Ultrasound-induced Cavitation enhances the efficacy of Chemotherapy in a 3D Model of Pancreatic Ductal Adenocarcinoma with its microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is supported by a complex microenvironment whose physical contribution to chemoresistance could be overcome by ultrasound (US) therapy. This study aims to investigate the ability of US-induced inertial cavitation in association with chemotherapy to alter tumor cell viability via microenvironment disruption. For this purpose, we used a 3D-coculture PDAC model partially mimicking the tumor and its microenvironment. Coculture spheroids combining DT66066 cells isolated from KPC-transgenic mice and murine embryonic fibroblasts (iMEF) were obtained by using a magnetic nanoshuttle method. Spheroids were exposed to US with incremental inertial cavitation indexes. Conditions studied included control, gemcitabine, US-cavitation and US-cavitation + gemcitabine. Spheroid viability was assessed by the reduction of resazurin and flow cytometry. The 3D-coculture spheroid model incorporated activated fibroblasts and produced type 1-collagen, thus providing a partial miniature representation of tumors with their microenvironment. Main findings were: (a) Gemcitabine (5 µM) was significantly less cytotoxic in the presence of KPC/iMEFs spheroids compared with KPC (fibroblast-free) spheroids; (b) US-induced inertial cavitation combined with Gemcitabine significantly decreased spheroid viability compared to Gemcitabine alone; (c) both cavitation and chemotherapy affected KPC cell viability but not that of fibroblasts, confirming the protective role of the latter vis-à-vis tumor cells. Gemcitabine toxicity is enhanced when cocultured spheroids of KPC and iMEF are exposed to US-cavitation. Although the model used is only a partial representation of PDAC, this experience supports the hypothesis that US-inertial cavitation can enhance drug penetration and cytotoxicity by disrupting PDAC microenvironment.

Assay of hydroxyl radicals generated by focused ultrasound.

Water sonolysis leads to the formation of hydroxyl radicals (OH*). Various techniques are used to detect the OH* production and thus to assess the level of ultrasound-mediated cavitation generated in vitro. In this study, we used terephthalic acid (TA) as an OH* trap. This method is based on the fluorescent properties of hydroxyterephthalic acid (HTA) formed by the reaction of TA with OH* and used as an indicator of the degree of inertial cavitation caused. The experimental system is comprised mainly of a focused piezoelectric ultrasound transmitter and a measurement cell containing 1X PBS/TA diluted solution. In the first part, we aimed to characterize the most appropriate experimental conditions (TA dosimeter solution, irradiation time) in order to optimize the resulting HTA fluorescence values. Then, we could determine that the HTA production increased with the level of the cavitation phenomenon caused by the acoustic power from which OH* production may be estimated.

Endothelial cell costimulation of T cell activation through CD58-CD2 interactions involves lipid raft aggregation.

Human endothelial cells (EC) costimulate CD4(+) memory T cell activation through CD58-CD2 interactions. In this study we tested the hypothesis that EC activate distinct costimulatory pathways in T cells that target specific transcription factors. AP-1, composed of fos and jun proteins, is a critical effector of TCR signaling and binds several sites in the IL-2 promoter. EC augment c-fos promoter activity in T cells; however, deletion analysis reveals no transcription factor binding sites in the promoter uniquely responsive to EC costimulation. Overexpression of AP-1 proteins in T cells augments the activity of an AP-1-luciferase reporter gene equally in the absence or the presence of EC costimulation. Interestingly, EC stimulate a similar 2- to 3-fold up-regulation of AP-1, NF-AT, NF-kappaB, and NF-IL-2-luciferase reporters. CD2 mAbs completely block EC effects on all of these pathways, as well as costimulation of IL-2 secretion. We conclude that EC costimulation through CD2 does not trigger a single distinct costimulatory pathway in T cells, but rather, it amplifies several pathways downstream of the TCR. Indeed, we find that early EC costimulation acts "upstream" of the TCR by promoting lipid raft aggregation, thus amplifying TCR signaling. Soluble CD2 mAbs block EC-induced raft aggregation, whereas cross-linking CD2 promotes aggregation. These data are consistent with the critical role of CD2 in organizing the T cell-APC contact zone.

Single-cell analysis of costimulation by B cells, endothelial cells, and fibroblasts demonstrates heterogeneity in responses of CD4(+) memory T cells.

Human endothelial cells (EC) express MHC class II molecules in vivo and are likely to be involved in presentation of antigens to CD4(+) T cells. We examined, at the single-cell level, EC presentation of superantigens to resting CD4(+) memory T cells. Within 2 h of adherence to class II+ EC early T cell activation is evidenced by translocation of nuclear factor of activated T cells (NFAT), surface expression of CD69, and synthesis of IFN-gamma and IL-2. Naive T cells are not activated. T cell activation is dependent on the prior induction of MHC class II molecules on EC and is blocked by antibodies to LFA-3 (CD58). Our data place EC along a spectrum of antigen-presenting ability. Activated B cells and macrophages trigger more cells to express cytokines than do EC and at lower antigen concentrations; EC are in turn, superior to fibroblasts or smooth muscle cells. Furthermore, the concept of activation thresholds for cytokine synthesis within T cells also extends to earlier activation events: NFAT translocation is relatively easy to trigger, as is CD69 expression; fewer cells can be triggered to express IFN-gamma and fewer still to express IL-2. EC may, therefore, contribute to a graded immune response by inducing qualitatively and quantitatively different responses than professional APC.

Motorised resection device for transurethral resection of the prostate: a laboratory evaluation.

Transurethral resection of the prostate is the most common method of relieving urinary outflow obstruction secondary to prostatic enlargement. However, this procedure can be responsible for various complications, including irrigant-fluid absorption and blood loss, both of which are strongly dependent on operation duration time. To reduce the latter, a new resection device has been designed for transurethral prostatectomy. The device basically consists of a rotating cutting loop controlled externally, with three degrees of freedom, to fit the adenoma shape. Its performance is assessed in vitro by drilling conical and semi-ellipsoidal cavities in agar gel models. The mean difference between the calculated and obtained cavity volumes is 3% (SD = 0.9%). The volume cutting rate, found to be independent of the type of cavity drilled, is equal to 2.9 +/- 0.3 cm3 min-1. The advantages of this motorised resection device prototype are reduction in operation duration and accuracy of the resected volume. In vivo resection of a 20 cm3 adenoma in less than 15 min can be expected.

High-efficiency shock-wave generator for extracorporeal lithotripsy.

In extracorporeal lithotripsy, the electro-acoustic efficiency of electrohydraulic generators is limited by the inductance of the electrical discharge circuit. A new shock-wave generator is described that uses a coaxial discharge line enabling electro-acoustic efficiency to be greatly increased. The line is built using a para-electric ceramic with a relative dielectric constant of 1700, manufactured for use in high-voltage impulse mode. A coaxial spark gap, with minimal inductance, has been developed to obtain the triggered breakdown of the discharge line. Shock waves are created with a coaxial electrode plugged directly into the spark gap and immersed in an electrolyte of degassed saline. Electrode gap and electrolyte resistivity are adjusted to match the resistivity of the electrolyte volume between the underwater electrodes to the characteristic impedance of the line. The discharge line generates in the medium a rectangular current pulse with an amplitude of about 6000 A and a rise time of 50 ns. Compared with conventional generators, measurements of the expansive peak pressure pulse show an increase of 105% at 10 kV, 86.5% at 12 kV and 34.5% at 14 kV charging voltage. Electro-acoustic efficiency is found to be 11% instead of 5.5% for a conventional discharge circuit.

Texture analysis of ultrasonic images of the prostate by means of co-occurrence matrices.

As speckle on ultrasonic B-scan may reveal information relative to tissue structure, the present work attempts to discriminate the various prostatic tissues (normal tissue, benign prostatic hypertrophy and cancer) by means of texture analysis. We select regions of interest by their homogeneous appearance. A pre-processing stage is required to obtain stationary samples. The method used measures the second-order statistics, namely co-occurrence matrices. Fairly good tissue signatures have been obtained with parameters derived from these matrices. Of 37 images processed, 78 percent of the samples were classified with success, which is a high score considering that the images cannot be discriminated visually. However, while such results are obtained when wide regions of interest are investigated (64 x 64 pixels), they are not as good with smaller sample sizes, i.e., when the pathological area is very small.

Volume measurement by ultrasonic transverse or sagittal cross-sectional scanning.

A technique is described that provides an accurate estimation of the volume of an organ from its ultrasonic cross-sectional images. The technique is applied to two types of ultrasonic investigation, one providing transverse and the other sagittal images. The organ outline has to be traced on each scan. The computer first calculates the area and then the volume from the vector areas and the centroids of a series of sections. The technique has been tested with phantoms of various shapes and volumes made with agar gel. These experiments show that the error in the volume estimation is less than 10% and the variability of measurements is less than 2%.

Influence of water conductivity on the efficiency and the reproducibility of electrohydraulic shock wave generation.

In an electrohydraulic generator, two underwater metal electrodes are connected with a capacitor charged to a high voltage. When the circuit is switched on, a plasma is generated reaching temperatures of thousands of K, resulting in a compressive pressure pulse. The formation of the plasma is a nonreproducible phenomenon inducing great variations of the pressure pulse. When the electrodes are immersed in an electrolyte instead of degassed water, the conditions of electrical discharge are dramatically modified. The latency time and the amplitude of the oscillations of the discharge current decrease as the conductivity of the electrolyte increases. For a conductivity of 7 omega.cm, there is no latency, and the critically damped discharge is achieved. The expanding pressure wave is increased by 10%, and the mean peak pressure value over 120 shocks at the second focus after focalization is increased by 50%. The relative standard deviation of the pressure value at the second focus is only 5%, while it is about 30% in ordinary water. The fragmentation efficiency is considerably increased because total fragmentation is obtained in 220 shocks instead of 450 shocks in ordinary water when standard stones are used, and in 131 shocks instead of 304 shocks when gallstones are used. Last, we show that the wear of the electrodes is reduced by a factor 8 when electrolyte is used. The improvement is supposed to have two causes: First, the energy is delivered into the medium in a shorter time, and, second, the center of the shock wave is always located at the same place. The decreased wear should make it possible to treat a much greater number of patients without changing electrodes, and the enhancement of the pressure should increase the efficiency of the fragmentation of the gallstones without aggravating the patient's pain.

Extracorporeal lithotripsy of bile duct stones using ultrasonography for stone localization.

In 19 patients, extraction of bile duct stones through the papilla using a Dormia basket or a mechanical lithotripter was not possible following endoscopic sphincterotomy. After the insertion of a nasobiliary drain, extracorporeal lithotripsy was performed with intravenous sedation using an ultrasonographic stone localization system. The number and location of stones were first determined by retrograde cholangiography. At the time of lithotripsy, saline was injected in the bile ducts to modify the acoustic impedance of tissues surrounding the stones, and subsequent ultrasonography was effective in localizing all stones present in 4 of 5 (80%) patients with intrahepatic stones, and 13 of 14 (93%) with common bile duct stones. In 10 patients (53%), fragmentation was satisfactory and the bile ducts were cleared completely. The mean single stone diameter was significantly smaller in successful cases of fragmentation compared with failures (22.8 +/- 6.6 mm vs. 40 +/- 10 mm). The results in patients with multiple stones were significantly worse than those in patients with single stones of similar size (25% vs. 100% successful fragmentation). Reasons for this difference in results included the small size of the focal area and the reduced ability of ultrasonography (1) to adequately visualize multiple calculi individually and (2) to assess the degree of stone destruction. Care was taken to first await the resolution of infection or the correction of coagulation abnormalities when present; no morbidity following extracorporeal lithotripsy was observed. Despite its 3-step approach (endoscopic sphincterotomy, lithotripsy, and endoscopic extraction), the need for only intravenous sedation and the absence of patient immersion in water render this technique attractive for elderly and frail patients.

The significance of the prostatic hypoechoic area: results in 226 ultrasonically guided prostatic biopsies.

A total of 666 patients with symptoms of urinary outflow obstruction underwent assessment of the patients 64 had a palpable abnormality suggestive of cancer (stages T1 to T4, or B to C). In the remainder the prostate was either palpably normal, firm or enlarged by benign prostatic hypertrophy. All 64 patients with a palpable abnormality and 162 of 602 with normal rectal examination findings had a hypoechoic area on transrectal ultrasound. Biopsy of the ultrasonic abnormality was done in 148 men by the transperineal route with linear array ultrasound guidance and in 78 by the transrectal route with a mechanical sector scanner in the sagittal plane. Of the 64 patients with a nodular prostate 34 (53%) had cancer (31% of those with stages T1 and 2, 83% with stage T3 and 100% with stage T4 disease). In 14% of the patients with stages T1 and T2 cancer the biopsy showed prostatic intraepithelial neoplasia grade 3. Of the 162 patients with a palpably normal prostate who underwent ultrasound-guided biopsy 11 (6.7%) had cancer and 6 (3.7%) had grade 3 prostatic intraepithelial neoplasia detected in the biopsy material. Patients with stages T1 to T2 cancer and those with ultrasound-diagnosed impalpable cancer were not significantly different with respect to patient age (67 versus 70 years), cancer size (3.0 +/- 1.6 versus 3.9 +/- 2.5 cm.2) or Gleason score (5.4 +/- 1.2 versus 6.5 +/- 0.9). The results demonstrate that ultrasound guidance improves the yield of prostate needle biopsy. Furthermore, it is suggested that prostate cancer found by ultrasound alone is not different from early palpable disease and should be treated in the same manner.

A novel method to control P+/P- ratio of the shock wave pulses used in the extracorporeal piezoelectric lithotripsy (EPL).

There is growing evidence that acoustic cavitation plays an important role in stone fragmentation during extracorporeal shock wave lithotripsy (ESL) treatment. In addition, side effects of the treatment, such as the hemorrhage and destruction of the tissue in the vicinity of the stone are also ascribed to cavitation phenomenon. Since cavitation is associated with the maximum negative pressure in the shock pulse, it would thus appear that possibility of controlling this pressure would be desirable in ESL applications. This paper describes a novel technique developed to control the ratio of compressional peak (P+) to rarefactional peak pressure (P-) of the shock wave for use in lithotripsy treatment. The procedure is based on the finite amplitude wave generation by focused piezoelectric transducers and subsequent interaction of the shocked waves in the common focal region. The highly asymmetrical shock wave is produced in the focal region by providing an appropriate time delay to each of the high voltage electrical excitation signals which drive the transducers. The degree of relative reduction of negative halfcycles and the corresponding positive halfcycles amplification increases with the number of the acoustic sources used. The practical implementation of the shock wave generator was obtained by using 5 cm diameter, focused 1 MHz transmitter, and additional transducers of identical construction having frequencies corresponding to the harmonics and subharmonics of the 1 MHz frequency. The importance of the results for the future development of lithotripters, and stone treatment efficiency is pointed out.

Gallstone disappearance after extracorporeal lithotripsy and oral bile acid dissolution.

Extracorporeal shock-wave cholelithotripsy was carried out in 135 symptomatic patients with radiolucent gallstones, followed by oral bile acid dissolution to assess the resultant stone disappearance rates. Fragmentation was satisfactory (all fragments less than 5 mm in diameter) in 34 patients (25%) after a single session of lithotripsy, and in 65 (48%) after multiple sessions. The overall satisfactory fragmentation rate was significantly higher in patients with single stones less than or equal to 20 mm in diameter when compared with those with larger solitary stones (71% vs. 38%, p less than 0.05), as it also was in all subjects with solitary stones when compared with those with multiple stones (60% vs. 34%, p less than 0.05). After 6, 9, and 12 mo of oral bile acid treatment, the stone-free rates were significantly higher in patients with satisfactory than in those with partial fragmentation (55% vs. 0%, 80% vs. 29%, and 90% vs. 33%, respectively; p less than 0.05). Only 1 of the 7 patients who had previously undergone endoscopic sphincterotomy for concomitant choledocholithiasis was free of stones after 1 yr of dissolution. During dissolution therapy, of the 102 patients in whom fragmentation had occurred, 1 (1%) developed mild acute pancreatitis, 23 (23%) suffered attacks of biliary colic, and 6 (6%) required cholecystectomy. We conclude that the result of fragmentation appears to be a major determinant of the success and rapidity of subsequent oral bile acid dissolution, and that when satisfactory, it allows for complete stone disappearance in most patients within the following year. A comparison of the present results with those of previous original studies suggests that to achieve such satisfactory fragmentation, patients should be selected on the basis of their stone characteristics, which optimally should present as solitary gallbladder calculi less than 20 mm in diameter. Furthermore, real-time ultrasonographic monitoring should be used during lithotripsy with a transducer centered along the shock-wave axis. Despite the innocuousness of the shock waves, the incidence of fragment migration and its possible complications, in our experience, emphasizes the need to restrict at present this nonoperative approach to the treatment of symptomatic gallstone disease.

Ultrasound stone localisation for extracorporeal shock wave lithotripsy.

An extracorporeal shock wave lithotriptor using an ultrasound scan head pantograph location system has been designed. The shock wave ellipsoid reflector position is adjusted to the stone with a computer assisted positioning device. Seven dogs with stones implanted into the renal pelvis were treated and stone fragmentation occurred in all cases. Subsequently, 45 patients with stones were treated. The stones ranged in size from 5 to 29 mm (mean 16). Radio-opaque as well as poorly opaque or radiolucent stones were treated and fragmentation was achieved in 85% of cases. An additional endoscopic procedure was performed in four cases. No fragmentation occurred in four patients. Further shock wave treatment was necessary in two patients who presented with stones larger than 2 cm. Both radio-opaque and poorly opaque stones can be treated with this system. Ultrasound localisation and the ellipsoid positioning device avoid the need for expensive fluoroscopic equipment and a hydraulic patient positioning system.