Monoclonal antibodies that cross-react with the E1 glycoprotein of different alphavirus serogroups: characterization including passive protection in vivo.

A panel of four monoclonal antibodies (mAb) were produced that cross-react with representatives of two different togavirus serogroups, namely sindbis (SIN) and Semliki Forest (SF) viruses, by ELISA and ADCMC assays. Three of these mAb, IgG2a and IgG2b isotypes, passively protected C3H/Hej mice against 10 and 100 LD50 of SF challenge and one, IgM, did not protect against either challenge dose, or even at 1 LD50. All these mAb were cross-reactive with the E1 glycoprotein of the viruses by immunoblotting in which three different patterns of reactivity were evident, suggesting that three epitopes were involved. The patterns depended upon whether the mAb recognized E1 extracted from purified virions or infected cells and whether SDS-PAGE and immunoblotting were done in the presence or absence of beta-mercaptoethanol. One mAb (IgM) reacted with nonreduced or reduced E1 from either virions or cells suggesting recognition of a linear epitope. The other three mAb reacted with nonreduced but not reduced E1 from virions suggesting that recognition depends upon conformational epitopes. These three mAb reacted also with nonreduced E1 extracted from SF-infected cells whereas only one reacted with nonreduced E1 extracted from SIN-infected cells.

Disobutamide: a model agent for investigating intracellular drug storage.

Disobutamide, a bis tertiary amine (pKa1 = 8.6; pKa2 = 10.2) cationic amphiphilic compound, and a putative cardiac antiarrhythmic drug induced clear cytoplasmic vacuoles in dogs and rats. Ultrastructurally, the vacuoles were membrane-bound vesicles containing primarily electron-lucent material. Some concentric lamellar bodies indicative of phospholipidosis were also present. Although numerous vacuoles were seen in one-year toxicity studies in dogs and rats, there was no apparent evidence of necrosis, inflammation, atrophy, hypoplasia, hyperplasia, or metaplasia. Clinical signs or laboratory findings indicative of functional impairment were also not apparent. The picture of the vacuolation in vivo was one of storage. In cultured cells vacuoles were shown to be storage sites for disobutamide and specifically in distended vesicles of the cytoplasmic acidic compartments, such as lysosomes, endocytic, and probably transport vesicles. Storage of the drug in acidic vesicles is compatible with the dibasic nature of the cationic moiety of disobutamide. The intrinsic cell chemicals which accumulate in the vacuoles along with disobutamide remain unknown. Disobutamide may be a useful agent for defining experimentally the borderline between physiologic limits (normal function) and toxicity (functional impairment) in the condition of intracellular drug storage abnormalities and for advancing knowledge of storage mechanisms.

Spontaneous disseminated panarteritis in laboratory beagle dogs in a toxicity study: a possible genetic predilection.

Disseminated panarteritis was found in 16 (9 males and 7 females) of 49 laboratory beagle dogs (25 males and 24 females) from one breeding kennel. The dogs had been used in a 6-month oral toxicity study. Panarteritis was not associated with clinical or gross abnormalities. The incidence was similar in the control and test article-treated groups. Mainly medium-sized arteries throughout the body, particularly intercostal arteries (at their aortic origin), and coronary, epididymal and thymic vessels were affected. Chronic mononuclear-cell periarteritis was the predominant feature. Mixed cellular inflammation of the wall, proliferation or degeneration of muscle cells, focal "fibrinoid" material in the tunica media, fragmented internal elastic lamina and intimal thickening associated with myointimal cellular proliferation also occurred. These histologic changes are compatible with those of immune arteritis. Round worm intestinal infestation and granulomas of visceral larva migrans were common in several organs. Statistical analyses suggested that the pedigree of dogs is related to panarteritis, but the presence or absence of parasitization alone is not. The possible roles of genetic predilection and/or parasites in the pathogenesis are discussed. This panarteritis is spontaneous and may complicate the interpretation of lesions in toxicity studies.

Hyperostosis of the marrow cavity caused by misoprostol in CD-1 strain mice.

Misoprostol, a synthetic prostaglandin E1, was administered to CD-1 mice daily by gavage for 21 months in a safety study. Hyperostosis of the marrow cavity in the sternum and femur was found predominantly in female mice of the medium (1,600 mcg/kg/day) and high dosage (16,000 mcg/kg/day) groups. Many of the mice with hyperostosis also had cystic ovaries and cystic endometrial hyperplasia indicative of hyperestrinism. It is postulated that the hyperostosis was the result not only of the effects of misoprostol but also of endogenous estrogen. Since misoprostol did not cause hyperostosis in either rats or dogs, it is probable that this effect in mice is unique.

Two-year evaluation of misoprostol for carcinogenicity in CD Sprague-Dawley rats.

The carcinogenic potential of misoprostol, a synthetic prostaglandin E1 analogue with anti-ulcer potential, was evaluated in CD Sprague-Dawley rats. The compound was given daily by gavage at 24, 240, and 2,400 micrograms/kg, up to 150 times the daily human dose for 2 years. Necropsies were done on all animals and the incidences of non-neoplastic and neoplastic changes analyzed for significance by life table methods. The only statistically significant non-neoplastic finding was epithelial hyperplasia and hyperkeratosis of the gastric mucosa. These changes, which are characteristic of some prostaglandins, were expected. Other non-neoplastic findings were typical of known spontaneous conditions in this strain of rats. The most frequent neoplasm was the pituitary adenoma, followed by the mammary fibroadenoma, mammary adenoma, mammary adenocarcinoma, and thyroid C-cell adenoma. A rare neoplasm, squamous cell carcinoma of the ovary was found in two rats. There was no evidence that misoprostol is carcinogenic for CD Sprague-Dawley rats.

Twenty-one-month evaluation of misoprostol for carcinogenicity in CD-1 mice.

Misoprostol, a synthetic prostaglandin E1 methyl ester analogue with anti-ulcer potential, was evaluated for its carcinogenic potential in CD-1 strain mice. The compound was given daily by gavage at 160, 1,600, and 16,000 micrograms/kg for 21 months. Necropsies were done on all animals and the incidences of non-neoplastic and neoplastic changes analyzed for significance by life table methods. The only statistically significant non-neoplastic compound-related findings were epithelial hyperplasia and hyperkeratosis of the gastric mucosa and hyperostosis of bone in the marrow cavity of sternebrae and femurs. The changes in the gastric epithelium are characteristic of some prostaglandins and were expected. The bone hyperostosis was associated with misoprostol in high dosages, and was considered unique to the mouse. Other non-neoplastic findings were typical of known spontaneous conditions in mice. The most frequent neoplasm was the hepatocellular adenoma followed by lymphosarcoma, lung alveolar carcinoma, and Harderian gland adenoma. Several proliferative lesions of the duodenum were considered to be spontaneous. These were focal avillous hyperplasia, focal atypical hyperplasia, and junctional polyp. There was no evidence that misoprostol is carcinogenic for CD-1 mice.

Protection by zinc against acetaminophen induced hepatotoxicity in mice.

The purpose of this investigation was to assess protection by zinc against acetaminophen induced hepatotoxicity and to evaluate possible mechanisms of protection. Mice were treated with zinc (3 mg/kg, ip) or saline (ip) 48 and 24 hr before and sacrificed 12 hr after acetaminophen administration (375, 500, or 750 mg/kg, po). Liver toxicity was then assessed by histological examination. The incidence of hepatotoxicity was significantly less at 375 and 500 mg/kg of acetaminophen in zinc treated animals. The same dosage of zinc was not hepatoprotective when given 1 hr after acetaminophen. Mice were also treated with 1 to 10 mg/kg of zinc (ip) 48 and 24 hr prior to sacrifice, and metallothionein, cytochrome P-450, glutathione, and UDP-glucuronosyl transferase (GT) were determined in the liver. Metallothionein and UDP-GT were increased and P-450 and glutathione decreased at the higher dosages of zinc; however, only metallothionein was significantly changed at the dosage of zinc (3 mg/kg) used in the hepatoprotection experiments. Further, mice were similarly treated with 3 mg/kg of zinc before administration of 375 mg/kg of [3H]acetaminophen (po) and the amount of acetaminophen and acetaminophen bound to metallothionein were determined in the liver for 0.5 to 24 hr. In addition, after 6 hr the subcellular distribution and covalent binding to protein of acetaminophen were also determined. Zinc treatment had no significant effect in any of the above determinations. These results indicate that zinc protects against acetaminophen induced hepatotoxicity and that the observed protection is probably due to an induced biochemical change, but it is apparently not the result of any of the commonly invoked mechanisms.

Testicular toxicity of a novel 1,4-benzodiazepine (SC-32855) in rats and dogs.

SC-32855, a novel 1,4-benzodiazepine, was administered orally to adult male Beagle dogs (75 mg/kg/day for 10 days) and rats (550 mg/kg/day for 28 days). In both species, SC-32855 caused arrest of spermatogenesis and degeneration of the germinal epithelium, however, it had no effect on Sertoli or Leydig cells. There were no meaningful effects on the weights of the testis, epididymis or prostate in either species. Mean serum luteinizing hormone concentrations in dogs, which were 1.6 +/- 0.2 and 1.5 +/- 0.5 ng/ml prior to dosing, and 3.9 +/- 1.1 and 2.3 +/- 0.5 ng/ml after dosing for the control and treatment groups, respectively, were not significantly changed. Mean serum testosterone concentrations in dogs, which were 1.11 +/- 0.13 and 0.95 +/- 0.10 ng/ml prior to dosing, and 1.05 +/- 0.15 and 1.17 +/- 0.16 ng/ml after dosing for the control and treatment groups, respectively, were also not significantly changed. Further, SC-32855 in vitro did not inhibit the synthesis of testosterone in testicular microsomes isolated from normal rats or dogs. These data indicate that SC-32855 causes testicular damage, which is not secondary to decreased testosterone.

Preclinical toxicology profile of misoprostol.

The toxicity of misoprostol has been extensively examined in a variety of in vitro and in vivo studies. Preclinical studies evaluated acute and chronic toxicity, mutagenicity and carcinogenicity, and reproductive toxicity. Single oral dose studies in rodents and non-rodents indicate a safety margin of at least 500 to 1000 fold between lethal doses in animals and therapeutic doses in humans. Chronic toxicity studies (52 weeks) have been performed at daily oral doses of up to 300 and 9000 micrograms/kg body weight in dogs and rats, respectively. Rectal temperatures were increased at 100 and 300 micrograms/kg in dogs and serum iron was increased at 9000 micrograms/kg in rats. Stomach weights were increased in dogs and rats in a dose-correlated manner related, at least in part, to an increase in the number of normal epithelial cells (gastric hyperplasia). When drug treatment was stopped rectal temperatures, serum iron and stomach weights reverted to normal. Electron microscope studies on hyperplastic tissue showed that the ultrastructure was not affected. Hyperostosis has been observed, mainly in female mice, following prolonged drug treatment at high doses. Histological studies of bone tissues of rats and dogs and radiological studies of long bones of dogs following chronic administration of misoprostol showed that bone development was normal in all respects. Mutagenicity studies were negative and misoprostol was not fetotoxic or teratogenic in rats at oral doses up to 10000 micrograms/kg body weight, or in rabbits at doses up to 1000 micrograms/kg body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic analysis of the synthesis and assembly of type 1 fimbriae of Escherichia coli.

The adhesive organelles (type 1 fimbriae) of K-12 and other isolates of Escherichia coli are composed of identical 17,000-dalton subunits. We examined the assembly of these subunits into fimbrial organelles. After synthesis, the nascent subunits were first processed and then assembled into the organelles; the assembly step took almost 3 min in log-phase cultures at 37 degrees C. Even during blockage of protein synthesis, the free subunits continued to assemble until the pool was depleted. This pool was small in comparison with the amount of total fimbrial protein already assembled into surface organelles and was not sufficient to regenerate new detectable organelles after the removal of preexistent ones by blending. Assembly appeared to slow when the metabolic rate of the bacterial cells slowed, since subunits took longer to appear in the organelles at lower than optimal temperatures or as a culture entered the stationary phase. The synthetic rate of subunits slowed sooner than that of total cellular proteins as a culture approached the stationary phase and ceased completely as the culture entered the stationary phase. The amount of fimbrial antigen expressed on the surface of the cells remained relatively constant during growth of a culture.

Dependence of secretion and assembly of type 1 fimbrial subunits of Escherichia coli on normal protein export.

The export of fimbrial subunits was found to be diminished at the restrictive temperature in a strain bearing a secA(Ts) mutation. Likewise, export was inhibited in a strain harboring a malE-lacZ protein fusion upon induction of hybrid protein synthesis. Both conditions resulted in the accumulation of a precursor protein ca. 2,000 daltons larger than the mature fimbrial subunit.

Two cases of corneal epithelial dystrophy in rabbits.

Two New Zealand white rabbits which were used in a teratology experiment had a unilateral corneal opacity. The affected eye had a raised opaque membrane that extended from the limbus toward the center of the cornea to form a ring at the corneal margin. Sections of cornea showed local areas of thickened, elevated epithelium interspersed with areas of abnormally thin epithelium. A diagnosis of corneal epithelial dystrophy was made.

Isolation and characterization of a monoclonal antibody directed against type 1 fimbriae organelles from Escherichia coli.

We have isolated a mouse immunoglobulin M (IgM) monoclonal antibody directed against type 1 fimbriae from the Escherichia coli K-12-derived strain CSH50. Antibody specificity was demonstrated by (i) the ability of fimbriate but not nonfimbriate bacteria to compete with solid-phase purified fimbriae for antibody binding in an enzyme-linked immunosorbent assay, (ii) the visualized binding of antibody to fimbriae alone by electron microscopy, and (iii) the appearance in a radioimmunoprecipitation assay of a single electrophoretic band comigrating with pure type 1 fimbriae. The monoclonal antibody was further characterized by immunoblot analysis and compared with previously prepared rabbit anti-fimbrial antisera. Whereas the monospecific but polyvalent antisera recognized both fimbrial monomeric subunits and non-disaggregated fimbriae organelles, the monoclonal antibody recognized only the intact organelles even when the samples were prepared under nondenaturing conditions. The monoclonal antibody, therefore, might be directed against an epitope spanning two (or more) adjacent fimbrial subunits.

Antigenic quantitation of type 1 fimbriae on the surface of Escherichia coli cells by an enzyme-linked immunosorbent inhibition assay.

Type 1 fimbriae from two strains of Escherichia coli, K-12-derived CSH50 and a clinical isolate VL-2, were purified by a simplified procedure, which should be applicable to a variety of bacterial strains. After mechanical removal from the cells, the fimbriae were sedimented in the ultracentrifuge and resuspended in 5 M urea to disaggregate cell membranes and flagella, leaving the urea-resistant fimbriae intact. After several hours at 37 degrees C, this crude fimbrial suspension was diluted to 1 M urea, and the intact fimbriae were sedimented through a 1 M urea-1 M sucrose cushion. The pellet was found to be pure fimbriae by sodium docecyl sulfate-polyacrylamide gel electrophoresis, with apparent subunit molecular weights of 17,000 for the fimbriae from K-12 strain CSH50 and 19,000 for those from the clinical isolate VL-2. High-titer rabbit antiserum raised against CSH50 fimbriae was specific for fimbriae by indirect ferritin labeling and immunoprecipitation and was used to develop an enzyme-linked immunosorbent assay. Competitive inhibition of antifimbrial antiserum in the enzyme-linked immunosorbent assay by a known amount of either purified fimbriae or fimbriae-bearing bacteria permitted precise quantitation of fimbrial antigen in cultures of strain CSH50, thereby providing a simple means of determining the effects of environmental conditions on the synthesis of type 1 fimbriae.

Pseudocatabolite repression of type 1 fimbriae of Escherichia coli.

Previous work on the control of fimbriation in bacteria has demonstrated the importance of environmental factors such as static versus shaking broth and the absence versus the presence of glucose on the degree of fimbriation. When the Pil+ K-12 strain of Escherichia coli CSH50 was grown in static broth, the bacteria grown with glucose were less fimbriate (as determined by electron microscopy) than those grown without glucose. In contrast, a derivative, the pil-lac operon fusion strain VL361, gave off similar proportions of Lac+ and Lac- colonies when grown with or without glucose. Introduction of delta cya into either CSH50 or VL361 did not affect synthesis of either fimbriae or beta-galactosidase, respectively. When total synthesis of fimbriae by strain CSH50 was assayed, using an enzyme-linked immunosorbent inhibition test, glucose-grown bacteria made less antigen when they were grown in static broth but not when they were grown in shaking broth. When results are taken together, we interpret them as showing that glucose does not suppress fimbrial synthesis by classic catabolite repression but rather merely prevents the outgrowth or fimbriate bacteria in static broth.