Immunoglobulin chain recombination among antidigoxin antibodies by hybridoma-hybridoma fusion.

Conditions necessary for in vitro chain recombination of high affinity (10(9) to 10(12) M-1) antidigoxin monoclonal antibodies resulted in decreased affinity for both intact "native" and chain recombinant molecules. Chain recombination by somatic cell fusion was used instead to study the effects on antigen specificity and idiotypy of recombinants in which an homologous light (L) chain substituted for the parental L chain. The antidigoxin antibody 26-10 utilizes a VL sequence highly homologous to that of antibody 40-20, an antidigoxin antibody which uses a different VH gene than does 26-10 and lacks significant reactivity with an anti-26-10 idiotypic serum. The drug-marked antidigoxin cell line 26-10 (gamma 2a, kappa) and a drug-marked light chain producing variant of antidigoxin hybridoma 45-20 (lambda 1) which lacks both digoxin binding and idiotypy were fused. The fusion progeny (gamma 2a, kappa, lambda 1) which binds digoxin and is idiotype-positive, was selected for kappa loss (resulting in loss of digoxin and idiotype binding) and then fused with a heavy (H) chain loss variant of antidigoxin hybridoma 40-20 (kappa, digoxin nonbinding, idiotype negative). The resultant cell line CR-57 (gamma 2a, kappa, lambda) secretes antibodies which assemble the 26-10 H chain with both the 40-20 kappa-chain and the 45-20 lambda 1-chain. The affinity purified recombinant species consisting of 26-10 H chain and 40-20 kappa-chain expresses complete 26-10 idiotypic determinants. However, this recombinant antibody binds digoxin with decreased affinity and altered specificity relative to native 26-10. The binding specificity pattern nonetheless is most similar to the H chain donor. Amino acid and nucleotide sequence analyses of the respective light chains demonstrate six variable region differences between them, two of which are in complementarity-determining regions and the remainder in the framework. Hybridoma-hybridoma fusion provides an alternative to in vitro chain recombination for studying the contribution of chain combinational diversity to antibody diversity, antigen binding, and idiotypy.

The importance of endogenous digoxin-like factors in rats with various forms of experimental hypertension.

The acute administration of anti-digoxin serum (ADS) caused a pronounced long-lasting blood pressure decrease in young DOCA-salt hypertensive rats. The decrease of blood pressure was only moderate in 1K-1C Goldblatt rats while there was no change of blood pressure after the ADS injection in spontaneously hypertensive rats. However, the blockade of endogenous digoxin-like factors lowered blood pressure only in those hypertensive rats which were treated with DOCA-saline from youth but not in animals treated in the same manner only in adulthood. The age period at which salt intake was increased, could be responsible for the susceptibility of animals to salt- and volume-dependent forms of experimental hypertension as well as for the participation of slow acting humoral pressor agents in the induction and/or maintenance of elevated blood pressure. It is evident that endogenous digoxin-like factor(s) participate in a greater response of young rats to the hypertensive stimuli.

Effects of discontinuing maintenance digoxin therapy in patients with ischemic heart disease and congestive heart failure in sinus rhythm.

To evaluate the importance of oral maintenance digoxin therapy in chronic congestive heart failure (CHF), 24 patients in sinus rhythm on maintenance digoxin for documented CHF were studied prospectively on and off the drug. The average duration of therapy was 39 months (range 2 to 180). All 24 patients had documented coronary artery disease (CAD): 22 were in New York Heart Association functional class III and 2 in class II. Twenty-one patients (88%) were receiving diuretic or vasodilator therapy, or both, before digoxin discontinuance. At 1 month off digoxin and with no increase in doses of other medications excepting minor increases in antianginal therapy in 2 patients, no difference was observed in the group as a whole in symptoms, resting heart rate, arterial blood pressure, physical findings, weight, cardiothoracic ratio, radiographic signs of pulmonary congestion, radionuclide left ventricular ejection fraction (LVEF), duration of symptom-limited treadmill exercise (14 patients), or CHF score, compared with evaluation during maintenance digoxin therapy. Similar results were obtained in a subgroup of 9 patients with a resting LVEF less than 0.35 (0.27 +/- 0.02; mean +/- standard error of the mean). Six patients had a decrease and 5 patients an increase in LVEF of greater than or equal to 0.05 units after cessation of digoxin. Off digoxin, the CHF score increased by only 1 point in 2 patients, but also decreased in 2 patients. Thus, in this study population comprised of patients with CAD with documented CHF, most of whom were receiving diuretics or vasodilators, or both, digoxin withdrawal had no adverse clinical or hemodynamic effects.

The physiologic effects of digoxin under steady-state drug conditions in newborn and adult sheep.

The physiologic response to the chronic administration of digoxin was studied in 12 adult and 13 newborn sheep. Vascular pressures, cardiac output, isovolumic contraction phase indexes and systolic time intervals were measured before and after 2 weeks of digoxin therapy. Physiologic measurements were correlated with drug levels in plasma and myocardium. Resting myocardial function in newborns exceeded that in ewes. In ewes, the heart rate decreased from 98 to 74 beats/min, the preejection period (PEP) decreased from 76 to 57 msec, the ratio of PEP to left ventricular ejection time (LVET) decreased from 0.323 to 0.223 and dP/dt max increased from 2415 to 3460 mm Hg . sec-1 as plasma concentrations of digoxin increased to a mean of 1.8 ng/ml. Although the final steady-state plasma concentration of digoxin in newborn lambs averaged 1.7 ng/ml, cardiac output, PEP, PEP/LVET and dP/dt max did not change significantly from baseline values. These studies suggest that developmental differences in the physiologic response to digoxin are due either to a limited capacity for improvement in myocardial contractility shortly after birth or to an age-related difference in the effect of digoxin on myocardial tissue.