Background: Signal transducer and activator of transcription 3 (STAT3) was strongly expressed and activated in psoriatic keratinocytes (KCs) and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA (siRNA) combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms. Methods: Psoriatic KCs were transfected under four experimental conditions: (1) STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group); (2) STAT3 siRNA only carried by Lipofectamine 3000 (L group); (3) the negative control of siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (siRNA-NC); (4) not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration ([Ca]). The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance. Results: STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the elevation of [Ca]of KCs with the highest calcium fluorescence intensity mean of 1213.67 ± 60.51 in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the downregulation of cyclin D1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58% ± 4.92% and 64.06% ± 7.78% at mRNA level and at protein level, respectively (P < 0.05 vs. L group, siRNA-NC, or Blank). Conclusions STAT3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca], and downregulating cyclin D1 and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Keratinocytes ; Microbubbles ; Phospholipids ; Psoriasis ; therapy ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor ; metabolism ; Sulfur Hexafluoride ; Ultrasonics

PURPOSE: The purpose of this study was to establish a method for ultrasound (US) contrast agent synthesis and to evaluate the characteristics of the synthesized US contrast agent. METHODS: A US contrast agent, composed of liposome and sulfur hexafluoride (SF₆), was synthesized by dissolving 21 μmol 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC, C₄₀H₈₀NO₈P), 9 μmol cholesterol, and 1.9 μmol of dihexadecylphosphate (DCP, [CH₃(CH₂)15O]₂P(O)OH) in chloroform. After evaporation in a warm water bath and drying for 12-24 hours, the contrast agent was synthesized using the sonication process by the addition of a buffer and SF₆ gas. The size distribution of the bubbles was analyzed using dynamic light scattering measurement methods. The degradation curve was evaluated by assessing the change in the number of contrast agent bubbles using light microscopy immediately, 12, 24, 36, 48, 60, 72, and 84 hours after synthesis. The echogenicity of the synthesized microbubbles was compared with commercially available microbubbles (SonoVue, Bracco). RESULTS: contrast agent was synthesized successfully using an evaporation-drying-sonication method. Most bubbles had a mean diameter of 154.2 nm and showed marked degradation 24 hours after synthesis. Although no statistically significant differences were observed between SonoVue and the synthesized contrast agent, a difference in echogenicity was observed between the synthesized contrast agent and saline (P<0.01). CONCLUSION: We successfully synthesized a US contrast agent using an evaporation-dryingsonication method. These results may help future research in the fields of anticancer drug delivery, gene delivery, targeted molecular imaging, and targeted therapy.
Baths ; Chloroform ; Cholesterol ; Contrast Media* ; Drug Delivery Systems ; Dynamic Light Scattering ; Liposomes ; Methods ; Microbubbles ; Microscopy ; Molecular Imaging ; Radiotherapy, Image-Guided ; Sonication ; Sulfur Hexafluoride ; Ultrasonography* ; Water

PURPOSE: Ultrasound (US) is highly sensitive in the detection of renal masses. However, it may not be able to differentiate benign and malignant lesions in smaller masses. The purpose of this study was to determine the diagnostic efficacy of contrast-enhanced ultrasound (CEUS) for small renal masses. MATERIALS AND METHODS: From January 2011 to December 2013, a total of 85 patients underwent CEUS for evaluation of renal masses. Of these patients, CEUS findings were retrospectively analyzed for small renal cell carcinoma (RCC) cases (n=38) and angiomyolipoma (AML) cases (n=11). The tumor echogenicity and enhancement patterns and degrees were evaluated. The diagnostic efficacy of CEUS in differentiating the two diseases was compared. RESULTS: On CEUS, the findings of diffuse heterogeneous enhancement (observed in 78.9% of RCCs and 27.3% of AMLs, p=0.003), washout from hyperenhancement or iso-enhancement to hypoenhancement in late phase (73.7% of RCCs and 18.2% of AMLs, p=0.001), and perilesional rim-like enhancement (57.9% of RCCs and 9.1% of AMLs, p=0.006) were significantly different between AML and RCC cases. The corresponding sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 86.8% (33/38), 63.6% (7/11), 89.2% (33/37), 58.3% (7/12), and 81.6% (40/49), respectively. CONCLUSIONS: Our results suggest that the characteristic CEUS features could have diagnostic value in the evaluation of small renal mass. CEUS showed a higher diagnostic efficacy than conventional US for differentiating RCC and AML.
Adult ; Aged ; Aged, 80 and over ; Angiomyolipoma/*ultrasonography ; Carcinoma, Renal Cell/*ultrasonography ; Contrast Media/diagnostic use ; Diagnosis, Differential ; Female ; Humans ; Kidney Neoplasms/*ultrasonography ; Male ; Middle Aged ; Reproducibility of Results ; Retrospective Studies ; Sensitivity and Specificity ; Sulfur Hexafluoride/diagnostic use ; Ultrasonography/*methods

PURPOSE: Ultrasound (US) is highly sensitive in the detection of renal masses. However, it may not be able to differentiate benign and malignant lesions in smaller masses. The purpose of this study was to determine the diagnostic efficacy of contrast-enhanced ultrasound (CEUS) for small renal masses. MATERIALS AND METHODS: From January 2011 to December 2013, a total of 85 patients underwent CEUS for evaluation of renal masses. Of these patients, CEUS findings were retrospectively analyzed for small renal cell carcinoma (RCC) cases (n=38) and angiomyolipoma (AML) cases (n=11). The tumor echogenicity and enhancement patterns and degrees were evaluated. The diagnostic efficacy of CEUS in differentiating the two diseases was compared. RESULTS: On CEUS, the findings of diffuse heterogeneous enhancement (observed in 78.9% of RCCs and 27.3% of AMLs, p=0.003), washout from hyperenhancement or iso-enhancement to hypoenhancement in late phase (73.7% of RCCs and 18.2% of AMLs, p=0.001), and perilesional rim-like enhancement (57.9% of RCCs and 9.1% of AMLs, p=0.006) were significantly different between AML and RCC cases. The corresponding sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 86.8% (33/38), 63.6% (7/11), 89.2% (33/37), 58.3% (7/12), and 81.6% (40/49), respectively. CONCLUSIONS: Our results suggest that the characteristic CEUS features could have diagnostic value in the evaluation of small renal mass. CEUS showed a higher diagnostic efficacy than conventional US for differentiating RCC and AML.
Adult ; Aged ; Aged, 80 and over ; Angiomyolipoma/*ultrasonography ; Carcinoma, Renal Cell/*ultrasonography ; Contrast Media/diagnostic use ; Diagnosis, Differential ; Female ; Humans ; Kidney Neoplasms/*ultrasonography ; Male ; Middle Aged ; Reproducibility of Results ; Retrospective Studies ; Sensitivity and Specificity ; Sulfur Hexafluoride/diagnostic use ; Ultrasonography/*methods

The application of ultrasound contrast agents (UCAs) is considered essential when evaluating focal liver lesions (FLLs) using ultrasonography (US). Microbubble UCAs are easy to use and robust; their use poses no risk of nephrotoxicity and requires no ionizing radiation. The unique features of contrast enhanced US (CEUS) are not only noninvasiveness but also real-time assessing of liver perfusion throughout the vascular phases. The later feature has led to dramatic improvement in the diagnostic accuracy of US for detection and characterization of FLLs as well as the guidance to therapeutic procedures and evaluation of response to treatment. This article describes the current consensus and guidelines for the use of UCAs for the FLLs that are commonly encountered in US. After a brief description of the bases of different CEUS techniques, contrast-enhancement patterns of different types of benign and malignant FLLs and other clinical applications are described and discussed on the basis of our experience and the literature data.
Contrast Media/chemistry/*diagnostic use ; Humans ; Liver Diseases/radiography/*ultrasonography ; Liver Neoplasms/radiography/*ultrasonography ; Phospholipids/chemistry/diagnostic use ; Practice Guidelines as Topic ; Sulfur Hexafluoride/chemistry/diagnostic use ; Tomography, X-Ray Computed

OBJECTIVETo assess the value of voiding urosonography (VUS) with SonoVue in evaluation of vesicoureteral reflux.

METHODSThirty-six pediatric patients (72 pyeloureter units [PUUs]) suspected of vesicoureteral reflux underwent both VUS and fluoroscopic voiding cystourethrography (VCUG). The sensitivity of VUS and VCUG and their consistency in detecting vesicoureteral reflux as well as in grading vesicoureteral reflux were compared.

RESULTSVesicoureteral reflux was detected in 26 of the 72 PUUs (36.1%) by VUS while in 21 PUUs (29.2%) by VCUG (P=0.347). The two modalities yielded the same results for 65 PUUs (κ=0.843), showing a very good consistency between them. VUS also detected post-urethral valve in 2 patients via transperineal scans.

CONCLUSIONVUS with Sonovue has at least comparable, if not better, sensitivity in detecting vesicoureteral reflux with VCUG, and therefore should serve as the primary screening and follow-up modality for vesicoureteral reflux. In addition, transperineal VUS can be helpful in evaluation of post-urethral lesions.

Child ; Child, Preschool ; Contrast Media ; Female ; Fluoroscopy ; Humans ; Infant ; Infant, Newborn ; Kidney Pelvis ; diagnostic imaging ; Male ; Phospholipids ; Sulfur Hexafluoride ; Ultrasonography, Doppler, Color ; Ureter ; diagnostic imaging ; Vesico-Ureteral Reflux ; diagnosis ; diagnostic imaging

OBJECTIVETo assess the application of gray-scale contrast-enhanced ultrasound (CEUS) and contrast-enhanced spiral computed tomography (CECT) in detection of residual tumor after high intensity focused ultrasound (HIFU) ablation with microbubbles on rabbit hepatic VX2 tumors.

METHODSForty rabbits with hepatic VX2 tumors were randomly divided into three groups before ablation. Group I (n=10) served as sham ablation controls, rabbits in group II (n=15) and group III (n=15) were ablated using HIFU under the manipulation of computer. A bolus of 0.2 ml SonoVue solution was injected via ear marginal vein of rabbits in group III before ablation. Tumors were examined with CEUS and CECT before and within 3h after HIFU ablation. Necropsy and histopathological assessment were performed immediately after the completion of images evaluation.

RESULTSBefore ablation, intense arterial feeding vessels was detected in the tumors (77.5%,31/40 Compared with 52.5%,21/40) or the periphery of the tumors (22.5%,9/40 Compared with 47.5%,19/40) by CEUS and CECT, respectively. The tumors were characterized by quick wash-in and wash-out (high and rapid peak of enhancement in the arterial phase,followed by a fast decrease in enhancement level). The dose parameters used to achieve therapeutic effect in group III were significantly lower than those in group II(P<0.01). There were local residual viable tumor tissues due to incomplete ablation in 60.0% (9/15) of group II and 13.3% (2/15) of group III revealed by histopathology(P<0.05). The concordance rate of CECT and CEUS with histopathology on residual tumor detection was 27.3% and 81.8% (P<0.05), respectively.

CONCLUSIONThe administration of microbubble agent enhances the efficacy of HIFU on rabbit hepatic VX2 tumors. CEUS is more sensitive than CECT in detection of residual viable rabbit VX2 tumor after HIFU.

Animals ; Female ; High-Intensity Focused Ultrasound Ablation ; Liver Neoplasms, Experimental ; therapy ; Male ; Microbubbles ; Neoplasm, Residual ; diagnostic imaging ; pathology ; Phospholipids ; Rabbits ; Sulfur Hexafluoride ; Tomography, Spiral Computed ; Ultrasonography

The feasibility of contrast-enhanced ultrasonography in the assessment of atherosclerotic plaque neovascularization and its relation to histological findings were investigated. Abdominal aortic atherosclerotic plaque model was induced in 25 New Zealand white rabbits by a combination of high cholesterol-rich diet and balloon aortic denudation. Standard and contrast-enhanced ultrasonography was performed at the 16th week of the model induction period. The plaques were classified as echogenic plaques or echolucent plaques according to their echogenicity at standard ultrasonography. The maximum thickness of plaque was measured in the longitudinal section. Time intensity curve was used to quantify the enhanced intensity of the plaque. Animals were euthanized and abdominal aortas were harvested for histological staining of CD31 to evaluate the neovascularization density of atherosclerotic plaque. The results showed that the echolucent plaques had higher enhanced intensity during contrastenhanced ultrasonography and higher neovascularization density at CD31 staining than the echogenic plaques. The enhanced intensity of atherosclerotic plaque and its ratio to lumen were well correlated with histological neovascularization density (r=0.75, P<0.001; r=0.68, P<0.001, respectively). However, the maximum thickness of plaque was not correlated with neovascularization density (r=0.235, P=0.081). These findings demonstrated that the enhanced intensity in the plaque and ratio of enhanced intensity to that in the lumen of abdominal aorta may be more accurate in the evaluation of plaque neovascularization than maximum thickness. Our study indicates that contrast-enhanced ultrasonography provides us a reliable method for the evaluation of plaque neovascularization.
Animals ; Aorta, Abdominal ; diagnostic imaging ; Image Enhancement ; methods ; Image Interpretation, Computer-Assisted ; methods ; Neovascularization, Pathologic ; diagnostic imaging ; etiology ; Phospholipids ; Plaque, Atherosclerotic ; complications ; diagnostic imaging ; Rabbits ; Reproducibility of Results ; Sensitivity and Specificity ; Statistics as Topic ; Sulfur Hexafluoride ; Ultrasonography ; methods

PURPOSE: The purpose of this study is to establish the methodology regarding synthesis of ultrasound contrast agent imaging, and to evaluate the characteristics of the synthesized ultrasound contrast agents, including size or degradation interval and image quality. MATERIALS AND METHODS: The ultrasound contrast agent, composed of liposome and SF6, was synthesized from the mixture solution of 21 micromol DPPC (1, 2-Dihexadecanoyl-sn-glycero-3-phosphocholine, C40H80NO8P), 9 micromol cholesterol, 1.9 micromol of DCP(Dihexadecylphosphate, [CH3(CH2)15O]2P(O)OH), and chloroform. After evaporation in a warm water bath and drying during a period of 12-24 hours, the contrast agent was synthesized by the sonication process by addition of buffer and SF6 gas. The size of the contrast agent was controlled by use of either extruder or sonication methods. After synthesis of contrast agents, analysis of the size distribution of the bubbles was performed using dynamic light scattering measurement methods. The degradation curve was also evaluated by changes in the number of contrast agents via light microscopy immediate, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, and 84 hours after synthesis. For evaluation of the role as an US contrast agent, the echogenicity of the synthesized microbubble was compared with commercially available microbubbles (SonoVue, Bracco, Milan, Italy) using a clinical ultrasound machine and phantom. RESULTS: The contrast agents were synthesized successfully using an evaporation-drying-sonication method. The majority of bubbles showed a mean size of 154.2 nanometers, and they showed marked degradation 24 hours after synthesis. ANOVA test revealed a significant difference among SonoVue, synthesized contrast agent, and saline (p < 0.001). Although no significant difference was observed between SonoVue and the synthesized contrast agent, difference in echogenicity was observed between synthesized contrast agent and saline (p < 0.01). CONCLUSION: We could synthesize ultrasound contrast agents using an evaporation-drying-sonication method. On the basis of these results, many prospective types of research, such as anticancer drug delivery, gene delivery, including siRNA or microRNA, targeted molecular imaging, and targeted therapy can be performed.
Baths ; Chloroform ; Cholesterol ; Contrast Media ; Light ; Liposomes ; Microbubbles ; MicroRNAs ; Microscopy ; Molecular Imaging ; Phospholipids ; RNA, Small Interfering ; Sonication ; Sulfur Hexafluoride ; Water

Contrast-enhanced ultrasound is one of method for evaluating renal perfusion. The purpose of this project was to assess perfusion patterns and dynamics in normal micropig kidney using ultrasonographic contrast media. Eight young healthy micropigs were included in this study. Micropigs were anesthetized with propofol and received an intravenous bolus of microbubble contrast media through an ear vein. Time/mean pixel value (MPV) curves were generated for selected regions in the right renal cortex and medulla. The parenchyma was enhanced in two phases. The cortex was first enhanced followed by a more gradual enhancement of the medulla. A significant difference in perfusion was detected between the cortex and medulla. Following the bolus injection, the average upslope was 0.68 +/- 0.27 MPV/sec, downslope was -0.27 +/- 0.13 MPV/sec, baseline was 73.9 +/- 16.5 MPV, peak was 84.6 +/- 17.2 MPV, and time-to-peak (from injection) was 17.5 +/- 6.6 sec for the cortex. For the medulla, the average upslope was 0.50 +/- 0.24 MPV/sec, downslope was -0.12 +/- 0.06 MPV/sec, baseline was 52.7 +/- 7.0 MPV, peak was 65.2 +/- 9.3 MPV, and time-to-peak (from injection) was 27.5 +/- 5.0 sec. These data can be used as normal reference values for studying young micropigs.
Animals ; Contrast Media/*diagnostic use ; Image Processing, Computer-Assisted ; Injections, Intravenous/veterinary ; Kidney/*blood supply/ultrasonography ; Kidney Function Tests/veterinary ; Linear Models ; Microbubbles/diagnostic use/veterinary ; Reference Values ; Renal Circulation ; Sulfur Hexafluoride/diagnostic use ; Swine ; Swine, Miniature/*physiology ; Ultrasonography/*methods/veterinary