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.

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.

[RT-PCR in clinical diagnosis]. / La RT-PCR en diagnostic clinique.

Application fields of RT-PCR (reverse transcription-polymerase chain reaction) in clinical diagnosis comprises the assessment of viral load for RNA viruses and the analysis of gene transcription products. RT-PCR is also helpful when large genes have to be sequenced. Developments of quantitative approaches using real-time PCR recently led to a major widening of RT-PCR applications in clinical diagnosis. However, RT reaction is delicate due to its lack of reproducibility and to RNA lability and frequent contamination by DNA. In some cases additional difficulties come from the need to obtain a specific amplification in the presence of homologous sequences which might be present in higher amounts than the sequence of interest. These caveats have to be taken into account, when designing the RT protocol, and when choosing PCR primers and internal and/or external references. This review is aimed at helping the experimental setup of a RT-PCR based assay according to the objectives.

The 18 kDa peanut oleosin is a candidate allergen for IgE-mediated reactions to peanuts.

BACKGROUND: Peanut allergy is one of the five most frequent food allergies in children and in adults. Recently, we purified and evaluated the allergenicity of peanut oleosins, a family of small-sized proteins involved in the formation of peanut oil bodies. METHODS: Allergenicity of the purified native protein and of the recombinant protein was tested by Western blot and by IgE-RIA. RESULTS: We found IgE-binding with oleosin in 3 of 14 sera of patients who had suffered an allergic reaction to peanuts. Two sera reacted weakly against 16-18 kDa proteins corresponding to oleosin monomers, in Western blot. The main reacting bands had a molecular size estimated at approximately 34 kDa, approximately 50 kDa and approximately 68 kDa and could therefore correspond to oleosin oligomers. IgE reactivity was higher in extracts from roasted peanuts. The same phenomenon occurred with crude soybean oil fraction, with two bands of 16.5 and 24 kDa corresponding to monomers, and two bands of 50 kDa and 76 kDa corresponding to dimers and trimers, respectively. The 18 kDa band was observed in the 3 Western blots of a membrane-enriched fraction of recombinant oleosin produced in the Sf9-baculovirus expression system (performed with the 3 patient sera). CONCLUSIONS: We have characterized a new peanut allergen which belongs to the oleosins, a family of proteins involved in the formation of oil bodies. The protein may be involved in some of the allergic cross-reactions to peanuts and soybeans.

Penetration of telithromycin (HMR 3647), a new ketolide antimicrobial, into inflammatory blister fluid following oral administration.

The penetration of telithromycin (HMR 3647), a novel ketolide antimicrobial, has been assessed in an open, single-dose study in eight healthy male subjects. Following a single, oral, 600 mg dose the mean ratio of the concentration of telithromycin in blister fluid over plasma was 1.38. Significant blister fluid concentrations were maintained up to 24 h post-dose. These results indicate that telithromycin penetrates well into inflammatory extracellular fluid, achieving high and sustained concentrations in this medium.

Paracellular transport of avidin saturated or not with biotinylated cobalamin through Caco-2 cell epithelium monolayer.

BACKGROUND: The cationic charge of molecules may promote their uptake across epithelia, which are rich in brush border anionic sites. The transport of unsaturated avidin and avidin saturated with a biotinylated compound was investigated across Caco-2 adenocarcinoma cell with fetal enterocyte phenotype. METHODS: The unsaturated avidin and avidin saturated with either biotin or a biotinyl-cobalamin conjugate (biotinyl-Cbl) were iodinated to follow their transport through the cell monolayer. Their apparent permeability coefficient (Papp) and transepithelial pathway were determined and compared to those for control radiolabeled markers [3H]-mannitol, [125I]-beta-lactoglobulin and [57Co]-cobalamin/intrinsic factor (Cbl/IF). RESULTS: The Papp of [125I]-avidin estimated at 2.8 x 10(-7) +/- 0.08 cm/s was close to that for mannitol that uses paracellular pathway. The binding of biotin or biotin conjugate to avidin enhanced its tetrameric conformation. The Papp for [125I]-avidin/biotin and [125I]- avidin/biotinyl-Cbl were respectively increased by 2-fold, compared to that for [125I]-avidin and 4-fold, compared to that for [125I]-beta-lactoglobulin and [54Co]-Cbl/IF. The protein was not accumulated in the cell and was found in intact form in the basolateral side, after its transport across the monolayer. Chloroquine (0.66 micromol/ml) did not significantly decrease the Papp for [125I]-avidin/biotinyl-Cbl. Conversely it decreased by 80% the Papp for Cbl/IF, that uses transepithelial pathway. CONCLUSIONS: Avidin (either saturated or not with biotin and biotinyl-Cbl) was able to cross the monolayer of Caco-2 cell line through a paracellular pathway. This study pointed out the interest for using this protein as a shuttle for increasing the transport rate of biotinylated compounds through fetal epithelial barriers.

Hyperhomocysteinemia is related to residual glomerular filtration and folate, but not to methylenetetrahydrofolate-reductase and methionine synthase polymorphisms, in supplemented end-stage renal disease patients undergoing hemodialysis.

Glomerular filtration is one of the major determinants of plasma total homocysteine (tHcy). To evaluate the respective roles of residual glomerular filtration (by measuring a specific protein marker, cystatin C), genetic polymorphisms and nutritional status in tHcy blood levels in end-stage renal disease patients (ESRD) under hemodialysis and supplemented with folate, we measured tHcy, folate, vitamin B12 (B12), creatinine, cystatin C, albumin and C-reactive protein and determined the polymorphism of methylenetetrahydrofolate reductase (MTHFR) (C677T and A1289C) and of methionine synthase (MS) (A2756G) in 114 ESRD patients before hemodialysis and 76 control subjects. All patients received a folate supplementation of 700 microg/day. Hyperhomocysteinemia was observed in all patients and exceeded the upper normal limit by 2-fold in 52.4% of the patients. Serum folate was significantly increased and the B12 level was not different from controls. Folate, Cystatin C and creatinine were significantly correlated to tHcy, while no correlation was found between tHcy, albumin and C-reactive protein. No difference in genotype frequency between ESRD patients and controls was found for MTHFR A1289C and MS A2756G. The MTHFR 677TT genotype was less frequent and was associated with a significantly higher tHcy level in patients. Folate and residual glomerular filtration estimated by cystatin C and creatinine levels were two independent determinants of tHcy in ESRD patients. These data suggest that hyperhomocysteinemia is a consequence as well as a complicating factor of renal failure.

Overexpression of folate binding protein alpha is one of the mechanism explaining the adaptation of HT29 cells to high concentration of methotrexate.

The human colon adenocarcinoma cell line HT29 can be adapted to 10(-7)- 10(-4) M concentrations of methotrexate (MTX). Cells adapted to 10(-4) M MTX have an enterocyte-like phenotype with DHFR gene amplification. Presently, we hypothetized that an increased expression of folate binding protein (FBP) may participate to the MTX resistance of 10(-4) MTX HT29 cells. The cDNA FBPalpha/beta-actin ratio of amplified transcripts was 4.8- and 1.5- fold higher in 10(-4) and in 10(-7) M MTX HT29 respectively, than in standard type HT29 cells. An increase of transcript level was observed when decreasing folic acid concentration. PI-PLC cleaved 7.7 times more membrane FBP in 10(-4) M than in 10(-7) M MTX and wild type HT29 cells. In contrast to 10(-7) M MTX cells, growth of 10(-4) M MTX cells was dependent on folic acid concentration and abolished at a concentration lower than 0.9 microM. In conclusion, the adaptive mechanism of HT29 cells resistant to 10(-4) M MTX is the result of the synergistic overexpression of both DHFR and FBPalpha. Overexpression of FBPalpha may be related to the enterocyte-like phenotype of the cells.

Comparison of effects of ioxaglate versus iomeprol on histamine and tryptase release in patients with ischemic cardiomyopathy.

We observed a release of histamine, but not of tryptase, in arterial blood from 64 patients with ischemic heart disease and 24 patients without coronary disease, which was provoked by ioxaglate, a ionic compound, but was not provoked by iomeprol, a non-ionic radiocontrast compound. The release of histamine in arterial blood after ionic contrast medium injection was higher in patients with ischemic heart disease compared with patients without coronary disease, suggesting that an increased release from heart mast cells previously observed exists also for systemic blood basophils.

Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood.

Transcobalamin (TC) is the plasma transporter that delivers vitamin B(12) to cells. We have already reported that HT-29 and Caco-2 cells secrete different TC variants. HT-29 secretes 2 TC isoproteins (codon 259-Pro/Arg [259-P/R]), exhibiting unequal concentrations (TC 259-P > TC 259-R), and Caco-2 cells only secrete the phenotype 259-R. We investigated the relation between phenotypic and genetic TC polymorphism in HT-29 cells transfected with Caco-2 TC complementary DNA and in 159 healthy Caucasians. We found that codon 259-R is buried and, thus, the genetic polymorphism provides no explanation why the TCs from HT-29 and Caco-2 cells have different isoelectric points in nondenaturing isoelectric focusing (IEF). The newly translated TC in HT-29 cells from the Caco-2 complementary DNA recombinant plasmid had the same isoelectric point as the TC constitutively expressed in HT-29 cells, suggesting that TC phenotypic variability involves a specific cell folding of the protein. The codon 259 polymorphism was found to have a biallelic distribution: homozygotes P = 34.6%, heterozygotes R/P = 47.8%, and homozygotes R = 17.6%. In heterozygous samples, the IEF showed that the TC 259-P/TC 259-R ratio = 1.6. The blood apo-TC concentration of 259-P homozygous Caucasians was significantly higher than that of homozygous 259-R (P <.0001) and heterozygous (P <.0006) Caucasians. The heterozygotes 259-R/P had homocysteine concentration significantly higher than the homozygotes 259-R and 259-P (P =.02 and P =.01, respectively). In conclusion, TC codon-259 polymorphism affects TC plasma concentration and may interfere in vitamin B(12) cellular availability and homocysteine metabolism.

Pharmacokinetics of the new ketolide telithromycin (HMR 3647) administered in ascending single and multiple doses.

Telithromycin (HMR 3647) is a novel ketolide antimicrobial with good activity against both common and atypical respiratory pathogens, including many resistant strains. This randomized, three-period crossover study determined the dose proportionality of telithromycin pharmacokinetics after single and multiple dosing in healthy subjects. In each treatment period, subjects received a single oral dose of 400, 800 or 1,600 mg of telithromycin followed 4 days later by the same dose once daily for 7 days. Blood and urine samples were taken throughout the study for determination of pharmacokinetic parameters for telithromycin and RU 76363, its main metabolite. Telithromycin and RU 76363 achieved steady state within 2 to 3 days of once-daily dosing. A slight accumulation of telithromycin was observed after 7 days of therapy, with values of the area under the concentration-time curve from 0 to 24 h approximately 1.5 times higher than those achieved with the single dose. The pharmacokinetics of telithromycin and RU 76363 deviated moderately from dose proportionality. At a dose of 800 mg/day, telithromycin attained mean maximal and trough plasma concentrations of 2.27 and 0. 070 mg/liter respectively. Elimination was biphasic; initial and terminal half-lives were 2.87 and 9.81 h for the 800-mg dose. Study medication was well tolerated, although adverse events tended to be more frequent at the 1,600-mg dose. This study showed that telithromycin was generally well tolerated and suggests that a once-daily 800-mg oral dose of telithromycin maintains an effective concentration in plasma for the treatment of respiratory tract infections involving the key respiratory pathogens.

Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48.

Quantitation of plasma apo B-48 is currently performed by densitometric analysis of SDS-PAGE zones stained with Coomassie Brilliant Blue, using standard solutions of purified apo B-48. Here, preparative gel electrophoresis with a continuous elution system was used for purifying apo B-48. A chylomicron fraction was isolated by 107,000 g ultracentrifugation of a chylous ascite. The proteins were delipidated and precipitated in ethanol-diethyl ether (3:1, v/v), subjected to preparative electrophoresis in a 5% polyacrylamide gel and eluted in 0.1% SDS. The peak containing apo B-48 was eluted at a retention time of 445-480 min. The purity of apo B-48 in this fraction was assessed by the detection of a single band (M(r) 260,000) after silver staining and Coomassie staining of 4-15% gradient SDS-PAGE. It was confirmed by the absence of apo B-100 contaminant in Western blot of the purified protein preparation. A linear relationship was observed between the densitometric analysis of SDS-PAGE bands and the apo B-48 in a protein range of 0-3 microg. In conclusion, preparative gel electrophoresis was used in a single step purification of apo B-48 that was adapted to the preparation of a standard solution.

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.

Analysis of riboflavin and riboflavin cofactor levels in plasma by high-performance liquid chromatography.

We describe an assay which determines simultaneously riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in plasma, using galactoflavin (GF) as an internal standard. The flavins were extracted on a C18 Sep-Pack cartridge after protein precipitation with 10% trichloroacetic acid, and were analyzed on a C18 RP-HPLC with 85% phosphate-magnesium acetate buffer (pH 3.4) and 15% acetonitrile. FAD, FMN, GF and RF extraction recoveries were 101.0-5.6, 97.0-6.5, 97.0-2.0 and 95.0-4.1%, and reproducibilities were 5.9, 6.8, 2.1 and 4.3%, respectively. FAD, FMN and RF values in infant and adolescent plasma were in the range 53.5-108.2, 9.0-25.1 and 12.7-53.4 nM, and 36.5-157.20, 7.1-24.6 and 8.2-57.8 nM, respectively. Using GF as an internal standard improved the quantification of these B2 vitamers.

Comparison between serum alpha-glutathione S-transferase and aminotransaminases in detecting cytolysis in hepatitis C-infected children.

BACKGROUND: alpha-Glutathione S-transferase (alphaGST) has been proposed as a more sensitive indicator than serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in detecting hepatocellular damage due to chronic hepatitis C virus (HCV) infection. METHODS: The accuracy of alpha-GST was compared with that of ALT and AST in detecting cytolysis in 103 blood samples issued from 31 children positive for HCV RNA. RESULTS: alpha-GST had a lower sensitivity than ALT or AST (32% vs. 54.4% for each aminotransferase). The sensitivity of ALT and/or AST was 60.2%, whereas that of ALT and/or alpha-GST and AST and/or alpha-GST was lower (58.3% and 57.3%, respectively). Among 41 serum samples with negative ALT and AST, only 2 had positive alpha-GST, whereas alpha-GST failed to detect cytolysis in 31 samples with elevated ALT and/or AST. No correlation was found between alpha-GST, ALT, or AST and the Knodell score. CONCLUSIONS: The combination of ALT with AST is actually the best compromise in detecting cytolysis in untreated HCV-infected patients.

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.