Interleukin 3-dependent mediator release in basophils triggered by C5a.

The anaphylatoxin C5a is a potent trigger for basophil degranulations, but in contrast to IgE-dependent basophil activation, it does not result in the synthesis of sulfidoleukotrienes (leukotriene C4/D4/E4). Thus, degranulation and the generation of lipid mediators are separately regulated cellular responses. Exposure of human blood basophils to the cytokine IL-3 alone does not induce the release of histamine in cells from most donors and never leads to the generation of LTC4, indicating that IL-3 is not a direct agonist for basophil mediator release. However, preincubation of basophils with IL-3 enhances the degranulation response to C5a. Most importantly, IL-3 "primes" basophils to release large amounts of leukotriene C4 after challenge with C5a (mean of 50 gp LTC4 per nanograms cellular histamine), while neither peptide alone is capable of inducing the formation of bioactive lipids. This effect is dose dependent, occurring at IL-3 concentrations considerably lower than are required to stimulate the growth of bone marrow progenitor cells. IL-3 affects the extent but not the time course of basophil degranulation, and leukotriene release of cells sequentially exposed to IL-3 and C5a occurs very rapidly concomitant with degranulation. A preincubation of the basophils with IL-3 is strictly required for C5a-induced LTC4 synthesis, but not for an enhancement of degranulation. Priming for C5a-induced lipid mediator generation occurs rapidly after exposure of the cells to IL-3, starting at 1 min and reaching maximal effects at 5 min, but this altered state of responsiveness is relatively long lasting. Cell fractionation studies indicate that the basophil is the source of lipid mediators and that IL-3 affects the basophil response directly. This study demonstrates that IL-3 is a potent modifier of effector functions of mature basophils; this is possibly of greater in vivo significance than its growth factor properties. The large amounts of LTC4 formed after triggering of IL-3-primed basophils may not only enhance but also qualitatively change the pathophysiological consequences of complement activation, and this might be important in the pathogenesis of immediate type hypersensitivity reactions, shock syndromes, and inflammation.

Histocompatibility antigens and genetic control of the immune response in guinea pigs. III. Specific inhibition of antigen-induced lymphocyte proliferation by strain-specific anti-idiotypic antibodies.

The in vitro T-cell proliferation induced by penicilloylated bovine IgG (BPO-BGG) in sensitized strain 2 and strain 13 guinea pigs could be specifically blocked by strain-specific antisera presumably directed against cell membrane-associated immunoglobulin idiotypes. The anti-idiotypic antisera were prepared in strain 2 and strain 13 guinea pigs against immunoadsorbent purified anti-BPO-BGG antibodies which had been raised in strain 2 and strain 13 animals. Strain 13 antistrain 13 anti-BPO-BGG (a strain 13 BPO-BGG) suppressed the in vitro BPO-BGG response of cells from immunized strain 13 animals but did not inhibit the response of cells from immune strain 2 animals. Conversely, the corresponding antiserum raised in a strain 2 combination (a strain 2 BPO-BGG) only inhibited the in vitro BPO-BGG response of strain 2 cells. Furthermore, the inhibitory activity of the antisera could only be absorbed by immune cells from the syngeneic strain. The activity of the a strain 13 BPO-BGG serum was highly specific; the inhibitory activity could only be absorbed by BPO-BGG-sensitive strain 13 cells. The inhibitory activity of the anti-idiotypic sera was predominantly associated with the 19S fraction. The data suggest that immune cells and in particular T lymphocytes from strain 2 and strain 13 guinea pigs possess strain-specific recognition structures from BPO-BGG with the same idiotypes as the corresponding strain-specific immunoglobulins. Furthermore, the production of such inhibitory anti-idiotypic sera was restricted to syngeneic combinations, which suggests a potential role of autoanti-idiotypic antibodies in the regulation of the immune response. The anti-idiotypic antisera used here are apparently directed against gene products not associated with the strain 2 or strain 13 major histocompatibility complex.

The immunogenicity of dinitrophenyl amino acids.

Numerous dinitrophenyl amino acid preparations injected intradermally induced contact hypersensitivity to dinitrochlorobenzene, delayed type skin reactions to DNP-amino acids, and anti-DNP antibodies in guinea pigs. Some DNP-amino adds induced precipitating anti-DNP antibodies in rabbits as well. Some of the DNP-ammo acids studied were regularly immunogenic, possible immunogenic impurities having been excluded by extensive purification procedures. Others were either constantly nonimmunogenic or irregularly immunogenic, e.g., their immunogenicity varying from one preparation lot to another. By means of extensive chemical analyses and the establishment of dose-response curves, we were able to demonstrate in most cases that the immunogenicity was not due to contamination with unreacted dinitrofluorobenzene or other DNP derivatives, to photodecomposition or other degradation products, or to DNP-protein contaminants. Nevertheless, the irregular immunogenicity of several DNP-amino acid preparations can only be explained by a highly immunogenic impurity (or impurities) which we were unable to detect analytically. The regular immunogenicity of some other DNP-amino acids (e.g. di-DNP-L-histidine) appears to be based on a "transconjugation" phenomenon, the DNP group being able to split off from its amino acid carrier and to conjugate secondarily with proteins in vivo and in vitro. Accordingly, the interpretation of some recent data concerning the immunogenicity of low molecular weight hapten-amino acids may have to be reevaluated.

Factor-dependent in vitro growth of human normal bone marrow-derived basophil-like cells.

A factor(s) present in supernatants from lectin-stimulated peripheral blood mononuclear cells promoted the production of basophil-like cells in liquid cultures of normal human bone marrow cells. The cultured basophil-like cells had lobulated or round nuclei, and the cytoplasmic granules stained metachromatically with toluidine blue and azurophilic with Giemsa. 20% of the metachromatically staining cells were peroxidase positive but not positive for nonspecific esterase. The histamine content was 0.5-2 pg/cell. The basophil-like cells released histamine upon challenge with calcium ionophore A23187 but not with compound 48/80. They also released histamine with anti-IgE when passively sensitized with human myeloma IgE. The development of basophil-like cells was promoted in a dose-dependent fashion by a factor(s) in the conditioned medium. Blocking of cell proliferation with hydroxyurea or X irradiation inhibited the development of basophil-like cells. The production of the factor was dependent on the presence of T cells. The factor was different from interleukin 2 and its molecular weight was estimated to be 25,000-40,000 by gel filtration on a Sephacryl S-200 column. Thus, human basophil-like cells derived from normal bone marrow cells can grow and differentiate in vitro under the regulation of T cells.

Interleukin 5 modifies histamine release and leukotriene generation by human basophils in response to diverse agonists.

Human interleukin 5 (IL-5), known as a selective colony-stimulating factor of the eosinophil lineage and activator of mature eosinophils, also profoundly influences the mediator release profile of human basophils. IL-5 by itself triggers neither granule release nor de novo synthesis of lipid mediators. However, at low concentrations (0.1-10 ng/ml), IL-5 rapidly primes basophils for enhanced histamine release and leukotriene C4 (LTC4) generation in response to all established basophil agonists. LTC4 generation is more strongly affected by IL-5 than histamine release. In particular, IL-5 renders basophils capable of producing large quantities of LTC4 in response to C5a, which, without the cytokine, induces histamine release only. Finally, IL-5 renders basophils responsive to agonists (neutrophil-activating peptide 1 and C3a), which are otherwise inefficient triggers for basophil mediator release. The effects are similar to the recently established bioactivity of IL-3 on basophils, with the exception of its influence on IgE-dependent basophil activation, which is less pronounced. Thus, IL-5 strongly modulates the function not only of eosinophils but also of basophils, the two major effector leukocyte types involved in allergic inflammatory processes, e.g., in asthma.

Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals.

Neutrophils (PMN) preincubated with recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) for 2 h and then stimulated with the chemotactic factors, C5a or FMLP, produce substantial amounts of the lipoxygenase products 5-Hete, LTB4, and omega-oxidised LTB4 metabolites (4.36 +/- 0.95 (SEM) pM (n = 21) LTB4 and LTB4 metabolites/10(6) PMN). No lipoxygenase metabolites are detected by HPLC and RIA if purified PMN are stimulated by either GM-CSF or chemotactic factors in the absence of exogenous arachidonate. The priming effect of GM-CSF upon chemotactic factor induced generation of lipid mediators is a relatively slow process, clearly evident after 1 h and optimal after 2 h. Leukotriene generation is measurable with 0.8 U GM-CSF/10(6) PMN and is maximal with 80 U (10(-11)-10(-9) M). Upon activation of primed PMN with chemotactic factors, leukotriene synthesis is induced very rapidly. Already 2.5 min after activation the major lipoxygenase metabolites present are 20-OH LTB4 and 20-COOH LTB4. Our study shows that the synthesis of lipoxygenase metabolites from endogeneous AA can be initiated in PMN through receptor mediated processes by the appropriately timed combination of biological soluble inflammatory mediator peptides. Furthermore, these results indicate that GM-CSF not only enhances effector cell functions but can qualitatively change the mediator profile formed after activation with a second triggering signal. Such a mechanism might be important in amplifying inflammatory responses. Alternatively, lipid mediators formed might also have an intracellular or autocoid role and be responsible for the enhancement of other PMN functions like oxygen radical release.

The neutrophil-activating peptide NAF/NAP-1 induces histamine and leukotriene release by interleukin 3-primed basophils.

IgE-independent mediator release from basophils is considered an important event in inflammation, particularly in nonallergic immediate hypersensitivity and in allergic late-phase reactions. This study demonstrates that after exposure to IL-3, basophils release histamine and leukotrienes in response to the neutrophil-activating peptide NAF/NAP-1. Thus, the sequential action of two pure cytokines can promote basophils mediator release. In the presence of IL-3, NAF/NAP-1 functions like a "histamine-releasing factor" and may therefore not only induce cellular infiltration but also provoke symptoms of hypersensitivity reactions.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome: a multicenter study. II. Basophil activation by nonsteroidal anti-inflammatory drugs and its impact on pathogenesis.

BACKGROUND: Patients who are clinically hypersensitive to nonsteroidal anti-inflammatory drugs (NSAIDs) sometimes present basophil activation in vitro, and in 50% of cases a parallel response to release of sulfidoleukotrienes (cellular allergen stimulation test) is observed. These phenomena occur not only in clinically hypersensitive patients, but also in some healthy controls who tolerate NSAIDs. MATERIAL AND METHODS: We studied 16 clinically hypersensitive patients, 22 controls tolerating NSAIDs, and 29 healthy blood donors (clinical NSAID status unknown) using 2 different basophil isolation techniques (buffy coat or plasma leukocytes). RESULTS: In a population of 13 aspirin-tolerant healthy controls and 29 healthy blood donors, basophil activation with aspirin, diclofenac, and naproxen was analyzed at 4 different concentrations. The results in the 2 groups were quite similar in qualitative terms. Choosing a cutoff of 5% and a stimulation index >2, the proportion of positive results increased with the concentration. There were more positive results at all concentrations using the plasma leukocyte technique. CONCLUSIONS: The most important finding of this study is that basophil activation by NSAIDs occurs not only in clinically hypersensitive patients but also, to a very variable extent and on an individual basis, in apparently normal healthy individuals who tolerate NSAIDs. The phenomenon is clearly dose-related, and hypersensitive patients seem to react to lower NSAID concentrations.

Pharmacovigilance of drug allergy and hypersensitivity using the ENDA-DAHD database and the GALEN platform. The Galenda project.

Nonallergic hypersensitivity and allergic reactions are part of the many different types of adverse drug reactions (ADRs). Databases exist for the collection of ADRs. Spontaneous reporting makes up the core data-generating system of pharmacovigilance, but there is a large under-estimation of allergy/hypersensitivity drug reactions. A specific database is therefore required for drug allergy and hypersensitivity using standard operating procedures (SOPs), as the diagnosis of drug allergy/hypersensitivity is difficult and current pharmacovigilance algorithms are insufficient. Although difficult, the diagnosis of drug allergy/hypersensitivity has been standardized by the European Network for Drug Allergy (ENDA) under the aegis of the European Academy of Allergology and Clinical Immunology and SOPs have been published. Based on ENDA and Global Allergy and Asthma European Network (GA(2)LEN, EU Framework Programme 6) SOPs, a Drug Allergy and Hypersensitivity Database (DAHD((R))) has been established under FileMaker((R)) Pro 9. It is already available online in many different languages and can be accessed using a personal login. GA(2)LEN is a European network of 27 partners (16 countries) and 59 collaborating centres (26 countries), which can coordinate and implement the DAHD across Europe. The GA(2)LEN-ENDA-DAHD platform interacting with a pharmacovigilance network appears to be of great interest for the reporting of allergy/hypersensitivity ADRs in conjunction with other pharmacovigilance instruments.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome. A multicenter study. I. Clinical findings and in vitro diagnosis.

BACKGROUND: We present the results obtained from the largest series of in vitro diagnostic tests ever reported in patients with clinically validated hypersensitivity to acetylsalicylic acid (ASA)/nonsteroidal anti-inflammatory drugs (NSAID) compared with various categories of controls tolerating ASA/NSAIDs. This multicenter study, which was performed within the framework of the European Network for Drug Allergy (ENDA) group, showed that the basophil activation test (BAT), particularly when used with the 3 NSAIDs aspirin (ASA), diclofenac (DIC), and naproxen (NAP), allows us to confirm the diagnosis of NSAID hypersensitivity syndrome. The results of the cellular allergen stimulation test (CAST) frequently correlate with those of the BAT, although not always. An unexpected finding was that basophil activation by NSAIDs is not an all-or-nothing phenomenon restricted to clinically hypersensitive patients, but that it also occurs in a dose-related manner in some NSAID-tolerant control individuals.Therefore, NSAID hypersensitivity appears as a shift in the normal pharmacological response to NSAIDs. These findings allow us to formulate a new rational hypothesis about the mechanism of NSAID hypersensitivity syndrome, a mechanism that most authors continue to describe as "unknown." METHODS: We enrolled 152 patients with a history of hypersensitivity to NSAIDs and 136 control participants in 11 different centers between spring 2003 and spring 2006. Flowcytometric BAT was performed. RESULTS: The most noteworthy results of our study were that 57% of 140 patients presented very clear-cut positive BAT results to multiple NSAIDs, and 16% were entirely negative. In about 27% of cases, positive results were obtained with 1 or 2 concentrations of a single NSAID. There is clearly a correlation between the results of BAT and CAST. CONCLUSIONS: BAT seems particularly indicated in patients with a clinical history of NSAID intolerance, and in whom a provocation test is not advisable for ethical, clinical, or other reasons. Clear-cut positive results can be considered as confirming a history of NSAID hypersensitivity, although negative results may not exclude it.

Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls.

For the diagnosis of allergy, cellular basophil activation tests (BAT), e.g. histamine or sulfidoleukotriene release tests, have long been introduced, but the expression of basophil activation markers such as CD63 and CD203c detected by flow cytometry has attracted more recent attention. A recent opinion paper in this Journal has stressed not only the potential but also the possible pitfalls of flow-cytometric BAT. We have applied clinical validation of various BAT in various ways for several years, and our experience shows that these new technologies have more potentials and perspectives than pitfalls. A comprehensive review of clinically validated studies on allergy to aeroallergens, insect venoms, latex, food allergens and drugs, e.g. myorelaxants, beta-lactams, pyrazolones and non-steroidal anti-inflammatory drugs, as well as chronic urticaria shows clearly that even with different protocols, reproducible and meaningful results can be obtained. Although the available technologies may still be optimized and better standardized, there are no serious reasons to deprive allergic patients of clinically indicated BAT, which can be performed reliably by any laboratory with allergy and flow-cytometric capacity and expertise.

Antigen specific quantification of sulfidoleukotrienes in patients allergic to Betalactam antibiotics.

BACKGROUND: After in vitro allergen-specific stimulation, basophils become activated and release sulfidoleukotrienes LTC4, LTD4 and LTE4. This can be detected by means of the CAST assay. We assessed the positivity criteria and the reliability of antigen-specific sulfidoleukotriene production (CAST) in the in vitro diagnosis of betalactam (BL) allergic patients. MATERIAL AND METHODS: We studied a sample of 67 patients (age 48.94 +/- 15.76 years) who had presented with anaphylaxis or urticaria-angioedema within the first 60 minutes after administration of Amoxicillin (54/67), Penicillin G (7/67), Cefuroxime (5/67) or Cefazoline (1/67). All of them had a positive skin test to at least one of the antigenic determinants of Penicillin. As control group 30 adults with negative skin tests who tolerated BL were included. All of them underwent skin tests, oral provocation tests, specific IgE (CAP-FEIA, Pharmacia) and CAST. RESULTS: Positivity criteria were established by means of ROC curves: a sLT release induced by Betalactams of at least 100 pg/ml and greater than or equal to 3 times the basal value. The overall sensitivity of CAST is 47.7% and specificity 83.3%. Sensitivity of specific IgE is 37.8% and specificity 83.3%. CONCLUSIONS: We have established validated positivity criteria for the CAST technique in patients allergic to Betalactams. This technique is a useful in vitro diagnostic method in patients with IgE-mediated allergy to Betalactam antibiotics.

Suppression of established IgE antibody responses with isologous anti-idiotypic antibodies in guinea pigs.

Guinea pigs of strains 2 and 13 can produce isologous anti-idiotypic (aIds) antibodies against anti-benzylpenicilloyl (anti-BPO) IgG, following immunization with affinity-purified anti-BPO antibodies of the same strain. The specificity of aId was determined by inhibition of binding of aId to Fab(t) in ELISA. The results showed that the reaction of strain 2 (anti-BPO)aId can be inhibited with syngeneic anti-BPO Fab(t) and to a smaller degree with anti-BPO Fab(t) of strain 13. On the other hand, strain 13 (anti-BPO)aId reacted exclusively with syngeneic anti-BPO Fab(t). In both cases, binding of aId to anti-BPO Fab(t) could not be inhibited with BPO-epsilon-aminocaproic acid, indicating that these aId are not directed against the antigen-combining site. The effect of isologous aId on both short- and long-time established IgE responses was studied in guinea pigs of strain 13. In both situations, administration of isologous aId resulted in suppression of the anti-BPO IgE antibody response. The suppressive effect was antigen-specific and lasted for several weeks: in the case of an early-response IgE remained suppressed despite additional booster injections of antigen. In contrast to the IgE response, the production of anti-BPO IgG antibodies was only slightly affected.

Induction of immunological tolerance to the penicilloyl antigenic determinant--IV. The effect of BPO-oligolysines and cholestanol-bearing BPO-oligolysines on murine IgE responses.

Penta-, deca- and eicosalysine carriers were synthesized in solution and conjugated with benzylpenicillin to give BPO-conjugates of high haptenic density. Each oligolysine conjugate was prepared in two forms--with a free C-terminus and with an esterified C-terminus carrying via a benzylester bridge in essence a lipophilic cholestanol moiety [p-oxymethylbenzylcholestan-3 beta-yl succinate (OSuco group)]. Decalysines that carried a single haptenic BPO group and succinyl groups on the other amino functions were also prepared. Suppression of IgE responses was studied in BALB/c mice. It was found that BPO-specific suppression could be induced by injecting OSuco-bearing deca- or eicosalysine conjugates before immunization with BPO-Asc in A1(OH)3. The pentalysine conjugate was only slightly effective as were all OSuco-deficient conjugates. Ongoing IgE responses were only slightly suppressed and OSuco-bearing conjugates were not more effective than OSuco-deficient derivatives. When the monohaptenic OSuco-bearing decalysine, which exhibited weak tolerogenic effects on primary as well as on ongoing responses, was applied under conditions that favour suppressor T-cell induction, a pronounced unresponsiveness resulted. Direct evidence for suppressor T-cell involvement in the abrogation of anti-BPO responses by OSuco-bearing BPO-conjugates was obtained from cell transfer experiments. The study shows that relatively small haptenic conjugates, the lower limit of effectiveness being approximately represented by decalysine conjugates, may be effective tolerogens depending on the immune status.

A RAST assay for monitoring specific IgE antibodies in the mouse.

Mouse monoclonal anti-ovalbumin IgE was isolated from serum of BALB/c mice implanted with the IgE secreting hybridoma (14-205). Purified IgE was used to immunize rabbits and for the preparation of an antiserum specific for mouse IgE. The latter was used to develop a RAST assay to replace the PCA technique in monitoring antigen-specific IgE response in the mouse.

Determination of rat mast cells by flow-cytometry.

Counting of rat mast cells and assessment of degranulation were performed by flow-cytometry using a cytograph. It was shown that the number of mast cells in a total cell population may be obtained rapidly and reproducibly following toluidine blue staining. A dose-response for degranulation by compound 48/80 was performed and it was shown that azure A staining (in 40% sucrose) discriminates degranulated from non-degranulated mast cells, while trypan blue staining discriminates between cytotoxic and non-cytotoxic degranulation. Values obtained by flow-cytometry and microscopic evaluation were in good agreement; assessment by flow-cytometry provides a convenient new approach to counting mast cells and assessing their degree of degranulation.

Flow-cytofluorometric determination of active and total rosettes.

A flow-cytofluorometric technique is described for determining active and total rosettes formed by sheep red blood cells with human peripheral blood lymphocytes. This technique correlates well with the microscopic determination of active rosettes, when these are defined as three or more erythrocytes per lymphocyte, and with the determination of total rosettes, when these are defined as four or more erythrocytes per lymphocyte. In the small group of tumor patients tested, the observed decrease in capacity to form active rosettes was found to be dependent on the individual sheep red blood cells used, while the levels of total rosettes were not affected by individual SRBC's and were not significantly different from those of normal blood donors. Flow-cytometry is a rapid, reproducible and objective technique, which permits better measurement than microscopy of total and active rosette levels in both normal donors and cancer patients.

A dual microtiter plate (192 sample) luminometer employing computer-aided single-photon imaging applicable to cellular luminescence and luminescence immunoassay.

In spite of the increasing applications of luminescence measurements, no instrumentation is yet commercially available that permits sensitive luminescence measurements to be performed simultaneously on a large number of samples. Based on the principle of single-photon imaging, we describe here the performance of a computer-aided image luminometer with a capacity for two microtiter plates. Applications to the study of chemiluminescence by a B lymphocyte cell line, and to an IgE luminescence immunoassay are used to exemplify the capabilities of the system, which we have termed 'chemiluminescence multiwell analyser'.

Dot-immunobinding assay with monoclonal anti-IgE antibodies for the detection and quantitation of human IgE.

This paper describes a dot immunobinding assay for determining total human IgE with a tandem of monoclonal anti-IgE antibodies. Minute quantities of monoclonal anti-IgE antibodies were adsorbed on nitrocellulose discs. IgE bound to this solid phase monoclonal anti-IgE antibody was detected by a second monoclonal antibody conjugated with horseradish peroxidase. Using 4-chloro-1-naphthol as a chromogen results in a stable colour reaction that can be semiquantitatively analysed by the naked eye. The colour intensities of the reaction were also analysed by densitometry, yielding a very reproducible quantitation of human serum IgE. Using a serum dilution of 1:50, IgE could be detected in the range of 12.5-2500 U/ml. Using non-diluted serum samples IgE levels between 0.05-50 U/ml were reproducibly measured. Total serum IgE as determined by this dot assay correlated very well with IgE determinations performed by the commercial PRIST assay.

Anti-amiodarone antibodies: detection and relationship to the development of side effects.

PURPOSE: It has become evident in the past few years that amiodarone, a powerful antiarrhythmic agent, induces considerable side effects. These may be due to an amiodarone-elicited lipid storage disease and to the iodine content of amiodarone, but might also be causally related to amiodarone-induced immune reactions. The latter possibility prompted us to develop a sensitive anti-amiodarone antibody detection assay based on the immunodot technique. PATIENTS AND METHODS: Sera were obtained from 10 untreated control subjects and 33 patients receiving amiodarone. Using serum dilutions of 1:500 and 1:1,000, the lower detection limit was 0.3 microgram/ml of anti-amiodarone antibodies as calculated from a simultaneously performed IgG standard curve. RESULTS: Screening of sera from the untreated control subjects and amiodarone-treated patients revealed that the untreated subjects had no anti-amiodarone antibodies, that only one of 16 patients without clinical side effects had elevated anti-amiodarone antibodies, but that seven of 12 patients with amiodarone-induced thyroid disease and four of five patients with other side effects had elevated anti-amiodarone antibody titers (1.2 to 2.5 micrograms/ml). The combined evaluation of anti-amiodarone antibody titers and cumulative dose was found to be a highly reliable indicator of side effects, as all patients with more than 100-g cumulative dose of amiodarone and more than 0.6 microgram/ml of anti-amiodarone antibodies had side effects. CONCLUSION: The detection of anti-amiodarone antibodies in patients with amiodarone-elicited side effects underscores the possible contribution of immunologic reactions to the development of certain side effects.