Vessel fractions in tumor xenografts depicted by flow- or contrast-sensitive three-dimensional high-frequency Doppler ultrasound respond differently to antiangiogenic treatment

High-frequency volumetric Power Doppler ultrasound (HF-VPDU) captures flow-dependent signals in blood vessels and can be used to assess antiangiogenic therapy effects in rodent tumors. However, the sensitivity is limited to vessels larger than capillaries. Contrast-enhanced HF-VPDU reveals all perfused vessels by assessing stimulated acoustic emissions from disintegrating microbubbles. Thus, we investigated whether flow-sensitive and contrast-enhanced HF-VPDU can depict different vessel fractions and assess their early response to antiangiogenic therapy. Mice with A431 tumors were scanned before and after administration of polybutylcyanoacrylate microbubbles by HF-VPDU. Animals received either antiangiogenic treatment (SU11248) or a control substance and were imaged repeatedly over 9 days. At each time point, tumors were removed for immunohistochemical analysis. During growth of untreated tumors, vascularization decreased correspondingly on flow-sensitive and contrast-enhanced scans. Treated tumors showed a significantly (P < 0.05) stronger decline in vascularization than controls, which was more pronounced in contrast-enhanced scans. Surprisingly, whereas vascularization remained low in contrast-enhanced scans, flow-sensitive ultrasound indicated a reincrease after day 6 with a higher vascularization than the controls at day 9. Histologic evaluation indicated that immature vessels degraded markedly on therapy, whereas large mature vessels on the tumor periphery were more therapy resistant and drew closer due to tumor shrinkage. In conclusion, contrast-enhanced HF-VPDU and flow-sensitive HF-VPDU are both capable of assessing the effects of antiangiogenic therapy. Because contrast-sensitive ultrasound is more sensitive for small immature vessels and flow-sensitive ultrasound mostly captures large vessels at the tumor periphery, the combination of both methods can provide evidence of vascular maturity in tumors.     

Magnetic marker monitoring of disintegrating capsules

Magnetic marker monitoring was studied for its applicability to investigate the in vivo fate and behavior of disintegrating magnetically marked dosage forms. As a model, hard gelatin capsules were filled with an effervescent mixture of lactose, ascorbic acid and sodium hydrogen carbonate containing 1.3 mg black iron oxide as a magnetic label. The accuracy of the localization procedure whilst calculating all parameters of the dipole in one fitting procedure was checked in phantom experiments where the capsules were moved in well-defined paths with respect to the measurement device. The calculated position coordinates of the capsules deviated between less than 2 mm up to 8 mm from the expected position values depending on the distance between the sensor area and the capsule's path. Further experiments on the in vitro disintegration of the capsules showed that the value of the magnetic moment of the capsules can serve as a measure for their disintegration behavior. In vivo monitoring of the capsules was performed in eight experiments where a healthy volunteer swallowed each time one of the capsules. It was found that the in vivo disintegration behavior of the capsules corresponds well to their disintegration observed in water of about 37 degrees C.     

Determination of Plasma Protein Adsorption on Magnetic Iron Oxides: Sample Preparation

Purpose. The purpose of this study was to investigate the influence of the sample preparation on the plasma protein adsorption pattern of polysaccharide-stabilized iron oxide particles by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Methods. The iron oxide particles were incubated in vitro in human plasma for five minutes. Thereafter, four different methods for particle recovery, including adsorbed proteins from surplus plasma, were investigated: centrifugation, magnetic separation, gel filtration and membrane-based static microfiltration. Adsorbed proteins were desorbed from the particle surfaces by surfactants and analyzed by 2-D PAGE, as described elsewhere (1,2). Results. All the techniques investigated were able to separate small-size iron oxides (approx. 110 nm) and adsorbed proteins from excess plasma. The gels obtained by the different separation procedures displayed almost identical adsorption patterns. Major proteins identified were: fibrinogen, IgG, albumin and an unclassified protein of about 70 kDa with a pI value of 6.5–7.5. Conclusions. Centrifugation was regarded as the most suitable separation method due to its speed and ease of use. In contrast to gel filtration, any washing media can be used. The magnetic separation process is restricted to particles with high inducible magnetic saturation, in particular, to iron oxides with overall sizes > 50 nm.     

Targeting of ultrasmall superparamagnetic iron oxide (USPIO) particles to tumor cells in Vivo by using transferrin receptor pathways

Human transferrin was covalently coupled to ultrasmall superparamagnetic iron oxide (USPIO) particles, and the trans-ferrin-USPIO obtained was investigated in vivo in experimental SMT/2A tumor-bearing rats (rat mammary carcinoma). Physicochemical characterization showed an overall size of 36 nm (DLS) with a core size of 5 nm (TEM). Relaxivities were R,₁ = 23.6 and R₂ = 52.1 liter/mmol · s (0.47 T). Bound transferrin was 280 μg/mg of iron. Pharmacokinetic investigations revealed a half-life of 17 min in normal rats. The MR evaluation of tumor signal intensity over time showed a 40% (range 25–55%) signal reduction 150 min after injection with the reduction persisting for at least 8 h. Control experiments using the parent USPIO compound or USPIO labeled with a nonspecific human serum albumin (HSA-USPIO) showed a change of only 10% (range 5–15%) in tumor signal intensity over time. The results demonstrate that a combination of the USPIO relaxivity properties with the specificity of transferrin-medi-ated endocytosis allows in vivo detection of tumors by MR imaging.     

The first milliseconds of the pore formed by a fusogenic viral envelope protein during membrane fusion.

Fibroblasts expressing the influenza virus hemagglutinin on their plasma membrane were patch clamped while they fused to erythrocytes. An increase in the fibroblast's membrane capacitance indicated the opening of the "fusion pore," the first aqueous connection between the fusing cells. We show here that the capacitance increase is preceded by a brief current transient, generated as the erythrocyte discharges its membrane potential through the nascent fusion pore. This signal allows one to calculate the pore conductance during the first milliseconds of its existence. The pore conductance jumps from 0 to approximately 150 pS and then grows more gradually over the subsequent tens of milliseconds until growth is arrested. The initial conductance is similar to that of a large ion channel and suggests that the pore is initially only 1-2 nm wide. Hence, we are probably observing events caused by only a small number of hemagglutinin molecules.     

Guidance of visual reaching with the aid of a TV monitor: The effects of monitor position and of left/right and up/down reversals of the image in relation to age

Children, aged 2-9 years, were seated alongside a wall containing an opening through which they could reach with their preferred hand. Targets ("ink blots"), which could not be felt, were attached to the other (rear) side of the wall, around the opening. The child could see directly neither the target nor its hand beyond the front side of the wall; but both were visible on a TV monitor. The image on the screen of the monitor originated from a camera viewing the rear (target) side of the wall at the level of the central opening. The monitor screen was either (a) parallel to the wall, but rotated 180 degrees with respect to the axis of the camera, i.e. facing the camera; or (b) at 90 degrees to the wall; or (c) in a position similar to (a) but visible in a mirror attached to the front side of the wall and therefore in effect "aligned" with the axis of the camera. The image the child saw was either (1) electronically unswitched, i.e. when the target was towards the east on the wall it was to the west on the monitor in monitor position (a), or to the north in position (b), or to the "east" in position (c); or (2) left/right reversed relative to (1); or (3) up/down reversed relative to (1); or (4) both left/right and up/down reversed. The dependent variable was the time taken for the child to place the palm of the hand over the target (time was measured to 0.1 sec on video-recorder with a superimposed time display). Position of the monitor, comparing conditions (a)-(c), gave only minor, perhaps age-related, effects. Left/right reversals were easier than up/down reversals under monitor positions (a) and (b), but not under (c); but both reversals could be achieved by age 3 or older; hardest was condition (4). However, with monitor positions (a) and (b) children, at all ages we tested, found condition (1) ("east gives west/north") easier than (2) ("east gives east/south"), whereas for adults these conditions were equally easy, or they found (2) easier than (1); but with monitor position (c) condition (1), now "east gives east", was easiest. Moreover, the claim that chimpanzees but not monkeys can achieve accurate reaching under the conditions varied in this study seems premature: the chimpanzees may have made use of strategies based on uncontrolled cues.     

Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells

After exocytosis, chromaffin granules release essentially all their catecholamines in small fractions of a second, but it is unknown how fast they release stored peptides and proteins. Here we compare the exocytic release of fluorescently labelled neuropeptide Y (NPY) and tissue plasminogen activator from single granules. Exocytosis was tracked by measuring the membrane capacitance, and single granules in live cells were imaged by evanescent field microscopy. Neuropeptide Y left most granules in small fractions of a second, while tissue plasminogen activator remained in open granules for minutes. Taking advantage of the dependence on pH of the fluorescence of green fluorescent protein, we used rhythmic external acidification to determine whether and when granules re-sealed. One-third of them re-sealed within 100 s and retained significant levels of tissue plasminogen activator. Re-sealing accounts for only a fraction of the endocytosis monitored in capacitance measurements. When external [Ca²⁺] was raised, even neuropeptide Y remained in open granules until they re-sealed. It is concluded that a significant fraction of chromaffin granules re-seal after exocytosis, and retain those proteins that leave granules slowly. We suggest that granules vary the stoichiometry of release by varying both granule re-sealing and the association of proteins with the granule matrix.     

Automatic passive tracking of an endorectal prostate biopsy device using phase-only cross-correlation.

MR-guided transrectal prostate biopsy is currently a time-consuming procedure because the imaging slice is often manually realigned with the biopsy needle during lesion targeting. In this work a pulse sequence is presented that automatically follows a passive marker attached to a dedicated MR biopsy device holder, thus providing an alternative to existing active tracking methods. In two orthogonal tracking FLASH images of the marker the position of the needle axis is automatically identified using a phase-only cross-correlation (POCC) algorithm. The position information is then used to realign a trueFISP imaging slice in real time. In phantom experiments the sensitivity of this technique to initial misalignments of the marker and to the signal-to-noise ratio was evaluated. In several puncture experiments the precision of the needle placement was analyzed. The POCC algorithm allowed for a precise identification of the marker in the images even under severe initial misalignments of up to 45 degrees. At a frame rate 1 image/s a precision of the needle placement of 1.5 +/- 1.1 mm could be achieved.     

Potential applications of flat-panel volumetric CT in morphologic and functional small animal imaging

Noninvasive radiologic imaging has recently gained considerable interest in basic and preclinical research for monitoring disease progression and therapeutic efficacy. In this report, we introduce flat-panel volumetric computed tomography (fpVCT) as a powerful new tool for noninvasive imaging of different organ systems in preclinical research. The three-dimensional visualization that is achieved by isotropic high-resolution datasets is illustrated for the skeleton, chest, abdominal organs, and brain of mice. The high image quality of chest scans enables the visualization of small lung nodules in an orthotopic lung cancer model and the reliable imaging of therapy side effects such as lung fibrosis. Using contrast-enhanced scans, fpVCT displayed the vascular trees of the brain, liver, and kidney down to the subsegmental level. Functional application of fpVCT in dynamic contrast-enhanced scans of the rat brain delivered physiologically reliable data of perfusion and tissue blood volume. Beyond scanning of small animal models as demonstrated here, fpVCT provides the ability to image animals up to the size of primates.