Pharmacokinetics of synthetic atrial natriuretic peptides in normal men.

The kinetics of atrial natriuretic peptides (ANP) and the kinetic profile of their effect on blood pressure and renal hemodynamic and electrolyte excretion were investigated in 20 salt-loaded healthy volunteers during and after constant rate infusion. At steady state, mean plasma concentrations of ANP were measured at 210, 430, and 2990 pg/ml and mean systemic clearance was 2.6, 2.5, and 1.7 L/min for ANP infusion rates of 0.5, 1, and 5 micrograms/min, respectively, which corresponds to the clearance rate of other vasoactive peptide hormones. The apparent volume of distribution averaged 17 L and the mean half-life was 4.5 minutes. ANP induced dose-related effects on systemic and renal hemodynamic, as well as urinary electrolyte excretion, albeit with a time lag between onset and full effect.

Plasma and atrial content of atrial natriuretic factor in cardiomyopathic hamsters.

Immunoreactive atrial natriuretic factor (IR-ANF) was measured in plasma and atrial extracts from normal and cardiomyopathic Syrian golden hamsters. Plasma IR-ANF was increased from 84.8 +/- 9.8 pg/ml (n = 17) to 234 +/- 23 (n = 25; P less than .0001) in hamsters with moderate failure, and to 1085 +/- 321 pg/ml (n = 10; P less than .02) in animals with severe failure. Plasma IR-ANF increased with increased atrial hypertrophy. Atrial IR-ANF content was essentially the same in normal animals and in those with moderate heart failure (3.06 +/- 0.28 vs. 3.17 +/- 0.19 microgram/100 g body wt.) and lower in the majority of those with severe failure (1.82 micrograms/100 g body wt., P less than .001). The elevations of IR-ANF in plasma are similar to those seen in patients with congestive heart failure. Our studies do not support bioassay results showing a deficiency of atrial ANF content as being important in the congestive heart failure associated with cardiomyopathy in the hamster.

Characterization of murine monoclonal antibodies to Escherichia coli J5.

Twenty-eight independently derived monoclonal antibodies (MAb) directed against Escherichia coli J5 endotoxin were produced and characterized. Each MAb exhibited a specific titer by both radioimmunoassay and passive hemagglutination assay. Most of the MAb were of the immunoglobulin G isotype; however, several immunoglobulin M antibodies and one immunoglobulin A antibody were produced. When characterized for their capacity to cross-react with purified endotoxin preparations from several gram-negative bacteria, 22 MAb exhibited no cross-reactivity; 6 demonstrated a limited capacity to cross-react with other endotoxin preparations. When characterized for their capacity to react with the intact organism instead of the purified endotoxin the pattern of cross-reactivity was quite different. Most of the MAb were able to react with Salmonella minnesota Re595. Eighteen were able to react with E. coli O111:B4 (the parent strain of E. coli J5), 13 MAb reacted weakly with Pseudomonas aeruginosa, and 3 reacted weakly with Klebsiella pneumonia. The data imply that MAb generated against E. coli J5 endotoxin demonstrate greater cross-reactivity when assayed against the whole bacterium than when assayed against the corresponding purified endotoxin. We were unable to demonstrate that any of the 28 MAb could passively protect mice against lethal endotoxin challenge.

Prophylaxis of Pseudomonas aeruginosa infections in leukopenic mice by a combination of active and passive immunization.

Mice rendered leukopenic with cyclophosphamide and then challenged with viable Pseudomonas aeruginosa were used to determine the protective efficacy of active immunization against exotoxin A and of passive immunization with human antiserum to Escherichia coli J5, a rough mutant of Escherichia coli O111:B4. Neither treatment alone provided a greater degree of protection than its respective control. However, the combination of these treatments produced a moderate, yet consistent, increase in the survival of infected immunosuppressed mice.

Characterization of synthetic atrial natriuretic factor: vasodilator profile and decreased vascular sensitivity in hypertensive rats.

Synthetic atrial natriuretic factor (ANF) relaxed agonist-induced tone in rabbit aortic rings as well as intrinsic, myogenic contractions in the isolated rabbit facial vein (IC50s = 0.1 to 5 nM). Tissues depolarized by high levels of K+ were refractory to ANF. A similar profile was obtained with extracts of rat atria and with sodium nitroprusside (NaNP) but not other vasodilators. Aortas from spontaneously hypertensive rats (SHR) were significantly less sensitive to the relaxant effects of ANF (and NaNP) than were aortas from Wistar-Kyoto (WKY) rats. However, atrial extracts from SHR were more effective than WKY rat atrial extracts in relaxing normotensive aortic rings. Radioimmunoassay analysis confirmed a small increase in ANF immunoreactive material in SHR compared with WKY rat atria. The similar vascular profile for both ANF and NaNP suggests that these agents share a common mechanism of action. A reduced end organ responsiveness in SHR may lead to an increased atrial content of ANF in these animals.

Toxoids of Pseudomonas aeruginosa exotoxin-A: photoaffinity inactivation of purified toxin and purified toxin derivatives.

For the preparation of greatly detoxified but highly immunogenic toxoids, two enzymatically active, low-toxicity derivatives of Pseudomonas aeruginosa exotoxin-A were further inactivated by photoaffinity labeling. These derivatives were formed during toxin purification, when a relatively crude toxin preparation was concentrated by ammonium sulfate precipitation and subsequently dialyzed. These derivatives, designated peak-1 protein (PK-1) and peak-2 protein (PK-2) were antigenically indistinguishable from native toxin, but had isoelectric points (5.00 and 4.90, respectively) that were different from that of the native toxin (4.95). Although the enzymatic activities and molecular weights of PK-1 and PK-2 were similar to those of native toxin, their toxicities were greatly reduced (ca. 500-fold). Photoaffinity labeling of fully active toxin-A, purified by a process which limits the formation of these derivatives, decreased its enzymatic activity (ca. 30-fold) and toxicity (ca. 100-fold). Likewise, photoaffinity labeling of purified PK-1 and PK-2 decreased their enzymatic activities and toxicities (ca. 30-fold and 100-fold, respectively) and, thus, yielded toxoids that were ca. 50,000-fold less toxic than unpurified native toxin. These toxoids were irreversibly detoxified and highly immunogenic during 9 months of storage at 4 degrees C.

Pseudomonas exotoxin A: toxoid preparation by photoaffinity inactivation.

A method for toxoid preparation has been developed in which toxins expressing enzymatic activity can be detoxified by photoaffinity labeling techniques. In the case of Pseudomonas aeruginosa exotoxin A, the method relies on the affinity of azido-substituted analogs of the substrate (NAD) for the proenzyme form of the toxin. Photolysis of the putative toxin-analog complex results in irreversible inactivation of the toxin without loss of antigenic character. It is proposed that this occurs by nitrene insertion into a chemical bond on the toxin molecule. This affinity photoinactivation process should be applicable to other ADP-ribosylating toxins.

Enzyme-linked immunosorbent assay for measurement of antibodies against pneumococcal polysaccharide antigens: comparison with radioimmunoassay.

An enzyme-linked immunosorbent assay (ELISA) for measuring antibodies against each of 14 polysaccharides in contemporary pneumococcal vaccine is described, and the findings of tests of paired sera from vaccinated human subjects are compared with those obtained by radioimmunoassay. The findings were in very poor agreement, and this appears to be due to the lesser ability of the ELISA procedure to measure antibody of low avidity. The ELISA procedure described here is not considered to be a satisfactory substitute for radioimmunoassay for measuring antibody responses to pneumococcal vaccine.

Passive protection by antitoxin in experimental Pseudomonas aeruginosa burn infections.

The protective effect of intravenously administered rabbit antitoxin serum was studied in lethal Pseudomonas aeruginosa burn infections in mice. Survival after infection with 2 median lethal doses of a toxigenic, low-protease-producing strain (PA103) was enhanced in antitoxin-treated mice, as compared with controls that had received anti-bovine serum albumin serum (P = 0.0004). Survival time was prolonged in other antitoxin-treated mice infected with toxigenic, high-protease-producing strains (PA86 and PA220, P = 0.0003 and P = 0.01, respectively). In contrast, antitoxin had no protective effect in mice challenged with a nontoxigenic strain (WR 5, P = 0.57). There were fewer viable bacteria in blood and liver of antitoxin-treated mice than in those of anti-bovine serum albumin-treated controls after infection with toxigenic organisms, whereas there were no significant differences between the two groups after challenge with the nontoxigenic strain. These data suggest that P. aeruginosa exotoxin A contributes to lethality in this burn infection model, and this effect is diminished by passive immunization with antitoxin.

Exotoxin production by clinical isolates of pseudomonas aeruginosa.

Seventy-five consecutive clinical Pseudomonas aeruginosa isolates were tested for in vitro exotoxin production. Exotoxin was demonstrated in culture filtrates biologically, by its ability to produce characteristic dermonecrotic lesions in guinea pigs, and seriologically, by counterimmunoelectrophoresis (CIE) with rabbit antiserum elicited with purified exotoxin. By these two methods, exotoxin was detected in 87 and 89% of P. aeruginosa strains, respectively (r = 0.48, P less than 0.001). Although less sensitive than CIE in detecting exotoxin immunodiffusion demonstrated a reaction of antigenic identity in most cases. Exotoxin was produced by all seven Fisher-Devlin immunotypes and by untypable strains. In contrast, exotoxin was not detected in the culture filtrates of 16 non-aeruginosa pseudomonas isolates and 48 non-pseudomonas organisms. The production of biologically similar antigenically closely related exotoxins is thus a characteristic of the majority of P. aeruginosa strains derived from diverse clinical sources.

Neutralizing antibody to Pseudomonas aeruginosa exotoxin in human sera: evidence for in vivo toxin production during infection.

Antibody to Pseudomonas aeruginosa exotoxin was detected in human sera by using a cytotoxicity-neutralization assay. Serum antitoxin was present in high titer in all 14 patients who recovered from serious pseudomonas infections (log2 of 50% neutralization titer, mean +/- standard deviation = 6.0 +/- 1.2). In contrast, serum antitoxin was present in lower titer in four of seven patients with fatal pseudomonas infections (3.3 +/- 2.7, P less than 0.005), in 3 of 7 patients with non-pseudomonas infections (1.4 +/- 0.6 P less than 0.001), and in 6 of 14 normal control subjects (2.0 +/- 1.3, P less than 0.001). Fourfold or greater serum antitoxin rises were demonstrated in two survivors of acute infections, and toxin-neutralizing activity was associated with the immunoglobulin G fraction of human sera. Immunization of rabbits with purified exotoxin also induced high antitoxin titers.

Pseudomonas aeruginosa exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of a highly specific antitoxin serum.

Pseudomonas aeruginosa exotoxin has been purified to a specific activity of 12,000 to 16,000 mouse median lethal doses/mg of protein. Total recovery was about 25%, and the degree of purification was approximately 3,000-fold. Preparative polyacrylamide gel electrophoresis greatly facilitated purification. As judged by analytical disc gel electrophoresis, the purified toxin contained one major band of protein and only a negligible amount of contamination. Antiserum prepared against the purified toxin neutralized the lethal activity of crude toxin preprations and reacted by double immunodiffusion with a single component of concentrated broth cultures of P. aeruginosa isolates obtained from a clinical source.

Use of microculture plates and the multiple automated sample harvester for in vitro microassay of bacterial toxins.

An in vitro cytotoxicity microassay for the measurement of nanogram quantities of Pseudomonas aeruginosa exotoxin and Vibrio cholerae enterotoxin is described.

Purification and characterization of Pseudomonas aeruginosa exotoxin.

A trypsin-sensitive, heat-labile exotoxin of Pseudomonas aeruginosa strain P-A-103 has been purified by a procedure that includes membrane ultrafiltration, hydroxylapatite chromatography, ion-exchange cellulose chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the exotoxin with a 40-fold increase in specific activity (micrograms of protein per median lethal dose [LD(50)]). The mean lethal dose of the purified toxin administered intravenously into mice weighing 20 g was approximately 6 mug of protein. The toxin contained virtually no nucleic acid, detectable pigment, or lipopolysaccharide. When subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, the toxin separated into at least six protein components which appeared to have similar molecular weights. The estimated molecular weight of the toxin is 54,000, and its isoelectric point is 5.0

Biochemistry of Vibrio cholerae virulence. 3. Nutritional requirements for toxin production and the effects of pH on toxin elaboration in chemically defined media.

The investigations described in this report concern the metabolic and physiologic parameters governing cholera enterotoxin production in chemically defined growth media. The results indicate that the minimal nutritional requirements for growth of pathogenic Vibrio cholerae are the same as those for toxin production, and that toxin production parallels growth of the organisms. Studies of the relationship between toxin accumulation and pH reveal that toxin biosynthesis can be separated from toxin release. Toxin is synthesized below pH 7.0, but release and accumulation of extracellular toxin occur only at neutral or alkaline pH values.

Biochemistry of vibrio cholerae virulence. II. Skin permeability factor-cholera enterotoxin production in a chemically defined medium.

A chemically defined medium capable of eliciting high titers of skin permeability factor (PF)/enterotoxin from three different strains of Vibrio cholerae has been developed. Toxin/antigen elaboration in synthetic and in complex media was monitored by a specific passive hemagglutination-inhibition test. A distinct temporal difference in the pattern of toxin/antigen elaboration was noted when the two types of media were compared. In complex media, PF activity and corresponding antigen release were coincident, whereas in the defined medium a biphasic pattern resulting in elaboration of nontoxic antigen during the second phase was seen. Possible reasons for the latter observation are discussed, and several experiments illustrating the unique utility of the defined medium are presented.