Production of radioactive particles for endovascular therapeutic interventions.

Recanalization is a common phenomenon that decreases the efficacy of embolization procedures. It can be inhibited by beta-radiation. Two novel ways of producing radioactive particles are described, by neutron beam irradiation of gold-containing microspheres, or by using the 32P binding capacity of zirconium-containing microspheres. Particles were tested in vivo, to assess their ability to deliver radioactivity locally, using canine renal artery, porcine rete mirabile, and rabbit ear embolization models. Both radioactive microspheres (198Au and 32P) showed no detectable activity outside the target territory. 32P microspheres demonstrated typical radiation changes in a porcine rete mirabile arteriovenous malformation model.

The effect of ricin B chain on the intracellular trafficking of an A chain immunotoxin.

Covalent linkage of the A chain of ricin to the LICR-LOND-Fib75 monoclonal antibody produced an immunotoxin, Fib75-SS-ricin A, which demonstrated immunospecific toxicity to human bladder carcinoma cells in tissue culture (Forrester et al., 1984). The present studies have shown that ricin B chain potentiates the toxicity of the immunotoxin by two orders of magnitude and also significantly increases the rate of protein synthesis inhibition. Using immunoelectron microscopy, the receptor-mediated endocytosis and intracellular routing of the immunotoxin was studied with and without ricin B chain treatment after immunolocalisation of the conjugate. Fib75-SS-ricin A was internalised by the EJ cells predominantly in uncoated pits and vesicles and directed to the endosomes. Some degradation of the complex appeared to take place in multivesicular endosomes at early timepoints and 24 h after internalisation, most of the immunotoxin was found in lysosomes. Some ricin A chain epitopes were detected in Golgi vesicles. Cells treated with immunotoxin and ricin B chain endocytosed the complex predominantly in coated pits and coated vesicles. Using pre-embedding immunoperoxidase techniques, ricin chains were found in the whole Golgi complex and most of the conjugate escaped lysosomal degradation. Internalised immunotoxin was recycled back to the plasma membrane in an active form associated with vesicles which appeared to be derived predominantly from multivesicular endosomes. A similar mode of recycling has recently been reported (McIntosh et al., 1990) for ricin holotoxin in the same cell line. These observations may explain the potentiating effect of toxin B chains in the antibody-directed targeting of toxin A chains.

The measurement of skin lymph flow by isotope clearance--reliability, reproducibility, injection dynamics, and the effect of massage.

The measurement of skin lymph flow was investigated using an isotope clearance technique (ICT). Multiple lymph flow determinations were undertaken in the skin of anaesthetized large white pigs to test for reproducibility, ascertain the most suitable tracer, study the influence of injection dynamics, and observe the effect of massage as a stimulus to lymph flow. Blood clearance of tracer was also investigated. Results demonstrated that lymphatic clearance is a monoexponential function with good reproducibility under controlled laboratory conditions. 99mTc-colloid (TCK17 Cis) compared favorably with 131I-human serum albumin as a tracer and both performed better than colloid gold (198Au). Lymph flow was significantly faster in one pig than in the other. No difference existed between left and right sides or between caudal and rostral sites on each flank, but clearance was significantly slower in thigh than flank skin. Sub-epidermal injections cleared faster and more consistently than either deep or subcutaneous injections. Neither injection volume nor needle tract backflow of tracer influenced results, but local massage significantly enhanced clearance. Escape of 99mTc-colloid by the blood was negligible. These results indicate that skin lymph flow can be reliably measured when conditions are controlled. Extrinsic factors such as massage strongly influence lymph flow. Greater sensitivity in detecting degrees of lymphatic insufficiency may be achieved if a standardized stimulus to lymph flow is administered during isotope clearance measurement.

Evidence for immunoglobulin Fc receptor-mediated prostaglandin2 and platelet-activating factor formation by cultured rat mesangial cells.

The possibility of Fc-dependent uptake of IgG immune complexes was examined in subcultured rat mesangial cells free of monocytes. 195Au-labeled colloidal gold particles were coated either with BSA only or with BSA followed by rabbit anti-BSA-IgG or the F(ab')2 fragment of the IgG. Mesangial cells preferentially took up 195Au particles covered with BSA-anti-BSA-IgG over those covered with BSA or the F(ab')2 fragment. This uptake was a time-dependent and saturable process inhibitable by sodium azide or cytochalasin B. Using phase-contrast microscopy in the light reflectance mode, it was established that essentially all mesangial cells took up IgG-coated gold particles. By electron microscopy the process was shown to consist of vesicular uptake with delivery to endosomes. Mesangial binding-uptake of the IgG-covered particles was associated with stimulation of PGE2 synthesis and production of platelet-activating factor, a lipid mediator of inflammation. To characterize the potential Fc receptor for IgG we used the rosetting technique with sheep red blood cells coated with IgG subclass-specific mouse monoclonal antibodies. 50% of mesangial cells exhibited rosetting with red cells coated with mouse IgG2a, whereas negligible rosetting was observed with IgG2b or IgG1. Competition experiments confirmed the specificity of IgG2a binding. We conclude that cultured rat mesangial cells exhibit specific receptors for IgG and that occupancy of Fc receptors results in endocytosis and is associated with generation of PGE2 and platelet-activating factor. These observations may be of significance for immune-mediated glomerular diseases.

Accumulation and toxicity of gold in the rabbit lens in vitro.

Gold compounds are used therapeutically and have been observed to accumulate in the human eye. In the present study, we have measured the accumulation and loss of gold (AuIII) in rabbit lenses in vitro using 195Au as a tracer. Accumulation increased progressively with time of incubation up to 24 hr. The highest concentration of Au3+ observed was about 16 mumol kg-1 wet wt. Rate of accumulation also rose with increase in the external concentration of Au3+, but the magnitude of the change exceeded the concentration difference. Autoradiography showed a high accumulation of 195Au in a central region of the anterior face of the lens, as has been observed in vivo in humans undergoing chrysotherapy. Loss (efflux) of accumulated 195Au was increased by the presence of Au3+ or dithiothreitol in the external media. Lanthanum (La3+), however, decreased the loss of such Au and promoted its accumulation. Gold can have a toxic effect on lenses as incubation for 7 days in a medium containing 10(-7) M Au3+ resulted in a marked gain of Na and loss of K. The results suggest that Au accretion occurs at surface sites associated with the anterior of the lens, but whether or not intracellular accumulation occurs is not clear. This Au, in excess, may have toxic effects on the tissue.

Biokinetics and dosimetry for 195Au, evaluated in an animal model.

The use of the generator produced radionuclide 195mAu, half life 30 s, has become feasible for several different investigations, e.g. cardiac studies. To assess the absorbed dose from the long lived radionuclide 195Au (the daughter of 195mAu, half life 183 days), a biodistribution study of 195Au was performed in animals. Seven rabbits were injected with eluate from a 195mHg-195mAu generator and the retention and the biodistribution of the long lived gold isotope was investigated. The activity was localized mainly in the liver and the kidneys. Transforming the data to man resulted in an absorbed dose from 195Au from 1 elution (approximately 925 MBq 195mAu) of 2.2 mGy to the kidney and 1.3 mGy to the liver and an effective dose equivalent of 0.26 mSv. The total effective dose equivalent from all radionuclides in the eluate (195mAu, 195Au, 195mHg and 195Hg), was estimated to be 0.65 mSv for a single injection (925 MBq 195mAu).

Correlation between the behavior of magnetic iron oxide particles in the lungs of rabbits and phagocytosis.

Five New Zealand white male rabbits were exposed (30 min; 300 mg/m3) to a submicrometric magnetic iron oxide aerosol (gamma-Fe2O3) produced by burning iron pentacarbonyl in a reducing atmosphere. After aerosol inhalation, an external magnetic field was applied to the rabbits to magnetize and align the ferrimagnetic particles within their lungs. After removal of the external field, a remanent magnetic field was detectable at the body surface. Using a flux-gate magnetometer probe in an enclosure shielded against external magnetic noise, the peak remanent field after magnetization was measured periodically during the next 6 weeks. After each magnetization, the strength of the remanent field decayed rapidly with time (relaxation). The mechanism responsible is particle rotation caused by tissue, cell, organelle, or Brownian movement. The rate of relaxation changed with time after particle inhalation, especially during the first day; changes in the relaxation rate correlated with an estimate of in situ particle phagocytosis during that time. Analysis of pulmonary lavage fluid from 15 rabbits into which radioactive gold-198 had been intratracheally instilled showed that, at 1 hr after instillation, 27% of the gold had been phagocytized, whereas at 16 hr 91% had been ingested. The strength of the magnetic field immediately after each magnetization (that is, before relaxation) was used to estimate the amount of iron oxide in the lungs. At 1 day after exposure, 96.8 +/- 8.8% (mean +/- standard error) of the initial dust was still present; at 10 days, 67.9 +/- 16.2%; and at 40 days, 16.0 +/- 4.6%. It is concluded that ferrimagnetic particles can serve as an easily measured, long-lasting marker that can be used for noninvasive studies of clearance and of particle phagocytosis and as a probe for intracellular processes such as organelle motion.