Mitra Azadniv

Correlation of ultrasound-induced hemolysis with cavitation detector output in vitro

A 20-MHz passive acoustic detector was used to quantify the amount of transient acoustic cavitation occurring in a sample exposed to intense pulsed ultrasound. A dilute suspension of human erythrocytes with and without a microbubble echo-contrast agent was exposed in vitro to 500 W/cm2 (SPPA) ultrasound of center frequency 1 MHz and tone burst duration 20, 100, 200, 500 and 1000 microseconds at a pulse repetition frequency of 20 Hz. Inertial cavitation occurring within the sample, as measured by the temporal average of the detector output, correlated well with hemolysis, suggesting that violent bubble collapse is responsible for cell damage. The result also raises the prospect of cavitation monitoring as a possible predictor of adverse bioeffects when echo-contrast agents are used clinically.

Systemic effects of inhaled ultrafine particles in two compromised, aged rat strains

Epidemiological studies associate morbidity and mortality with exposure to particulate air pollution in elderly individuals with existing cardiopulmonary disease. These associations led to the hypothesis that inhaled particles can exert adverse effects outside of the lung, particularly on the cardiovascular system. We tested this hypothesis by examining the pulmonary and peripheral effects of inhaled ultrafine carbon particles in old rats that were injected with endotoxin (lipopolysaccharide, LPS) to model systemic gram-negative bacterial infection. Fischer 344 rats (23 mo) and spontaneously hypertensive (SH) rats (11–14 mo) were injected with LPS (2 mg/kg, ip) immediately before being exposed to inhaled ultrafine carbon particles for 6 h (150 μg/m3, CMD = 36 nm). Controls were injected with sterile saline or were sham exposed. Twenty-four hours after LPS injection, bronchoalveolar lavage (BAL) fluid, cells, and blood were obtained to assess endpoints of inflammation, oxidant stress, coagulability, and the acute-phase response. LPS did not cause an influx of neutrophils (PMNs) into the alveolar space, but did increase the number and percentage of circulating PMNs and the concentration of plasma fibrinogen in both rat strains. Inhaled ultrafine particles did not induce lung inflammation in either rat strain. In both strains, ultrafine particles (UFP) were found to decrease the number of blood PMNs, increase the intracellular oxidation of a fluorescent dye (DCFD) in blood PMNs, and affect plasma thrombin–anti-thrombin (TAT) complex and fibrinogen levels. UFP were also found to interact with ip LPS with respect to plasma TAT complex levels and blood PMN DCFD oxidation. Differences between the two rat strains were also found for TAT complex levels, BAL cell reactive oxygen species release, and DCFD oxidation in both BAL macrophages and blood PMNs. These results suggest that inhaled ultrafine carbon particles inhaled at concentrations mimicking high episodic increases in urban air can exert extrapulmonary effects in old rats and that they can change the systemic response to an inflammatory stimulus.

Effect of a Stabilized Microbubble Contrast Agent on CW Ultrasound Induced Red Blood Cell Lysis In Vitro

Human red blood cells (RBCs) in vitro at various fractional hematocrits (HCTs) were exposed for 60–120 seconds in a dialysis tubing vessel to 1 MHz continuous wave ultrasound (0–5 W/cm2 SPTA intensity); exposure vessels were either rotated at 200 rpm or stationary. Some RBC suspensions also contained Albunex® (ALX; a commercially‐produced microbubble clinical ultrasound contrast agent) at final concentrations ranging from 0–41 μL/mL RBC suspension. Isonation was either by one transducer or by two opposing, continuously‐gated, balanced transducers. For the vessel rotation / no rotation experiments, ultrasound‐induced cell lysis was always increased with the ALX regimen relative to that of the no‐ALX regimen at HCTs up to about 10%; at higher HCTs (including whole blood), ultrasound‐induced hemol‐ysis essectially ceased to occur. The data are consistent with reports of the ineffectiveness of continuous wave ultrasound at 3–5 W/cm2 to lyse cells in vitro at physiological cell densities with or without echocontrast medium supplemantation.

Hemolysis of albunex-supplemented, 40% hematocrit human erythrocytes in vitro by 1-MHz pulsed ultrasound: Acoustic pressure and pulse length dependence

The tested hypothesis was that ultrasound-induced hemolysis in blood supplemented with a microbubble contrast agent varies with ultrasound intensity and pulse duration. Human erythrocytes in autologous plasma containing 3.6% v:v Albunex microspheres were exposed to 1.07-MHz ultrasound pulses of 5 to 1000 mus at SPTP intensities of 0 to 1100 W/cm2. The dependence of hemolysis on the mechanical index (MI) value of the exposures was also examined. Ultrasound-induced hemolysis: (1) was evident at all pulse/intensity combinations; (2) increased generally with increasing pulse duration at constant intensity; and (3) increased with increasing MI at constant pulse duration. For pulses of 10 to 30 mus, ultrasound-induced hemolysis remained low (< or = 2%) at MI values < approximately 2 and increased sharply with further increase in MI; for 5-mus pulses, this abrupt increase in hemolysis was associated with a larger MI (approximately 3).

Effect of a Stabilized Microbubble Echo Contrast Agent on Hemolysis of Human Erythrocytes Exposed to High Intensity Pulsed Ultrasound

Microbubble contrast agents have been shown to enhance ultrasonic cell lysis in vitro when exposed to continuous‐wave ultrasound having spatial peak temporal average (SPTA) intensities of a few W/cm2. The response is strongly dependent upon the hematocrit (HCT) of the cell sample; detectable cell lysis essentially disappears as the HCT approaches 5%‐10%. This study was conducted to determine whether high intensity pulsedsound is an effective lytic agent in the presence of preexisting potential cavitation nuclei (Albunex® contrast agent). Human erythrocytes weresuspended in autologous plasma to HCTs ranging from 1%–40%. Suspensions were exposed or sham exposed for 60 seconds to focused, pulsed ultrasound. The pulse duration was 1 msec, and the pulse repetition frequency was 20 Hz. The pressure amplitudes, spatial peak pulse average (SPPA) intensity, and SPTA intensity were 4.7 MPa peak positive pressure, ‐2.7 MPa peak negative pressure, 420 W/cm2, and 8.5 /cm2, respectively. Samples were exposed to ultrasound in a dialysis membrane exposure vessel rotating at 200 rpm. When included in the erythrocyte samples, the Albunex concentration was 35 μL/mL suspension. Significant ultrasound‐induced hemolysis in the absence of Albunex was observed only at the lowest HCT value tested (1%). In the presence of Albunex significant cell lysis was observed at all tested HCT values. The relative fraction of cells lysed by the combination of ultrasound exposure and Albunex diminished with increasing HCT, but the number of cells lysed per sample was nearly constant over the range of 5%–40% HCT. The ultrasound exposure parameters used in this study differ substantially from those associated with diagnostic imaging equipment; it is not valid to infer from the present results that the use of Albunex in diagnostic applications will induce or enhance hemolysis in vivo.

Galactosylated LDL nanoparticles: a novel targeting delivery system to deliver antigen to macrophages and enhance antigen specific T cell responses.

We aim to define the role of Kupffer cells in intrahepatic antigen presentation, using the selective delivery of antigen to Kupffer cells rather than other populations of liver antigen-presenting cells. To achieve this we developed a novel antigen delivery system that can target antigens to macrophages, based on a galactosylated low-density lipoprotein nanoscale platform. Antigen was delivered via the galactose particle receptor (GPr), internalized, degraded and presented to T cells. The conjugation of fluoresceinated ovalbumin (FLUO-OVA) and lactobionic acid with LDL resulted in a substantially increased uptake of FLUO-OVA by murine macrophage-like ANA1 cells in preference to NIH3T3 cells, and by primary peritoneal macrophages in preference to primary hepatic stellate cells. Such preferential uptake led to enhanced proliferation of OVA specific T cells, showing that the galactosylated LDL nanoscale platform is a successful antigen carrier, targeting antigen to macrophages but not to all categories of antigen presenting cells. This system will allow targeted delivery of antigen to macrophages in the liver and elsewhere, addressing the question of the role of Kupffer cells in liver immunology. It may also be an effective way of delivering drugs or vaccines directly at macrophages.

The E2F-1 Transcription Factor Promotes Caspase-8 and Bid Expression, and Enhances Fas Signaling in T Cells

The immune system depends on the extensive proliferation of rare Ag-specific precursor T lymphocytes, followed by their differentiation, the delivery of effector function, and finally death by apoptosis. T cells that lack the E2F-1 transcription factor, which is activated as cells pass the restriction point and enter S phase, show defects in activation-induced cell death. We now report that E2F-1 increases the activity of an apoptotic pathway that is important in murine primary T cells. Thus, E2F-1 promotes the transcription of Bid, a molecule that links death receptor signaling to the activation of apoptotic mechanisms in mitochondria. It also promotes the transcription of caspase-8, the enzyme that cleaves and activates Bid. Enforced expression of Bid can partially restore apoptosis in E2F-1-deficient T cells. Thus, E2F-1 integrates cell cycle progression with apoptosis.

Effect of static pressure on acoustic transmittance of Albunex(R) microbubble suspensions

Albunex (ALX), an albumin-stabilized microbubble echo contrast agent, is sensitive to pressures similar to those produced by the heart. The tested hypothesis was that the acoustic transmittance of ALX suspensions will increase with increasing hydrostatic pressure (Ps). The test involved an acoustic setup analogous to a spectrophotometer. The acoustic transmittance of microbubble suspensions was strongly Ps dependent. Transmittance at 1 MHz was essentially zero at ambient pressure, increasing to approximately 50%, approximately 63%, and nearly 100% at Ps of 80, 120, and 400 mm Hg, respectively. The ultrasound pulses used to interrogate samples were without measurable effect on the acoustic transmittance of suspensions maintained at ambient pressure during experimental measurements. The data indicate that many of the microbubbles are destroyed at Ps comparable to those produced by the heart.

Temporality in Ultrasound-Induced Cell Lysis In Vitro

To test the hypothesis that the full extent of in vitro cell lysis due to ultrasound becomes evident with time lapse following insonation, human erythrocytes (2% hematocrit) in autologous plasma were mixed with Albunex®, a pulse echo contrast agent, and exposed to 1‐MHz, continuous‐wave ultrasound (US) (5 W/cm2 SPTA intensity) for 60 seconds while in a rotating (200 rpm) dialysis membrane vessel. Exposed and sham‐exposed samples were subsequently assayed for hemolysis colorimetrically, either immediately or after a delay of 3 hours. Hemolysis was dependent on the interval between US exposure and assay, with significantly greater lysis evident with delayed assay. There was also temporality in lytic yield with sample number, i.e., with time postpreparation of the blood sample, US‐induced cell lysis decreased. The temporality of lytic yield was eliminated by maintenance at ice water temperatures, or by waiting about 1 hour before beginning treatments. The collective data indicate that the full extent of US‐induced cell lysis is not evident upon assay immediately after insonation, and that with time postpreparation and preinsonation, erythrocytes may undergo changes in sensitivity to US.

Bubble recycling and ultrasonic cell lysis in a stationary exposure vessel

An exposure system producing interleafed pulses of 1-MHz ultrasound from balanced, opposing transducers was used to test the bubble recycling hypothesis (as formulated with respect to rotating exposure vessels) in a stationary exposure vessel containing P388 cell suspensions. Cell lysis was measured in cell suspensions which had been exposed or sham-exposed to 1-MHz ultrasound (5 W/cm2 spatial peak pulse average intensity) delivered in continuously alternating, opposing pulses. The opposing pulse cycle frequency (i.e., gating frequency) varied from 0.1 Hz to 100 kHz, corresponding to single pulse durations ranging from 5 microseconds to 5 s. Unequivocal support for the bubble recycling hypothesis was obtained. Lytic yield increased as the gating frequency increased, passing through a maximum at gating frequencies of 100 Hz-1 kHz. As the gating frequency increased further, lytic yield declined rapidly. The results are discussed in relation to recent theoretical estimates of bubble translation speeds in an ultrasound field, and are found to be consistent with those estimates.