Antibody drug carrier for immunotherapy of superficial bladder cancer: ultrastructural studies.

Superficial bladder cancer represents a promising target for intravesical, antibody-guided therapy. The construction of an optimum antibody-cytotoxic drug conjugate depends mostly on the appropriate selection of a monoclonal antibody (mAb). We have used immunogold labeling and SEM to specifically map the distribution of antigens expressed on three bladder cancer cell lines and on the luminal surface of biopsies from human transitional cell carcinoma of various grades and from normal bladder mucosa. The 48-127 mAb, which recognizes a M(r) 54,000 surface glycoprotein (gp54), was found to be very promising as a potential drug carrier. This antibody reacts with the surface of cells from low- and high-grade tumors; it does not react with the normal urothelium. Labeling of normal bladder mucosa was observed, however, on microvillous intermediate urothelial cells occasionally exposed by small areas of desquamation. The 48-127 mAb could target drugs to all areas of transformed urothelium while avoiding drug delivery to the normal, undesquamated bladder mucosa. Kinetics of gp54/48-127/gold complexes were tested in vitro with T24 and RT4 human bladder carcinoma cell lines incubated in the presence of the 48-127 mAb directly conjugated with 17.7-nm gold particles. Internalization of the gp54/48-127/gold complex was readily demonstrated by transmission electron microscopy. These results suggest that the 48-127 mAb represents a valuable drug carrier for intravesical therapy, allowing specific tumor targeting and internalization of various cytotoxic agents.

Immunogold labeling of the low-affinity (55 kd) IL2 receptor on the surface of IL2 receptor-bearing cultured cells and mitogen-activated peripheral blood lymphocytes.

Foreign antigens or mitogens initiate activation of T lymphocytes through antigen-specific receptors. After antigen-receptor interaction, T cells release a lymphotrophic hormone, interleukin 2 (IL2), and high-affinity IL2 receptors (IL2R) are progressively expressed on their surfaces. When evaluated under the scanning electron microscope after specific immunolabeling with colloidal gold markers, the density of expression of the 55 kd low-affinity IL2 receptor (Tac) increases in a time-dependent manner, reaching a maximum 72 hours after the onset of phytohemagglutinin (PHA) activation. In contrast, the number of cells expressing the 55 kd molecule reaches a maximum by 24 hours and remains constant through 72 hours. Immunogold labeling allows simultaneous determination of IL2 receptor density at the level of each individual cell and of the percentage of IL2 receptor-positive cells in any cell population under study. The rank order of receptor expression on the surface of established culture cell lines correlates with results reported from other laboratories with immunofluorescence and Scatchard analysis of binding of radiolabeled IL2. Although at high density of receptor expression immunogold labeling with 40 nm gold markers detects significantly fewer sites than radiolabeling, the method appears very sensitive in identifying low levels of expression. This results from 1) the extremely low non-specific background of the immunogold labeling technique and 2) the analysis of single cells rather than cell populations. Our results indicate that 2 cell lines, reported as negative for the expression of the 55 kd IL2 receptor (YT2C2 and MLA144), express low numbers of this molecule as demonstrated by immunogold labeling.

Scanning electron microscopic study of immunogold-labeled human leukocytes.

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25 degrees C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.

Effects of halothane on arrhythmias induced by myocardial ischaemia.

The effect of halothane on arrhythmias induced by ischaemia was investigated in rats, isolated perfused rat hearts, and pigs. Responses to the occlusion of the left anterior descending coronary artery were determined in groups (n = 9) of chronically prepared rats treated with no halothane, 0.5, or 1.0 per cent halothane immediately after occlusion; in isolated rat hearts (n = 10) treated with no halothane, 0.5, 1.0, 2.0, or 4.0 per cent halothane for 15 min before and after occlusion; and 20-25 kg pigs (n = 11) anaesthetised with halothane or pentobarbital. The ECG, arrhythmias, blood pressure (BP), heart rate (HR) and extent of infarction were determined in each model. In pigs, left ventricular pressure, dp/dtmax and cardiac output were also measured. In chronically prepared rats, halothane anaesthesia started after occlusion was antiarrhythmic and decreased the incidence of ventricular fibrillation and resulting mortality. In isolated rat hearts, 0.5 or 1.0 per cent halothane had little effect on occlusion-induced arrhythmias. The highest concentration of halothane increased the incidence of ventricular fibrillation both before and after occlusion. Halothane decreased developed ventricular pressure in a dose-dependent manner. In acutely prepared pigs, halothane pre-treatment had no appreciable effect upon occlusion-induced arrhythmias when compared with pentobarbital anaesthesia. Thus, halothane is antiarrhythmic when treatment is initiated after occlusion in the rat but this action is not seen in isolated hearts or intact pigs. The antiarrhythmic action of halothane is, therefore, species and model dependent.