Immunogenicity studies with haptenated melittin peptides: implication for membrane involvement during the recognition step.

Melittin peptides carrying 2,4-dinitro-6-carboxyphenyl (Dncp) haptenic groups regularly evoked anti-hapten IgG responses in mice or guinea pigs when the hapten was C-terminally attached. Single haptens on the N-terminal helix in several positions gave poor or no responses in the early stages but adequate titres after prolonged immunization. Peptides with Dncp at the C-terminus as an invariant feature and a second Dncp in various positions along the peptide chain did not fail to produce adequate responses. The hampering effect is not due to a defect at the T-cell level but involves the recognition step on the B-cell. It is implied that the haptenic interaction with the paratope of the recognizing immunoglobulin on the B-cell involves the cell membrane in an important way. It is also suggested that late antibody responses should not be overlooked during the development of proteinaceous immunogens for vaccination.

Heterogeneous T cell responses to beta-lactam-modified self-structures are observed in penicillin-allergic individuals.

To investigate the role of T cells in drug allergy, we stimulated PBMC from penicillin-allergic patients with reactive penicillin G itself or penicillin G coupled with human serum albumin (BPO-HSA). T cell clones specific for penicillin G or BPO-HSA were established and their phenotype and reactivity to both forms of the beta-lactam were analyzed. T cell clones stimulated by penicillin G were CD4 and CD8 positive, whereas BPO-HSA stimulated the growth of CD4+ T cells. The penicillin G-specific clones were HLA class I or class II restricted and processing was not required as fixed APC could still present penicillin G. In contrast, BPO-HSA has to undergo processing to stimulate BPO-HSA-specific T cell clones. In addition to classical APC, activated MHC class II expressing T cells could also restimulate the penicillin G-specific clones, indicating that various cell types might serve as APC. Penicillin G and BPO-HSA-specific T cell clones produced a heterogeneous cytokine pattern as most clones produced high amounts of IL-2, IFN-gamma, TFN-alpha, and rather variable levels of IL-4 and IL-5. Since no Ag processing was required, penicillin G may stimulate T cells by binding directly to MHC molecules on the cell surface or to their embedded peptide. Alternatively, it may bind to soluble proteins like HSA, which are processed and subsequently presented in an immunogenic form. These different modes of presentation, which elicit a variety of immunological reactivities, may explain the great heterogeneity of the clinical pictures seen in penicillin allergy.

Immunogenicity of dinitrocarboxyphenylated melittin: the influence of C-terminal chain shortening, N-terminal substitution and prolin insertion at positions 5 and 10.

Peptides derived from the bee-venom melittin were fitted with the haptenic group dinitrocarboxyphenyl (Dncp) and tested in out-bred guinea pigs for immunogenicity by measuring the IgG anti-Dncp antibody response by ELISA. Dncp-conjugates comprising virtually the entire melittin proved to be strong immunogens producing antibody responses comparable to those of proteins. Weak responses were obtained with considerably shortened sequences. Conjugates with N-terminal Dncp gave markedly reduced antibody responses compared to peptides with C-terminal Dncp. An N-terminal biotinyl substituent abolished the immune response whereas N-terminal lauryl and caprylyl had little effect. Insertion of L-proline into a hexadecapeptide conjugate abolishing the possibility of helix formation gave an immunogen to which individual animals clearly responded on a low level. Oligomerisation, but not the cytolytic activity of melittin peptides, may contribute to the immunogenicities observed.

Anaphylactic properties of monohaptenic dinitrophenylated tripalmitoyl-S-glyceryl-cysteinyl lipopeptides.

Tripalmitoyl-S-glycerylcysteinyl lipopeptides are B-cell and macrophage activating and may be used as low molecular weight immunogens of considerable potency and even as vaccines when conjugated with suitable epitopic structures. Selected lipopeptides carrying single Dnp haptens were found to evoke mild passive cutaneous anaphylaxis in guinea pigs sensitized against Dnp. The reactions were observed after intravenous injection whereas intradermally applied antigen was negative. The anaphylactogenicity seems unrelated to micelle or aggregate formation of the insoluble peptides which require lecithin additions as well as sonication to become solubilized. The dinitrophenylated lipopeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-lysine produced toxic reactions which were not observed with the lipopeptide devoid of Dnp. Dinitrophenylated tripalmitoyl-S-glycerylcysteiny-1,6-diaminohexane and tripalmitoyl-S-glyceryl-cysteinyl-lysine did not show these toxic reactions.

On the immunochemical specificity of pyrazolinone and pyrazolidinedione reactions.

Haptenic groups based on the 1-phenyl-2,3-dimethyl-3-pyrazolin-5-one and 1,2-diphenyl-pyrazolidine-3,5-dione structures conjugated to human serum albumin gave antibody responses in rabbits which were compared with those raised against the same haptens conjugated via spacer bridges to the protein carrier. The spacing up to 12.4 A had no marked influence on the antibody specificity evaluated by haptenic inhibition of ELISA. The specificities are more pronounced than those found with e.g. anti-penicilloyl antibodies which seems in line with clinical experience involving e.g. phenylbutazone, sulfinpyrazone, propyphenazone and metamizole. It is argued that such strict specificities may lead to false negative tests in diagnostic procedures in vitro as well as in skin testing.

Potency of hexavalent and decavalent anaphylactogens: affinity enhancement by epitope clusters.

Two deca-L-lysine conjugates, one carrying ten, the other carrying six D-benzylpenicilloyl haptenic groups as epitope models, were used as elicitors of passive cutaneous anaphylaxis in guinea pigs. It was found that equal haptenic doses yielded virtually indistinguishable responses, i.e., the potencies were close to 10:6. Interpretation of these data appears possible by using established immunochemical concepts of preequilibria and equilibria in free solution as starting point. The discussion is simplified due to the fact that the two conjugates being decavalent and hexavalent, respectively, in free solution, are both hexavalent on cell membranes. It was found that the clusters of haptenic groups displayed by the conjugates enhance the affinity for antibody binding and that the haptenic densities are directly related to potencies. Epitope clusters of enhanced affinity interacting with cell-bound antibody may be significant and useful in a number of ways.

A single-photon imaging system for the simultaneous quantitation of luminescent emissions from multiple samples.

A combination of a two-dimensional photon detector (double-microchannel plate) with single-photon sensitivity and an optical projection system that allows space-resolved quantitation of luminescent emissions from spatially extended objects is described. A "luminescent image" of the object focused onto the detector is accumulated over a preset time and stored in a digital frame memory from which photon counts over areas of interest can be read. In this study, the object consisted of a microtiter plate containing luminescent samples which was placed below a projecting lens (2.0/21 mm, 36 X 24-mm format camera lens) at a distance of 38.5 cm. Although geometry substantially limited photon collection, the sensitivity achieved was only 10X less than that obtained with a dedicated photon-counting luminometer. A slightly diminished photon collection from peripheral wells was apparently caused by the projection system and could be corrected arithmetically. Both chemically generated luminescence (ATP bioluminescence) and cell-derived, superoxide-dependent luminescence (with lucigenin as chemilumigenic probe) were detected with excellent spatial resolution and linearity of response over a wide range.

Diagnosis of antibody-mediated drug allergy. Pyrazolinone and pyrazolidinedione cross-reactivity relationships.

Based on the 1-phenyl-2,3-dimethyl-3-pyrazolin-5-one series and on the 1,2-diphenyl-pyrazolidine-3,5-dione series of drugs, haptenic reagents and conjugates were synthesized and evaluated by passive cutaneous anaphylaxis in guinea pigs, and by ELISA tests using rabbit antisera against the haptens. No cross-reactivity between pyrazolinone and pyrazolidinedione haptenic reagents was found in any of the test systems. But also within each series, the animal antibodies showed rather strict specificities upon interaction with related haptens or drugs. These results are somewhat unexpected, because the haptens were used in connection with long and flexible spacer arms. Furthermore, they are not typical for certain other drug allergies. The strict specificity reduces the diagnostic potential of the haptenic reagents when used in serological tests or in skin testing.

Diagnostic reagents in drug allergy: immunochemical specificity in the 1,2-diphenyl-pyrazolidinedione series.

Chemically defined haptenic reagents and haptenic conjugates were synthesized to be used for skin tests in allergic patients and for serological tests. One series of reagents is based on an open-chain derivative which is formed by reaction of the oxidation product of phenylbutazone, 4-hydroxyphenylbutazone, with amino functions. A second series uses the intact 1,2-diphenyl-pyrazolidine-3,5-dione molecule which is substituted in the 4-position with acetic acid. Both haptens are used in conjunction with spacer molecules which provide considerable distances between haptenic moiety and carriers. The skin test reagents are hexavalent conjugates based on the bis-penta-L-lysine carrier "PAL". Rabbit and guinea-pig antisera against the haptens were obtained by immunizations with human serum albumin conjugates. Data obtained from passive cutaneous anaphylaxis and from ELISA tests show that there is generally only slight cross-reactivity between the two series of haptenic reagents. Also, there is only modest cross-reactivity between intact drugs and haptenic reagents. No measurable crossreactions were noted between 1-phenyl-2,3-dimethyl-3-pyrazolin-5-one derivatives and haptenic reagents of the 1,2-diphenyl-pyrazolidinedione series.

Penamaldate assay: improved methodology for the quantitation of penicilloic acids, penicilloyl amides and penicilloyl esters.

Conversion of penicilloic acids or their derivatives with modified alpha-carboxyl function to penamaldic acid derivatives involves formation of strong absorption bands in the UV. The penamaldate absorbances generated in the optical cell by HgCl2 addition can be used for quantitation and detailed accounts on the procedures are given. Methodological improvements allow determinations of penicilloyl derivatives within a range of 3% (+/- 1.5%). Free penicilloic acid determinations fall within a range of 8%. The penamaldate band at 282 nm from penicilloyl derivatives decreases to 95% within 10 min depending on conditions. The band from penicilloic acid at 275 nm decreases to 25%. The decay appears to be complex and measurements fall within a range of 15% even under carefully controlled conditions. The penamaldate stability can be used for characterization and for semiquantitative assessments of penicilloic acid/penicilloyl derivative mixtures.

Synthesis of carboxyl terminal segments of beta-subunit of human chorionic gonadotropin. Application of the two-phase method.

The synthesis in solution of carboxyl terminal peptide segments of the beta-subunit of human chorionic gonadotropin is described. The protected segments include sequences 119-131, 132-137, and 138-145. The syntheses were based on a standardized liquid-liquid extraction program for routine purification of intermediates (two-phase method). Condensation of terminally deblocked segments afforded protection peptides 132-145, 126-145, 120-145, and 119-145. Protected peptides 126-145 and 120-145 were deprotected in liquid hydrogen fluoride and used in conjugated form for immunization of rabbits. Data on the specificity of the antibody response are reported.

Liquid-liquid extraction in peptide synthesis.

An efficient procedure of peptide synthesis in solution based on liquid-liquid extraction for the purification of intermediates (two-phase method) is described. Generally, peptide segments of 10 and in favorable cases 15 amino acids can be prepared by using conventional blocking groups. Special blocking groups adapted for optimal performance are discussed. Simple equipment enabling rapid synthesis is also described.