An in vitro study of a microbubble contrast agent using a clinical ultrasound imaging system

Optimal insonation settings for contrast imaging are yet to be specified, mainly due to the lack of good understanding of the behaviour of the microbubbles. A satisfactory model that explains the behaviour of individual contrast agent scatterers has not yet been reported in the literature. An in vitro system based on a commercial scanner (ATL HDI3000) has been developed to investigate the backscatter of such agents. Suspensions of Definity were introduced in an anechoic tank. The frequency of transmitted ultrasound varied from 1 to 5 MHz, pulse period from 2 to 10 periods and peak negative acoustic pressure from 0.08 to 1.7 MPa. The backscatter at the fundamental and second harmonic frequency windows from the agent was normalized in terms of the corresponding components of backscatter from a blood mimicking fluid suspension. The agent provided a dominant resonance effect at 1.6 MHz transmit frequency. Second harmonic normalized backscatter averaged around 9 dB higher than the fundamental. The normalized fundamental backscatter intensity was linear with peak negative pressure. The second harmonic at resonance peaked at 0.5 MPa suggestive of bubble disruption above such pressure. The system proved capable of illustrating the ultrasonic behaviour of Definity in vitro, and the investigation suggested particular insonation conditions for optimal image enhancement using Definity.     

微泡造影剂联合超声辐照短暂性增加血管内皮细胞膜通透性

目的探讨微泡造影剂联合超声辐照对体外培养的人脐静脉血管内皮细胞(HUVEC)膜通透性改变及作用持续时间.方法用连续多普勒超声辐照HUVEC,照射条件为频率1.9 MHz,软组织热指数(TIS)0.8,增益80%,输出声强I SATA为80.0 mW/cm2,照射时间60 s.悬浮HUVEC,加入2%微泡造影剂SonoVue,于照射后0、1、5、30 min、2 h时点分别加入大分子荧光素物质FD500,荧光显微镜下观察FD500进入细胞内的情况,用流式细胞仪测FD500荧光染色阳性的细胞.锥虫蓝染色观察符组样本细胞活性.结果0、1 min组荧光显微镜下大部分HUVEC荧光染色阳性,贴壁后观察可见细胞质内吴现不均质的绿色荧光显影,而细胞核不显影;5、30 min、2 h组仅见少数HUVEC胞质内绿色荧光显影.0、1、5、30 min、2 h组流式细胞仪测荧光染色阳性率分别为(61.63±18.35)%、(57.26±13.53)%、(14.66±6.07)%、(4.12±0.96)%、(5.93±1.45)%,前两组与后者比较,差异有统计学意义(P<0.01).结论微泡造影剂联合诊断级别超声辐照具有增加HUVEC膜通透性的作用,其作用持续时间较为短暂,可能的机制为超声空化导致细胞膜"声穿孔"效应.     

Use of a model for simulating individual aortic dynamics in man

A simulation model is suggested for the analysis of aortic dynamics in man. The aortic model consists of six segments and is part of a larger model of the closed-loop human circulation. The model is simulated on a special-purpose analogue computer. Three parameters are employed to characterise the arterial system; peripheral resistance, aortic compliance and peripheral damping. Using a method for adapting the model to the individual patient, measurements of aortic pressure, cardiac output and pulse transmission time from 29 patients were used to test the validity of this approach. The model is able to simulate the pressure course along the aorta satisfactorily. The compliance calculated from the transmission properties of the aorta was compared with the complicance calculated from the stroke volume and pressure pulse. An adequate correlation (r=0.98) was found between these two independent methods. The mean compliance of the total aorta was 0.6 ml/mm Hg at a mean pressure of 104 mm Hg. The compliance showed large individual variations and decreasing values with increasing age of the patient. It is concluded that the model enables simulation of the individual aorta.     

Characterization of plaque components and vulnerability with intravascular ultrasound elastography

Intravascular ultrasound elastography is a method for measuring the local elastic properties using intravascular ultrasound (IVUS). The elastic properties of the different tissues within the atherosclerotic plaque are measured through the strain. Knowledge of these elastic properties is useful for guiding interventional procedures (balloon dilatation, ablation) and detection of the vulnerable plaque. In the last decade, several groups have applied elastography intravascularly with various levels of success. In this paper, the approaches of the different research groups will be discussed. The focus will be on our approach to the application of intravascular elastography. Elastograms were acquired in vitro and in vivo using the relative local displacements between IVUS images acquired at two levels of intravascular pressure with a 30 MHz mechanical or a 20 MHz array echo catheter. These displacements were estimated from the time shift between gated radiofrequency echo signals using cross-correlation algorithms with interpolation around the peak. Experiments on gel-based phantoms mimicking atherosclerotic vessels demonstrated the capability of elastography to identify soft and hard tissues independently of the echogenicity contrast. In vitro experiments on human arteries have demonstrated the potential of intravascular elastography to identify different plaque types based on their mechanical properties. These plaques could not be identified using the IVUS image alone. In vivo experiments revealed that reproducible elastograms could be obtained near end-diastole. Partial validation using the echogram was performed. Intravascular elastography provides information that is frequently unavailable or inconclusive from the IVUS image and which may therefore assist in the diagnosis and treatment of atherosclerotic disease.     

超声辐照微泡对肝细胞生长因子转染大鼠损伤面神经的促进作用

目的 观察超声微泡介导肝细胞生长因子(HGF)基因促进大鼠受损面神经修复的可行性及有效性.方法 将40只SD大鼠随机分成4组,A组:单纯手术组(PBS);B组:HGF+微泡组;C组:HGF+超声组;D组:HGF+超声+微泡组,每组10只.建立大鼠面神经损伤模型,以HGF作为治疗基因.基因转染后28 d,观察各组大鼠的一般情况,检测面神经传导速度、潜伏期及神经电位波幅的变化.处死各组大鼠,采用Westem blot和RT-PCR检测损伤面神经HGF蛋白和mRNA的表达.结果 经行为学检测,D组大鼠在触须摆动及鼻尖位置均明显优于其余3组.D组面神经传导速度、神经电位波幅明显高于其余3组,潜伏期明显低于其余3组(p＜0.05).D组中HGF蛋白和mRNA的表达量明显高于其他3组(P＜0.05).结论 微泡在一定频率和强度的超声作用下能够安全、有效的将目的基因转染进入损伤后的面神经,有利于受损面神经的修复.     

Characterization of cell seeding and specific capture of B cells in microbubble well arrays

Development of micro-well array systems for use in high-throughput screening of rare cells requires a detailed understanding of the factors that impact the specific capture of cells in wells and the distribution statistics of the number of cells deposited into wells. In this study we investigate the development of microbubble (MB) well array technology for sorting antigen-specific B-cells. Using Poisson statistics we delineate the important role that the fractional area of MB well opening and the cell seeding density have on determining cell seeding distribution in wells. The unique architecture of the MB well hinders captured cells from escaping the well and provides a unique microenvironmental niche that enables media changes as needed for extended cell culture. Using cell lines and primary B and T cells isolated from human peripheral blood we demonstrate the use of affinity capture agents coated in the MB wells to enrich for the selective capture of B cells. Important differences were noted in the efficacy of bovine serum albumin to block the nonspecific adsorption of primary cells relative to cell lines as well as the efficacy of the capture coatings using mixed primary B and T cells samples. These results emphasize the importance of using primary cells in technology development and suggest the need to utilize B cell capture agents that are insensitive to cell activation.     

Ultrasound molecular imaging of tumor angiogenesis with a neuropilin-1-targeted microbubble

Ultrasound molecular imaging has great potential to impact early disease diagnosis, evaluation of disease progression and the development of target-specific therapy. In this paper, two neuropilin-1 (NRP) targeted peptides, CRPPR and ATWLPPR, were conjugated onto the surface of lipid microbubbles (MBs) to evaluate molecular imaging of tumor angiogenesis in a breast cancer model. Development of a molecular imaging agent using CRPPR has particular importance due to the previously demonstrated internalizing capability of this and similar ligands. In vitro, CRPPR MBs bound to an NRP-expressing cell line 2.6 and 15.6 times more than ATWLPPR MBs and non-targeted (NT) MBs, respectively, and the binding was inhibited by pretreating the cells with an NRP antibody. In vivo, the backscattered intensity within the tumor, relative to nearby vasculature, increased over time during the ∼6 min circulation of the CRPPR-targeted contrast agents providing high contrast images of angiogenic tumors. Approximately 67% of the initial signal from CRPPR MBs remained bound after the majority of circulating MBs had cleared (8 min), 8 and 4.5 times greater than ATWLPPR and NT MBs, respectively. Finally, at 7–21 days after the first injection, we found that CRPPR MBs cleared faster from circulation and tumor accumulation was reduced likely due to a complement-mediated recognition of the targeted microbubble and a decrease in angiogenic vasculature, respectively. In summary, we find that CRPPR MBs specifically bind to NRP-expressing cells and provide an effective new agent for molecular imaging of angiogenesis.     

Virus load dynamics of individual CMV-genotypes in lung transplant recipients with mixed-genotype infections

Human cytomegalovirus (CMV) is a major cause of disease and transplant dysfunction in lung transplant recipients. Simultaneous emergence of more than one CMV-genotype can occur, and appears to be disadvantageous for the patient. In this study, the dynamics of individual CMV-genotypes in blood and lung was assessed within mixed CMV-genotype populations emerging after lung transplantation. In 69 plasma and 76 bronchoalveolar lavage samples of 16 lung transplant recipients with mixed CMV-genotype infections within the first year posttransplantation each of the major glycoprotein B (gB) and glycoprotein H (gH) genotypes was selectively quantified by genotype-specific quantitative TaqMan assays. The data obtained revealed that individually different genotype dynamics occurred for the individual patients and that the relative levels of the genotypes to each other may change over time. The quantitative development was independent of the specific gB-gH-genotype. In 10 of the 16 lung recipients the patient's individual genotype composition was the same in blood and lung. Genotype development during the follow-up was influenced by antiviral treatment. These data show for the first time that the CMV load used as diagnostic tool after transplantation is not always a constant entity but reflects the sum of the individual CMV-genotype dynamics developing over time.     

Characterization of a new antigen-antibody system associated with hepatitis B.

The occurrence of a new antigen-antibody system was recently described in hepatitis B surface antigen (HBsAg) positive sera with different specificities, one of which was designated e. These specificities are related as demonstrated by the presently utilized e reagents not discriminating between them in immunodiffusion tests. Hence, they are collectively referred to as e in this report. The occurrence of e antibodies in sera containing antibodies against hepatitis B surface antigen (anti-HBs) is reported. The e antibodies were found to move as 7S globulin on rate zonal centrifugation. The e precipitate was demonstrated on the cathode side of the HBsAg precipitate on immunoelectrophoresis consistent with e antigen migrating in the gamma-globulin region. The e antigen is reported to have an S value of 11-6 +/- 0-6 (s.d.) and a buoyant density of 1-291 +/- 0-003 (s.d.), hence differing from HBsAg in all respects studied thus far.     

Photoacoustic-guided ultrasound therapy with a dual-mode ultrasound array

Photoacoustics has recently been proposed as a potential method to guide and/or monitor therapy based on high-intensity focused ultrasound (HIFU). We experimentally demonstrate the creation of a HIFU lesion at the location of an optical absorber, by use of photoacoustic signals emitted by the absorber detected on a dual mode transducer array. To do so, a dedicated ultrasound array intended to both detect photoacoustic waves and emit HIFU with the same elements was used. Such a dual-mode array provides automatically coregistered reference frames for photoacoustic detection and HIFU emission, a highly desired feature for methods involving guidance or monitoring of HIFU by use of photoacoustics. The prototype is first characterized in terms of both photoacoustic and HIFU performances. The probe is then used to perform an idealized scenario of photoacoustic-guided therapy, where photoacoustic signals generated by an absorbing thread embedded in a piece of chicken breast are used to automatically refocus a HIFU beam with a time-reversal mirror and necrose the tissue at the location of the absorber.     

The dynamics of quantifiable homeostasis I. The individual

In this field of enquiry, “homeostasis” is understood in the sense of Bernard, Cannon, and Wiener, that is, with the system of continuous adjustments, in a trait not inherently stable, to meet the challenges of the environment. It is shown that the clinical fitness of a trait may depend on factors other than the mean or the variance. In particular the broad pattern of variation of the phenotype may be of importance and hence the characteristics of the homeostatic mechanisms for the control of traits subject to variation: notably the homing value (the “setting” of the homeostat), and the strength with which the organism responds to departures from this value. These parameters are related, but perhaps only remotely, to the traditional notion of the value of a phenotype. In general, where the environmental value is variable, there exist circumstances in which the optimal control would be neither extremely tight nor extremely loose. For instance, anticipatory action on the part of the body may be vitiated by too tight a control. Some illustrations are given of genetic disorders in which there is a fault in the strength of control. While neither the pattern of inheritance nor the impact on the species is explored in detail, the broad implications are indicated. Compromise adjustments are explored where two or more traits are regulated through the same homeostatic device.     

Characterization of a new HLA-A allele,A*0256, identified in a Caucasian individual

HLA-A typing by the PCR-SSP method in a male 21-year-old Caucasian individual revealed a very rare allele combination corresponding to a unique reaction pattern. Therefore, the result was examined using sequence-based typing. Sequencing of exons 2 and 3 of the HLA-A locus after allelic separation with specific primers revealed the sequence of a new allele, similar to A*0245. Sequencing of exons 1 and 4 resulted in no additional inconclusive positions. The sequence pattern of the new allele HLA-A*0256 might have been generated as a result of a double crossing over recombination of an A*0201 and either an A*03 or an A*11.     

Investigation of a scanned cylindrical ultrasound system for breast hyperthermia

This paper investigates the feasibility of a scanned cylindrical ultrasound system for producing uniform heating from the central to the superficial portions of the breast or localized heating within the breast at a specific location. The proposed system consists of plane ultrasound transducer(s) mounted on a scanned cylindrical support. The breast was immersed in water and surrounded by this system during the treatment. The control parameters considered are the size of the transducer, the ultrasound frequency, the scan angle and the shifting distance between the axes of the breast and the system. Three-dimensional acoustical and thermal models were used to calculate the temperature distribution. Non-perfused phantom experiments were performed to verify the simulation results. Simulation results indicate that high frequency ultrasound could be used for the superficial heating, and the scan angle of the transducer could be varied to obtain an appropriate high temperature region to cover the desired treatment region. Low frequency ultrasound could be used for deep heating and the high temperature region could be moved by shifting the system. In addition, a combination of low and high frequency ultrasound could result in a portion treatment from the central to the superficial breast or an entire breast treatment. Good agreement was obtained between non-perfused experiments and simulation results. The findings of this study can be used to determine the effects of the control parameters of this system, as well as to select the optimal parameters for a specific treatment.     

Establishment of a pharmacogenomic testing system for the realization of individual pharmacotherapy

The metabolism of and sensitivity to drugs differ from individual to individual, and one base polymorphism of drug-metabolizing enzymes is known to play an important role in this difference between individuals. The genotyping of drug-metabolizing enzymes prior to drug administration would help to predict individual reactivity to drugs and possible adverse reactions that may occur, which is essential to realize tailor-made therapy for individual patients. In July 2005, the Pharmacogenomics Working Group, composed of members of the Clinical Genomic Medicine Unit, Pharmaceutical Department, Medical Informatics Department, Clinical Laboratory, Gastroenterology, Cardiovascular Medicine, and Department of Neurology, was established in our university hospital in an attempt to introduce such pharmacogenomic testing. The project was approved by the Institutional Research Ethics Committee of the Faculty of Medicine, the University of Tokyo, and, in August 2006, testing for CYP2C19*2 and CYP2C19*3, enzymes involved in the metabolism of proton pump inhibitors, was started. Furthermore, in August 2007, testing for CYP2C9*3, the enzyme involved in the metabolism of Warfarin, and vitamin K epoxidereductase1 (VKORC1) 6484 C>T was started. In the CYP2C19 genotyping, the high incidence of poor metabolizers has been demonstrated; it was speculated that the test could confirm the adverse effects of the drug, i.e., after administration of the drug to patients. Moreover, testing for CYP2C9*3 and VKORC1 6484 C>T was shown to be useful for the safe administration of warfarin. The pharmacogenomic testing system was successfully established in our university hospital, and the Pharmacogenomics Working Group is still active, playing an important role in this project.     

Characterization of a novel HLA-Cw*02 variant, Cw*0208, in a Caucasian individual

We describe an additional HLA-Cw*02 variant, HLA-Cw*0208, which has been identified in a renal transplant recipient of Caucasian origin (Italy). After performing preliminary serological typing, we analyzed exons 2 and 3 of the HLA-C locus polymorphism by cloning the amplified DNA and using a sequence-based typing method. The new allele differs from Cw*020202 by one nucleotide substitution at nucleotide 61 (G-->A) of exon 2, which translates to a difference of one amino acid at residue 21 (His-->Arg) of the HLA-C heavy chain. We propose that Cw*0208 was generated by a random point mutation in codon 21 from the Cw*020202 allele, or through gene conversion of Cw*020202 with another allele, probably the Cw*1205 and Cw*1602 alleles.     

Characterization of a novel HLA allele HLA-B*15:25:03 in a Chinese individual

Nucleotide sequence of HLA-B*15:25:03 allele was different from that of HLA-B*15:25:01 at position 819 G to A.     

Characterization of a novel HLA allele, HLA-B*40:128, in a Chinese individual

Nucleotide sequence of HLA-B*40:128 has a single nucleotide difference at position 539 T>G compared with that of HLA-B*40:01:01, with an amino acid change from Leu to Arg at codon 156.     

基于有限元分析和集中参数模型微血管与超声微泡声学响应的模拟

BACKGROUND: Exploration on nonlinear acoustic response of the contrast agent microbubble contained in microvessel under ultrasound excitation is of great significance to maximizing ultrasonic energy deposition, promoting the development of quantitative imaging algorithm, revealing the damage mechanism or evaluating the targeted therapy, and overcoming the limitations of the traditional methods that are mainly used in large-size vessels, and measuring microvessel elasticity. OBJECTIVE: To build a microvessel containing an ultrasound microbubble, revealing the internal mechanism among ultrasound, microbubble, blood flow and microvessel. METHODS: Based on the finite element analysis and the lumped parameter model, three-dimensional microvessel containing microbubble model was built and simulated on Comsol Multiphysics 4.4 platform. RESULTS AND CONCLUSION: Microbubble exhibited slower radial motion compared with axial motion due to vascular wall limitation, but maximum displacement and stress were found near the microbubble center because of the oscillation coupling of the microbubble with the vascular wall. Under the same ultrasound pressure, the excitation frequency increased, accompanied by decreased and stabilized microvessl constriction and dilation; under the same frequency, with the enhancement of ultrasound pressure, the local microbubble oscillation lasted longer. With the increase of Young’s modulus of the microvessel wall, the frequency of microbubble oscillation was reduced, while the amplitude increased. All these findings indicate that the frequency of microbubble oscillation increased with the reduction of microvessel size, while its amplitude decreased. The frequency of microbubble oscillation increased with the enhancement of ultrasound excitation, while the amplitude decreased. On the contrary, ultrasound pressure affected the dynamic characteristics of microbubble and microvessel. In particular, it was the first to demonstrate that the elasticity of microvessel has approximate linear positive correlation with the amplitude of microbubble oscillation, which reveals the relationship between microvessel elasticity and microbubble response so as to provide theoretical basis for indirect measurement of microvessel elasticity. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Blood oxygen flux estimation with a combined photoacoustic and high-frequency ultrasound microscopy system: a phantom study

The metabolic rate of oxygen consumption, an important indicator of tissue metabolism, can be expressed as the change of net blood oxygen flux into and out of a tissue region per 100 g of tissue. In this work, we propose a photoacoustic and Doppler ultrasound method for imaging local blood oxygen flux of a single vessel. An imaging system for combined photoacoustic and high-frequency ultrasound microscopy is presented. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and Doppler ultrasound images are co-registered. Since the mean flow speed can be measured by color Doppler ultrasound, the vessel cross-sectional area can be measured by power Doppler or structural photoacoustic imaging, and multi-wavelength photoacoustic methods can be used to estimate oxygen saturation (sO(2)) and total concentration of haemoglobin (C(Hb)), all of the parameters necessary for oxygen flux estimation can be provided. The accuracy of the flow speed and sO(2) estimation has been investigated. In vitro sheep blood phantom experiments have been performed at different sO(2) levels and mean flow speeds. Blood oxygen flux has been estimated, and the uncertainty of the measurement has been quantified.