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.

Effects of L-arginine and phosphodiesterase-5 inhibitor, sildenafil, on inflammation and airway responsiveness of sensitized BP2 mice.

Nitric oxide (NO) levels are elevated in the exhaled breath of asthmatic patients and NO is considered as a biomarker of airway inflammation. However, the functions of NO in the airways are not completely understood. L-arginine, as the substrate of NO synthases, is the precursor of NO which stimulates guanylate cyclase and leads to the formation of cyclic GMP (cGMP). Sildenafil, a phosphodiestérase-5 (PDE-5) inhibitor, prevents the degradation of cGMP. In this study the effects of L-arginine and sildenafil treatment, alone or in combination, were evaluated in ovalbumin-sensitized BP2 mice. These effects concerning the airway responsiveness to inhaled methacholine (MCh) were evaluated by whole-body plethysmography (WBP), the inflammatory response evaluated by bronchoalveolar lavage fluid (BALF) analyses and lung tissue biopsies (eosinophilic inflammation associated with lung remodelling), and NO metabolite measurements (by Griess reaction) in BALF. Ovalbumin sensitization induced: (a) an inflammatory reaction with eosinophil and neutrophil influx in BALF and lung; and (b) an increased bronchial responsiveness to MCh. L-arginine treatment [50 mg/kg intraperitoneally (i.p.), for 7 days] increased the relative amount of eosinophils and neutrophils in BALF, had a tendency to increase the airway responsiveness to inhaled MCh and increased the NO metabolite level in BAL. Sildenafil treatment (20 mg/kg i.p. for 7 days) did not affect the airway responsiveness to MCh and had a lower effect compared with L-arginine on inflammatory reactions. The combination of the two treatments resulted in a dramatic enhancement of the airway responsiveness to inhaled MCh. The relative amount of eosinophils was increased and lung histology showed obvious worsened tissular lesions such as epithelial shedding and hypertrophy, hyperplasia of smooth muscle cells, and fibrosis. These findings are consistent with the notion that NO production plays a role in the development of airway inflammation and hyperresponsiveness of sensitized mice and highlighted the potential risk of the L-arginine dietary complement or PDE5 treatment in asthmatic patients.

Clinical presentation and time course in hypersensitivity reactions to beta-lactams.

BACKGROUND: beta-lactam hypersensitivity reactions are classified as immediate or nonimmediate. Diagnosis is usually based upon skin tests and provocation challenges. OBJECTIVE: The time course of the reactions in proven beta-lactam hypersensitivities was studied and then correlated with the symptoms to determine the relationship between the clinical presentations and the time course. METHOD: All of the patients who consulted between 1996 and 2004 for a suspected beta-lactam hypersensitivity reaction were studied. Two hundred and ten patients with a proven hypersensitivity reaction diagnosed according to the European Network on Drug Allergy were included in the present study. RESULTS: Of the patients, 36.7% had urticaria as a single symptom, 19.1% anaphylaxis without shock, 17.6% anaphylactic shock and 19.1% maculopapular exanthema. Anaphylactic shock and anaphylaxis mostly occurred within 1 h after drug administration. Exanthema mainly occurred after 24 h. Urticaria as a single symptom occurred at any time. A firm diagnosis was determined using immediate-reading skin prick (10.0%) and intradermal tests (38.1%), late-reading skin tests (19.1%) or provocation tests (32.9%). CONCLUSION AND CLINICAL IMPLICATION: Depending on the time course of the reaction, three clinical groups were identified: anaphylaxis and anaphylactic shock (immediate reaction); maculopapular exanthema (late reaction) as well as urticaria (immediate and late reaction).

Relevance of the determination of serum-specific IgE antibodies in the diagnosis of immediate beta-lactam allergy.

BACKGROUND: Allergic reactions to beta-lactams are the most frequent cause of adverse drug reactions mediated by specific immunologic mechanisms. They can be explored by in vivo and/or in vitro tests. The measurement of serum-specific immunoglobulin E (IgE) presents several advantages: safety, simplicity, and availability to nonallergologist physicians. OBJECTIVES: To establish the diagnostic value of specific IgE determination in the diagnosis procedure of immediate beta-lactam allergy. METHODS: The in vitro determination of beta-lactam-specific IgE antibodies was compared in three well-defined groups of patients (n=45): one with negative skin tests and a positive drug provocation test, another with positive skin tests, and a third control exposed population with good tolerance. Two techniques were used: the CAP-FEIA system (Phadia) commercially available and a homemade radioallergosorbent test (RAST). RESULTS: The specificity of CAP-FEIA ranged from 83.3% to 100% and sensitivity from 0% to 25% depending on initial clinical manifestations. The specificity of RAST was between 66.7% and 83.3% and sensitivity 42.9% and 75%. In the subgroup of patients with an anaphylactic shock and negative skin tests, the sensitivity and specificity of RAST were 75%. Positive and negative predictive values were 45.5% and 77.1% with CAP-FEIA and 38.5% and 81.5% with RAST, respectively. CONCLUSION: These results indicate that, although the specificity of beta-lactam-specific IgE measurement is good, sensitivity is low. Immunoglobulin E measurement should be limited to patients with a clinical history of anaphylactic shock and negative skin tests in order to avoid a drug provocation test. More sensitive assays should be developed.

Diagnosis of neuromuscular blocking agent hypersensitivity reactions using cytofluorimetric analysis of basophils.

BACKGROUND: Immunoglobulin E (IgE)-mediated hypersensitivity reactions to neuromuscular blocking agents (NMBA) are common and life threatening. Basophil activation based upon the expression of CD63 in the presence of specific allergens was found to be of importance for the diagnosis of IgE-mediated hypersensibility. METHODS: The Basotest was evaluated for the diagnosis of NMBA in 47 patients with proven NMBA anaphylaxis, 40 atopic subjects nonallergic to NMBA and five healthy volunteers. Diagnosis of NMBA was made according to international standards on clinical history, skin tests and provocation tests when needed. RESULTS: In the NMBA allergic patients, sensitivity of Basotest was 36.1%, but it increased to 85.7% for reactions which occurred within the last 3 years. The specificity was 93.3%. CONCLUSION: Basotest may be useful for the diagnosis of NMBA allergy in patients with a suspicion of recent IgE-mediated hypersensitivity reaction to NMBA.

Allergen-induced mediator release tests.

The diagnosis of allergic reactions in clinical practice is based on both clinical history and the determination of specific immunoglobulin E (IgE), either in the serum or on skin mast cells. However, for various reasons, identification of the causative factors is not possible in all the cases. Moreover, not all allergies are IgE-dependent. In an attempt to find sensitive, specific and cost-effective methods to investigate hypersensitivity reactions, in vitro tests were developed at a very early stage. Allergen-induced mediator release assays analyze the mediator released from effector cells, mainly peripheral blood cells, when stimulated in vitro with serial dilutions of the putative allergens. Described initially as research tools, they could well become diagnostic tests. However, relatively few high quality reports have been published so far. In this review, we will detail allergen-dependent histamine, tryptase, arachidonic acid metabolite, e.g. cysteinyl leukotrienes and 15-hydroxyeicosatetraenoic mediator release tests.

Increased bronchoalveolar lavage CD8 lymphocyte subset population in wheezy infants.

Episodes of wheezing are very common in infancy but, despite their high prevalence, their mechanism is still poorly understood. To better understand the airway inflammation of wheezing infants, we examined cells of the bronchoalveolar lavage (BAL), focusing on the phenotype of lymphocytes and macrophages by using cytofluorimetry. Twenty-one wheezers (mean age 15.4 months) and seven non-wheezers (mean age 24.1 months) were studied. BAL was collected at fiberoptic bronchoscopy. Total and differential cell counts were similar in both populations. Eosinophils were not detected in the BAL fluid. The cell-surface markers CD2, CD3, CD4, CD7, CD8, CD19, and CD45 were studied for the lymphocyte sub-population analysis. The cell-surface markers CD14, CD54, CD62L, and human leucocyte antigen (HLA)-DR were studied for the macrophage sub-population analysis. A significant increase in the CD8(+) lymphocyte population (p = 0.03) was observed in wheezers (median 43.1%, 25-75% percentile: 30.1-54.9%), as compared to non-wheezers (median 29.3%, 25-75% percentile: 13.5-34.7%). A significantly (p = 0.04) decreased expression of HLA-DR (mean fluorescence intensity [MFI]) was detected in the macrophage population of the wheezers (median MFI, 7,016; range 2135-7986), as compared to non-wheezers (median MFI, 8,369; range: 6478-8860). The results of the present study suggest that viral infection may have induced a CD8(+) response in BAL cells.

Two-dimensional structure of the native light-harvesting complex LH2 from Rubrivivax gelatinosus and of a truncated form.

The light-harvesting complex LH2 of Rubrivivax gelatinosus has an oligomeric structure built from alpha-beta heterodimers containing three bacteriochlorophylls and one carotenoid each. The alpha subunit (71 residues) presents a C-terminal hydrophobic extension (residues 51-71) which is prone to attack by an endogenous protease. This extension can also be cleaved by a mild thermolysin treatment, as demonstrated by electrophoresis and by matrix-assisted laser desorption-time of flight mass spectrometry. This cleavage does not affect the pigment binding sites as shown by absorption spectroscopy. Electron microscopy was used to investigate the structures of the native and thermolysin cleaved forms of the complexes. Two-dimensional crystals of the reconstituted complexes were examined after negative staining and cryomicroscopy. Projection maps at 10 A resolution were calculated, demonstrating the nonameric ring-like organization of alpha-beta subunits. The cleaved form presents the same structural features. We conclude that the LH2 complex is structurally homologous to the Rhodopseudomonas acidophila LH2. The hydrophobic C-terminal extension does not fold back in the membrane, but lays out on the periplasmic surface of the complex.

Basophil activation during pollen season in patients monosensitized to grass pollens.

BACKGROUND: Basotest is a new basophil-activation test based upon the expression of CD63 (gp53) in the presence of allergens. It is an effective diagnostic test for pollen-allergic patients. However, it is not known whether Basotest results differ during the pollen season. METHODS: We examined the activation of basophils by Basotest in 13 patients sensitized only to grass pollen, before and during the pollen season, in order to assess whether Basotest could be used as a diagnostic test during the pollen season. Dose-response curves with 10-fold increasing concentrations of timothy grass pollen (10-4 to 100 AU) were carried out. RESULTS: Basophils were not activated spontaneously during the pollen season since the CD63 expression was below detectable levels before in vitro cell activation. A decreased percentage of activated basophils at the peak of activation was found in comparing the pre- and in-season tests, but all patients had a positive test. When basophil activation was at its peak, the allergen concentration was similar during the two periods. Moreover, the median area under the curve was significantly (P < 0.02) reduced during the season as compared to before the season. CONCLUSION: Basotest can therefore be used as a diagnostic test during the pollen season, but the allergen exposure needs to be characterized if quantitative studies are performed.

In vitro diagnosis of cypress pollen allergy by using cytofluorimetric analysis of basophils (Basotest).

BACKGROUND: Cupressaceae pollen allergy is a worldwide pollinosis, but its in vitro diagnosis is notoriously difficult. The Basotest is a newly available in vitro test for the detection of allergen-specific IgE based on the level of cellular activation of basophils by using flow cytometry. OBJECTIVES: The Basotest was compared with the measurement of cypress pollen-specific IgE in highly selected patients. METHODS: We analyzed 34 patients allergic to cypress pollen selected on the basis of a suggestive clinical history and positive skin test and nasal challenge responses to cypress pollen extract. We also analyzed 8 patients with positive skin test responses to cypress pollen extract who did not present symptoms during the pollen season (intermediate group) and 33 control subjects. Sensitivity, specificity, and efficiency of the Basotest and serum-specific IgE levels measured by using the CAP System were determined in patients allergic to cypress pollen. Histamine release was studied in a selected group of patients. RESULTS: The Basotest was more sensitive (91.2%) than the CAP System (76%) for the in vitro diagnosis of cypress pollen allergy. A dose-response curve was observed in basophils obtained from patients allergic to cypress pollen. There were no false-positive results with either test (specificity 100%). The results of the Basotest or those of the CAP System did not correlate with the patients' in vivo threshold sensitivity assessed by skin tests and nasal challenge. CONCLUSIONS: The Basotest was found to be an effective diagnostic test in patients allergic to cypress pollen.

High resolution x-ray analysis of two mutants of a curaremimetic snake toxin.

A previous mutational analysis of erabutoxin a (Ea), a curaremimetic toxin from sea snake venom, showed that the substitutions S8G and S8T caused, respectively, 176-fold and 780-fold affinity decreases for the nicotinic acetylcholine receptor (AchR). In view of the fact that the side-chain of Ser8 is buried in the wild-type toxin, we wondered whether these affinity changes reflect a direct binding contribution of S8 to the receptor and/or conformational changes that could have occurred in Ea as a result of the introduced mutations. To approach this question, we solved X-ray structures of the two mutants S8G and S8T at high resolution (0.18 nm and 0.17 nm, with R factors of 18.0% and 17.9%, respectively). The data show that none of the mutations significantly modified the toxin structure. Even within the site where the toxin binds to the receptor the backbone conformation remained unchanged. Therefore, the low affinities of the mutants S8T and S8G cannot be explained by a large conformational change of the toxin structure. Although we cannot exclude the possibility that undetectable structural changes have occurred in the toxin mutants, our data support the view that, although buried between loop I and II, S8 is part of the functional epitope of the toxin.

1.2 A refinement of the Kunitz-type domain from the alpha3 chain of human type VI collagen.

The recombinant Kunitz-type domain (C5) of human collagen alpha3(VI) chain was previously described at 1.6 A resolution at room temperature. By changing the crystallization conditions and using synchrotron radiation, we are able to record diffraction data to 1.2 A resolution for crystals of the same space group at 291 K. The protein-water-ion model has been refined anisotropically against these new data using the program SHELXL93; the results converged to an R factor of 15.0%, with all data between 7 and 1.2 A. The final electron-density map reveals a clear chain tracing with a few disordered residues and five residues out of 58 that present alternate conformations. The Cys14-Cys38 bond presents the less frequently observed left-hand conformation (chi1 = -60 degrees). The solvent molecules and a phosphate ion are well ordered with an average B of 38 A2. The high-resolution structure reveals the N and C termini which were missing from the 1.6 A structure.

Modification of surface marker expression on CD14 monocytes of allergic patients after lysis or Ficoll purification.

It has been observed that peripheral blood monocytes are often in a primed or activated state in inflammatory diseases such as asthma. However, the majority of these studies have been performed using cells which have been purified by density gradient centrifugation on Percoll or Ficoll-Hypaque. Using cytofluorimetry, we compared the expression of monocyte surface markers of monocytes from untreated blood with monocytes purified by erythrocyte lysis or density centrifugation using the Ficoll technique. Monocytes from two groups of subjects were analyzed: healthy subjects and allergic patients. When compared with untreated blood, the percentage of CD16-positive cells, and the sMFI was significantly greater after monocyte purification (lysis or Ficoll). The expression of CD62L (percentage and sMFI) was modified after monocyte purification. Such modification of these two surface markers was predominantly observed on monocytes from allergic patients, and not on monocytes from healthy subjects. This study suggests that surface marker analysis should be performed on unfractionated whole blood in order to avoid modification of monocyte antigens.

Cells and mediators from pharyngeal secretions in infants with acute wheezing episodes.

An acute wheezing episode is the most common feature of severe lower respiratory tract infection during infancy. Respiratory syncytial virus (RSV) is the major causative agent. In order to study inflammation during acute wheezing episodes in infants, we wanted to assess the feasibility and contribution of induction of pharyngeal secretions. We therefore compared inflammatory markers in the pharyngeal secretions of 27 infants suffering from acute wheezing episodes with an RSV infection (RSV+) and 18 infants suffering with acute wheezing episodes without RSV infection (RSV-). Pharyngeal secretions were recovered by physiotherapy using isotonic saline. The safety of the procedure was carefully checked. Pharyngeal secretions were homogenized with dithiothreitol. Total cells and eosinophils were counted and levels of eosinophil cationic protein (ECP) and histamine were measured. Induction of pharyngeal secretion was always well tolerated. Eosinophils were present in five RSV+ and seven RSV- patients. ECP levels were not significantly different between the groups. Histamine levels after protein adjustment were significantly increased in RSV+ patients (p<0.01) in comparison to RSV- patients. In this study, we have shown, that pharyngeal secretion can be safely recovered from infants suffering from acute wheezing episodes, and that it can be analysed for enumeration of inflammatory cells and measurement of inflammatory mediators.

Ex vivo pharmacologic modulation of basophil histamine release in asthmatic patients.

Histamine is one of a range of mediators which play an important role in asthma, and the "releasability" of basophils has been shown to be upregulated in this disease. In vitro, beta 2-agonists and to a lesser extent corticosteroids have been shown to reduce histamine release. The ex vivo effects of salmeterol and inhaled corticosteroids on histamine release were studied in 78 asthmatic patients with variable disease severity and 20 control subjects. Spontaneous and anti-IgE-induced histamine release was measured in all subjects. Fifteen patients were not receiving any form of treatment, 42 were treated with inhaled corticosteroids, and 21 received inhaled corticosteroids and salmeterol. Seven patients treated with inhaled corticosteroids and seven patients treated with inhaled corticosteroids and salmeterol were tested twice to assess the effect of salmeterol on histamine release. Nine patients treated with inhaled corticosteroids were tested before and after 1 month of salmeterol treatment to determine the possible inhibition by salmeterol. Patients who were treated with inhaled corticosteroids and salmeterol showed significantly lower levels of spontaneous histamine release (median: 2.5%) than untreated (5.2%) and inhaled corticosteroids-treated asthmatics (3.4%). No tachyphylaxis to salmeterol was observed when patients were tested twice at a 3-month interval. This study suggests that salmeterol may have an additive anti-inflammatory effect with inhaled corticosteroids, although this hypothesis must be tested by further studies involving cells obtained by bronchoalveolar lavage and studies with bronchial biopsies.

Biochemical and cellular effects of heparin-protamine injection in rabbits are partially inhibited by a PAF-acether receptor antagonist.

The origin of the thrombocytopenia and leucopenia induced by protamine-heparin complexes is unknown. We studied the biochemical and cellular effects of protamine (6 mg x kg-1, i.v.) injected after heparin (5 mg x kg-1, i.v.) in New Zealand rabbits. After protamine injection (0.5 min) increases in blood platelet-activating factor (PAF-acether, PAF) (27.6 +/- 27.6 to 148.2 +/- 48.9 pg x ml-1, P < 0.05), thrombocytopenia (403 +/- 64 to 166 +/- 13 cells x 10(-3) x mm-3, P < 0.05) and leucopenia (7650 +/- 930 to 4300 +/- 668 cells x mm-3, P < 0.05) were noted. Plasma thromboxane B2 increased at 1 min (125.6 +/- 24.4 to 879.7 +/- 141.0 pg x ml-1, P < 0.01). Protamine alone induced no change. Indomethacin (3 mg x kg-1, i.v.) did not counteract the effects of heparin-protamine. Pretreatment with the PAF receptor antagonist BN 52021 [9H1, 7a-(epoxymethano)-1 H,6aH-cyclopenta[c]furo[2,3-b]furo-[3',2',3,4]cyclopenta[1,2-d]fur an-5,9, 12(4H)trione,3-tert-butylhexahydro-4,7b,11 hydroxy-8 methyl] alone (3 mg x kg-1, i.v.) delayed thrombocytopenia and reduced plasma thromboxane B2 concentration but did not modify leucopenia. Thus thrombocytopenia and thromboxane B2 release triggered by heparin-protamine may be potentiated by the release of PAF.

1.8 A structure of Hypoderma lineatum collagenase: a member of the serine proteinase family.

Collagenase from the fly larvae Hypoderma lineatum cleaves triple-helical collagen in a single region. It was crystallized at neutral pH in the absence of inhibitor and 1.8 A data were collected using synchrotron radiation and a Mark II prototype detector. The structure was solved by combining multiple isomorphous replacement methods and rotation translation function in real space. Refinement between 7 and 1.8 A using the program X-PLOR led to a final R factor of 16.9%. The overall fold is similar to that of other trypsin-like enzymes but the structure differs mainly by the presence of a beta-sheet at position 31-44. The two embedded molecules of the asymmetric unit are related by a pseudo twofold axis. The beta-sheet 31-44 of one molecule is involved in hydrogen bonds with binding-pocket residues of the other molecule. It thus completely prevents access to the active site. The specificity of this enzyme probably results from the position of Phe192 and Tyr99 at the entrance of the active site.