Rubin H. Flocks and colloidal gold treatments for prostate cancer.

In the early 1950s, Rubin H. Flocks of the University of Iowa began to treat prostate cancer patients with colloidal gold (Au(198)) therapy, evolving his technique over nearly 25 years in 1515 patients. We reviewed the long-term outcomes of Flocks' prostate cancer patients as compared to those patients treated by other methods at the University of Iowa before Flocks' chairmanship. We reviewed archived patient records, Flocks' published data, and long-term survival data from the Iowa Tumor Registry to determine short- and long-term outcomes of Flocks' work with colloidal gold. We also reviewed the literature of Flocks' time to compare his outcomes against those of his contemporaries. The use of colloidal gold, either as primary or adjunctive therapy, provided short- and long-term survival benefit for the majority of Flocks' patients as compared to historical treatment options (p < 0.001). Flocks' use of colloidal gold for the treatment of locally advanced prostate cancer offered short- and long-term survival benefits compared to other contemporary treatments.

Evidence for a cytolytic factor released by macrophages.

Mouse peritoneal macrophages cultivated in vitro acquire a strong extracellular cytotoxic activity towards isotope labeled syngeneic erythrocytes as demonstrated by isotope release to the medium. This lytic process is mediated by an extremely labile macrophage cytolytic factor (MCF) which is not detected under ordinary tissue culture conditions with serum present in the medium. By the use of serum-free medium containing low doses of 2-mercaptoethanol MCF is stabilized and found to be an easily dialysable, low molecular substance which resists heating at 60 degrees C for 30 min.

Further observations on the mechanism of reticuloendothelial blockade.

The mechanism of the reticuloendothelial "blockade" which followed injection of large quantities of chromic phosphate without exogenous stabilizing material was investigated in Wistar rats. The RE blockade observed for several hours after induction appeared related to the continuing circulation of the chromic phosphate-blockading dose, and a reduction in the size of the particles used enhanced blockade. RE blockade appeared to be particles specific and was not related to a generalized depression of RE-phagocytic cell function. Studies in isolated perfused rat livers appeared to eliminate saturation of particle-specific macrophage clones as a likely explanation of blockade, and blockade could not be explained on the basis of depletion of serum opsonins. In the system employed, it is postulated that blockade occurs when large numbers of circulating particles saturate specific macrophage cell membrane-binding sites rather than from physical stuffing of RE-phagocytic cells.

The isolation and selected properties of blood monocytes.

A technique is described for the quantitative recovery of monocytes from horse blood by means of flotation on dense albumin solutions. Monocytes are concentrated in a surface pellicle along with a few lymphocytes which are then removed when the monocytes adhere to a glass surface. The in vitro cultivation of homogeneous populations of monocytes results in an increase in (a) cell size, (b) number of mitochondria, and (c) phase-dense granules of the centrosphere. The phase-dense granules are osmiophilic and acid phosphatase positive. Quantitative biochemical analysis during cultivation have revealed increased levels of cytochrome oxidase, acid phosphatase, arylsulfatase, and BPN hydrolase. In addition, glucose utilization and lactic acid production are stimulated under the same conditions. The uptake of both bacteria and colloidal gold is stimulated during in vitro cultivation. The phagocytic activity of cultured monocytes may be enhanced by a purified bacterial lipopolysaccharide. These data are consistant with the in vitro maturation of monocytes to macrophages, a cell with greater metabolic and functional potentional.

A novel approach for scanning electron microscopy of colloidal gold-labeled cell surfaces.

A method is described for the use of scanning electron microscopy on the surface of gold-labeled cells. It includes the use of 45- or 20-nm colloidal gold marker conjugated with Staphylococcal protein A. The marker is best recognized on the basis of its atomic number contrast by using the backscattered electron imaging mode of the scanning electron microscope. When the backscattered electron signal is mixed with the secondary electron signal, an optimum correlation between the distribution of the labeled sites and the cell surface structures is demonstrated. The method is illustrated by its application to the identification of human circulating granulocytes. Its good resolution, high contrast, and good labeling efficiency offers a promising approach to the specific localization of cell surface antigenic sites labeled with particles of colloidal gold.

Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes.

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold-particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin-binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.

Reversible pinocytosis in polymorphonuclear leukocytes.

Since pinocytosis has only been recently recognized in polymorphonuclear leukocytes (PMNs), little is known about the fate of pinosomes. Here we report that pinosomes can fuse with the cytoplasmic granules of PMNs. We also find that at least for a short period of time after formation, pinosomes can fuse with the plasma membrane and release their contents to the outside. We present a morphological description and biochemical data on the kinetic parameters of a steady state pool of reversible pinosomes in PMNs. In addition, we have developed conditions under which pinosomes continue to form and fuse with the plasma membrane but fail to fuse with the cytoplasmic granules, i.e., only "reversible" pinocytosis occurs. This inhibition of fusion with the granules is not due to an inability of the pinosomes to move from the surface since under these conditions pinosomes labeled with an electron-dense marker can be seen in the cell interior.

Liver sinusoidal cells. Identification of a subpopulation for erythrocyte catabolism.

Sequestration and degradation of red blood cells (RBC) are believed to occur in part in the liver, but the magnitude and cellular localization of this process remain uncertain. This problem was studied in rats by investigating isolated parenchymal and sinusoidal cell populations of the liver. After digesting the perfused liver with pronase, hepatic sinusoidal cells were isolated free of RBC and debris. Of the isolated cells, 90% were phagocytic, as judged by their uptake of colloidal (198)Au or of aggregated albumin-(131)I administered in vivo After administration of spherocytic (heat-treated) RBC, however, only about one quarter of the isolated cells were found to contain phagocytized RBC. This apparently distinct population of RBC-phagocytizing cells is designated as "erythrophagocytic (EP)" cells. The EP cell population was further characterized functionally by its specific phagocytosis of colloidal carbon and of (99m)technetium-sulfur colloid and histochemically by its peroxidase activity. The role of the EP population in the catabolism of RBC-hemoglobin was studied in isolated hepatic sinusoidal cells by assay of microsomal heme oxygenase (MHO), which is the inducible enzyme system that converts heme to bilirubin. The MHO activity of individual sinusoidal isolates was related directly to their content of EP cells Assay of the MHO activity of the whole spleen and of the total EP cell population of the liver suggested that these two tissues may be of comparable importance in their ability to degrade RBC-hemoglobin.

Structure-function relationships in the adipose cell. II. Pinocytosis and factors influencing its activity in the isolated adipose cell.

Pinocytic activity in the adipose cell has been examined by measuring the uptake of colloidal gold. Pinocytic activity occurs in the isolated adipose cell under all experimental conditions; a portion of the vesicular elements of the cell can be identified by electron microscopy as pinocytic in origin. The isolated adipose cell appears to take up serum albumin by pinocytosis. Pinocytic activity in the isolated adipose cell is enhanced by epinephrine, but not by insulin. The relationship between pinocytosis and the metabolic activity of the adipose cell has been studied by measuring simultaneously the uptake of radioactive colloidal gold, the incorporation of (14)C-counts from U-glucose-(14)C into CO(2), total lipid, triglyceride glycerol and triglyceride fatty acids, and the release of nonesterified fatty acids in the absence of hormones and in the presence of insulin or epinephrine. Correlations between hormone-produced alterations in lipid metabolism and in pinocytic activity suggest that intracellular nonesterified fatty acid levels are a factor in the regulation of both the cell's pinocytic activity and its metabolism and that pinocytosis in the adipose cell functions in the extracellular-intracellular transport of nonesterified fatty acids.

Noncytokinetic radiation injury: anticoagulants as radioprotective agents in experimental radiation hepatitis.

Rats that have been irradiated with a single dose of 1500 roentgens directed to the liver show the following abnormalities: increased clearance of colloidal gold by the liver at 30 days, a decreased albumin space index at 30 days, and increased alkaline phosphatase at 60 days. In each of these cases, animals receiving Depo-heparin in dose ranges reasonable for human application had essentially normal values. There was no radioprotective effect with salicylates at the doses used. Higher doses of salicylates resulted in death of the animals.

Effect of Corynebacterium parvum, methanol-extraction residue of BCG, and levamisole on macrophage random migration, chemotaxis, and pinocytosis.

Three parameters of macrophage function: random migration, chemotaxis, and pinocytosis, were studied in the guinea pig after administration of Corynebacterium parvum, methanol-extraction residue of BCG, and levamisole (LMS), a synthetic anthelmintic. Macrophage migration studies were performed with a modified Boyden chamber. Pinocytosis was assessed by the uptake of colloidal 198Au. After ip administration, each of the three immunostimulators induced an increase in macrophage chemotactic responsiveness and, to a lesser extent and duration, in random motility. Kinetic, dose-response, and time course data for the effect of each agent on macrophage movement were explored. LMS was the most effective stimulator of macrophage activation, which occurred earlier and persisted longer than it did with the other agents. Macrophages from animals receiving each of the agents showed enhanced pinocytosis. Measurement of macrophage random migration, chemotaxis, and pinocytosis appeared to provide a rapid and quantitative assessment of several parameters of macrophage function and, when studied with other immunologic parameters, may provide useful tools for the evaluation of potential immunoadjuvants.

Ca2+-independent, protein-mediated fusion of chromaffin granule ghosts with liposomes.

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093-4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca2+, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.

Metallic colloid nanotechnology, applications in diagnosis and therapeutics.

In recent years the fields of medicine and biology assist to an ever-growing innovation related to the development of nanotechnologies. In the pharmaceutical domain, for example, liposomes, polymer based micro and nanoparticles have been subjects of intense research and development during the last three decades. In this scenario metallic particles, which use was already suggested in the first half of the '80, are now experiencing a real renaissance. In the field of diagnosis, magnetic resonance imaging is one of the first and up to now the most developed application of metallic particles. But beside this application, a very new generation of biosensors based on the optical properties of colloidal gold and fluorescent nanocrystals, called quantum dots seems to be ready to be implemented in diagnosis and medical imaging. Concerning therapeutic applications, the potentialities of metal nanoparticles to help fulfilling the need of time and space controlled release of drugs has been intuited for a long time. Nowadays, magnetically guided carriers or thermal responsive matrices, in which drug release is triggered by the heating of metal nanoparticles, are effective examples of their application in drug delivery, while more recently efforts to develop metallic nanoobjects to be used as vectors of nucleic acids for vaccination and transfection have been multiplied. In the future, one of the most interesting challenges is certainly the use of metallic nanoparticles for an innovating, effective and selective physical treatment of solid tumors via targeted intracellular hyperthermia.

Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation.

We have developed a rapid and precise electron microscope technique for the quantitation of gold particles in suspension using latex microspheres as a reference (EM latex technique). This technique allowed us to determine the specific absorption of colloidal gold at its absorption maximum (520 nm) and the average number of ligands ([125I]IgG) bound to one gold particle. On the basis of these values important binding characteristics of protein-gold complexes to cell surfaces were analyzed in a model system consisting of Staphylococcus aureus with protein A on the cell wall as a specific binding site for IgG-Au. Our observations showed that the number of binding sites represented by one IgG-gold complex depended primarily on the particle size, with one 20-nm IgG-Au corresponding to 15 and one 6-nm IgG-Au to 2.5 binding sites. Hence, the efficiency of binding of IgG-Au complexes increased with decreasing gold particle size. Saturation of binding sites, however, was not achieved. The technique also made possible the determination of the affinity between IgG-Au complexes and the cell surface; this affinity can either be regarded as a characteristic of the ligand IgG or of the gold particle. We observed that the affinity of IgG decreased with the size of the gold particles to which IgG was bound, whereas the affinity of the entire gold particle increased with particle size. The EM latex technique for quantitation of gold particles extends the general use of protein-gold complexes to the quantitative characterization of their interaction with cell surface constituents.

Dynamics of low-density lipoprotein receptors in the plasma membrane of cultured human skin fibroblasts as visualized by colloidal gold in conjunction with surface replicas.

The aim of this study was to characterize further the human skin fibroblast membrane receptor for LDL. We herein report that LDL particles bound to colloidal gold in conjunction with the surface replication technique can be used to visualize steps in the movement of their receptors in the plane of the lipid bilayer and provide the first clear demonstration of the sequential clustering of LDL-receptors into coated pits. Competition experiments have shown that recycled LDL-receptors are inserted in plaques and not at random into unspecialized regions of the plasma membrane. The site of replacement of LDL-receptors into the plasma membrane corresponds to the site of formation of coated pits and their subsequent internalization.

Intracellular pathways of electron-opaque gonadotropin-releasing hormone derivatives bound by cultured gonadotropes.

A metabolically stable GnRH agonist (D-Lys6-GnRH) has been coupled to electron-opaque markers (colloidal gold and ferritin) to characterize the intracellular pathway of the releasing hormone bound by pituitary gonadotropes. This approach has the advantage of increasing the resolution of localization to a "circle of uncertainty" about 10- to 20-fold smaller than that which can be obtained by autoradiography. After an initial uniform distribution on the cell surface, the derivatives were taken up individually as well as in small clusters in coated and uncoated membrane invaginations and moved to the lysosomal compartment either directly or after passage through the Golgi apparatus. The results suggest that labeled GnRH or GnRH-receptor complex may be routed to two distinct intracellular compartments: the lysosome and the Golgi cisternae. It is unclear whether each releasing hormone-marker conjugate must be transported through both compartments before degradation.

Effects of particle size and perfusate composition on the uptake of colloidal gold by the rabbit thoracic aorta perfused in situ.

The influence has been investigated of particle size on the uptake of radioactive gold colloid by the rabbit thoracic aorta perfused in situ. Particles ranging in diameter from 14 nm to 40 nm were suspended in 0.9% NaCl and infused either at a pressure of 15 mm Hg for times of between 2 1/2 and 60 min or at pressure of between 15 and 160 mm Hg for 5 min. Uptake by the whole intima-media increased with perfusion time and hydrostatic pressure but did not depend on particle size. Radioactive assay of serial sections across the aortic wall also showed that particle size did not influence the distribution of tracer. An effect of perfusate composition on uptake was demonstrated in further experiments in which particles either 14 or 40 nm in diameter were suspended in pooled rabbit serum and infused at pressures of between 15 and 140 mm Hg for 5 min. Uptake and transmural distribution were again independent of particle size, but uptake was 4-5-fold less than when the particles were perfused in saline. Under all perfusion conditions radioactivity fell steeply across the intima and then rose gradually across the media and adventitia. Radioactivity in the outer media and adventitia increased with perfusion time but little change could be detected in intimal activity. In transmission electron micrographs, particles in the intima were not seen to penetrate the internal elastic lamella and in the outer media particles remained extracellular and did not enter collagen bundles. Autoradiographs showed that particles in the intima were uniformly distributed around the circumference of the vessel but in the outer media and adventitia particles usually clustered close to the vasa vasorum.

Immunoperoxidase-immunogold double labelling in immunoelectronmicroscopy of large granular lymphocytes.

A method for better characterization of mononuclear cell subpopulations using detection of 2 surface antigens simultaneously in electron microscopy was developed with immunoperoxidase-immunogold double labelling. Monoclonal antibodies of IgG and IgM classes were used in the first step, and colloidal gold-labelled anti-mouse IgG antibody and peroxidase-labelled anti-mouse IgM antibody (mu chain-specific) in the second step. Immunoelectronmicroscopy with such double labelling improves ultrastructural analysis of mononuclear cell subpopulations.

Transpalatal insertion of radioactive gold grain for the treatment of persistent and recurrent nasopharyngeal carcinoma.

PURPOSE: To evaluate the efficacy of radioactive gold grain implant via the split palate approach in the control of locally recurrent or persistent nasopharyngeal carcinoma. METHODS AND MATERIAL: Forty-three patients, 10 for persistent NPC, 28 for first relapse in the nasopharynx, and five for second relapse in the nasopharynx, were treated. The diameter of the tumors at the time of gold grain implant ranged from 0.5 to 5 cm, the number of gold grains inserted varied from 4 to 14, the median number was seven. RESULTS: There was no significant difference in the control of the primary tumor for persistent disease (80% at 5 years), first relapse (61% at 5 years) and second relapse (80% at 3 years), p = 0.8845. The difference in survival between the three subgroups of patients, however, was highly significant (p = 0.0040). Thirty patients had CT evaluation before gold grain implant and the tumor was found confined to the nasopharynx in 21, in the remaining nine patients erosion of the sphenoid sinus or other parts of the base of skull was noted. The difference in the control between those patients with tumors confined to the nasopharynx and those patients with extranasopharyngeal extension of tumor almost reached statistical significance (81% and 44% respectively at 5 years, p = 0.0554). For the six patients who developed local recurrence after gold grain implant and were evaluable for the pattern of failure, the recurrent tumors were considered originating from another region of the nasopharynx in four, and in-field failure in the other two cases. CONCLUSION: Radioactive gold grain implant as salvage treatment provides satisfactory control of persistent and recurrent nasopharyngeal carcinoma. The local control was better when the tumor was localized to the nasopharynx, thus underlines the importance of close follow-up for early recognition of relapse and persistent tumor. However, such patients still suffered from high incidence of regional and distant failure, the pathophysiology and management of which require further investigation.

A method for the identification of human peripheral blood T lymphocytes by sequential immunogold and esterase double staining.

A double staining method involving the sequential use of monoclonal OKT hybridoma antibodies applied in the colloidal immunogold method and followed by a simultaneously capturing azo dye method for the detection of acid alpha-naphthyl acetate esterase (ANAE) is described. Mononuclear leukocytes isolated from human peripheral blood using a Ficoll-Hypaque density gradient were stained. M-pattern ANAE-positive monocytes (diffuse staining) were excluded from the lymphocyte counts. 80 +/- 5% of all lymphocytes were T-pattern ANAE positive (dot-like staining) and 77 +/- 3% were OKT3 positive. 86 +/- 6% of all ANAE-positive T-pattern lymphocytes were also OKT3 positive, and 89 +/- 6% of all OKT3-positive lymphocytes were also ANAE positive. This indicates that ANAE is a good marker for total human T lymphocytes. 53 +/- 10% of human peripheral blood lymphocytes were OKT4 positive and 87 +/- 8% of all OKT4-positive lymphocytes were also ANAE positive. 30 +/- 6% of all lymphocytes were OKT8 positive, and only 26 +/- 18% of all OKT8-positive lymphocytes were ANAE negative. This indicates that ANAE cannot be used to distinguish T-helper and T-suppressor lymphocytes as identified by monoclonal antibodies.