β-Lactam drug allergens: Fine structural recognition patterns of cephalosporin-reactive IgE antibodies

Lack of experimental findings on the spectrum of cephalosporin allergenic determinants has hindered diagnosis of adverse reactions to these drugs and retarded understanding of allergenic cross-reactions between cephalosporins and between cephalosporins and penicillins. Subjects allergic to the widely used cephalosporin antibiotic cefaclor have serum immuno globulin (Ig) E antibodies that react with the drug. Quantitative hapten inhibition studies employing sera from subjects allergic to cefaclor revealed fine structual recognition differences between the combining site specificities of cefaclor-reactive IgE antibodies in the sera of different subjects. Unlike penicillins, where discrete side chain or thiazolidine ring determinants alone may be recognized, IgE binding determinants on cefaclor encompassed the entire molecule. Fine structural recognition specificity differences at positions R₁ (side-chain) and R₂ (substituent attached to dihydrothiazine ring) were detected between IgE antibodies in different sera. Some antibodies showed clear preferential recognition of the aminobenzyl group at position R₁ and Cl at R₂ while with others, a greater degree of recognition tolerance was seen at R₁ where, for example, the aminohydroxybenzyl or aminodihydrobenzyl groups were recognized, and at R₂ where a methyl or even an ester group was tolerated. As with the penicillins, cephalosporins as allergens cannot simply be considered as a group of compounds with a common allergenic determinant structure. IgE antibodies that bind to cefaclor show great heterogeneity indicated by clear, fine structural differences in recognition of the R₁ and R₂ groups on the drug.     

Detection and side-chain specificity of IgE antibodies to flucloxicillin in allergic subjects

Unlike studies on the antigenicity of penicillins in laboratory animals, limited information is available on the allergenicity of penicillins in man, especially with regard to fine structural allergenic differences between the many different penicillins. Inconsistent with the earlier conclusions of others, our studies suggest that side-chain structures to flucloxacillin-reactive IgE antibodies. Quantitative hapten inhibition studies revealed potent inhibition by flucloxacillin and three structurally related penicillins: oxacillin, cloxacillin and dicloxacillin. Analysis of the results showed that the side-chain group of flucloxacillin, 3-2(-chloro-6-fluorophenyl)-5-methyl-4-isoxazolyl, is recognized by some antibodies and that the 5-methyl-3-phenyl-4-isoxazolyl group, with or without halogen substituents, accounts for the reactivity of other antibodies and for the cross-reactions seen with some other penicillins. Since it is the side-chain group that distinguishes the many different types of penicillin, and since IgE antibodies in many of the allergic reactions recognize the side-chain groups, it is becoming clear that specific assays are required for the detection of IgE antibodies to each of the different penicillins.     

Penicillin acylase-catalyzed synthesis of beta-lactam antibiotics in highly condensed aqueous systems: beneficial impact of kinetic substrate supersaturation

Advantages of performing penicillin acylase-catalyzed synthesis of new penicillins and cephalosporins by enzymatic acyl transfer to the beta-lactam antibiotic nuclei in the supersaturated solutions of substrates have been demonstrated. It has been shown that the effective nucleophile reactivity of 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids in their supersaturated solutions continue to grow proportionally to the nucleophile concentration. As a result, synthesis/hydrolysis ratio in the enzymatic synthesis can be significantly (up to three times) increased due to the nucleophile supersaturation. In the antibiotic nuclei conversion to the target antibiotic the remarkable improvement (up to 14%) has been gained. Methods of obtaining relatively stable supersaturated solutions of 6-APA, 7-ADCA, and D-p-hydroxyphenylglycine amide (D-HPGA) have been developed and syntheses of ampicillin, amoxicillin, and cephalexin starting from the supersaturated homogeneous solutions of substrates were performed. Higher synthetic efficiency and increased productivity of these reactions compared to the heterogeneous "aqueous solution-precipitate" systems were observed. The suggested approach seems to be an effective solution for the aqueous synthesis of the most widely requested beta-lactam antibiotics (i.e., amoxicillin, cephalexin, cephadroxil, cephaclor, etc.).     

Measurement of absolute amounts of antigen-specific human IgE by a radioallergosorbent test (RAST) elution technique.

A technique for the absolute quantification of antigen-specific human IgE is described. It employs elution of a calculable amount of antigen-specific IgE from an allergosorbent-antibody complex by means of alkaline pH treatment, followed by measurement of the IgE content of the eluate with a modified radioimmunosorbent test (RIST). With this method IgE antibody directed against the benzylpenicilloyl determinant of penicillin (BPO) was measured quantitatively in sera from seven penicillin allergic patients. IgE specific for ragweed antigen E was measured in sera from 33 ragweed allergic patients. Values obtained for IgE anti-BPO ranged from 19 to 1806 ng/ml and comprised from 1.3 to 27.5% of total serum IgE. Values of IgE anti-antigen E ranged from 9 to 1807 ng/ml, comprising from 3 to 84% of total serum IgE. Excellent correlation (r = 0.99; p less than 0.001) was obtained for both antigen systems between values determined by the RAST elution technique and by simple RAST assay with interpolation from a reference serum of known specific IgE content as determined by the elution technique may be needed only for primary standardization of reference sera.     

Lymphocyte transformation studies in drug hypersensitivity

In a group of patients with clinically diagnosed drug hypersensitivity the in vitro lymphocyte response to the suspected drug was assessed by the lymphocyte transformation test. The test gave positive results in all 15 patients with penicillin-induced immediate or accelerated allergic reactions and positive immediate skin-test reactivity to the major or the minor antigenic determinant of penicillin, or both, but in only 3 of the 12 patients with delayed-onset maculopapular rashes induced by penicillin, despite positive immediate reactivity to the skin-test reagents.Lymphocyte stimulation greater than five times the control level was demonstrated for five patients with penicillin-induced erythroderma, Stevens-Johnson syndrome or a serum-sickness-like illness, or with methicillin-induced interstitial nephritis, all of whom had negative reactions to the appropriate skin-test reagents. A low level of stimulation was seen in eight other skin-test-negative patients with possible allergic reactions induced by penicillins. However, in all subjects tested the stimulation was significantly greater than the mean for control subjects.For 9 of 11 patients with isoniazid-induced hepatitis or maculopapular rashes, but for only 8 of 31 patients with eruptions induced by a variety of drugs other than penicillins and isoniazid, significant stimulation occurred in the lymphocyte transformation test.It is concluded that the lymphocyte transformation test is useful in the detection of hypersensitivity to the penicillins (although in IgE-mediated reactions skin testing is clearly preferable) and isoniazid but is of limited value in the demonstration of hypersensitivity to other drugs.     

Measurement of IgG blocking antibodies by interference in the radioallergosorbent test

We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.     

Theoretical studies on penicillins,penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues: binding specificities of transeptidases and penicillinases

Empirical potential energy calculations have been carried out to determine the preferred conformations of penicillins and penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues. With the exception of 1-oxa-1-dethia penicillins, all the other compounds favour C₂ and the C₃ puckered conformations of their five-membered rings. Replacement of C2 methyl groups by hydrogen atoms as in bisnorpenicillin V or oxidation of sulphur in position 1 as in sulphones, makes the C₃ puckered form much less favourable. Addition of an amino-acyl group at the C6 atom, however, makes the C₃ puckered form more favoured in penicillin G or V and in 1-carba-1-dethia penicillins. Through the replacement of the sulphur atom at position 1 by an oxygen atom or by a -CH₂ group increases the non-planarity of the lactam peptide bond, it significantly affects the relative disposition of the C3 carboxyl group with respect to the β-lactam ring. These conformational differences have been correlated with the biological activities of these compounds. The present study suggests that the conformation of the bicyclic ring system may be more important for initial binding with the crosslinking enzyme(s) involved in the biosynthesis of bacterial cell-wall peptidoglycan and that the mode of binding is influenced by the nature of the side-group at the C6 atom. These studies predict, in agreement with experimental results, that the 1-oxa-1-dethia penicillin nulceus is an inhibitor of penicillianses. The study also suggests that the stereospecificities of the crosslinking enzyme(s) and penicillinases are very similar with regard to the nature of the side-group at the 6 atom and the confirmation of the bicyclic ring system. However, the confirmational requirement for the bicyclic ring system appears to be more specific in the former enzyme than in the latter.     

Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

New Type of Allergic Asthma due to IgG “Reaginic” Antibody

This study was undertaken to examine the possibility that IgE is not the only immunoglobulin responsible for immediate allergic reactions. A group of asthmatics were investigated in whom immediate allergic reactivity of the bronchi to common inhalant allergens had been confirmed by provocation tests. Their sera were fractionated and the reaginic activity of the immunoglobulin classes was studied by passive cutaneous anaphylaxis testing in monkeys. The results showed that the immediate allergic reactions were due to IgE antibodies in most patients, but there was a group with reactions due to short-term anaphylactic IgG antibodies. It was not possible to inhibit the IgG-mediated responses with disodium cromoglycate. As these two groups had clearly different serum IgE levels the estimation of IgE provided an important guide to the management of these patients.     

Synthesis,characterization and immunochemical evaluation of cephalosporin antigenic determinants

Lack of knowledge of the exact chemical structure of cephalosporin antigenic determinants has hindered clinical interpretation of adverse reactions to these drugs and delayed understanding of the mechanisms involved in the specific recognition and binding of IgE molecules to these antigenic determinants. We further resolve the relationship between structure and activity of proposed antigenic chemicals, including the rational design and synthesis of these haptenic structures. Comparative RAST inhibition studies of the synthesized molecules revealed that they were recognized by IgE antibodies induced by cephalosporin antibiotics. Thus, these data indicate that recognition is mainly directed to the acyl side chain and to the beta-lactam fragment that remains linked to the carrier protein in the cephalosporin conjugation course.     

Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay

An immunogold assay (IGA) was developed to detect IgG and IgE antibodies to Aspergillus fumigatus. Sixteen sera from patients with allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and normal controls were studied. All sera were also evaluated for antibodies against A. fumigatus by biotin-avidin linked enzyme immunosorbent assay (BALISA) and by agar gel double diffusion method. A. fumigatus specific IgG and IgE antibodies could be detected by IGA in all the patients' sera but not in the sera of normal controls. Both IgG and IgE antibodies to A. fumigatus could be demonstrated in all the sera by BALISA and normal controls showed only low levels of these antibodies. There was a positive correlation between the degree of reactivity detected by IGA, the BALISA titer and the precipitins by agar gel diffusion. It can be concluded that IGA is a reliable, sensitive and simple method capable of detecting both IgG and IgE antibodies against A. fumigatus in patient serum.     

Crystal structure of the allergen Equ c 1. A dimeric lipocalin with restricted IgE-reactive epitopes

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 A resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a beta-barrel flanked by a C-terminal alpha-helix. The space between the two beta-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.     

Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) is used for the commercial production of semi-synthetic penicillins. It hydrolyses the amide bond in penicillin producing 6-aminopenicillanic acid and phenylacetate. 6-Aminopenicillanic acid, having the beta-lactam nucleus, is the parent compound for all semi-synthetic penicillins. Penicillin G acylase from Kluyvera citrophila was purified and chemically modified to identify the role of arginine in catalysis. Modification with 20 mM phenylglyoxal and 50 mM 2,3-butanedione resulted in 82% and 78% inactivation, respectively. Inactivation was prevented by protection with benzylpenicillin or phenylacetate at 50 mM. The reaction followed psuedo-first order kinetics and the inactivation kinetics (V(max), K(m), and k(cat)) of native and modified enzyme indicates the essentiality of arginyl residue in catalysis.     

Study on major mite allergens in Italian homes

Summary There has always been a great interest for allergologists in the relation between the exsposure to allergic pollens a/o perennials and allergic symptomatology. Indeed the allergologist is constantly looking for clear evidence to what is shown or suggested by clinical history and routine allergologic tests.At present the availability of reliable quantitative analytical methods has given us the possibility of making a study on the presence of house mite and cat allergens in Italian homes.The study involved 5 Italian towns and was carried out in autumn months and the collection of samples has been made with a simple retail electric vacuum cleaner. The dosage of the allergenic content of the dust has been carried out by an immunoassay using specific monoclonal antibodies and a mixture of allergic patients' sera and a monoclonal anti IgE antibody coupled with an enzyme. The results suggest a wide variability of the allergenic content of the samples coming from the same geographical area but a constant ratio between the major mite allergens (Der I/Der II) ranging around a value 21.The prevalence of one mite species over another is variable, and depends on the geographical locality.     

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.     

The role of anti-CCD antibodies in grape allergy diagnosis

Background:

Allergens are mostly composed of glycoprotein structures. It is believed that glycan-specific antibodies may lead to false-positive reactions in immunoassays. In this study we investigated the glycosylation state of grape allergens as well as the presence of antibodies to cross-reactive carbohydrate determinants (anti-CCDs) in sera from grape-sensitive individuals.

Methods:

Grape extract proteins were electrotransferred onto PVDF membranes and their glycosylation states were analyzed by blotting methods. To assess the presence of anti-CCDs, natural and mildly deglycosylated proteins were immunoblotted with grape-allergic subjects'' sera. We also measured the IgE reactivity of each subject’s sera with other fruit extracts via an indirect ELISA.

Results:

Immunoblotting studies showed that mildly deglycosylated grape proteins had lower IgE-binding capacity than their intact natural counterparts, which could be due to the presence of anti-CCDs. Biotinylation studies confirmed that the glycosylation levels of the 24, 32, and 60 kDa IgE-reactive proteins were higher than those of the 38 and 45 kDa ones. Lectin blotting showed that the 24 and 60 kDa bands were highly mannosylated, with the highest level of mannosylation on the 24 kDa allergen.

Conclusion:

This study showed that some grape allergens are glycosylated and that anti-CCD antibodies may cause weakly false-positive results during assessment of IgE reactivity to grape allergens.Key Words: Allergy, Grape, Cross-reactive carbohydrate determinants, Antibody     

The major human structural IgE epitope of the Brazil nut allergen Ber e 1: a chimaeric and protein microarray approach

A protein microarray system containing different dilutions of 77 related and non-related proteins was used to show that IgE from subjects allergic to Brazil nut specifically recognise the seed 2S albumin protein (Ber e 1). Further, correctly folded chimaeric 2S albumin proteins containing structural epitope replacement were constructed and directed to the secretion pathway of the methylotropic yeast Pichia pastoris. Through the use of a chimaeric protein microarray system together with sera from a panel of 18 well-characterised Brazil nut allergic subjects, a structural IgE epitope of Ber e 1 was mapped to a helix-loop-helix region. The same structural region has been previously reported as the immunodominant region in related food allergens by different techniques. In conclusion, the combination of chimaeric proteins and protein microarrays will greatly facilitate the screening of a large number of individuals for a particular structural epitope and help to further our understanding of how proteins are recognised by the adaptive immune system.     

Improved Synthesis of 3-14C-3-Hydroxy-3-methylglutaryl Coenzyme A

A new polysaccharide with a molecular weight of 5.0×10⁴ was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with β-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.     

The expression of a mountain cedar allergen comparing plant-viral apoplastic and yeast expression systems

Jun a 3, a major allergenic protein in mountain cedar pollen, causes seasonal allergic rhinitis in hypersensitive individuals. Recombinant Jun a 3 was expressed in Nicotiana benthamiana interstitial fluid (300 microg/g leaf material) and Pichia pastoris (100 microg/ml media). Polyclonal anti-Jun a 3 and IgE antibodies from the sera of allergic patients both reacted with the recombinant protein. Of the two systems, recombinant protein from the plant apoplast contained fewer contaminating proteins. This method allows for a more convenient and inexpensive expression of the recombinant allergen, which will allow for further structural studies and may prove useful in diagnostic and/or immunotherapeutic strategies for cedar allergy.