Allergic reactions to ampicillin. Studies on the specificity and selectivity in subjects with immediate reactions

Background and Objective Ainpicillin (AMP) is a drug that has been prescribed extensively. Reactions that have been reported include exanthema. desquamative contact eczema, urticaria and anaphylaxis. Experimental evidence indicates that the side chain of AMP is a structure that may induce a selective immune response either at the humoral or lymphocyte T-cell level. With regard to IgE reactions, the selectivity and specificity of the response needs to be studied in humans. Objective To study tbe specificity of tbe IgE response in a group of subjects who had an immediate allergic reaction after the administration of AMP. Methods Subjects developing an immediate response (anapbylaxis or urticaria) after the administration of AMP or an aminopenicillin derivative witb the same side chain as AMP were studied. Skin tests were made to determinants generated from benzyl penicillin (BP): benzyl penicilloyl (BPO) and minor determinant mixture (MDM), as well as amoxicillin (AX) and AMP. Specific IgE antibodies were determined to benzyl penicilloyl polylisine (BPO-PLL), amoxicilloyl-polyllsine (AX-PLL) and ampicilloyl-polylisine (AMPPLL). The specificity of the IgE antibody response was studied by RAST and RAST inbibition. Subjects were classified in three categories: group A: those who were skin test and/or RAST positive to determinants derived from benzylpenicllin, group B: those who were negative to determinants derived from benzylpenicillin but were skin lest and/or RAST positive to determinants derived from AX and AMP and group C: those who were exclusively positive to determinants derived from AMP. Results A total of 48 subjects was included in the study. In group A there were 35 cases, in group B 10 cases, and in group C tbree cases. RAST inhibition studies showed that in some instances tbe side chain of AMP could induce specific responses with a variable degree of crossreactivity between BP and AX. Conclusions Atbough AMP can induce an immediate IgE response in subjects allergic to betalactams and tbe structure of the side chain may contribute to the specificity of the response, our results indicate tbat in most instances crossreactivity with the other penicillins exists and that in the groups studied selective reactions to just AMP derived determinants were uncommon.     

Underestimation of heritability using a mixed model with a polygenic covariance structure in a genome-wide association study for complex traits

Recently, the use of a mixed model methodology in genome-wide association studies (GWAS) has been considered effective for controlling population stratification and explaining the polygenic effects of complex traits. However, estimating polygenic variance components and heritability was biased when the mixed model was used. This bias results from a diluted genetic relationship covariance structure, particularly with a limited number of underlying causal variants. We simulated disease and quantitative phenotypes with a variety of heritabilities (0.1, 0.2, 0.3, 0.4, and 0.5), prevalence rates (0.1, 0.2, 0.3, and 0.5), and causal variant numbers (10, 30, 50, and 100). Heritabilities from the simulated data using restricted maximum likelihood were underestimated in many populations (P<0.05). The underestimation increased with a large heritability, a small prevalence, and a small number of causal variants. The underestimation was larger in analyzing disease traits compared with quantitative traits. This study suggests an underestimated heritability in GWAS upon using the mixed model methodology with an excessively larger number of variants versus causal variants.     

Estimation of epistasis among finite polygenic loci for complex traits with a mixed model using Gibbs sampling

Epistasis among loci is important factor behind the expression of many complex traits, but many analyses have ruled out its possibility. A method to estimate epistasis was introduced with a mixed model using Gibbs sampling (MMGS). The posterior mean estimate for every possible genotype combined from multiple loci was calculated as the mean of the conditional expected values of the parameters in post warming-up rounds from Gibbs sampling. A simulation study was performed to compare MMGS with restricted partition method (RPM). Mean square prediction error (MSPE) using MMGS was smaller than that using RPM (P<0.05), which might be due to information loss introduced by grouping of genotypes in RPM. This was also supported by the result that MSPE increased as the number of merged groups decreased. The simulation study implied that MMGS was more plausible in estimating epistatic effects than the RPM.     

Detection of antidrug IgG antibodies in patients with adverse drug reactions to amodiaquine

Antidrug IgG antibodies have been detected in some patients receiving amodiaquine (AQ). Antidrug antibodies were detected in 6/7 patients who experienced serious well-defined adverse drug reactions during malaria prophylaxis and in 7/22 patients who received comparable doses of the drug (at least 400 mg weekly x 6) but did not present with clinical adverse drug reactions. In contrast antidrug antibodies were not detected in 7 patients who received the drug for treatment (1.0-1.2 g total over 3 days). The specificity of the IgG response was defined by hapten inhibition experiments (IC50 value for AQ ranged between 0.050 and 0.282 microM) which suggest that the antibody recognised the drug linked to cysteine residues in protein via the 4-hydroxyanilino side chain. The data show that AQ is immunogenic in man and are consistent with the hypothesis that idiosyncratic adverse reactions to the drug have an immunological aetiology.     

Human autologous mixed lymphocyte reaction as an in vitro model for autoreactivity to apoptotic antigens

Recent studies have indicated that cells undergoing apoptosis are the source of autoantigens which drive autoimmune responses in systemic lupus erythematosus (SLE). It has been recognized for many years that in vitro stimulation of T cells with irradiated major histocompatibility complex (MHC) class II-bearing autologous cells results in T-cell proliferation with immunological specificity and memory, namely the autologous mixed lymphocyte reaction (AMLR). The nature of the major stimulants in the AMLR is still unclear. We investigated whether apoptotic fragments from irradiated cells act as antigenic stimulators for AMLR or nucleohistone-primed T cells. T-cell proliferation in the primary AMLR was significantly suppressed by the presence of a caspase inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD.fmk), indicating that apoptotic antigens released from irradiated autologous feeder cells act as stimulators of AMLR T cells. This inhibitory effect of Z-VAD was not caused by toxic effects, because the T-cell response to the mitogen phytohaemagglutinin (PHA) was not inhibited by Z-VAD. A nucleohistone preparation was shown to contain antigens that are important in the AMLR, as culture with nucleohistone (but not with thyroglobulin or hen-egg lysozyme) primed T cells to respond with secondary kinetics in a subsequent AMLR that was also suppressed by Z-VAD. Our data provide evidence that the AMLR constitutes a model for the evaluation of cellular and molecular mechanisms that may be relevant to the pathogenesis of SLE and similar autoimmune diseases.     

Monoclonal antibodies generated against an affinity-labeled immune complex of an anti-bile acid metabolite antibody: an approach to noncompetitive hapten immunoassays based on anti-idiotype or anti-metatype antibodies

Conventional immunoassays for haptens such as steroids and synthetic drugs are dependent on the competitive reaction between an unlabeled antigen (analyte) and a labeled antigen against a limited amount of anti-hapten antibody. Although noncompetitive immunoassay procedures such as two-site immunometric assays offer a much higher sensitivity, direct application of this principle to haptens has been difficult due to their small molecular mass precluding simultaneous binding by two antibody molecules. Here, we have attempted to develop a noncompetitive immunoassay system based on anti-idiotype or anti-metatype antibodies. Ursodeoxycholic acid 7-N-acetylglucosaminide (UDCA 7-NAG), which is a bile acid metabolite (molecular weight, 595.8), was selected as the model hapten. A/J mice were immunized with a monoclonal antibody against UDCA 7-NAG, which had been affinity-labeled with a relevant hapten derivative. The fusion between the immune spleen cells and P3/NS1/1-Ag4-1 myeloma cells yielded four kinds of alpha-type and two kinds of beta-type monoclonal anti-idiotype antibodies, each recognizing the framework region and paratope of the anti-hapten antibody. The use of a selected combination between alpha-type and beta-type antibodies together with the anti-hapten antibody provided a noncompetitive assay system with a subfemtomole order sensitivity (detection limit, 118 amol) and a practical specificity.     

Allergic reactions to inhalant glucocorticosteroids: a hot topic for pneumologists and allergologists

Allergic contact dermatitis to topical glucocorticosteroids (GCS) is a delayed type cell-mediated hypersensitivity reaction; it is frequently observed in dermatological and allergological practice, although its incidence is likely underestimated. By contrast, allergic contact sensitization to inhalant GCS is virtually unknown to most pneumologists. Here, we review some cases of adverse reactions to inhalant GCS in terms of pathogenetic mechanisms, risk factors, epidemiology, and allergic cross-sensitivity. In fact, this particular form of sensitization to drugs that have a wide spectrum of use in pneumological practice deserves more attention than in the past.     

Tethered spheroids as an in vitro hepatocyte model for drug safety screening

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.     

Failure to demonstrate an advantage in combining sulphamethoxazole with trimethoprim in an experimental model of urinary infection

Co-trimoxazole was found to have a predominantly bacteriostatic effect upon 28 urinary isolates of Enterobacteriaceae in nutrient broth and was never bactericidal in artificially infected urine. The components of co-trimoxazole were tested individually and trimethoprim was found to be at least as effective as co-trimoxazole in nutrient broth and in urine. Trimethoprim alone produced some bactericidal effect in urine but this was antagonized by sulphamethoxazole.Laboratory tests for evaluating these drugs may give a misleading impression of their activity in vivo. Further clinical comparisons should therefore be made between trimethoprim and cotrimoxazole to determine when trimethoprim should be used in preference to the combination.     

ELISAs for free light chains of human immunoglobulins using monoclonal antibodies: comparison of their specificity with available polyclonal antibodies

For the measurement of human immunoglobulin free light chains (LCs) in clinical samples, highly specific assays for free LCs are required to discriminate them from LC portions of intact immunoglobulins (bound LCs). To develop specific enzyme-linked immunosorbent assays (ELISAs) for free LCs, two anti-free LC kappa and lambda monoclonal antibodies (MAbs) were raised by a mouse/mouse hybridoma technique. We compared the specificities of these two MAbs with those of six commercially available anti-free LC antisera, which are widely used in free LC immunoassays. Comparative titrations against free LCs and intact IgGs showed the MAbs had less cross-reactivity to intact IgGs, while the four of six antisera had high reactivity to intact IgGs. Using these MAbs, we developed LC kappa and LC lambda ELISAs with ranges from 7.8 to 500 micro g/l of free LCs and less cross-reactivity to intact IgGs (less than 0.12%). On the other hand, ELISAs with anti-free LC antisera showed low specificity and/or sensitivity. Thus, the use of these MAbs may provide reliable methods for specific measurements of free LCs in clinical samples.     

Fibrin-antibiotic mixtures: an in vitro study assessing the possibility of using a biologic carrier for local drug delivery

An in vitro study assessing the kinetics of drug release from antibiotic-fibrin seal compounds and the antibacterial efficacy of the delivered drug has been performed. Antibiotic sensitivity and the amount of drug released was measured by means of agar diffusion test. Standard and experimental curves were established for each antibiotic and each bacterial test in order to evaluate the quantities of the drug released during each 24 h period. The reconstitution of lyophilized human fibrin with an aqueous solution containing an antibiotic resulted in only minor modification of the clotting process, with the exception of those solutions containing cefotaxim and mezlocillin which failed to clot altogether and were excluded from further study. The evaluation of the kinetics of elution of the antibiotics from the fibrin clots showed that all of the antibiotics had been almost completely released by 96 h. The delivered amount of each drug was enough to maintain the Minimal Inhibitory Concentration (MIC) until the 4th day of culture for the most of antibiotics, resulting in a prolonged release of the drug.     

Use of a predictive model derived from in vivo endophenotype measurements to demonstrate associations with a complex locus, CYP2A6

This study demonstrates a novel approach to test associations between highly heterogeneous genetic loci and complex phenotypes. Previous investigations of the relationship between Cytochrome P450 2A6 (CYP2A6) genotype and smoking phenotypes made comparisons by dividing subjects into broad categories based on assumptions that simplify the range of function of different CYP2A6 alleles, their numerous possible diplotype combinations and non-additive allele effects. A predictive model that translates CYP2A6 diplotype into a single continuous variable was previously derived from an in vivo metabolism experiment in 189 European Americans. Here, we apply this model to assess associations between genotype, inferred nicotine metabolism and smoking behaviors in larger samples without direct nicotine metabolism measurements. CYP2A6 genotype is not associated with nicotine dependence, as defined by the Fagerstr?m Test of Nicotine Dependence, demonstrating that cigarettes smoked per day (CPD) and nicotine dependence have distinct genetic correlates. The predicted metric is significantly associated with CPD among African Americans and European American dependent smokers. Individual slow metabolizing genotypes are associated with lower CPD, but the predicted metric is the best predictor of CPD. Furthermore, optimizing the predictive model by including additional CYP2A6 alleles improves the fit of the model in an independent data set and provides a novel method of predicting the functional impact of alleles without direct metabolism measurements. Lastly, comprehensive genotyping and in vivo metabolism data are used to demonstrate that genome-wide significant associations between CPD and single nucleotide polymorphisms are the result of synthetic associations.     

Role of dietary corn oil in the function of hepatic drug and carcinogen metabolizing enzymes of starved-refed rats: response to the mixed function oxidase inducer, 3-methylcholanthrene

Hepatic microsomes from rats starved 48 hours and refed diets containing zero, 3 or 20% corn oil metabolized benzo(a)pyrene, aniline and N-nitrosodimethylamine in proportion to the quantity of corn oil in the diet. No diet-related changes in apparent Km for these reactions were evident. The content of microsomal cytochrome P-450 was also clearly dependent upon the content of corn oil in the refed diets. When metabolism of these three substrates is expressed as product formed per unit of cytochrome P-450, the activities are least in microsomes from rats fed the 20% corn oil diet, suggesting that P-450 species responsible for metabolizing substrates other than these are enhanced preferentially. Cytosolic glutathione S-transferase activities are also increased with increasing corn oil in the diet. The administration of 3-MC increased cytochrome P-448 content of microsomes from all rats, regardless of diet, however highest content was present in microsomes from rats fed the 20% corn oil diet. Induction of benzo(a)pyrene hydroxylase was not influenced by dietary corn oil and, as anticipated, 3-MC caused significant repression of DMN N-demethylase in microsomes from rats fed the 20% corn oil diet. In like manner, 3-MC induced glutathione S-transferase only in cytosol from rats fed the fat-free diet.     

In vitro tests for drug hypersensitivity reactions: an ENDA/EAACI Drug Allergy Interest Group position paper

Drug hypersensitivity reactions (DHRs) are a matter of great concern, both for outpatient and in hospital care. The evaluation of these patients is complex, because in vivo tests have a suboptimal sensitivity and can be time‐consuming, expensive and potentially risky, especially drug provocation tests. There are several currently available in vitro methods that can be classified into two main groups: those that help to characterize the active phase of the reaction and those that help to identify the culprit drug. The utility of these in vitro methods depends on the mechanisms involved, meaning that they cannot be used for the evaluation of all types of DHRs. Moreover, their effectiveness has not been defined by a consensus agreement between experts in the field. Thus, the European Network on Drug Allergy and Drug Allergy Interest Group of the European Academy of Allergy and Clinical Immunology has organized a task force to provide data and recommendations regarding the available in vitro methods for DHR diagnosis. We have found that although there are many in vitro tests, few of them can be given a recommendation of grade B or above mainly because there is a lack of well‐controlled studies, most information comes from small studies with few subjects and results are not always confirmed in later studies. Therefore, it is necessary to validate the currently available in vitro tests in a large series of well‐characterized patients with DHR and to develop new tests for diagnosis.     

Compliance and adverse drug reactions: a prospective study with ethinylestradiol using continuous compliance monitoring

Summary This study examined the relationship between adverse reactions and patient compliance with ethinylestradiol at 40 g twice daily versus 20 g four times daily. In a randomized study 61 female patients with primary- infertility were prescribed the drug twice daily (n = 31) or four times daily (n = 30). Ethinylestradiol was administered for 7 days before the sperm cervical mucus penetration-test was performed for hormonal standardization of the cervical mucus quality. Drug compliance was measured by continuous monitoring using the Medication Event Monitoring System. Two parameters were evaluated: percentage of prescribed doses taken (administration compliance) and adherence to the prescribed dose schedule (regimen compliance, number of days with two or four dosing events recorded). Adverse drug reactions were assessed using a standardized questionnaire. Fourty-four women experienced side effects, of which 81% were rated by patients as being mild. Patient compliance was higher with the twice daily than with the four times daily regimen: 85% versus 65% prescribed doses taken (P<0.05). There was no significant difference in compliance comparing patients with and without adverse reactions (82% versus 72%, respectively), but compliance was lower and more irregular with at least 3 versus one or two adverse reactions reported: 54% versus 84% in administration compliance and 31% versus 58% in regimen compliance (P<0.05). Compliance was also lower in patients with nausea and vomiting than in those without these symptoms, 59% versus 91% and 34% versus 66% (P<0.005), respectively, and lower with moderate or severe compared to mild side effects; 48% versus 85% and 25% versus 59% (P<0.005). Thus the mere occurrence of side effects was not associated with low compliance. However, the number and nature of symptoms and their intensity as perceived by patients may have considerably influenced drug use behavior.Abbreviations ADR adverse drug reactions - SCMPT sperm cervical mucus penetration-test Prof. Dr. E. Weber died 7 December 1992     

Allergic reactions to vespids: comparison of sensitivities of two species in a Mediterranean area

We have studied a group of twenty-seven patients who suffer allergic reactions to vespids stings. Specific IgE antibodies to venom extracts from Polistes gallicus and Vespula germanica were measured by RAST, and the crossreactivity between the two venoms was compared using the RAST inhibition technique. We concluded that, in southern Spain, sensitization to P. gallicus was more prevalent than that to V. germanica , with 44% of the subjects in this study reacting to P. gallicus compared with 33% to V. germanica. However, there was a considerable degree of crossreactivity between the two species. It is evident that Polistes is an important species in this area; however, both in Spain and other Mediterranean countries, V. germanica venom is used almost exclusively for diagnosis and immunotherapy.     

Ceftazidime-induced hemolysis in a patient with drug-dependent antibodies reactive by immune complex and drug adsorption mechanisms

A 35-year-old woman developed acute intravascular hemolysis within five days of beginning a course of ceftazidime. The direct antiglobulin test became strongly positive with both anti-IgG and anticomplement. The serum contained an antibody that, in the presence of ceftazidime, sensitized unmodified reagent cells with IgG and complement (immune-complex type). The serum also agglutinated ceftazidime-pretreated cells at room temperature and 37 degrees C (drug-adsorption type). Retrospective testing disclosed that the drug adsorption antibody, which had been present before the current course of antibiotics, was not demonstrable during the hemolysis. The reactivity of the immune complex antibody, which developed by the second day of ceftazidime, paralleled the degree of hemolysis and the strength of the direct antiglobulin test. The authors believe that this patient had two separate ceftazidime-dependent antibodies and that the antibody reactive by immune complex mechanism mediated an episode of acute intravascular hemolysis.