Development of an in vitro macrophage screening system on the immunomodulating effects of feed components.

BACKGROUND: While feed components capable of modulating the immune system are highly sought after and marketed, often little evidence is available to support functional immune response claims. Thus, a high-throughput in vitro cell screening system was developed to test these compounds for innate immune signaling effects, using Saccharomyces cerevisiae and its cell wall components in addition to lauric acid and its esters as models in two separate experiments. This screening system utilized RAW 264.7 murine macrophages to assess live S. cerevisiae cells and S. cerevisiae-derived cell wall components ß-glucan, mannan, and zymosan (a crude cell wall preparation containing both ß-glucan and mannan). D-mannose was also evaluated as the monomer of mannan. We also examined the effect of a saturated fatty acid (C12:0, lauric acid) and its esters (methyl laurate and glycerol monolaurate) on innate immune cell activation and cellular metabolism. RAW cells were transfected with a vector that drives expression of alkaline phosphatase upon promoter activation of nuclear factor κ-light-chain-enhancer of activated B cells (NFκB), a major inflammatory/immune transcription factor. RAW cells were incubated with 0.01, 0.1 or 1 mg/mL of yeast compounds alone or RAW cells were challenged with LPS and then incubated with yeast compounds. In a separate experiment, RAW cells were incubated with 0, 0.5, 2.5, 12.5, 62.5, and 312.5 µmol/L of lauric acid, methyl laurate, or glycerol monolaurate alone, or RAW cells were challenged with LPS and then incubated with fatty acid treatments. RESULTS: Treatment with zymosan or ß-glucan alone induced NFκB activation in a dose-dependent manner, whereas treatment with D-mannose, mannan, or live S. cerevisiae cells did not. Post-treatment with mannan after an LPS challenge decreased NFκB activation, suggesting that this treatment may ameliorate LPS-induced inflammation. Slight increases in NFκB activation were found when fatty acid treatments were applied in the absence of LPS, yet substantial reductions in NFκB activation were seen when treatments were applied following an LPS challenge. CONCLUSIONS: Overall, this cell screening system using RAW macrophages was effective, high-throughput, and sensitive to feed components combined with LPS challenges, indicating modulation of innate immune signaling in vitro.

Surface integration of velocity vectors from 3D digital colour Doppler: an angle independent method for laminar flow measurements.

BACKGROUND: The study was designed to test the angle independence of a dynamic three-dimensional digital colour Doppler method for laminar flow measurement. The technique acquired three-dimensional data by rotational acquisition and used surface integration of Doppler vector velocities and flow areas in time and space for flow computation. METHOD: A series of pulsatile flows (peak flow 55-180 ml/s) through a curved tube were studied with reference flow rates obtained using an ultrasonic flow meter. Colour Doppler imaging was performed at three angles to the direction of flow (20 degrees, 30 degrees, 40 degrees), using a multiplane transoesophageal probe controlled by an ATL HDI5000 system. Integration of digital velocity vectors over a curved three-dimensional surface across the tube for each of the 11 flow rates at each angle was performed off-line to compute peak flow. RESULTS: Peak flow rates correlated closely (r=0.99) with the flow meter with the mean difference from the reference being -0.8+/-2 x 4 ml/s, 0.9+/-2.6 ml/s, 1.0+/-2 x 3 ml/s for 20 degrees, 30 degrees and 40 degrees respectively. Comparison of the three angle groups showed no significant differences (P=0.15, ANOVA). When sampled obliquely, the flow area on the curved surface increased while the velocities measured decreased. CONCLUSION: Surface integration of velocity vectors to compute three-dimensional Doppler flow data is less angle dependent than conventional Doppler methods.

Noninvasive assessment of left ventricular isovolumic contraction and relaxation with continuous wave Doppler aortic regurgitant velocity signals: an in vivo validation study.

The purpose of this study was to provide fundamental in vivo validation of a method with the use of aortic regurgitant (AR) jet signals recorded with continuous wave (CW) Doppler for assessing left ventricular (LV) isovolumic contraction and relaxation. Preliminary studies have suggested that analysis of CW Doppler AR velocity signals permits the estimation of LV positive and negative dP/dt. We studied 19 hemodynamically different states in 6 sheep with surgically induced chronic aortic regurgitation. CW AR velocity spectra and high-fidelity LV and aortic pressures were recorded simultaneously. Rates of LV pressure rise and fall (RPR and RPF) were calculated by determining the time interval between points at 1 m/s and 2.5 m/s in the deceleration and acceleration slopes of the CW Doppler AR velocity envelope (corresponding to a pressure change of 21 mm Hg). RPR and RPF calculated by CW Doppler analysis for each state were compared with the peak positive dP/dt and negative dP/dt, obtained from the corresponding high-fidelity LV pressure curve, respectively. The LV peak positive and negative dP/dt derived by catheter ranged from 817 to 2625 mm Hg/s and from 917 to 2583 mm Hg/s, respectively. Multiple regression analysis showed that Doppler RPR correlated well with catheter peak positive dP/dt (r = 0.93; mean differences, -413 +/- 250 mm Hg/s). There was also good correlation and agreement between Doppler RPF and the catheter peak negative dP/dt (r = 0.89; mean difference, -279 +/- 239 mm Hg/s). Both Doppler-determined RPR and RPF underestimated their respective LV peak dP/dt. CW Doppler AR spectra can provide a reliable noninvasive estimate of LV dP/dt and could be helpful in the serial assessment of ventricular function in patients with aortic regurgitation.

Validation of a digital color Doppler flow measurement method for pulmonary regurgitant volumes and regurgitant fractions in an in vitro model and in a chronic animal model of postoperative repaired tetralogy of Fallot.

OBJECTIVES: The purpose of this study was to validate a digital color Doppler (DCD) automated cardiac flow measurement method for quantifying pulmonary regurgitation (PR) in an in vitro and a chronic animal model of the right ventricular outflow tract of postoperative tetralogy of Fallot (TOF). BACKGROUND: There has been no reliable ultrasound method that can accurately quantitate PR. METHODS: We developed an in vitro model of mild pulmonary stenosis and wide-open PR that mimics the patterns of flow seen in patients with postoperative TOF. Thirteen different forward and regurgitant stroke volumes (RSVs) across the noncircular shaped cross-sectional outflow tract flow area were estimated using the DCD method in two orthogonal planes. In six sheep with surgically created PR, 24 different hemodynamic states with PR strictly quantified by electromagnetic probes were also studied. RESULTS: The RSVs and regurgitant fractions (RFs) obtained by the DCD method using average values from two orthogonal planes correlated well with reference values (RSV: r = 0.99, mean difference = 0.02 +/- 0.39 ml/beat for in vitro model; r = 0.97, mean differences = 1.79 +/- 1.84 ml/beat for animal model, RF: r = 0.98, mean difference = -1.10 +/- 4.34% for in vitro model; r = 0.94, mean difference = 2.73 +/- 6.75% for animal model). However, the DCD method using a single plane had limited accuracy for estimating pulmonary RFs and RSVs. CONCLUSIONS: The DCD method using average values from two orthogonal planes provides accurate estimation of RSVs and RFs and should have clinical importance for serially quantifying PR in patients with postoperative TOF.

Impact of harmonic imaging and transducer frequency on 'ventricular volume' measurements using real-time three-dimensional echocardiography: studies in an in vitro model.

AIMS: Harmonic imaging has increased the yield of quantifiable scans in two-dimensional echocardiography. Although real-time three-dimensional echocardiography avoids geometric assumptions in volume analysis, accurate measurement can be limited by image quality. This study compared volumes from a balloon model mimicking the left ventricle, scanned with and without harmonic imaging, using real time three-dimensional echocardiography. METHODS: Two balloons separated by ultrasound gel were suspended in a water bath. To mimic different chamber volumes, 12 volumes of water within the inner balloon (40-180ml) were scanned using a 3.5MHz probe at fundamental frequency and using a 2.5MHz probe with and without harmonic imaging. RESULTS: Scanning at 3.5MHz, the long axis (B) scans did not significantly underestimate the balloon volume but the 'short axis' (C) scans did (mean difference from actual volumes -0.7-1.4ml, P=0.14 - 3.9 +/- 1.2 ml,P < 0.0001 for B and C scans, respectively). Scanning at 2.5MHz both B and C scans significantly underestimated even more the true volume, C scans to a greater extent (mean difference -6.9 +/- 2.4ml and -11.2 +/- 4.0ml for B and C scans respectively,P < 0.0001 in both cases). However with harmonic imaging, transmitting at 1.7MHz and receiving at 2-4MHz, there was no significant difference of either B or C scans from the reference values (mean difference of B scans -1.2 +/- 1.9ml, P=0.06 and C scans -0.6 +/- 2.2ml, P=0.4). CONCLUSION: The enhanced resolution provided by harmonic imaging improves accuracy of volume analysis by real-time three-dimensional echocardiography.

Quantification of flow volume with a new digital three-dimensional color Doppler flow approach: an in vitro study.

OBJECTIVE: The quantification of flow stroke volume is important for evaluation of patients with cardiac dysfunction and cardiovascular disease. Three-dimensional digital color Doppler flow imaging allows the acquisition of flow data in an orientation approximately parallel to flow and analysis of the Doppler flow velocities perpendicular to flow (cross-sectional flow calculation). This in vitro study assessed the applicability of this method for quantifying cardiac output in a funnel-shaped tube model similar to mitral inflow or the left ventricular outflow tract. METHODS: A new digital three-dimensional color Doppler method was used to acquire Doppler flow information. Raw scan line data with digital velocity assignments were obtained on a conventional Doppler color flow imaging system with a 180 degrees rotating multiplanar transesophageal probe connected to a computer workstation. Nine stroke volumes (20-60 mL) with flow rates ranging from 1.5 to 5.28 L/min in a funnel-shaped pulsatile laminar flow model were studied. Three-dimensional flow rates were compared with standard-of-reference measurements of flow obtained from timed collection in a graduated cylinder and with an ultrasonic flow meter. RESULTS: Within the funnel tube, the flow volumes that were calculated from the first, second, and third depths and the average of all 3 depths correlated well with the actual flow rate (r = 0.97-0.99). Results from the middle and second levels and from the average of all 3 depths provided the closest fit to the actual flow rates (r = 0.99; y = 0.96x + 0.14; and r = 0.98; y = 1.14x - 0.43, respectively). CONCLUSIONS: Although a work in progress, this digital three-dimensional color Doppler flow measurement method is feasible, accurate, and simple, and it may offer in vivo evaluation of blood volume flow given a favorable orientation between the valve orifice and the scanning device.

Comparison of ventricular volume and mass measurements from B- and C-scan images with the use of real-time 3-dimensional echocardiography: studies in an in vitro model.

BACKGROUND: Real-time 3-dimensional (3D) echocardiography avoids geometric assumptions in volume analysis and permits immediate visualization in any plane without the need for cardiac or respiratory gating or computation time. This study compared the accuracy of volume and mass assessments between standard long-axis (B-scan) and short-axis (C-scan) views in a simplified but quantifiable left ventricular phantom. METHODS AND RESULTS: The model comprised an inner balloon within an outer balloon separated by ultrasonographic gel. First, to mimic different chamber volumes, 12 volumes (40 to 180 mL) of water within the inner balloon were scanned with a real-time 3D system. Second, 10 volumes (80 to 170 mL) of gel were inserted between the balloons to mimic varying cardiac mass, and the gel volume space (mass) was calculated by subtracting the inner from the outer balloon volume. "Chamber" and "mass" measurements for both B and C scans correlated closely with the actual values (r = 0.99). However, chamber volumes from C scans were consistently less than B-scan values (mean difference from reference for C scans: -5.2 +/- 1.2 mL, P <.0001; for the 2 orthogonal B scans: 0.03 +/- 1.4 mL and -0.9 +/- 1.5 mL, respectively, P = NS). Similarly, for gel volume measurements, B-scan results were closer to actual mass volumes (mean difference 0. 3 +/- 2.5 and 1.7 +/- 2.9 mL) than those of C scans, which tended to underestimate (-4.5 +/- 2.5 mL, P <.0001). CONCLUSION: Our study suggests that real-time 3D echocardiography should provide an accurate means of determining chamber volumes and cardiac mass. However, measurements performed from B-scan views may be closer to the actual values than those from C-scan views, presumably since they are less highly influenced by distortions related to lateral resolution.

A digital 3-dimensional method for computing great artery flows: in vitro validation studies.

BACKGROUND: Conventional 2-dimensional Doppler large vessels are prone to inaccuracy. Three-dimensional (3D) volume imaging provides the opportunity to make cross-sectional flow calculations through digital spatiotemporal integration of flow velocity, area, and profile. METHODS: A new digital 3D color Doppler reconstruction method was used to generate radially acquired flow data sets. Raw scanline data with digital velocity assignments, obtained by scanning parallel to flow, were transferred from a specially programmed but otherwise conventional ultrasonographic system, which controlled a multiplane transesophageal probe, to a computer workstation via an Ethernet link for assimilation into color 3D data sets. This configuration was used to study 20 pulsatile laminar flows (stroke volumes 30 to 70 mL and peak flow rates 65 to 205 mL/s) in a curved tube model with an oval cross-sectional geometry. After generation of the color 3D data set, flow velocity values from cross sections perpendicular to the tubes were analyzed to determine flow rate and stroke volume. RESULTS: The flows from 3D digital velocity profiles showed close correlation with peak instantaneous flow rates (r = 0.99, y = 1.01x-0.9, standard error of estimate 4.1 mL/s). When interpreted with pulsed wave Doppler data obtained through the cardiac cycle, they also allowed computation of stroke volume (r = 0.98, y = 1.44x-2.5, standard error of estimate 3.8 mL). CONCLUSION: The ability to compute laminar flows from 3D digital data sets obtained parallel to the direction of flow and without the need for geometric assumptions represents an important opportunity for and advantage of 3D color Doppler echocardiography.