6-O-sulfo sialylparagloboside and sialyl Lewis X neo-glycolipids containing lactamized neuraminic acid: synthesis and antigenic reactivity against G159 monoclonal antibody.

Synthesis and antigenic reactivity of 6-O-sulfo sialylparagloboside (SPG) and sialyl Lewis X (sLeX) neo-glycolipids containing lactamized neuraminic acid are described. The suitably protected GlcNAc-beta (1-->3)-Gal-beta (1-->4)-GlcOSE derivative was glycosylated with NeuTFAc-alpha (2-->3)-Gal imidate to give NeuTFAc-alpha (2-->3)-Galbeta (1-->4)-GlcNAc-beta (1-->3)-Gal-beta (1-->4)-GlcOSE pentasaccharide. The partial N,O-deacylation in the NeuTFAc-alpha (2-->3)-Gal part afforded N-deacetylated SPG derivative which was converted to the desired oligosaccharide containing lactamized neuraminic acid. Similar treatment of the sLeX hexasaccharide derivative, NeuTFAc-alpha (2-->3)-Gal-beta (1-->4) [Fuc-alpha (1-->3)]-GlcNAc-beta (1-->3)-Gal-beta (1-->4)-GlcOSE, gave the key hexasaccharide intermediate containing lactamized neuraminic acid. These suitably protected SPG and sLex oligosaccharides were converted stepwise into the desired neo-glycolipids (GSC-551 and GSC-552) by the coupling with 2-(tetradecyl)hexadecanol, 6-O-sulfation at C-6 of the GlcNAc residure, and complete deprotection. Both lactamized-sialyl 6-O-sulfo SPG (GSC-551) and sLex (GSC-552) neo-glycolipids were clearly recognized with G159 monoclonal antibody showing that both the lactamized neuraminic acid and the 6-O-sulfate at C-6 of GlcNAc would be involved in the G159-defined determinant. However, the Fuc residue and the lipophilic (ceramide) part may not be critical for this recognition.

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.

Alergia a beta-lactámicos / Allergy to betalactams

La alergia a b-lactámicos es la primera causa de alergia medicamentosa en el mundo. En nuestro país, el estudio in vivo (pruebas cutááneas) realizado regularmente no se efectúa de acuerdo a patrones internacionales establecidos. Este artículo revisa el diagnóstico y manejo de estas reacciones. Las reacciones alérgicas a b-lactámicos pueden clasificarse, según su perfil temporal, en inmediatas, aceleradas y tardías, lo que se relaciona con las manifestaciones clínicas y mecanismos patogénicos. La mayor parte de las reacciones inmediatas y aceleradas son mediadas por IgE, con expresión clínica de hipersensibilidad inmediata. Entre las reacciones tardías destacan las toxidermias y exantemas máculo-papulares, mediadas probablemente por hipersensibilidad retardada. Los alergenos implicados en las reacciones de hipersensibilidad inmediata a b-lactámicos pueden ser los determinantes mayores (75 por ciento de los casos), determinantes menores o las cadenas laterales de los fármacos sospechosos. El estudio de estos pacientes incluye IgE específicas, pruebas cutáneas y pruebas de provocación. Los objetivos de estos estudios son diagnosticar reactividades cruzadas o monosensibilizaciones, y autorizar o prohibir la utilización de todos los b-lactámicos o sólo algunos de los fármacos del grupo, además de asegurar la tolerancia a fármacos alternativos.

Studies on the endogenous L-selectin ligands: systematic and highly efficient total synthetic routes to lactamized-sialyl 6-O-sulfo Lewis X and other novel gangliosides containing lactamized neuraminic acid.

Systematic syntheses of lactamized neuraminic acid-containing gangliosides GM4, sulfated sialylparagloboside, and sulfated/nonsulfated sialyl Lewis X are described. The highly efficient, one-step lactamization of neuraminic acid was accomplished by treatment of the N-deacetylated sialic acid (neuraminic acid)-containing gangliosides with HBTU and HOBt in DMF at 65 degrees C. Both the lactamized neuraminic acid residue and the sulfate group at O-6 of the GlcNAc residue were found to be involved in the antigenic determinant defined by G159 monoclonal antibody, while the fucose residue may not be critical for the recognition by G159 mAb.

Sera from patients with multiple drug allergy syndrome contain circulating histamine-releasing factors.

BACKGROUND: A subset of drug-intolerant patients show a marked propensity to react to several chemically unrelated antibacterial drugs. This condition is termed multiple drug allergy syndrome (MDAS). The pathogenesis of MDAS is still unclear. A possible mechanism is that a nonspecific patient-related factor leading to direct histamine release from mast cells and basophils is involved. We investigated whether a patient-related facilitating factor such as the clinically unapparent presence of circulating histamine-releasing factors may represent a nonspecific mechanism underlying drug-induced histamine release in patients with MDAS. METHODS: 38 otherwise healthy adults with a history of acute urticaria following the ingestion of antibacterial drugs [18 subjects with MDAS (patients) and 20 monosensitive subjects (drug-allergic controls) on the basis of both clinical history and single-blind peroral challenges with alternative substances] and 20 subjects without a history of drug allergy (normal controls) underwent an autologous serum skin test (ASST). IgE specific for beta-lactams was measured in sera from 25 subjects (11 patients and 14 drug-allergic controls) with a history of amoxicillin intolerance. Sera from 13 patients and 5 drug-allergic controls (all positive on ASST) were used in the in vitro histamine release assay using basophils from 3 normal donors. RESULTS: 17 of 18 patients (94%) versus 8 of 20 drug-allergic controls (40%) showed an unequivocal wheal-and-flare reaction on ASST (p < 0.05). Skin reactions were generally more intense in the patient group. In one MDAS patient, the ASST was not assessable due to dermographism. No normal control was positive on ASST. Sera from 3 of 13 patients (23%) versus 0 of 6 drug-allergic controls (not significant) induced significant histamine release from basophils of normal donors. IgE specific for beta-lactams was detected in sera from 1 of 11 patients (9%) versus 5 of 14 drug-allergic controls (36%) (not significant). CONCLUSION: Most patients with MDAS and more than one third of subjects with a history of hypersensitivity to a single antibacterial drug were characterized by the presence of circulating histamine-releasing factors. Such factors might play a role in drug-induced adverse reactions observed in these patients.

Memory to the hapten in non-immediate cutaneous allergic reactions to betalactams resides in a lymphocyte subpopulation expressing both CD45RO and CLA markers.

The cutaneous lymphocyte-associated antigen (CLA) is a homing receptor expressed in a subpopulation of memory T lymphocytes that migrates to the skin and participates in different inflammatory processes. The aim of the study was to compare the T cell response to betalactams in both CLA+ and CLA- memory T cell subsets from subjects with non-immediate allergic reactions to these drugs. Three patients with a non-immediate reaction to penicillins were studied during their acute episodes. Peripheral blood mononuclear cells were isolated by Ficoll density gradient and were used for flow cytometry and lymphocyte transformation test assays. CD3+ cells were purified via high affinity negative selection columns. CD45RA+ and CD45RO+ subpopulations were obtained by magnetic sorting and the memory subpopulation was subdivided into CLA+ and CLA- fractions. These were cultured in triplicate together with feeder cells and different concentrations of amoxicillin and benzylpenicillin. In all cases, the proliferative responses to the drugs were confined to the CD45RO+CLA+ subpopulation. The CD45RO+CLA- subset showed no proliferative response to either drug at any concentration. We have shown that the in vitro memory to penicillins in non-immediate cutaneous allergic reactions to these drugs resides in the CD45RO+CD3+ subset expressing CLA, which enables these T cells to migrate to the skin. These findings may have relevance to understanding the involvement of T cells in allergic reactions to penicillins.

Comparative in vitro activity of penicillin G, levofloxacin, moxifloxacin, telithromycin, pristinamycin, quinupristin-dalfopristin and linezolid against ofloxacin-intermediate and -resistant Streptococcus pneumoniae.

Screening by ofloxacin disk was carried out on 1158 strains of Streptococcus pneumoniae in order to investigate the in vitro bacteriostatic activity of penicillin G, levofloxacin, moxifloxacin, telithromycin, linezolid, pristinamycin and quinupristin-dalfopristin against ofloxacin-intermediate and -resistant S. pneumoniae strains. It was concluded that these new antimicrobial agents could be useful for the treatment of pneumococcal infections caused by penicillin-sensitive and -resistant S. pneumoniae, and would represent a valid therapeutic option for patients allergic to beta-lactams, should they prove to be potent in vivo.

Flow cytometric basophil activation test by detection of CD63 expression in patients with immediate-type reactions to betalactam antibiotics.

BACKGROUND: In this study, we used flow cytometry to determine the percentage of activated basophils that expressed the CD63 marker after in vitro stimulation by different betalactam antibiotics. The diagnostic reliability of the technique was assessed, as well as its correlation with specific IgE. METHODS: Fifty-eight patients with clinical allergy to betalactam antibiotics and presenting positive skin tests to at least one of the allergens (minor determinant mixture (MDM), benzylpenicilloyl-polylysine (PPL), penicillin, ampicillin, amoxicillin, cephalosporins) were tested. Thirty subjects non-allergic to betalactams were also studied as controls. The flow assay stimulation test (FAST) uses flow cytometry to determine the percentage of basophils that express CD63 as an activation marker after in vitro stimulation with allergen. Double labelling with monoclonal antibodies anti-CD63-PE and anti-IgE FITC was used. RESULTS: The allergic patients show a statistically greater number of activated basophils than the control subjects, after the incubation of cells with all the betalactams at various concentrations. The sensitivity of the technique is 50%, the specificity 93.3%, the likelihood ratio for a positive value 7.46 and the likelihood ratio for a negative value 0.54. In spite of having a greater sensitivity (37.9%) and specificity (86.7%) than CAP, differences between sensitivity and specificities of both techniques (CAP and FAST) do not reach statistical significance. CONCLUSION: The basophil activation test is a particularly useful technique in the diagnosis of patients with IgE-mediated allergy to betalactams and allows the identification of 50% of patients. Used in conjunction with CAP, it allows the identification of 65.5% of such patients.

Allergy to betalactams: relationship between chemical structure and antigenicity of molecules.

As betalactams (penicillins, cephalosporins, carbapenems, monobactams, betalactamase inhibitors) are the most widely used antibiotic drugs worldwide, allergy to betalactams is a frequent problem encountered in clinical practice, concerning from 0.7-8% of treated patients. It is now clear that the antigenicity of these drugs is strictly related to the chemical structure of the various molecules and of their constituent parts. In this paper we first describe the chemical structure of betalactams and then try to establish a correlation between this structure and their antigenicity.

Evaluation of drug-related hypersensitivity reactions in children.

Patients with drug reactions are often referred to allergists for "allergy". Skin testing and clinical history seem to have a good negative predictive value, however, although drug challenge could be dangerous, it is the only way to confirm the diagnosis. We aimed to demonstrate that most children with a history of non-life-threatening drug reactions do not have a true drug allergy and examined the use of drug challenge in childhood. Patients with reactions were referred to our clinic by pediatricians. In 1 year, 354 reactions were studied in 239 children. Patients were classified according to their positive or negative history of drug allergy. Skin prick testing was done in all cases. Exclusion criteria for challenge included drug anaphylaxis, Stevens-Johnson syndrome, systemic reactions with severe concomitant illness, beta-inhibitor drug therapy or positive skin test to the implicated drug with a positive history. It was found that the beta-lactam antibiotics were involved in 50% of suspected reactions, aspirin in 10% and sulfonamides in 9%. Histories were considered positive only in 25%. Drug challenges confirmed only 4% of all reactions. It was concluded that drug challenge may be the gold standard for most childhood reactions that are considered to be allergic, non-life-threatening and drug-related. Only 4% of these suspected reactions were exclusively caused by drug allergy.

New aspects in betalactam recognition.

The data presented in this review confirm that penicillin continues to be the most well defined model for studying drug allergy. The identification of new specificities has improved the understanding of allergy to penicillins, and different well defined subgroups now exist. The capacity of humans to respond to unique penicillin determinants has shown that although penicillin is a very small molecule it can be recognized in different ways by different IgE antibodies. These well defined models have left open the possibility that other betalactams can also induce specific reactions which implies that for diagnostic purposes, in addition to classical determinants, others are required for in vitro and/or in vivo evaluation. When the different subgroups now recognized are compared, not only are there differences in the manner of hapten recognition but also in the evolution of the natural sensitivity and in the capacity for recognizing other structures. The recognition of betalactams by T cells is also important and a number of studies have shown that subjects respond specifically to some aminopenicillins or cephalosporins with good tolerance to benzylpenicillin. The confirmation that these responses can be a T-cell-mediated reaction have been reported not only in vitro by the generation of T cell lines and T cell clones but also in vivo doing skin biopsies in subjects who have developed different types of delayed cutaneous reactions [44]. More studies are needed to determine the structure of T cell epitopes and this will help for a better understanding of both the IgE and IgG-mediated reactions.

Nonimmediate reactions to betalactams: prevalence and role of the different penicillins.

In patients treated with penicillins, adverse cutaneous reactions can occur within minutes or may take several days to develop. IgE antibody-mediated reactions are well documented, but other mechanisms may also be involved. In particular, nonimmediate reactions have not been studied extensively, and the purpose of the present work was to establish the incidence of such reactions among a large group of patients and to study the penicillins involved. A total of 380 subjects with a history of a cutaneous reaction following administration of a penicillin antibiotic was included in the study. Skin tests and specific IgE measurements (RAST) were carried out using various penicillins and penicillin-related reagents, and patients were also challenged with various penicillins. In some patients with delayed skin test responses, skin biopsies were carried out. The tests confirmed that 74 subjects (19.4% of total investigated) had suffered a cutaneous reaction to a penicillin derivative, and 29 of these subjects (7.6% of total or 39% of confirmed) showed evidence of having suffered a nonimmediate reaction. The latter group were identified by giving a positive delayed direct challenge, and in 65% of the cases a delayed skin test response was detected. In most cases, these responses were to amino penicillins. Skin biopsies showed a lymphomonocytic cell infiltrate. Nonimmediate reactions to penicillins are a reproducible phenomenon, suggesting that a specific mechanism is responsible. By direct challenge, 93% of responders were positive to amino penicillins (10.3% ampicillin, 82.7% amoxicillin), indicating a major role for these penicillins in nonimmediate reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

[Chemotherapeutic intolerance]. / Chemotherapeutische Unverträglichkeiten

Incombatibility reactions can be observed in almost all antibiotics and chemotherapeutics. Kind and severity oftthe side effects depend on the terrain component and on the particular antibiotics. Clinical distinctions were made between allergic, toxic and dysmicrobial reactions. Special attention was given to nephrotoxicity and to the side effects of the combination of trimethoprim and sulfomethoxazol.