Brief Report: Drugs Implicated in Systemic Autoimmunity Modulate Neutrophil Extracellular Trap Formation.

OBJECTIVE: Aberrant neutrophil extracellular trap (NET) formation has been implicated as a mechanism to induce autoreactivity in individuals at risk of autoimmune diseases. The objective of this study was to assess whether medications implicated in cases of drug-induced autoimmunity (hydralazine and procainamide) and medications less commonly associated with drug-induced autoimmunity (minocycline and clozapine) induce NET formation and/or prevent NET degradation. METHODS: Human neutrophils were incubated with the drugs of interest and resultant NET formation was quantified by fluorescent microscopy. The ability of these drugs to interfere with NET degradation by serum nuclei was assessed. Pathways of drug-induced NET formation were studied with pharmacologic inhibitors of reactive oxygen species (ROS), peptidylarginine deiminases (PADs), and muscarinic receptors, and by assessment of intracellular calcium levels by flow cytometry. To determine if NET protein cargo varies by drug stimulus and/or neutrophil source, proteomic analysis of NET lysates induced by specific medications was compared using neutrophils from healthy donors and from patients with autoimmune diseases. RESULTS: Hydralazine and procainamide significantly induced NET formation while minocycline and clozapine did not. None of the medications significantly impaired NET degradation. NETosis induced by these drugs required NADPH oxidase and PAD4 activation. Procainamide triggered NETs via muscarinic receptor engagement on neutrophils, while hydralazine modulated calcium release from intracellular stores. Differences in protein cargo, particularly histone content, were observed in NETs induced by hydralazine and procainamide. CONCLUSION: Medications commonly implicated in drug-induced autoimmunity trigger NET formation displaying distinct protein cargo, via common and specific pathways. NETosis may play a role in the pathogenesis of drug-induced autoimmunity.

Atrial Fibrillation in Anaphylaxis.

BACKGROUND: The relationship between anaphylaxis and cardiovascular events has been reported in the past. While skin and respiratory symptoms are usually the most common and the first to appear, cardiovascular complications play a key role and represent the leading cause of death in anaphylaxis. METHODS: We report 3 episodes of atrial fibrillation triggered by anaphylaxis. Allergy and cardiology studies were performed. In both patients, the etiological agent was identified: Anisakis simplex hypersensitivity and food allergy. RESULTS: The heart is the source and target of chemical mediators released during an allergic reaction. In the heart, there are plenty of mast cells, and they are predominantly located around the coronary adventitia and in close contact with small vessels in the muscle wall. The release of mediators can influence ventricular function, heart rate, and coronary artery tone. Anaphylaxis can trigger any kind of arrhythmia. In these cases, the very interesting point of discussion was: which should be first, treating anaphylaxis or cardiac events? The other controversial point was the use of epinephrine, the first line of treatment for anaphylaxis. Recommendations about epinephrine in cardiac patients during an anaphylactic event are still a major dilemma. CONCLUSIONS: We emphasize the importance of the priority of establishing protocols between cardiologist and allergist in treatment of cardiac complications during anaphylaxis, and we warn about the correct diagnosis of arrhythmias in anaphylaxis in order to treat them as soon as possible, to prevent other consequences and complications.

Digoxin--'a friend or foe'.

The authors report about a patient who was admitted after developing nausea, vomiting, change in vision and lethargy. She was on digoxin 250 mcg once daily among all her other medications in the wake of a recent stroke that was accompanied by atrial fibrillation (AF). Her digitalis levels shortly before and on admission were 3.4 and 2.9 ng/ml, respectively. Her admission rhythm was slowly conducted AF at an average of 35 bpm. After a careful assessment by the cardiology consultant in charge, she received Digibind infusion for a chronic digitalis toxicity with the digoxin immune Fab dose based on the formula recommended in the product literature.(3) A few days observation on the ward ensured that her resting heart rate rose to 65 bpm and that she did not need a pacemaker for a slow AF. Her functional status remained reasonably good as she enjoyed a satisfactory recovery postthrombolysis for her recent stroke.

Interference between eplerenone and digoxin in fluorescence polarization immunoassay, microparticle enzyme immunoassay, and affinity column-mediated immunoassay.

Digitalis-like immunoreactive substances have crossreactivity with antidigoxin antibodies and the interference between digoxin and spironolactone/canrenone has been reported. The structure of eplerenone is similar to that of spironolactone/canrenone. Therefore, we hypothesized that eplerenone might also interfere with the measurement of digoxin by immunoassay. We performed three types of assays (fluorescence polarization immunoassay [FPIA], microparticle enzyme immunoassay [MEIA], and affinity column-mediated immunoassay [ACMIA]) to determine crossreactions between eplerenone and antidigoxin antibodies. Furthermore, we used FPIA, MEIA, and ACMIA to measure the apparent digoxin concentration in mixed solutions of eplerenone (1-100 µg/mL) and digoxin (1-3 ng/mL). In the crossreaction tests, eplerenone was detected as digoxin by FPIA and ACMIA. By FPIA, a known concentration of 1 µg/mL of eplerenone was measured as 0.33 ± 0.11 ng/mL of digoxin (crossreaction rate, 0.03%). By ACMIA, a known concentration of 10 µg/mL of eplerenone was measured as 0.13 ± 0.05 ng/mL of digoxin (crossreaction rate, 0.001%). No crossreaction between eplerenone and digoxin was determined by MEIA. In the interference of eplerenone coadministered with digoxin, the apparent concentration of digoxin was increased in FPIA, but decreased in MEIA and ACMIA. The results suggest that eplerenone crossreacts with antidigoxin antibodies in FPIA, MEIA, and ACMIA, but that the interference of eplerenone might be smaller than that of spironolactone/canrenone.

Emergency step-by-step specific immunotherapy in severe digoxin poisoning: an observational cohort study.

OBJECTIVE: To evaluate the efficacy and safety of a step-by-step fixed dose of specific immunotherapy protocol in case of severe digoxin poisoning in an open uncontrolled prospective study. METHODS: Twenty consecutive patients were admitted because of severe digoxin poisoning. The inclusion criteria were: digoxin overdose and either life-threatening arrhythmia; high-degree atrioventricular block, ventricular arrhythmia, or bradycardia less than 50 bpm and hyperkalaemia (>5.5 mmol/l). A two-step protocol of antidigoxin antibodies treatment was carried out. At admission, every patient received two vials of specific Fab-fragments. If after 1 h following infusion ECG signs regressed, no more treatment was given. If ECG signs did not regress, patients were given two more vials. At inclusion and 6 h after immunotherapy, clinical (cardiac rhythm, ECG records) and biological (serum digoxin concentration, potassium) findings were recorded. RESULTS: Patients had a median (interquartile range) age of 83 (75-90) years. Four patients had acute poisoning and 16 chronic overdoses. Eleven patients showed ventricular arrhythmia, and five had high-degree atrioventricular block. Seventy percent of the patients needed only the first step. Significant decreases were observed in the number of cardiac dysrhythmia (16 vs. three patients), in the median (interquartile range) of serum digoxin concentration [5 microg/l (3.8-6.2) vs. 0.4 microg/l (0.3-2.2)] and in serum potassium [4.6 mmol/l (4.1-5.5) vs. 3.85 mmol/l (3.7-4.55)] before and after immunotherapy. The digoxin-related mortality was 5%. CONCLUSION: This protocol of step-by-step digoxin-specific immunotherapy seems to be as effective as the equimolar treatment, and there was significant cost reduction in case of acute poisoning.

[Immunochemical properties of conjugated antigens from N-substituted phenothiazines and dibenzazepines with antiarrhythmic activity].

The use of metabolites of antiarrhythmic drugs (ethmozine, ethacizine, and bonnecor) as haptens in the synthesis of conjugated antigens allowed us to induce the formation of antibodies with different specificity for certain metabolites. A new enzyme immunoassay was developed for the detection of phenothiazine and dibenzazepine derivatives (ethmozine, ethacizine, and bonnecor). Nanogram and subnanogram quantities of these substances may be detected in biological fluids.

Antibodies to non-bilayer phospholipid arrangements induce a murine autoimmune disease resembling human lupus.

Antibodies recognizing non-bilayer phospholipid arrangements (NPA) in membrane models and in cell membranes in vivo, triggered an autoimmune-like disease in mice. This exhibited features similar to human lupus and was induced by injecting mice either with the H308 monoclonal antibody specific to NPA, with sera from mice which already had developed the autoimmune disease, or with liposomes treated with the NPA inductors chlorpromazine or procainamide; or with these NPA inductors alone. All these procedures revealed the involvement of antibodies to non-bilayer phospholipids in inducing this autoimmune-like disease. Unraveling the mechanisms of these antibodies might contribute to a better understanding of the molecular and immunological basis of autoimmune diseases like lupus and, hopefully, towards the development of better therapeutic strategies.

A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs.

We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin. The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H:100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10:digoxin co-crystal structure. Several mutant Fabs obtained following panning on digoxin-BSA showed increased affinity for digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs than to digoxin. This specificity change was unexpected, as C16 lies on the opposite side of digoxin from H:CDR3. Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a target glycoprotein in drug-induced thrombocytopenia.

Drug-induced immune thrombocytopenia (DITP) is a serious complication of drug treatment. Previous studies demonstrated that most drug-dependent antibodies (DDAbs) react with the platelet membrane glycoprotein (GP) complexes IIb/IIIa and Ib/IX/V. We analyzed the sera from 5 patients who presented with DITP after intake of carbimazole. Notably, thrombocytopenia induced by carbimazole was relatively mild in comparison to patients with DITP induced by quinidine. The sera reacted with platelets in an immunoassay on addition of the drug. In immunoprecipitation experiments with biotin-labeled platelets and endothelial cells, reactivity with the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) could be demonstrated, whereas neither GPIIb/IIIa nor GPIb/IX was precipitated in the presence of the drug. These results could be confirmed by GP-specific immunoassay (MAIPA) using monoclonal antibodies (mabs) against PECAM-1. In addition, the binding of DDAbs could be abolished by preincubation with soluble recombinant PECAM-1. Carbimazole-dependent antibodies showed similar reactivity with platelets carrying the Leu(125) and Val(125) PECAM-1 isoforms, indicating that this polymorphic structure, which is located in the first extracellular domain, is not responsible for the epitope formation. Binding studies with biotin-labeled mutants of PECAM-1 and analysis of sera with mabs against different epitopes on PECAM-1 in MAIPA assay suggested that carbimazole-dependent antibodies prominently bound to the second immunoglobulin homology domain of the molecule. Analysis of 20 sera from patients with quinidine-induced thrombocytopenia by MAIPA assay revealed evidence that DDAbs against PECAM-1 are involved in addition to anti-GPIb/IX and anti-GPIIb/IIIa. We conclude that PECAM-1 is an important target GP in DITP. (Blood. 2000;96:1409-1414)

[Severe digoxin poisoning. The successful use of the classic treatment]. / Intoxicación grave por digoxina. Utilización exitosa del tratamiento clásico.

In our environment, the use of Fab antibodies for digoxin intoxication is often difficult due to the low availability of this drug in most centers. We present a case of massive digoxin intoxication that was successfully managed with the classic treatment. Later, we discuss the need to individualize the management of this kind of intoxications in order to reduce, when possible, sanitary costs.

Treatment of severe chronic digoxin toxicosis in a dog with cardiac disease, using ovine digoxin-specific immunoglobulin G Fab fragments.

Severe digoxin toxicosis in a 13-year-old mixed-breed dog with dilated cardiomyopathy was successfully treated with i.v. administration of ovine digoxin-specific IgG Fab fragments. Management of this dog following treatment with Fab fragments was complicated by hypomagnesemia and loss of the positive inotropic effect of digoxin.

Analytic performance of two automated nonpretreatment digoxin immunoassays.

The analytic performance of two automated nonpretreatment digoxin methods, AxSYM Digoxin II and Vitros digoxin immunoassays, was assessed. Both assays had analytic sensitivities of less than 0.2 microg/L, were linear from digoxin concentrations of 0.5 to 4.0 microg/L, and showed acceptable precision, with a maximum total coefficient of variation (CV) of 8.9% and 6.4% for the AxSYM and Vitros, respectively. Comparison of the two methods using samples from patients receiving digoxin gave the following relationship: Vitros = 0.91 x AxSYM + 0.23 (r = 0.97, Sy,x = 0.12). Digoxinlike immunoreactive factor (DLIF) crossreactivity was examined in specimens from patients who had hepatic disease, renal insufficiency, had undergone cardiac surgery, and in neonatal cord blood samples. Minimal crossreactivity was observed for most samples and the average crossreactivity for each group of samples was comparable for the two methods. The recovery of digoxin added to samples from each group of DLIF was similar, except for that from cord blood samples, for which recovery was significantly lower with the AxSYM method. Titration of a digoxin-spiked serum pool with digoxin-immune Fab showed a similar decrease in the measured digoxin concentration for both methods. Overall, the analytic performance characteristics of these two methods were comparable.

Amiodarone-induced severe hepatitis mediated by immunological mechanisms.

A 64-year-old man developed a fulminant hepatitis 4 days after initiation of amiodarone treatment and a total dose of 7.1 g. The direct Coombs test was positive and became negative again soon after stopping treatment. Immediately after stopping treatment the extremely increased parameters of hepatic failure returned to normal again. A rechallenge with 200 mg of amiodarone was accompanied by a positive Coombs test which again became negative after several days. We conclude that the occurrence of an acute hepatitis soon after initiation of amiodarone treatment is mediated by immunological mechanisms. There should be high vigilance with respect to this rare life-threatening adverse drug reaction.

Genetic and immunologic studies of patients on procainamide.

Forty (40) patients with cardiac arrhythmias receiving procainamide (PA) therapy and 24 patients who were receiving other drugs for their cardiac disorders were investigated for class II HLA phenotypes and their DRB1*04 and DQB1*03 subtypes. Other genetic marker evaluations in the PA patients included: 1) class III MHC C4A and C4B null alleles of complement; and, 2) acetylation phenotype. Twenty (20) of the PA patients were also tested for the ability of their stimulated cells to secrete Interleukin-1 (IL-1 beta) and tumor necrosis factor (TNF alpha). We also examined the spontaneous production of these cytokines by peripheral blood leukocytes (PBL) from patients who were receiving chronic PA treatment. The results revealed no association of acetylation phenotypes with the class II HLA phenotypes nor class III MHC C4 allotypes in these patients. The results did show a significant increase in class III C4 complement allotypes in the PA patients when compared to the controls. The results also showed a significant increase in autoantibodies and DQw3 phenotypes in the PA patient group when compared to control populations. Results of spontaneous IL-1 and TNF production suggested there may be an association of select class II HLA phenotypes in some patients and this may be relevant to host responsiveness to PA treatment.

[Digitalis poisoning treated with a specific antidote]. / Digitalisforgiftning behandlet med spesifikt antidot.

Toxicity by digitalis is a common problem in everyday clinical practice. In this paper three cases of severe poisoning with digitoxin with maximal S-digitoxin levels of 115, 150 and 239 nmol/l are described. All patients received specific digoxin Fab-fragment intravenously. Administration of antidotes resulted in a favourable outcome in all three patients. So far, the use of digoxin-specific antibodies has been limited to a few cases of severe intoxication where life-threatening arrhythmias and hyperkalaemia were present. We discuss whether a more liberal indication should be accepted.

A case of nondigitalis cardiac glycoside toxicity.

A case is presented of cardiac glycoside poisoning in a 1-year-old patient from the plant Nerium oleander (common oleander). The patient had bradycardia, vomiting, altered level of consciousness, and no history of ingestion. Antibody-based digoxin assays may cross-react with other cardiac glycosides nonquantitatively. Chromatographic techniques can be used in the specific diagnosis.