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Gold enhancement of gold-labeled probes: gold-intensified staining technique (GIST).

In this report we present a staining method in which gold chloride is used to enhance the size of gold colloids. We show the utility of this technique when used in conjunction with small gold colloids, i.e., 5 nm, 4 nm, and 2.6 nm. Post-embedding staining of epoxy-embedded, gold-labeled mouse LM fibroblasts showed that staining with 0.1% gold chloride facilitated the visualization of the smallest gold colloids.

Animals

Characterization of gold in urine and bile following administration of gold sodium thiomalate with chelating agents to rats.

Gold was characterized in the urine and bile of rats treated with D-penicillamine (D-PEN), 2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercaptopropane sulphonate (DMPS), or N-(2-mercapto-2-methylpropanoyl)-L-cysteine (bucillamine) immediately after gold sodium thiomalate (AuTM) injection by both gel chromatographic and electrophoretic methods. It is suggested that the gold in the urine and bile after AuTM administration was predominantly bound to high molecular weight compounds. The characterization of gold in the urine after administration of AuTM with D-PEN, DMSA, or DMPS showed that most of the gold was bound to the chelating agents. In the treatment with the chelating agents such as D-PEN and DMPS, the gold was mainly excreted as a gold-chelating agent compound in the bile and a minor portion of the gold was present in the form of a gold-L-cysteine compound and high molecular weight compounds. DMSA treatment showed that a major portion of the gold was bound to high molecular weight compounds in the bile and a minor portion of the gold was present in the forms of gold-DMSA and gold-L-cysteine compounds. The administration of AuTM and bucillamine indicated that the gold was mainly present as a gold-Me-bucillamine compound in the urine and a gold-bucillamine compound in the bile.

Animals

Activation of gold-reactive T lymphocytes in rheumatoid arthritis patients treated with gold.

OBJECTIVE: To assess the role of T lymphocyte sensitization in the etiology of side effects of gold therapy in patients with rheumatoid arthritis (RA). METHODS: Lymphocyte proliferation induced by gold(III) and gold(I) salts was measured in 53 subjects: 30 RA patients with gold-induced side effects (17 with dermatitis, 9 with proteinuria, 3 with hematologic complications, and 1 with colitis), 9 RA patients without side effects despite prolonged chrysotherapy, 4 RA patients who had never received gold, and 10 healthy controls. Peripheral blood lymphocytes were cultured with the different gold salts and proliferation was measured by 3H-thymidine incorporation. RESULTS: Thirteen of the 17 RA patients who developed gold-induced dermatitis showed significant T lymphocyte proliferation in response to gold(III) salts, and this proliferation could be completely blocked by monoclonal antibodies directed at the HLA-DR molecule. Such proliferative responses were not seen in patients with other gold-induced side effects, in patients who had never received gold, or in healthy controls. Only 1 of 9 patients who had not developed side effects despite long-term maintenance chrysotherapy showed significant lymphocyte activation with gold(III) salts. Lymphocyte proliferation could not be induced with gold(I) salts or with other metal salts. CONCLUSION: Patients with RA who develop dermatitis following treatment with sodium aurothiomalate [gold(I)] have T cells which proliferate in an HLA-DR-restricted manner in response to HAuCl4 [gold(III)]. We believe this observation can lead to more accurate diagnosis and treatment of side effects, which currently limit the use of one of the most effective antirheumatic drugs.

Adult

Inter-strand cross-links and single-strand breaks produced by gold(I) and gold(III) coordination complexes.

The ability of gold coordination complexes to bind to DNA and produce inter-strand cross-links in DNA was assessed in an assay system based on the fluorescence properties of the DNA intercalative dye, ethidium bromide. Results from these studies using a variety of gold(I) and gold(III) complexes suggest that the ability of gold complexes to bind to and produce inter-strand cross-links in DNA is not dependent on the oxidation state of gold in the complex but is influenced by the nature of the coordinating ligands. Those complexes in which the gold was ligated through one or more weakly coordinating ligands showed evidence for DNA binding. However, only those complexes with two or more of these relatively weak coordinating ligands produced inter-strand cross-links. Both the amount of binding to and cross-linking of DNA by these compounds were decreased by treatment of the gold-DNA complex with 2-mercaptoethanol and other thiol containing agents. As shown by agarose gel electrophoresis, 2-mercaptoethanol caused a dissociation of the gold-DNA complexes and a regeneration of closed circular superhelical pBR322 DNA. DNA strand breakage also resulted from treatment of a number of gold-DNA complexes with 2-mercaptoethanol; this was observed with the gold compounds which were shown to produce inter-strand cross-links in DNA. The amount of DNA strand breakage produced by treatment of gold-DNA complexes with 2-mercaptoethanol was influenced by the initial conformation of the DNA; gold-DNA complexes which resulted from the binding of gold compounds to covalently closed superhelical DNA were more sensitive to the breakage induced by 2-mercaptoethanol treatment than those complexes in which closed circular, relaxed DNA was used as substrate. The DNA breakage was not reduced in partially anaerobic conditions or by free-radical scavengers, suggesting that it is not mediated by oxygen. The results are discussed with respect to the potential for the interaction of gold complexes with intracellular DNA and chromatin and their biological implications.

Anaerobiosis

Effective diameters of protein A-gold and goat anti-rabbit-gold conjugates visualized by field emission scanning electron microscopy.

High-voltage (15-30 kV) field emission scanning electron microscopy (FESEM) was used to evaluate the effects of gold particle size and protein concentration on the formation of protein-gold complexes. Six colloidal gold sols were prepared, ranging in diameter from 7.6 to 39.8 nm. The minimal protecting amounts (m.p.a.) of protein A and goat anti-rabbit antibody (GAR) were experimentally determined. Gold particles were conjugated at the m.p.a., one half the m.p.a., and ten times the m.p.a. for both proteins, and protein-gold complexes prepared for FESEM. The smallest colloidal gold particles required the most protein per milliliter of gold suspension for stabilization. Transmission electron microscopy was found to be the preferred method for accurate sizing of gold particles, whereas FESEM of protein-gold complexes permitted visualization of a protein halo around a spherical gold core. Protein halo width varied significantly with changes in gold particle size. Measurements of protein halos indicated that conjugation with the m.p.a. of protein A resulted in the thickest protein layers for all gold sizes. GAR conjugation with the m.p.a. again produced the thickest protein layers. However, GAR halos were significantly smaller than those obtained with protein A conjugation. The proteins used showed similar adsorption patterns for the larger gold particles. For smaller gold particles, proteins may act differently, and these complexes should be further characterized by low-voltage FESEM.

Animals

[A case of gold lung with positive lymphocyte stimulation test to gold, using bronchoalveolar lymphocytes].

Gold lung, a gold-induced pneumonitis, is considered to be caused by hypersensitivity reaction to gold. We performed lymphocyte stimulation test (LST) to determine the response to gold, using lymphocytes obtained by bronchoalveolar lavage (BAL) from a patient with gold lung. A 57-year-old man was admitted with progressive shortness of breath following a skin eruption. He had been receiving weekly sodium gold thiomalate (Shiosol) for rheumatoid arthritis, with a cumulative dose of 485 mg. Chest roentgenogram showed diffuse interstitial infiltrates. LST for the response to gold, using peripheral lymphocytes, was positive. T cell lymphocytosis was observed in BAL, and transbronchial lung biopsy showed lymphocytic alveolitis and granulation tissue in alveolar ducts. From these findings, we diagnosed gold lung. Prednisolone (PSL) was started with an initial dose of 30 mg/day and resulted in a rapid improvement. As the dose of PSL was tapered, the patient's condition deteriorated and he was treated with a maintenance dose of 10 mg PSL. The second BAL revealed persistent lymphocytosis, and LST using bronchoalveolar lymphocytes for response to gold was positive. LST using peripheral lymphocytes was also positive, but was weaker than that using bronchoalveolar lymphocytes. This is the first report in Japan of a positive LST for response to gold, using bronchoalveolar lymphocytes from a patient with gold lung. This case suggests that the presence of activated lymphocytes against gold in the lung is cumulative, and that cell-mediated hypersensitivity is related to gold lung.

Arthritis, Rheumatoid

Streptococcal protein G-gold complex: comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling.

Protein G, a cell wall protein isolated from human group G streptococci strain G148, binds in a similar manner as protein A from Staphylococcus aureus to the Fc portion of IgG molecules. Indeed, protein G has been proposed as a superior Fc binding protein due to its broader species reactivity. Thus, we have prepared a complex of protein G with particles of colloidal gold and determined its applicability for spot-blot analysis and postembedding immunolabeling by comparing it with protein A-gold complex. By spot-blot analysis no difference in binding of protein G-gold or protein A-gold to IgG molecules from a whole spectrum of animal species was observed. Moreover, using rabbit, sheep, or goat anti-rat albumin antibodies to detect nitrocellulose-immobilized rat albumin or antigenic sites in paraffin and Lowicryl K4M thin sections from rat liver, no difference was found with protein G-gold or protein A-gold. Similarly, no difference in binding to protein G-gold or protein A-gold was observed with a battery of monoclonal antibodies. However, in contrast to expectations, protein A-gold reacted well with both sheep and goat IgG molecules; indeed, for the light and electron microscopic localization of albumin with sheep or goat antibodies it was as efficient as protein G-gold. These results demonstrate, therefore, that both protein G-gold and protein A-gold are useful second step reagents for immunolabeling and that protein G-gold was not a superior probe in the systems tested.

Albumins

Factors affecting efficiency of colloidal gold staining: pH-dependent stability of protein-gold conjugates.

The preparation of stable protein-gold conjugates is important in the quantitative colloidal gold staining. The protein adsorption onto colloidal gold particles and pH-dependent stability of protein-gold conjugates were examined using albumin and fibrinogen as model proteins. Albumin stabilized gold sols at pH values higher than 5.0. The amount of albumin necessary to stabilize gold sols was lowest at pH 5.6 and slightly increased as the pH of the adsorption medium was increased. Albumin was also able to stabilize gold particles even at acidic pH values ranging from 3.5 to 4.5, if a sufficient amount of albumin was added. Stable fibrinogen-gold conjugates were obtained at pH values above 6.5. Unlike albumin, the same amount of fibrinogen was able to stabilize gold sols as pH was increased from 7 to 10. The stability of the prepared protein-gold conjugates was very sensitive to the pH of the storing medium. The protein-gold conjugates were least stable at the isoelectric pH of the protein. The results suggest that protein-gold conjugates should be prepared at pH where conjugates are most stable, instead of pH close to the isoelectric point.

Colloids

Probe size and bound label conformation in colloidal gold-ligand labels and gold-immunolabels.

Colloidal gold can be produced in sizes ranging from 1.0nm to 150nm. All sizes of gold can be conjugated, principally by hydrophobic bonding, to a variety of molecules including ligands, enzymes and antibodies, as well as lectins and polysaccharides. The activity of most of these biological molecules is retained on conjugation with gold particles irregardless of size range, although the ratio of protein surface area to gold particle surface area varies widely depending on particle and protein size. We have employed low voltage high resolution scanning electron microscopy to compare, microscopically, the shapes of biological molecules unbound, bound to very small (3nm) gold particles, and bound to larger (18nm-30nm) gold particles. When very small gold particles are conjugated to large protein molecules, several particles bind along the length of each molecule, while smaller protein molecules often wrap around a single small gold particle. With larger gold particles, several biological molecules bind to a single gold particle. In addition, the shape of protein molecules bound to larger gold particles differs from that of molecules bound to small gold particles.

Animals

Skin contact with gold and gold alloys.

3 types of reaction to gold merit discussion. First, there is the effect known as black dermographism, in which stroking with certain metals immediately produces well-defined black lines on the skin. Some gold alloys are amongst such metals. The evidence indicates that the effect is the result of impregnation of the skin with black metallic particles generated by mechanical abrasion of the metal by contaminants of the skin. There is no positive and unequivocal evidence of the ability of metals to mark uncontaminated skin so rapidly that it is possible to write upon it. Secondly there are the 2 related phenomena of the wear of gold jewelry, and the susceptibility to certain individuals to blackening of the skin where it is in contact with such jewelry. The occurrence of smudge, as it is often called, is not very common, but is brought to the attention of most jewelers from time to time. In extreme cases it may make it embarrassing for the person concerned to wear metallic jewelry. It would appear as if gold smudge also results mainly from mechanical abrasion of jewelry, though this may be aided and/or supplemented in some instances by corrosion of gold or gold alloy induced by certain components of the sweat. Finally, there is the question of true allergic responses to contact of the skin with gold and its alloys. Judging from the very few cases which have been recorded, such responses are extremely rare. Some recent observations on the reactions of metallic gold with amino acids and of reaction to contact of the skin with gold on the part of rheumatoid arthritis patients undergoing gold therapy, are, however, relevant in this connection.

Corrosion

Interaction of D-penicillamine with gold salts: in vivo studies on gold chelation and in vitro studies on protein binding.

Serum and urinary gold levels were monitored in 18 patients previously treated with gold salts for rheumatoid arthritis and the effects of D-penicillamine studied. There was no statistically significant change in urinary gold levels on D-penicillamine therapy although there were some individual variations. Serum gold levels fell during D-penicillamine therapy but the rates of fall did not differ from those seen in patients not treated. In vitro studies on protein binding of gold salts suggest that a high affinity exists between gold salts and albumin with low levels of unbound gold even at concentrations far exceeding those seen in vivo. These preliminary results suggest that at therapeutic levels only small amounts of gold are available for chelation by penicillamine. It is concluded that penicillamine at low dosage is an unreliable chelator of gold salts in vivo and its use in the management of gold toxicity remains speculative.

Arthritis, Rheumatoid

Ultrastructural localization of gold particles within neural grafts labeled with gold-filled Sendai viral envelopes.

The ability to discriminate between host and donor cells is required to interpret the organization of neural grafts at the electron microscopic (EM) level. Using light microscopy, Ardizzoni et al. (Ardizzoni, S.C., Michaels A., and Arendash, G.W. [1988] Science 239:635-637) described a method, using gold-filled Sendai viral envelopes, for labeling cell suspensions prior to grafting. As the colloidal gold used in this procedure is especially attractive for use with EM, we have examined the ultrastructural distribution and character of this label with transplanted cells. Cell suspensions taken from the nucleus basalis of fetal rats were labeled using gold-filled Sendai viral envelopes and grafted into the dorsal neocortex of adult host rats with nucleus basalis lesions. After varying survival times ranging from 1 to 14 months, grafts and surrounding host tissue were examined using standard EM techniques. Within the graft site, gold particles ranging from 10-200 nm were found associated with various membranes throughout the cytoplasm of both neurons and glia. Gold particles of similar size were also found within the nuclei of neuronal and non-neuronal cells. Host cells near the graft site contained some small gold particles (10-40 nm). Control injections of non-viable, gold-labeled cells or colloidal gold alone resulted in similar patterns of small gold particles which were readily discriminable from the larger virally inserted gold particles found in viable labeled donor cells. We conclude that this method allows discrimination between closely associated host and donor cells.

Animals

Gold-specific T cells in rheumatoid arthritis patients treated with gold.

Gold-specific T lymphocyte clones were isolated from a patient with rheumatoid arthritis who developed delayed type hypersensitivity reactions to gold. All of the isolated T cell clones required histocompatible antigen presenting cells as well as gold for induction of proliferation. Using a panel of HLA-homozygous Epstein Barr virus-transformed B (EBV-B) cells and anti-HLA antibodies, the clones were shown to recognize gold in the context of DR1 molecules. Gold recognition did not require active antigen processing since specific proliferation was not affected by glutaraldehyde fixation of the DR1 homozygous antigen presenting cells. Furthermore, we could show that gold salts inhibited peptide-induced responses of a peptide-specific T cell clone. In addition to providing evidence for gold-specific T cells in gold-treated RA patients exhibiting delayed type hypersensitivity responses, these data suggest that gold can alter MHC-peptide complexes. The latter observation may in part explain the mechanism/s responsible for both the therapeutic and the toxic effects of gold.

Antigen-Presenting Cells

Gold concentrations and toxicity during oral gold treatment with auranofin.

The concentrations of gold in whole blood, serum, urine and blood cells of patients with rheumatoid arthritis were measured during 6 months of oral treatment either with Auranofin or placebo. Any adverse effects attributable to the treatments were also recorded. Although the time course of gold during Auranofin therapy was similar to that of injected gold, the blood and serum gold concentrations were significantly lower than those measured in patients receiving injected gold. Between 45 and 58% of Auranofin gold in the blood was associated with blood cells. In comparison, following injections of gold thiomalate, only about 4% of the gold was present in the blood cells. During the 6-month period, 2 patients receiving Auranofin withdrew because of diarrhoea and another because of rash. 1 placebo patient withdrew because of headaches. No laboratory evidence of haematological, renal or hepatic abnormality was encountered. It is suggested that the markedly lower concentrations of gold in the body sustained during treatment with Auranofin may be the critical factor towards a greater tolerance of the drug in the treatment of rheumatoid arthritis.

Arthritis, Rheumatoid

[A study on the inhibition of non-specific neutral proteinase by gold salt. Part 1. Gold sodium thiomalate].

The inhibitory effect of gold sodium thiomalate on non-specific neutral proteinase (EC 3.4.24.4) was investigated in vitro, in order to clarify the suppressive mechanism of the inflammatory process of joints by gold salt. The neutral proteinase was inhibited time- and concentration-dependently by gold salt. When the enzyme was incubated with 1 x 10(-2)M gold sodium thiomalate for 60 minutes, almost 100% of the activity was lost. Even at a lower concentration (1 x 10(-5)M, gold sodium thiomalate inhibited the enzyme time-dependently after longer incubation (3 or 9 hours). However, the same concentration of gold sodium thiomalate previously incubated with alpha-casein did not inhibit it. The results obtained here suggest that gold sodium thiomalate may inhibit the enzyme by binding it. The delay of neutral proteinase inhibition by gold sodium thiomalate seemed to be dependent on the concentration of gold salt.

Antirheumatic Agents

Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation of the enzyme-gold and the lectin-gold approaches.

Mannoside residues were revealed at the ultrastructural level in different cellular and extracellular compartments by means of the enzyme-gold and the lectin-gold approaches. For the enzyme-gold technique, an alpha-mannosidase-gold complex was prepared and conditions for the preparation of this complex as well as for its application were determined. Labeling was found over the rough endoplasmic reticulum mainly at the level of the membranes, the lumen of the cisternae being devoid of labeling. In the nucleus, the dense chromatin and the edge of the fibrillar threads in the nucleolus were intensely labeled. Few gold particles were present over the Golgi apparatus and mitochondria. The secretory granules in pancreatic cells, the peroxisomes in liver and the mucin in duodenal goblet cells were devoid of labeling. In the extracellular space, the basal lamina was labeled. Over the glomerular basal lamina, the labeling was mainly towards the epithelial side, in close contact with the podocytes. The results with the concanavalin A horseradish peroxidase (Con A-HRP)-gold technique were similar to those found with the enzyme-gold approach. Some differences were, however, detected at the level of the rough endoplasmic reticulum and the nucleus. In the endoplasmic reticulum, Con A-HRP-gold labeling was present over both the membranes and the lumen of the cisternae. In the nucleus, the labeling was mainly over the dispersed chromatin. These differences may be due to the binding of Con A not only to mannoside but also to other sugar residues as well as to the affinity of HRP-gold for some nucleoplasmic components.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

A comparison of tissue gold levels in guinea-pigs after treatment with myocrisin injected intramuscularly and triethylphosphine gold chloride and myocrisin administered orally.

A comparative study of tissue gold levels produced in guinea-pigs after the oral administration of either triethylphosphine gold chloride or Myocrisin (sodium aurothiomalate) or after the injection of Myocrisin intramuscularly is reported. Gold concentrations were determined 5, 24 and 168 hours after administration in stomach, small intestine, large intestine, kidney, liver and spleen and 5 and 24 hours after administration in skin, adrenals, heart, lung and brain. In gastrointestinal tissues, tissue gold concentrations were highest with triethylphosphine gold chloride. The stomach gold level 5 hours after oral administration of triethylphosphine gold chloride is particularly high and, taken in conjunction with the other gastrointestinal gold levels measured, suggests that a stomach rather than an intestinal absorption mechanism may predominate. A more extensive time-course study on kidney and liver is reported and the possible relationship between tissue concentration and toxicity is discussed.

Administration, Oral