A potential krimpsiekte vaccine.

Krimpsiekte, a chronic form of cardiac glycoside poisoning, is an important plant-induced intoxication of small stock in South Africa. It is caused by cumulative, neurotoxic bufadienolides, such as cotyledoside. A cotyledoside-bovine serum albumin conjugate was synthesized to immunize animals. The efficacy of the cotyledoside-conjugate in inducing an immunological response was ascertained in rabbits (n = 4) and sheep (n = 4) by determining cotyledoside antibody titres with an ELISA using cotyledoside-hen ovalbumin as antigen. The formation of anticotyledoside antibodies was induced in both rabbits and sheep following immunization with the cotyledoside-protein conjugate. Protection provided by the vaccine was demonstrated by challenging sheep (n = 4) with repeated, daily doses of cotyledoside (0.015 mg/kg) administered intravenously, commencing 45 days after the initial vaccination. One control animal died on Day 3 of the challenge period and the other was severely affected after administration of the third cotyledoside dose. The immunized ewes (n = 2) remained clinically unaffected and the challenge was suspended following six daily injections. Vaccination as a means of preventing krimpsiekte seems to be quite feasible and deserves further investigation.

Isolation and characterization of novel endogenous digitalis-like factors in the ovary of the giant toad, Bufo marinus.

We have previously described the structures of four novel unconjugated bufadienolides in the ovary of the toad, Bufo marinus. In this study, we report the separation and characterization of three novel bufadienolide conjugates. These compounds were purified by HPLC, and their structures were determined to be 11alpha, 19-dihydroxytelocinobufagin-3-(12-hydroxydodecanoic acid) ester, 11alpha,19-dihydroxytelocinobufagin-3-(14-hydroxy-7-tetra decenoic acid) ester, and 11alpha, 19-dihydroxytelocinobufagin-3-(14-hydroxytetradecanoic acid) ester on the basis of NMR and MS data. Numerous dicarboxylic acid esters of bufadienolides have previously been described, but the three bufadienolide conjugates described in this report differ from previously described esters in that they contain hydroxylated monocarboxylic acids. The function of these three conjugates is not known but they are, like bufotoxins, potent inhibitors of Na+, K+-ATPase and may play a developmental role in the differentiation of toad oocytes.

Ouabain-sensitive Na+,K(+)-ATPase activity in toad brain.

Toads of the genus Bufo are highly resistant to the toxic effects of digitalis glycosides, and the Na+,K(+)-ATPase of all toad tissues studied to date has been relatively insensitive to inhibition by digitalis and related compounds. In studies of brain microsomal preparations from two toad species, Bufo marinus and Bufo viridis, inhibition of ATPase activity and displacement of [3H]ouabain from Na+,K(+)-ATPase occurred over broad ranges of ouabain or bufalin concentrations, consistent with the possibility that more than one Na+,K(+)-ATPase isoform may be present in toad brain. The data could be fitted to one- or two-site models, both of which were consistent with the presence of Na+,K(+)-ATPase activity with high sensitivity to ouabain and bufalin. Ki (concentration capable of producing 50% inhibition of activity) values for ouabain in the one-site model were in the 0.2 to 3.7 microM range, whereas Ki1 values in the two-site model ranged from 0.085 to 0.85 microM, indicating that brain ATPase was at least three orders of magnitude more sensitive to ouabain than B. marinus bladder ATPase (Ki = 5940 microM). Ouabain was also an effective inhibitor of 86Rb+ uptake in B. marinus brain tissue slices (Ki = 3.1 microM in the one-site model; Ki1 = 0.03 microM in the two-site model). However, the relative contribution of the high ouabain-sensitivity site to the total activity was 17% in the transport assay as compared with 63% in the Na+,K(+)-ATPase enzymatic assay. We conclude that a highly ouabain-sensitive Na+,K(+)-ATPase activity is present and functional in toad brain but that its function may be partially inhibited in vivo.

A novel bufadienolide, marinosin, in the skin of the giant toad, Bufo marinus.

We have identified a novel cardiac steroid, 11,19-epoxy-19-methoxytelocinobufagin, named marinosin (1), in the skin of the toad, Bufo marinus (L.) Schneider. The treatment of compound 1 with 50% CH3CN containing 0.1% trifluoroacetic acid yielded a 11 alpha-hydroxyhellebrigenin (2), which has not previously been isolated from animals or plants. The structures of both compounds were established from spectral data obtained by NMR and MS, which were compared with those of a reference bufadienolide, 11 alpha-hydroxytelocinobufagin. Compounds 1 and 2 have A/B cis and C/D cis configuration, which is characteristic of bufadienolides such as bufalin and marinobufagin. However, the stereo-structure of compound 1 was characterized by a boat form of the B ring, which is different from the chair form in typical bufadienolides such as compound 2. Compounds 1 and 2 both exhibited activity, as demonstrated by inhibition of Na+, K(+)-ATPase enzymatic activity and by inhibition of the binding of [3H]ouabain to Na+, K(+)-ATPase; however, marinosin (1) was as less effective inhibitor than 2, 11 alpha-hydroxyhellebrigenin. We have identified compound 2 in toad venom, but not in the skin.

Heterogeneity and lability of endogenous digitalis-like substances in the plasma of the toad, Bufo marinus.

Three major groups of endogenous digitalis-like substances (EDLS) have been identified in the plasma of the toad, Bufo marinus. One group of compounds, present in fresh plasma, is composed of chromatographically homogeneous polar conjugates, principally bufadienolide 3-sulfates, which exhibit relatively weak Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitory activity. A second and larger group of compounds, also found in fresh plasma, includes chromatographically heterogeneous conjugates, which are effective inhibitors of Na(+)-K(+)-ATPase; these compounds possess properties similar to those of bufotoxins. The third group of EDLS consists of free unconjugated bufadienolides, which are also effective Na(+)-K(+)-ATPase inhibitors. These unconjugated bufadienolides are present in relatively low concentrations in fresh toad plasma, but appreciable quantities are enzymatically generated from conjugates (believed to consist principally of bufotoxins) during the in vitro incubation of plasma. We suggest that the extent to which circulating polar EDLS are enzymatically deconjugated in vivo may be important in the regulation of the digitalis-sensitive Na(+)-K(+)-ATPase of toad brain, the only known digitalis-sensitive Na(+)-K(+)-ATPase in the toad.

Marinoic acid, a novel bufadienolide-related substance in the skin of the giant toad, Bufo marinus.

We found a novel substance, 3 beta-hydroxy-11,12-seco-5 beta, 14 beta-bufa-20,22-dienolide-11,14-olide-12-oic acid (1), which we called marinoic acid, in the skin of the toad, Bufo marinus. The structure was established from NMR and MS data. Like bufadienolides, marinoic acid contained an A/B ring structure in the cis configuration and a D/alpha-pyrone ring structure, but the structure of the C ring differed considerably from that of bufadienolides. Marinoic acid exhibited biological activity, as demonstrated by inhibition of Na+, K(+)-ATPase enzymatic activity, and by inhibition of [3H]ouabain binding to the digitalis receptor site on Na+, K(+)-ATPase, although marinoic acid was a less effective inhibitor than typical bufadienolides. Although marinoic acid cannot be classified as a bufadienolide, its chemical structure and its Na+, K(+)-ATPase inhibitory activity suggest that it is bufadienolide-related.

Digitalis-induced visual disturbances with therapeutic serum digitalis concentrations.

OBJECTIVE: To assess the role of digitalis in the development of visual symptoms severe enough to warrant ophthalmologic consultation in patients who received digitalis and who had no other clinical or laboratory evidence of digitalis toxicity. DESIGN: Clinical case study. SETTING: Neuro-ophthalmology referral practice. PATIENTS: Six elderly patients (aged 66 to 85 years) who received digitalis were referred to ophthalmologists for evaluation of photopsia (five patients) or decreased visual acuity (one patient). No patient had chromatopsia or nonvisual clinical manifestations of digitalis intoxication at the time of examination. MEASUREMENTS: All patients had serum digitalis concentrations within or below the therapeutic range. In most patients, the electroretinographic cone b-wave implicit time was longer than normal. RESULTS: Discontinuation of digitalis therapy, which was possible in five patients, was followed by resolution of visual symptoms and by shortening of the b-wave implicit time. Characteristic features of digitalis-induced photopsia were its dependence on illumination and its tendency to be localized in peripheral visual fields. CONCLUSIONS: In an elderly patient receiving digitalis, the development of photopsia characterized by innumerable points of light in the peripheral visual fields or a decrease in visual acuity raises the possibility that the patient's visual disturbance may have been digitalis induced. Digitalis-induced visual disturbances other than chromatopsia or disturbances of color vision may occur in elderly patients who have no other clinical manifestations of digitalis intoxication and who have a serum digitalis concentration within or below the therapeutic range.

Bile salts of the toad, Bufo marinus: characterization of a new unsaturated higher bile acid, 3 alpha,7 alpha,12 alpha,26-tetrahydroxy-5 beta-cholest-23-en-27-oic acid.

The bile salts present in gallbladder bile of the toad, Bufo marinus, were found to consist of a mixture of bile alcohol sulfates and unconjugated bile acids. The major bile alcohol was 5 beta-bufol; 5 alpha- and 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 26-tetrols occurred as the minor bile alcohols. Bile acids of Bufo marinus were cholic acid, allocholic acid, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha- and 5 beta-cholestan-26-oic acids, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha- and 5 beta-cholest-23-en-26-oic acids, 3 alpha,7 alpha,12 alpha, 26-tetrahydroxy-5 beta-cholestan-27-oic acid, and a C27 bile acid which has not been previously described. By chromatographic behavior, mass spectral data, and identification of the products of catalytic hydrogenation and ozonolysis, the structure of the new higher bile acid was elucidated as 3 alpha,7 alpha,12 alpha,26-tetrahydroxy-5 beta-cholest-23-en-27-oic acid. The bile salt pattern of Bufo marinus closely resembles that of Bufo vulgaris formosus, except for the absence of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-22-ene-24-carboxylic acid, the major bile acid of the latter toad.

Relationships of Na+ and K+ concentrations to GRP, CGRP, and calcitonin immunoreactivities and Na+,K(+)-ATPase (NKA) inhibitory activity in human breast cyst fluid.

BACKGROUND: Although the etiology of gross cystic disease of the breast is unknown, elevated cyst concentrations of potassium (K+) (> 60 mM/L) may be related to symptoms. The purpose of this study was to clarify the mechanism(s) of K+ accumulation in breast cysts. METHODS: We assayed cyst fluids for factors known to exert effects on K+ transport, namely, endogenous digitalis-like inhibitors of Na+,K(+)-ATPase (NKA) and the neuropeptides gastrin-releasing peptide (GRP), calcitonin (CT), and calcitonin gene-related peptide (CGRP). RESULTS: Cyst fluid K+ was directly correlated with cyst volume, cyst NKA inhibitory activity (in ouabain equivalents), and cyst concentrations of calcitonin, GRP, and CGRP. Cyst fluid Na+ was inversely correlated with cyst fluid K+, cyst NKA inhibitory activity, cyst volume, and cyst fluid concentrations of calcitonin, GRP, and CGRP. NKA inhibitory activity correlated directly with GRP and CGRP. Immunocytochemistry localized GRP to breast cyst lining cells and areas of ductal and lobular epithelial hyperplasia in biopsies of 15 of 15 cysts and in 5 of 5 breast carcinomas, but not in (0 of 5) normal breast biopsies. Specificity of GRP staining was demonstrated by total abolition of reactivity after adsorption with synthetic GRP, but not after adsorption with synthetic substance P, neurokinin A, or neurokinin B. CONCLUSIONS: We conclude that both the concentrations of endogenous digitalis-like factors and the neuropeptides calcitonin, GRP, and CGRP in human breast cyst fluids are related to the concentrations of K+ and Na+ in breast cysts and to cyst volume.

Structures of novel bufadienolides in the eggs of a toad, Bufo marinus.

In this paper, we report chemical structures of five compounds including four novel polyhydroxylated cardiac steroids in the eggs of a toad, Bufo marinus. These cardiac steroids were purified by high-performance liquid chromatography, and their structures were determined to be 11 alpha,19-dihydroxy-telocinobufagin (I), 11 alpha-hydroxytelocinobufagin (II), 11 alpha,19-dihydroxymarinobufagin (III), 11 alpha-hydroxymarinobufagin (IV) and 19-hydroxytelocinobufagin (V) on the basis of spectral data of nuclear magnetic resonance and mass spectroscopy. All the five compounds showed biological activity, as tested by inhibition of Na+,K(+)-ATPase activity and of [3H]ouabain binding to the receptor on Na+,K(+)-ATPase. This is the first finding of bufadienolides as cardiac steroids in animal eggs.

Human gamma delta T cells amplify IgE production by Epstein-Barr virus-activated B cells.

In summary, these data document that gamma delta T cells regulate B cell differentiation, antibody synthesis, and IgE secretion in EBV-activated B cells. The classical "helper" subset of CD4+ alpha beta T cells, although potent inducers of other Ig isotypes, are not as efficient in augmenting IgE synthesis. However, interactions between alpha beta and gamma delta cells enhance the IgE response further. The precise mechanism by which gamma delta T cells function to augment IgE secretion is under study. It will be important, for example, to determine whether gamma delta T cells, or products of these cells, directly regulate class switching or mediate the clonal expansion of IgE-expressing B cells. In this regard, we ruled out the possibility that IL-4 release alone could replace gamma delta cells in increasing IgE secretion. Finally, we think it is of potential interest that gamma delta cells migrate to skin epithelia as well as gastrointestinal and pulmonary mucosa, sites of invasion by parasitic organisms and contact with allergens. If gamma delta T cells were specifically activated, either directly or indirectly, by these foreign pathogens, then the data presented here might elucidate the cellular basis for the events leading to IgE secretion, a critical step in the immune response to both parasites and allergens.

Treatment of 150 cases of life-threatening digitalis intoxication with digoxin-specific Fab antibody fragments. Final report of a multicenter study.

One hundred fifty patients with potentially life-threatening digitalis toxicity were treated with digoxin-specific antibody fragments (Fab) purified from immunoglobulin G produced in sheep. The dose of Fab fragments was equal to the amount of digoxin or digitoxin in the patient's body as estimated from medical histories or determinations of serum digoxin or digitoxin concentrations. The youngest patient received Fab fragments within several hours of birth, and the oldest patient was 94 years old. Seventy-five patients (50%) were receiving long-term digitalis therapy, 15 (10%) had taken a large overdose of digitalis accidentally, and 59 (39%) had ingested an overdose of digitalis with suicidal intent. The clinical response to Fab was unspecified in two cases, leaving 148 patients who could be evaluated. One hundred nineteen patients (80%) had resolution of all signs and symptoms of digitalis toxicity, 14 (10%) improved, and 15 (10%) showed no response. After termination of the Fab infusion, the median time to initial response was 19 minutes, and 75% of the patients had some evidence of a response by 60 minutes. There were only 14 patients with adverse events considered to possibly or probably have been caused by Fab; the most common events were rapid development of hypokalemia and exacerbation of congestive heart failure. No allergic reactions were identified in response to Fab treatment. Of patients who experienced cardiac arrest as a manifestation of digitalis toxicity, 54% survived hospitalization.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular electrophysiologic effects of vertebrate digitalis-like substances.

Substances structurally and functionally similar to digitalis glycosides are produced by several vertebrate species. There also is evidence for a digitalis-like substance of human origin. Standard microelectrode techniques were used to study the direct effects on the cellular electrophysiology of canine Purkinje fibers of 1) bufalin, an unconjugated cardiotonic steroid molecule that is produced by the toad Bufo marinus, and 2) an extract of human bile that showed digitalis-like immunoreactivity on radioimmunoassay. The goal of this study was to determine whether these substances have arrhythmogenic effects comparable with those seen with toxic doses of digitalis glycosides. Bufalin, 2 x 10(-8) M, significantly (p less than 0.05) reduced maximal diastolic potential, action potential amplitude and duration and maximal rate of rise of phase 0 (Vmax) within 40 min of onset of exposure. All six fibers developed delayed afterdepolarizations and two developed triggered rhythms. Ouabain was less potent, in that a 2 x 10(-7) M concentration was required to comparably reduce maximal diastolic potential, action potential amplitude and duration and Vmax within 30 min. These Purkinje fibers also developed delayed afterdepolarizations and triggered rhythms. A sample of an extract of human bile that showed digitalis-like immunoreactivity with an antibufalin serum also reduced maximal diastolic potential, action potential amplitude and duration and Vmax, and produced delayed afterdepolarizations and triggered activity. In contrast, immunologically unreactive bile extracts had no appreciable effect on the action potential. In summary, the cardiac toxicity of digitalis substances produced by lower vertebrates is comparable with that induced by the glycosides. Moreover, it appears that humans may produce digitalis-like substances that may be cardiotoxic.

Digoxin inactivation by the gut flora in infancy and childhood.

Inactivation of digoxin by reduction of the lactone ring has recently been shown to occur in one third of adults and to be mediated by anaerobic intestinal bacteria. Children from birth through adolescence were studied to determine the pattern of development of this gut flora-mediated process. None of 36 digitalized infants 8 months of age or less excreted reduced digoxin metabolites in the urine. The adult pattern of digoxin reduction product excretion by one third of patients was observed after 16 months of age; however, high levels of digoxin reduction products such as are found in 10% of adults were not encountered in children less than 9 years of age. Even though reduced metabolites were not formed in vivo early in life, stool cultures of 20 of 73 infants younger than 8 months of age contained digoxin reduction product-forming bacteria at high concentrations, in some instances as early as the second week of life. Maturation of the gut flora with respect to digoxin metabolism appears to be a protracted process. The relative digoxin resistance of infants and children is not due to bacterial inactivation.

Treatment of 63 severely digitalis-toxic patients with digoxin-specific antibody fragments.

Sixty-three patients with life-threatening digitalis intoxication were treated with purified fragments of digoxin-specific antibodies (Fab) obtained from sheep. Twenty-eight patients developed toxicity as the result of digitalis ingestion in a suicide attempt, 5 ingested a large amount of digoxin accidentally and 30 developed toxicity in the course of treatment for underlying heart disease. The dosage of digoxin-specific Fab was calculated to be equimolar to the amount of cardiac glycoside in the patient's body. Digitalis toxicity was completely reversed in most cases, with onset of effect usually within 30 minutes of administration of Fab fragments. Unbound and, therefore, active digoxin serum concentrations decreased to undetectable levels within minutes after administration of the fragments. In all patients who had elevated serum potassium concentrations caused by massive digitalis toxicity, treatment with the Fab fragments reversed the hyperkalemia. There were no obvious adverse reactions to treatment. Potentially life-threatening digitalis intoxication can be rapidly and safely reversed by treatment with purified digoxin-specific Fab fragments.

Use of fab fragments of digoxin-specific antibodies in the therapy of massive digoxin poisoning.

A case of massive digoxin ingestion with multiple arrhythmias, consisting of high grade A-V block and ventricular ectopy not responsive to lidocaine, is described. The arrhythmias ceased following administration of digoxin-specific Fab fragments. The patient improved and was transferred to the psychiatric unit.

Studies on human low serum IgD phenotype and Gm markers.

The human "low serum IgD phenotype" was studied by simultaneous Gm typing and IgD immunoassay of several populations. An association between Gm (f+b+) haplotype and low human IgD was confirmed and extended to the "low serum IgD phenotype"--as defined from population distribution and genetic studies by Dunnette et al. 1978. Further, it was shown that Black American sera determined by Gm haplotype, had a similar percentage of "low serum IgD phenotype" samples (16%) although they lacked the "associated" Gm(f+b+) haplotype of White American samples. Sardinian sera showed a low incidence of the "low serum IgD phenotype" which was not correlated with Gm haplotype distribution. Familial aggregation of the "low serum IgD phenotype" was observed. No association was found between "low serum IgD phenotype" and serum IgE values. Age related abiotrophy of IgD could not be attributed to selective survival of "low serum IgD phenotype" persons.

Digoxin toxicity in a premature infant: treatment with Fab fragments of digoxin-specific antibodies.

The first use of Fab fragments to treat digoxin toxicity in a premature infant with renal failure, 18 h after the onset of severe arrhythmias, is reported with dramatic results. The development of digoxin toxicity in the context of accepted therapeutic dosing to treat heart failure due to a cerebral arteriovenous malformation is discussed.