[Alpha-1 antitrypsin deficiency caused by Null mutation]. / Déficit en alpha-1 antitrypsine secondaire à une mutation Null.

INTRODUCTION: Alpha-1 antitrypsin deficiency is a hereditary disease defined at the biological level by a serum alpha-1 antitrypsin level below 11µM/L. The null variants are characterized by undetectable circulating alpha-1 antitrypsin levels. Suspicion of a null variant requires the use of appropriate diagnostic techniques. CASE REPORT: We report the case of a 33-year old patient presenting with dyspnea on exertion, associated with a moderate airflow obstruction, incompletely reversible. His tobacco use was less than 3pack-years. The thoracic CT-scan showed emphysema. The serum alpha-1 antitrypsin level was collapsed. Phenotyping by isoelectrofocusing on agarose gels did not show any band. The study of the SERPINA1 gene, by PCR-sequence of the II, III, IV and V exons and the flanking intronic sequences, allowed identification of the NullQ0ourém allele in homozygous state. This mutation was found in heterozygous state in both parents of the index case and in one of his brothers. The index case showed a rapid aggravation of the airflow obstruction. CONCLUSION: In the case of a serum alpha-1 antitrypsin deficiency, the analysis of the phenotype of the protein by isoelectrofocusing must be performed as a first-line investigation. The detection of an atypical profile may suggest the presence of deficient alleles other than the PI S and PI Z alleles that can only be characterized by sequencing of the whole SERPINA1 gene. The patients carrying a null mutation have a high risk of severe chronic obstructive pulmonary disease.

Comparison of cytokine gene polymorphism in drug-induced maculopapular eruption, urticaria and drug reaction with eosinophilia and systemic symptoms (DRESS).

BACKGROUND: Polymorphisms of genes controlling cytokine production have not been studied in the genetic susceptibility to cutaneous adverse drug reactions (CADR). OBJECTIVES: The objective was to determine whether polymorphisms in nine cytokine genes were associated to the occurrence of drug reaction with eosinophilia and systemic symptoms (DRESS) compared to drug-induced maculopapular eruption or urticaria and to controls without drug intolerance. METHODS: Results from 118 patients with a well-defined CADR were compared to 236 controls without drug intolerance living in the same area of France. We assessed nine polymorphisms: interleukin (IL)1-alpha-889C>T (rs 1800587), IL1-beta-511C>T (rs 16944), IL1-RN intron-2-VNTR (rs2234663), IL2-330T>G (rs 2069762), IL4-33C>T (rs 2070874), IL5-745C>T (rs 2069812), IL10-592C>A (rs 1800872), IL16-295T>C (rs 4778889) and tumour necrosis factor-alpha-308G>A (rs 1800629). RESULTS: Three polymorphisms exhibited a significant association with CADR (P < 0.05). The combination of the IL1-RN-A2 and IL1-beta-511C alleles was statistically different between cases and controls (P = 0.007) and the A2C haplotype was associated with susceptibility to CADR, particularly in drug reaction with eosinophilia and systemic symptoms (DRESS) patients (odds ratio = 3.22; 95% confidence interval = 1.23-8.41; P = 0.016). The frequency of the IL10-592A allele was higher in DRESS patients than in controls (dominant model CC vs. CA + AA: P = 0.035). These abnormalities were not evident in maculopapular eruptions or urticaria. CONCLUSIONS: This is the first study showing that IL1-cluster polymorphisms and haplotypes and the IL10-592A allele (a low IL10 producer) are associated with DRESS. These gene variants may decrease drug tolerance and promote herpes virus reactivation.

[Does hypersensitivity to multiple drugs really exist?]. / La polysensibilisation médicamenteuse systémique existe-t-elle ?

BACKGROUND: Multiple-drug hypersensitivity (MDH) in the literature concerns different entities. Our objective was to define its frequency and characteristics in patients examined for cutaneous adverse drug reaction (CADR) before studying genetic predisposition. MATERIALS AND METHODS: From a database comprising all patients referred for CADR between 2000 and 2010, we selected those meeting the following criteria: sensitisation to at least two chemically unrelated substances, as confirmed by positive skin tests or challenge tests. The following were excluded: patients with haematological diseases, HIV or chronic wounds and sensitization to the excipients. RESULTS: Of the 1925 patients included, 11 (0.6%) were classed as polysensitized: eight women and three men, of mean age 62 years, presenting 2.5 episodes of drug hypersensitivity per patient. Four cases of DRESS were noted. DISCUSSION: The strict criteria stipulated for this study enabled us to select patients with MDH, and to affirm that while it does in fact exist, it seems rare. Compared to polysensitized patients described in the literature, we preferred to distinguish between three groups of MDH: one occurring with different substances in separate episodes of CADR, one occurring with different substances during the same episode of CADR, and one occurring during DRESS and correlating with viral replication. CONCLUSION: MDH exists and genetic predisposition could be investigated by studying cytokine polymorphism in such patients. However, because of its rarity, it is impossible to rule out fortuitous association of two episodes of CADR in the same patient.

Recognition of iodixanol by dendritic cells increases the cellular response in delayed allergic reactions to contrast media.

BACKGROUND: Delayed reactions to iodine contrast media (CM) account for 1-3% of patients with adverse reactions to iodine CM. The cellular and molecular mechanisms of these reactions remain poorly documented. Although most of these reactions are T cell mediated, the involvement of dendritic cells (DC) has not been investigated sufficiently. OBJECTIVE: To determine whether the T cell response to iodixanol requires DC as antigen-presenting cell and, more particularly, to evaluate the changes induced by iodixanol on DC maturation and in vitro production of cytokines after drug stimulation in patients with maculopapular exanthema. METHODS: Peripheral blood lymphocytes, immature monocyte-derived DC (imDC) and skin biopsies were obtained from patients with delayed reactions to iodixanol and tolerant subjects. We studied the consequences of the interaction between DC, lymphocytes and iodixanol by phenotype analysis, proliferation and cytokine production. RESULTS: A T-cell-mediated reaction was evidenced in patient biopsies, with a lymphocyte-rich, peri-vascular infiltrate. Iodixanol induced maturation of imDC from patients but not from controls, with expression of the co-stimulatory markers CD83, CD86 and CD40 and an increase in mean fluorescence intensity of CD80, CD86 and HLA-DR. In the absence of DC, positive cell proliferation to iodixanol was detected in only one patient while the addition of DC produced a positive test in five of the six patients. Similarly, the increase in cytokines (IFN-γ, IL-2, IL-6, IL-1b and TNF-α) was higher when imDC were introduced into the culture together with the culprit drug. CONCLUSION AND CLINICAL RELEVANCE: These results provide evidence for a DC-mediated mechanism in delayed allergic reactions to CM, influencing T cell proliferation and cytokine production. These new insights will be helpful for designing immunotherapeutic strategies and in vitro diagnostic tests of CM-delayed reactions.

Perioperative anaphylaxis.

The incidence of immune-mediated anaphylaxis during anesthesia ranges from 1 in 10,000 to 1 in 20,000. Neuromuscular blocking agents represent the most frequently involved substances, followed by latex and antibiotics, but every drug or substance used may be involved. Diagnosis relies on tryptase measurements at the time of the reaction and skin tests and specific IgE or basophil activation assays.

Hypersensitivity reactions to neuromuscular blocking agents.

Neuromuscular blocking agents are the leading drugs responsible for immediate hypersensitivity reactions during anaesthesia. Most hypersensitivity reactions represent IgE-mediated allergic reactions. Their incidence is estimated to be between 1 in 3,000 to 1 in 110,000 general anaesthetics. However striking variations have been reported among countries. The mechanism of sensitisation seems to implicate the presence of a substituted ammonium ion in the molecule. Due to lack of exposure prior to the reaction in a large number of reactors, it has been hypothesised that sensitisation may involve other, as yet undefined, substituted (quaternary and tertiary) ammonium ion containing compounds such as pholcodine, present in the environment of the patient. This hypothesis is still under investigation. The mechanism of non-IgE mediated hypersensitivity reactions is less well known. Identified mechanisms correspond to direct histamine release or interactions with muscarinic and nicotinic receptors. Allergic reactions cannot be clinically distinguished from non-IgE-mediated reactions. Therefore, any suspected hypersensitivity reaction must be investigated using combined pre and postoperative testing. Because of the frequent but not systematic cross-reactivity observed with muscle relaxants, every available neuromuscular blocking agent should be tested, using intradermal tests to confirm the responsibility of the suspected drug which should be definitely excluded. Cross-sensitivity investigation will also try to identify the safety of drugs that can be potentially used in future anaesthesia. The determination of basophil activation investigations using direct leukocyte histamine release test or flow cytometry would be of particular interest to investigate cross sensitisation in complement to skin tests. There is no demonstrated evidence supporting systematic pre-operative screening in the general population at this time. However, since no specific treatment has been shown to reliably prevent anaphylaxis, allergy assessment must be performed in all high-risk patients. In view of the relative complexity of allergy investigation, and of the differences between countries, an active policy to identify patients at risk and to provide any necessary support from expert advice to anaesthetists and allergologists through the constitution of allergo-anaesthesia centres in every country should be promoted.

Genomic DNA extraction from small amounts of serum to be used for alpha1-antitrypsin genotype analysis.

If laboratory diagnosis of alpha1-antitrypsin (alpha1-AT) deficiency is usually based on its phenotype identification by isoelectric focusing, alpha1-antiprotease inhibitor (Pi)S and PiZ genotypes can also be determined by deoxyribonucleic acid (DNA)-based methods. Recently, several methods have been described for preparing genomic DNA from serum. The aim of the current study was to determine the Pi allele from serum extracted DNA by polymerase chain reaction (PCR) and to compare these results with those obtained with whole blood extracted DNA. Serum alpha1-AT concentration and phenotypic identification were systematically performed in 43 hospitalised patients. Genomic DNA was simultaneously purified from whole blood and from serum. The mutation detection was found using a PCR-mediated site-directed mutagenesis method. Concerning phenotypic identification, 29 patients were MM homozygotes, 11 were heterozygotes for S (MS = 7) or for Z (MZ = 4) and three showed a ZZ phenotype. Genotyping analyses gave identical results with serum and whole blood extracted DNA and all the results were in agreement with the phenotyping results. The authors found that the deoxyribonucleic acid-based test proved to be a reliable tool for alpha1-antitrypsin deficiency diagnosis and appears to be an alternative for the labour intensive alpha1-antitrypsin determination by isoelectric focusing. The authors also concluded that this method yields good quality deoxyribonucleic acid from serum, equal to that extracted from whole blood and is helpful in retrospective studies of multiple genetic markers.

The increased histamine release in ischaemic heart disease patients undergoing coronaroangiography is not mediated by specific IgE.

BACKGROUND: The release of histamine by iodinated contrast media (ICM) is higher in coronary artery disease patients than in noncoronary patients during coronary angiogram. METHODS: Eighty-eight patients who underwent a coronary angiography were classified either as having coronary artery disease or as noncoronary patients. Histamine concentration was higher than the 6.8 nM upper limit in 7 cases (group 1), of whom six were coronary artery disease patients. We compared the IgE and complement fractions in plasma of these patients to two control groups with normal histamine blood level, one (group 2) with and the other (group 3) without coronary artery disease. RESULTS: No difference of total IgE and C(3c) and C(4) complement fractions was found among the three groups. Anti-ioxaglate IgE-RIA was positive in only one patient from group 1. The affinity of drug-IgE binding in the serum of this patient was very low (Kd: 18.7 mM). The level of anti-ICM IgE detected by ioxitalamate- and iomeprol-Sepharose RIA was significantly higher in groups 2 and 3 than in group 1. CONCLUSIONS: The higher histamine release in ischaemic heart disease patients undergoing coronaroangiography is not mediated by IgE or complement activation. Further studies are needed to investigate the implication of histamine release factors.

Homocysteine, vitamins B6, B12, folate, and risk of coronary artery disease in patients undergoing diagnostic coronary angiography.

Homocysteine and vitamins B were correlated with coronary artery disease in patients undergoing diagnostic coronary angiography. 160 patients having > or =1 stenosis (G1), 55 patients having normal coronary arteries (G2) and 171 healthy volunteers (G3) were prospectively recruited. Homocysteine levels were significantly higher in patients, particularly in those with normal coronary angiograms, than in healthy subjects (13.8 +/-6.3 micromol/L in G1 (p < 0.0001) and 15.2 +/- 8.8 micromol/L in G2 (p < 0.0001) versus 10.1 +/- 3.1 micromol/L in G3). Homocysteine levels were not related to the extent of coronary artery disease. In patients with normal angiogram, vitamin B12 and folate levels were significantly higher compared with the other groups (p < 0.05 and p < 0.001, respectively) showing that vitamin B deficiency was not involved in the hyperhomocysteinemia. In conclusion, homocysteine and vitamins B levels do not contribute to discriminate for the presence of coronary artery disease in patients undergoing diagnostic coronary angiography. Homocysteine levels, however, were higher in patients referred for coronary angiography than in healthy controls.

Assimilation of [57Co]-labeled cobalamin in human fetal gastrointestinal xenografts into nude mice.

Cobalamin (Cbl) and its Cbl-binding proteins are present in amniotic fluid. Because amniotic fluid is swallowed by the embryo-fetus, we studied the ability of Cbl to be transported and metabolized across the embryo-fetal digestive tract. Human embryonic stomachs and intestines were transplanted into nude mice. The basal secretion of Cbl-binding proteins was studied by gel filtration of the graft juices. Intrinsic factor (IF) was looked for in gastric mucosa by immunohistochemistry. The uptake of [57Co]-labeled Cbl by the intestinal graft was studied by Schilling tests and HPLC. IF, haptocorrin, and a transcobalamin-like protein were detected in gastric juice, with concentration ranges of 5.0-26.4, 1.9-27.1, and 5.2-12.6 pmol/mL, respectively. The IF [57Co]Cbl complex had a single isoprotein with a pI at 5.6, which was maintained after incubation with neuraminidase. Urine excretion percentages (Schilling tests) ranged from 5.5 to 21.2% and from 0.3 to 1.6% when cyano-[57Co]Cbl-IF or cyano-[57Co]Cbl, respectively, was instilled in intestinal grafts. Chloroquine reduced significantly the percentage of excreted [57Co]Cbl. The [57Co]Cbl was mainly excreted as cyano-[57Co]Cbl in urines, showing a low coenzyme conversion. In conclusion, IF is secreted by the nonstimulated embryonic stomach and lacks sialic acid. Cbl binds to it and is subsequently transported across the xenografted embryo-fetal intestine. This suggests that amniotic fluid may contribute to Cbl delivery to the embryo-fetus.

C-Reactive protein and coronary artery disease: additional evidence of the implication of an inflammatory process in acute coronary syndromes.

BACKGROUND: Inflammation might promote the development of atherosclerosis, and high levels of C-reactive protein (CRP) and fibrinogen are associated with an increased risk of acute coronary events. OBJECTIVE: We assessed the levels of CRP and other risk factors in patients with angiographically documented coronary artery disease compared with healthy volunteers and patients undergoing coronary angiography who had normal coronary angiograms. METHODS: Ultrasensitive immunoassay was used to measure CRP levels in 142 patients with coronary disease (group 1), 37 patients with normal coronary angiograms (group 2), and 37 control healthy subjects (group 3). RESULTS: CRP levels were higher in group 1 (7.1 +/- 11.2 mg/L) compared with group 2 (4.8 +/- 4.0 mg/L) and group 3 (2.3 +/- 3.6 mg/L). In group 1, CRP levels were higher for patients with previous myocardial infarction (8.7 +/- 9.2 mg/L) or unstable angina (11.6 +/- 18.8 mg/L). Though CRP levels in patients with coronary artery disease and stable symptoms were higher compared with healthy volunteers (5.15 +/- 7.2 mg/L vs 2.3 +/- 3.6 mg/L, P <.05), they were similar to those observed in the control population of patients with normal coronary angiograms (4.8 +/- 4.0 mg/L). Furthermore, CRP levels were positively correlated to plasma fibrinogen but not to Chlamydia pneumoniae or Helicobacter pylori serology. CONCLUSION: These results suggest that CRP has a strong association with acute coronary events but do not support the hypothesis that CRP is a potent determinant of chronic stable coronary disease.

Anaphylactoid and anaphylactic reactions to iodinated contrast material.

Some adverse reactions to iodinated contrast material (ICM) are considered allergy-like, with cutaneous, cardiovascular, respiratory, and digestive symptoms. Allergy-like reactions are usually unpredictable. Reactions are more frequent with ionic than with nonionic material, but the frequency of deaths is almost identical. In a recent study, 20 severe unexpected reactions to ICM, including 10 life-threatening reactions and one death, were investigated by measuring mediators in blood, within the first minutes or hours of reaction. The responsible ICMs were mostly ionic materials. Histamine and tryptase release correlated with the severity of the reaction. Specific IgE against the responsible ICM was significantly higher in reactors than in controls. A few patients had positive skin tests to the administered ICM, suggesting type-I allergic reaction. Only 2.4% and 3.1% of the cases yielded a positive IgE-RIA, in a second retrospective study which included 165 patients recruited during a 4-year period. In conclusion, IgE-mediated anaphylaxis is rare, but it may be one of the possible mechanisms of severe adverse reactions to ICM.

Criteria of evaluation and of interpretation of Sepharose drug IgE-RIA to anaesthetic drugs.

The detection of antidrug specific IgE in serum is usually performed by a sandwich-type immunoassay in which the serum IgE is first adsorbed to a reactive phase and subsequently quantified via the binding of an anti-IgE tracer. The preparation of a new drug-reactive phase requires one to establish carefully different steps of validation: 1) criteria of positivity of control sera 2) competitive inhibition assays with the soluble drug, which should include the determination of the inhibition constant rather than estimation of a single inhibition percentage, especially when the assay is performed for the identification of determinants 3) estimation of nonspecific binding of IgE to the solid phase, including hydrophobic binding. The competitive inhibition depends on the concentration of the competitor and of IgE in the test-tube and the concentration of reactive drug bound to the solid phase. We have improved the inhibition assay by performing the Dixon test for calculating the inhibition constant (Ki) of the competitor. The Ki of six different muscle relaxants was determined in 12 patients who experienced an anaphylactic reaction to muscle relaxants. The values ranged between 1.5 nM and 2.5 microM. This confirmed the great heterogeneity of drug IgE cross-reactivity among patients. The Ki value of the incriminated drug was the lowest (affinity, the highest) in eight of the 12 patients. It was better correlated to clinical data than the classical inhibition assay. A hydrophobic environment seemed to be necessary, close to the quaternary ion, to allow IgE binding to the muscle relaxant. By contrast, in tiemonium, a hydroxyl group present at a distance of about 3 A from the quaternary ion may explain why this molecule had a high Ki (microM). In conclusion, it should be recommended, in molecular-recognition studies, that the inhibition constant of the soluble drug and of the related compounds be determined to complement the experiments based only on hapten inhibition assays.

Mechanisms of severe, immediate reactions to iodinated contrast material.

PURPOSE: To measure and elucidate the mechanisms of presumed mediators of unexpected severe, immediate reactions to iodinated contrast materials. MATERIALS AND METHODS: In a multicenter study, 20 patients with mild to severe reactions to iodinated contrast material and 20 control subjects without reactions were evaluated. Ionic contrast material was associated with 18 (90%) of 20 reactions. Concentrations of plasma histamine, tryptase, urinary methylhistamine, specific immunoglobulin E (IgE) against ioxitalamate or ioxaglate, and the anaphylatoxins C3a and C4a were measured with radioimmunoassays; complement C3 and C4 levels were measured with nephelometry. RESULTS: Histamine levels were increased in 14 patients; tryptase levels, in 16; and methylhistamine levels, in six. Histamine and tryptase values correlated with the severity of the reaction (P < .02 and P < .004, respectively). Significantly higher levels of specific IgE against ioxaglate (P < .005) and ioxitalamate (P = .045) were found in patients. No differences were found for complement fractions. Skin test results in two patients with life-threatening reactions were positive for the administered contrast material. CONCLUSION: Histamine release and mast cell triggering are related to severe reactions. An IgE-related mechanism is strongly suspected. Radiologists should be trained to identify and treat anaphylactic shock in patients who react to iodinated contrast material.

Diagnosis and pathogenesis of the anaphylactic and anaphylactoid reactions to anaesthetics.

Immediate adverse reactions to anaesthetics have an immune mechanism in more than 50% of the cases. They are mainly due to muscle relaxant drugs. A prospective evaluation of tryptase, histamine and serotonin for diagnosing anaphylaxis to anaesthetics was performed over 2 years. The sensitivity of each marker was at 60-70% and it reached 80% when combining tryptase and histamine. Specific IgE have been already observed in serum from patients allergic to muscle relaxant, thiopentone, morphine, phenoperidine, propofol and radio-contrast media. However, the recent progress in the identification of drug epitopes by Sepharose-solid drug phase IgE radioimmunoassay has to be reconsidered as non-specific binding of hydrophobic drugs such as propofol to hydrophobic serum IgE has been observed recently in patients with drug allergy. In addition, association of drugs such as propofol and muscle relaxant may potentiate the mediator release by a non-elucidated mechanism.