Transplantation of a fetus with paternal Thy-1(+)CD34(+)cells for chronic granulomatous disease.

A fetus diagnosed with X-linked chronic granulomatous disease was transplanted with Thy-1(+)CD34(+) cells of paternal origin. The transplant was performed at 14 weeks gestation by ultrasound guided injection into the peritoneal cavity. The fetus was delivered at 38 weeks gestation after an otherwise uneventful pregnancy. Umbilical cord blood was collected and used to determine the level of peripheral blood chimerism as well as levels of functional engrafted cells. Flow cytometry was used to detect donor leukocytes identified as HLA-A2(-)B7(+) cells, whereas recipient cells were identified as HLA-A2(+)B7(-) cells. No evidence of donor cell engraftment above a level of 0.01% was found. PCR was used to detect HLA-DRB1*15(+) donor cells among the recipient's HLA-DRB1*15(-) cells, but no engraftment was seen with a sensitivity of 1:1000. The presence of functional, donor-derived neutrophils was assessed by flow cytometry using two different fluorescent dyes that measure reactive oxygen species generated by the phagocyte NADPH oxidase. No evidence of paternal-derived functional neutrophils above a level of 0.15% was observed. Peripheral blood and bone marrow samples were collected at 6 months of age. Neither sample showed engraftment by HLA typing using both flow cytometry and PCR. Functional phagocytes were also not observed. Furthermore, no indication of immunological tolerance specific for the donor cells was indicated by a mixed lymphocyte reaction assay performed at 6 months of age. While there appears to be no engraftment of the donor stem cells, the transplant caused no harm to the fetus and the child was healthy at 6 months of age. Analyses of fetal tissues, obtained from elective abortions, revealed that CD3(+) T cells and CD56(+)CD3(-) NK cells are present in the liver at 8 weeks gestation and in the blood by 9 weeks gestation. The presence of these lymphocytes may contribute to the lack of donor cell engraftment in the human fetus.

Cardiotonic agents. 7. Prodrug derivatives of 4-ethyl-1,3-dihydro- 5-[4-(2-methyl-1H-imidazol-1-yl)benzoyl]-2H-imidazol-2-one.

The cardiotonic agent 4-ethyl-1,3-dihydro-5-4-(2-methyl-1H-imidazol-1-yl)benzoyl]-2H- imidazol-2-one (1) was found to have low bioavailability when administered orally to rats and dogs. A series of N-acyl derivatives, an underutilized prodrug of acidic NH compounds, has been synthesized and tested for their ability to improve the oral bioavailability of 1. Reaction of the monosodium salt of 1 with various anhydrides afforded the N-1 monoacylimidazolones with surprisingly high regioselectivity. In addition to the prodrugs, acylation of 1 with propionic or phenylacetic anhydride led to the novel 3H-pyrrolo[1,2-c]imidazole-3,5(2H)-diones 6. The prodrugs showed a significant increase in the partition coefficients with a minor decrease in the aqueous solubility. The benzoyl derivative 4b exhibited the highest stability in both pH 1.5 and 7.4 buffer solutions. Further evaluation of 4b showed rapid conversion to 1 in canine plasma (t1/2 = 38 min), and human plasma (t1/2 = 10 min). Oral studies indicated that the bioavailability of 4b was increased to greater than 75% (compared to less than 20% for 1), and hemodynamic studies demonstrated that the selective inotropic profile of 1 was retained.

Hemodynamic effects of Gd-DTPA administered via rapid bolus or slow infusion: a study in dogs.

The hemodynamic effects of rapid (10 sec) versus slow (1 min) IV injection of Gd-DTPA were compared in normal, pentobarbital-anesthetized mongrel dogs. Hypertonic mannitol and isotonic saline were used as equiosmotic and equivolumetric controls. Two dose levels of Gd-DTPA were administered: 0.1 mmol/kg and 0.5 mmol/kg, corresponding to dose volumes of 0.2 and 1.0 ml/kg, respectively. The rapid injection of a high dose (0.5 mmol/kg) produced a transient decrease in arterial blood pressure (21 mm Hg, p less than .001), which returned to control values within 1 min after the injection. Slow IV infusion at this high dose had no statistically significant hemodynamic effects. There were no statistically significant effects of the lower dose (0.1 mmol/kg) administered as a bolus or by infusion, nor of the saline or osmotic control solutions. These results suggest that acute hemodynamic effects of Gd-DTPA are not observed at clinically relevant doses and the vasodilation observed at a higher dose may result from a direct action of the contrast agent.

Antiarrhythmic peptide has no direct cardiac actions.

The electrophysiologic, inotropic, and muscarinic effects of antiarrhythmic peptide (AAP) were examined in canine cardiac Purkinje fibers, ferret papillary muscle, and canine cardiac membranes, respectively. Aside from a prolongation of time to peak force in papillary muscle, no biologically significant effects of AAP could be determined in any preparation, suggesting that its antiarrhythmic effects are not mediated by direct membrane actions.

Cardiotonic agents. 5. Fragments from the heterocycle-phenyl-imidazole pharmacophore.

To examine the role of each component in the heterocycle-phenyl-imidazole inotropic pharmacophore, several imidazolone derivatives, an arylimidazole, a substituted 3,4-dihydro-4-oxopyrimidine, and a quinolin-2(1H)-one derivative were prepared as structural fragments or representatives from this relationship. Tests for cardiac inotropic activity in ferret papillary muscle strips (FPM) and for inhibition of crude cAMP phosphodiesterase obtained from canine cardiac tissue suggest that, while all three components contribute significantly toward potent activity (active at less than 1 microM concentrations in FPM), any combination of two components, in approximately a preferred geometry, represents the minimal requirements for weak activity (active at less than 25 microM concentrations). No single component appears to be requisite in an absolute sense.

Implications of altered inotropic effects of phenylephrine in pressure-overloaded cat ventricular muscle.

The positive inotropic action of phenylephrine in cardiac muscle is mediated by alpha- and beta-adrenoceptors. Data suggest the responsiveness of myocardium to inotropic agents is altered in cardiac disease. We evaluated the actions of phenylephrine on isometric contraction and K+-induced contracture in isolated cat right ventricular muscle from normal hearts and hearts with partial pulmonary artery ligation-induced pressure overload of 5-11 days in duration. Peak contractile force (Po) and rate of force development (dP/dt) were lower (p less than or equal to 0.005) in pressure-overloaded (0.59 +/- 0.2 g/mm2 and 4.6 +/- 1.7 g/s/mm2, respectively) than in normal (1.33 +/- 0.2 g/mm2 and 10.5 +/- 1.6 g/s/mm2, respectively) muscle. Phenylephrine (10(-6), 5 X 10(-6), and 10(-5) M) significantly (p less than or equal to 0.01) increased Po and dP/dt in normal but not in pressure-overloaded muscle. Phenylephrine (5 X 10(-6) M) reduced peak K+-induced contracture force (Pc) similarly in normal (-30 +/- 8%) and pressure-overloaded (-36 +/- 11%) muscles. beta-Adrenergic blockade (nadolol, 10(-4) M) reduced, but did not abolish, the "relaxant" action of the drug on Pc in both muscle groups. The lack of a positive inotropic effect of phenylephrine in pressure-overloaded muscle suggests a derangement in both alpha- and beta-adrenoceptor function in this model of cardiac disease.

Amrinone relaxes potassium-induced contracture of failing right ventricular muscle of cats.

The effects of amrinone (5.3 X 10(-4) M) on isometric contraction and K+-induced contracture force of right ventricular muscle isolated from normal cats (n = 6) and cats in right ventricular failure (RVF, n = 6), 3-14 days after partial pulmonary artery ligation, were studied. Peak isometric contractile force (Po) and maximal rate of force development (dP/dt) of RVF muscles (1.38 +/- 0.21 g/mm2; means +/- SEM, and 11 +/- 1 g/s/mm2, respectively) were significantly lower than normal muscles (2.46 +/- 0.41 g/mm2, p less than 0.025, and 24 +/- 3 g/s/mm2, p less than 0.005, respectively). Duration of contraction (DC) was significantly longer in RVF muscles (442 +/- 32 ms) than in normal muscles (361 +/- 11 ms, p less than 0.025). Times to peak twitch force (TTP) and peak K+-induced contracture force (Pc) of RVF and normal muscles were not different. Amrinone increased Po and dP/dt significantly in normal muscles (+78 +/- 24%; means +/- SEM; p less than 0.01, and +53 +/- 17%, p less than 0.025, respectively), but not RVF muscles (+11 +/- 16% and +8 +/- 19%, respectively). TTP and DC of normal muscle were unchanged by amrinone, whereas TTP was unchanged while DC was shortened (-12 +/- 3%, p less than 0.025) in RVF muscle. Amrinone relaxed Pc similarly and significantly in normal (-28 +/- 7%, p less than 0.005) and RVF (-26 +/- 4%, p less than 0.025) muscles. These results suggest that part of amrinone's salutary action in the failing heart occurs through modification of myocardial relaxation.

Inotropic actions of isoproterenol in cat ventricular muscle. Effects of extracellular potassium.

The inotropic actions of isoproterenol in cat papillary muscles or trabeculae bathed in a salt solution containing 4 mM KCl were compared to those in similar muscles bathed in a salt solution containing 22 mM KC1. Although isoproterenol evoked the same increase in force of contraction in both groups of muscles, the time course of contraction differed markedly. In muscles bathed in 4 mM KC1, isoproterenol caused a concentration-dependent decrease in time-to-peak force, but in muscles bathed in 22 mM KCl, isoproterenol caused a concentration-dependent increase in time-to-peak force. These data suggest that ventricular muscle activated by slow response action potentials may utilize a different mechanism of excitation-contraction coupling than do muscles activated by Na-dependent action potentials.

Oximes: 'enzymatic' slow channel antagonists in canine cardiac purkinje fibers?

In mammalian cardiac muscle voltage-dependent activation of slow channels, e.g., the slow inward current channel, may be possible only when the channels are phosphorylated. We examined the electrophysiological actions of oximes, mile nucleophilic agents which show 'phosphatase-like' activity in isolated enzyme systems, to assess their actions on slow channels in cardiac Purkinje fibers. Diacetyl monoxime (DAM) and pyridine-2-aldoxime (NorPAM) produced a marked, reversible and concentration-dependent reduction in the action potential (AP) plateau duration and abolished spontaneous phase 4 depolarization, but produced only minimal effects on resting potential, dV/dt max, action potential amplitude, duration of phase 3, or membrane resistance. Slow response action potentials evoked in the presence of elevated potassium plus isoproterenol or in Na-free solution were abolished by DAM. The effects of DAM on the AP plateau were antagonized by epinephrine, but an increase in Ca was relatively ineffective. The results suggest that oximes may act as surrogate phosphatases to remove phosphate groups which regulate the availability of slow current channels for voltage-dependent activation.

Sodium dependence of the positive inotropic effect of cardiac glycosides.

We have investigated the inotropic effects of digoxin, digitoxin and ouabain in cat ventricular muscle under conditions of reduced Na influx in an effort to determine the role of Na in the positive inotropic effect of these cardiac glycosides. When the normal, Na-dependent action potential is inactivated by potassium depolarization, these glycosides retain a positive inotropic effect. In contrast, when muscles are bathed in Na-poor or Na-free solutions, these glycosides do not influence contraction. This suggests that the cardiac glycosides are dependent on Na for their positive inotropic effects.

A comparison of the mechanical performance of papillary muscle and trabeculae carneae of cat ventricle.

The mechanical performance of papillary muscle and of trabeculae carneae from cat ventricle were compared in physiologic salt solutions using two different buffers, bicarbonate and HEPES. The results show that there are no significant differences between the muscle types and that, under the conditions of these studies, HEPES may be used interchangeably with bicarbonate.

Depressant effects of phenoxygenzamine on potassium contracture in cat ventricular muscle.

Phenoxybenzamine has been shown to have a depressant action on K+-depolarization contracture in cat ventricular muscle. In the present study, we show that this depressant action is specific for phenoxybenzamine and occurs in the presence of beta-adrenergic receptor blockade with nadolol. K+-contracture is not depressed by phentolamine nor augmented by phenylephrine. Thus, the depressant action of phenoxybenzamine is not mediated by its effects on cardiac adrenergic receptors.

Paradoxical actions of quinidine in hyperpermeable heart cells.

The effect of quinidine on the inotropic state was examined using small fragments of hyperpermeable ventricular muscle in vitro. In concentrations of 10(-5) and 10(-4) M, quinidine had no effect on the force of spontaneously contracting fibers. At a concentration of 10(-3) M, contractile force increased by at least twofold. We conclude that the negative inotropic action of quinidine is not by a direct action on intracellular organelles of the heart.