultrasound (US)-mediated gene transfer in three cell lines, 3T3-MDEI, C2C12 and CHO. The polymers showed differential effects on cell viability and transfection efficiency in a dose-dependent manner. All the polymers were unable to facilitate gene transfer when used alone, but enhanced US-mediated gene transfer significantly at concentrations around the critical micelle concentration in the three cell lines. F127 showed no significant toxicity at any concentration and protected the cells against US-mediated damage at a high concentration. L61 decreased cell viability significantly in a dose-dependent manner, whereas P85 showed mild toxicity when its concentration was at or above 0.05%.

Therapy with naked oligodeoxynucleotides (ODNs, molecular weight: 3000 to 7500) provides an elegant means of modulating gene expression without the problems associated with conventional gene therapy, but the relatively low transfer efficiency on intravascular administration is a limitation to clinical application. Ultrasound, which can be potentiated by microbubbles, shows promise as a method of delivering macromolecules such as plasmid DNA and other transgenes into cells. Since uptake of molecules into cells depends on their molecular weight, it might be expected that the delivery of ODNs, which are relatively small, will be facilitated by ultrasound and microbubbles. In the present study, we delivered ODNs into veins using ultrasound and microbubbles. First, we quantified the uptake of fluorescent-labeled ODNs into intact ex vivo human saphenous veins and isolated smooth muscle cells from the veins, evaluating the effect of ultrasound and microbubbles on uptake. Ultrasound potentiated the delivery of ODN in cells, except at high concentrations. When intact veins were studied, we achieved nuclear localization of fluorescent-labeled ODNs in cells. This increased with increasing concentration and incubation time and was not potentiated by ultrasound, even when microbubbles were used. We then applied a therapeutic ODN (antisense to intercellular adhesion molecule 1, ICAM-1) to vein samples and documented a functional inhibition of gene expression in a sequence-specific manner at the protein level with immunohistochemistry and western blot analysis. Again, no significant difference was seen with adjunct ultrasound. These observations suggest high diffusion of ODNs into human saphenous veins in this ex vivo model, indicating potential applications to inhibition of vascular bypass graft occlusion and other vasculopathies. Although microbubble-ultrasound was of value with cells in culture, it was not beneficial with intact veins.

Objective: Many authors have claimed that Doppler sonography indexes are of value in grading and assessing diffuse liver disease. However, there is much controversy regarding the reliability and reproducibility of these techniques. We performed a prospective study to evaluate whether these methods can grade disease in a well-stratified cohort of patients with hepatitis C virus (HCV)-related liver disease., Subjects and Methods: Sixty-five patients with biopsy-proven HCV-related liver disease were recruited, and Doppler sonography was performed by one operator. The patients were classified into one of the following three groups on the basis of the Ishak-modified histologic activity index (HAI) fibrosis (F) and necroinflammatory (NI) scores: mild hepatitis (F < or = 2 and NI < or = 3), moderate or severe hepatitis (3 < or = F < 6 or NI > or = 4), or cirrhosis (F = 6/6). We measured the following Doppler indexes: main hepatic artery peak velocity (Vmax) and resistive index, main portal vein peak velocity (Vmax), and maximal portal vein diameter and circumference that allowed calculation of the portal vein congestive index (portal vein area and portal vein velocity). The ratio of the hepatic artery velocity (Vmax) to the portal vein velocity (Vmax) was also calculated, and the phasicity (triphasic, biphasic, or monophasic) of the hepatic veins of each patient was recorded. We also measured the maximal spleen length longitudinally., Results: A total of 65 patients with liver disease (mild hepatitis, n = 20; moderate or severe hepatitis, n = 25; cirrhosis, n = 20) with biopsy-proven HCV-related liver disease were studied. Optimal hepatic arterial traces were obtained in only 30 patients and portal vein circumference in 18 patients. No significant differences were observed in the Doppler indexes with increasing severity of liver disease. Five (29%) of 17 patients with mild hepatitis had an abnormal hepatic vein trace (i.e., biphasic or monophasic) compared with 11 (55%) of 20 patients with moderate or severe hepatitis and 12 (60%) of 20 patients with cirrhosis. The only index to show a significant intergroup difference was splenic length (analysis of variance, p < 0.001), but there was still overlap between the groups., Conclusion: Doppler-derived indexes, which have previously been recommended for the assessment of severity in chronic liver disease, are difficult to reproduce reliably and therefore have a limited clinical role in the noninvasive assessment of hepatic fibrosis or inflammation.

Ultrasound (US) is a promising tool for facilitating direct gene transfer to skeletal muscle, but no systematic optimisation study has been performed. We exposed H2K myoblast cells to US with varying intensity of exposure and duration to evaluate its effect on cell viability and transfection efficiency using as endpoints transfection rate, average fluorescence intensity (fluorescence normalised by the number of transfected cells) and overall expression (the product of transfection rate and average fluorescence intensity) as indices. Cell viability decreased with exposure time and intensity, consistent with previous findings. Optimal setting of US was observed at the range of 0.5 to 1 W cm(-2) with duration of 20 s, producing maximum efficiency (transfection = 4.5%) in gene transfection with minimum cell toxicity (cell viability = 83%). Higher intensity alone or in combination with low intensity and long duration did not improve cell viability and transfection. The increase of eGFP (enhanced green fluorescence protein) plasmid concentration up to 200 microg per mL was related to an increase in average fluorescence intensity and overall expression. However, transfection rate saturated when DNA concentration reached 50 microg per mL despite initial increase with DNA concentration. The average fluorescence intensity was linearly proportional to the logarithm of DNA concentration, suggesting a diffusion-based model for DNA uptake under sonoporation. We conclude that low-intensity US irradiation provides a safe and effective alternative for gene delivery.

Background: We quantified changes in prostate carcinoma vascularity treated with anti-androgens using color Doppler and power transrectal ultrasound in combination with microbubble contrast agent Levovist., Methods: Thirty-six men with prostate carcinoma were studied at baseline and at intervals during treatment. At each attendance, Levovist((R)) (10 ml, 300 mg/ml) was given as an iv bolus. Using quantitative analysis, we calculated the pre-enhancement scores, arrival time, time to peak, peak value, and area under the time-enhancement curve (AUC). These were compared to pre-treatment values and serial PSA measurements., Results: The pre-enhancement, peak value, and AUC each showed a marked response with reductions within one week. The average AUC declined to 68% +/- 9% (mean +/- standard error) by week 1, 56% +/- 9% by week 3, and 20% +/- 4% by week 6. A strong correlation with changes in the mean PSA (r = 0.95, P < 0.001) was also measured. In four patients, Doppler indices did not fall with PSA: two patients with the most marked discrepancy relapsed at 6 months., Conclusion: The vascular enhancement declined with therapy, similar to PSA. Microbubble enhanced ultrasound can show early response to treatment., (Copyright 2002 Wiley-Liss, Inc.)

The aim of this pilot study was to see if the biodistribution of the microbubble Levovist (SHU 508 A; Schering AG, Berlin) during its liver specific phase is altered by radiotherapy. The mechanism of this liver-specific phase of this agent remains poorly understood. One way of investigating this is to see what effect radiotherapy has on liver uptake, as both Kupffer cell function and vascular endothelial integrity are selectively damaged by irradiation. The regional liver specific uptake of Levovist was evaluated in eight patients undergoing radiotherapy to the hepatic area. Ultrasound (US) sweeps were made 4 min after Levovist injection using the phase inversion mode (PIM) which is specific for microbubbles. Differences between irradiated and non-irradiated areas were observed in 2/8 subjects completing the study. Both subjective and objective evaluations in these subjects showed a significantly reduced grey scale unit in non-irradiated versus irradiated liver regions (average values 99 vs. 89, P < 0.0045 and 75 vs. 62, P < 0.0001). These findings are somewhat inconclusive, but given the difficulty in defining areas of irradiated and non-irradiated liver, because multiple radiotherapy portals were used in all patients, tentatively suggests a radiotherapy induced effect in at least some patients. The two likely mechanisms would be damage to the Kupffer cells and or the vascular endothelium, although the relative contribution of these is unclear.

We investigated the reproducibility of Doppler enhancement indices following intravenous bolus injections of Levovist (Schering AG, Berlin) microbubbles. We also aimed to determine whether observations from animal studies suggesting that aspirin potentiates microbubble enhancement were reproducible in humans. In five healthy volunteers, time enhancement profiles of Doppler intensity following repeated bolus injections of Levovist were acquired from the common carotid artery, hepatic vein and kidney using spectral and power Doppler before and after oral aspirin (600 mg). Peak enhancement (PE), area under the curve (AUC) and decay slopes (lambda) were calculated. Hepatic vein contrast arrival time (AT) was determined subjectively. Well-defined carotid enhancement was seen in 19/20 injections. Reproducibility was high (r > 0.8). PE and AUG were unaffected by aspirin, but lambda was slightly reduced (P = 0.02). Renal power Doppler profiles were well defined (10/10) with no significant changes of AUC, PE or lambda after aspirin. Our study demonstrates good reproducibility of carotid spectral Doppler time intensitometry with Levovist in man. Aspirin does not have a significant effect on enhancement indices except carotid spectral Doppler decay. We conclude that aspirin is unlikely to potentiate microbubble enhancement, as seen in animal studies.

Ultrasound (US) has undergone dramatic changes since its inception three decades ago; the original cumbersome B-mode gantry system has evolved into a high resolution real-time imaging system. This review describes both recent advances in ultrasound and contrast media and likely future developments. Technological advances in electronics and computing have revolutionized ultrasound practice with ever expanding applications. Developments in transducer materials and array designs have resulted in greater bandwidths with improvements in spatial and contrast resolution. Developments in digital signal processing have produced innovations in beam forming, image display and archiving. Technological advances have resulted in novel imaging modes which exploit the non-linear behaviour of tissue and microbubble contrast agents. Microbubble contrast agents have dramatically extended the clinical and research applications of ultrasound. Not only can Doppler studies be enhanced but also novel non-linear modes allow vessels down to the level of the microcirculation to be imaged. Functional and quantitative studies allow interrogation of a wide spectrum of tissue beds. The advent of tissue-specific agents promises to improve the sensitivity and specificity of ultrasound in the detection and characterization of focal liver lesions to rival that of computed tomography (CT) and magnetic resonance imaging (MRI). Ultrasound has recently moved into therapeutic applications with high intensity focused ultrasound (HIFU) and microbubble assisted delivery of drugs and genes showing great promise., (Copyright 2002 The Royal College of Radiologists.)

Objectives: Microbubble echo-enhancers help in the assessment of focal liver masses by enhancing the signal from blood vessels. A variety of linear and nonlinear scanning modes are now available, but it is unclear which is optimal. A controlled comparison was performed during the infusion of such an agent (SonoVue: Bracco, Milan, Italy)., Methods and Materials: Ten patients with known focal liver lesions were studied. The diagnoses, confirmed on dual phase helical computed tomography (CT) at the same attendance were metastasis (n = 7), haemangioma (n = 2) and focal nodular hyperplasia FNH (n = 1). A dose of 12 ml SonoVue concentrated at 5 mg/ml was infused intravenously at a rate of 1 ml/min. The enhancement level was monitored with a continuous wave (CW) Doppler probe over the right radial artery and the intensity of the signal was registered at 1 s intervals. When a plateau of enhancement was reached, a single lesion in each patient was imaged using five different continuous scanning modes, fundamental grey scale (FGS); fundamental colour Doppler (FCD); fundamental power Doppler (FPD); second harmonic grey scale (HGS); and pulse inversion mode (Pim) using an HDI5000 scanner and C5-2 probe (ATL, Bothell, WA). The order of scanning modes was varied between patients using a predefined randomisation protocol. The videos (super video home system (SVHS)) were analysed offsite by two blinded readers, both experienced in contrast ultrasound of the liver. The readers were asked to score each mode in terms of its ability to detect vessels within/around the lesion at optimal enhancement. This was done using a ranking system (1, worst; 5, best) for each patient., Results: Both observers scored FPD as the optimal imaging method, followed by Pim. (Scores summed across all patients, observer 1: FPD 48, Pim 42, FCD 37, HGS 21, FGS 10; observer 2: FPD 49, Pim 40, FCD 38, HGS 21, FGS 10). The differences from FPD were significant for FCD, HGS and FGS using a unpaired analysis of variance (ANOVA) comparison, with Bonferroni multiple corrections, (P<0.01, both observers). The differences between FPD and Pim were also significant both for observer 2 and for both observers combined (P<0.01), but did not reach significance for observer 1 (P = 0.19)., Conclusions: In this study, FPD performed best, and the non-linear modes, performed continuously (pulse inversion and second HGS), showed no clear advantage.

Rationale and Objectives: Two algorithms can be used to measure portal venous perfusion (PVP) with contrast material-enhanced single-level liver computed tomography. The "direct" and "indirect" algorithms use data from the portal vein and aorta, respectively. This study compared PVP values obtained with direct and with indirect algorithms in a series of patients., Materials and Methods: Both techniques were applied in 27 patients with cirrhosis (14 men and 13 women; mean age, 56.1 years +/- 9.4) and 18 control patients (seven men and 11 women; 52.8 years +/- 12.3). A single section through the liver was scanned after intravenous injection of ioversol (40-mL bolus; 320 mg of iodine per milliliter)., Results: Both techniques showed reduced PVP in patients with cirrhosis (0.63 for direct and 0.17 for indirect method) compared with control patients (1.06 and 0.26, respectively), but only the direct method agreed with physiologic expectations based on animal and human studies. In separating cirrhotic and control patients, the area under the receiver operating characteristic curve was significantly greater for the direct method (0.91 vs 0.78; P = .03)., Conclusion: Both direct and indirect methods are feasible and distinguish well between cirrhotic and control patients, but the direct method is more physiologic and is preferable if portal venous data are available.