Polymorphisms in eicosanoid-related biosynthesis enzymes associated with acute urticaria/angioedema induced by nonsteroidal anti-inflammatory drug hypersensitivity.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are the main triggers of drug hypersensitivity, with NSAID-induced acute urticaria/angioedema (NIUA) the most frequent phenotype. NSAID hypersensitivity is caused by cyclooxygenase 1 inhibition, which leads to an imbalance in prostaglandin (PG) and cysteinyl leukotriene (CysLT) synthesis. As only susceptible individuals develop NSAID hypersensitivity, genetic factors are believed to be involved; however, no study has assessed the overall genetic variability of key enzymes in PG and CysLT synthesis in NSAID hypersensitivity. OBJECTIVES: To evaluate simultaneously variants in the main genes involved in PG and CysLT biosynthesis in NIUA. METHODS: Two independent cohorts of patients were recruited in Spain, alongside NSAID-tolerant controls. The discovery cohort included only patients with NIUA; the replication cohort included patients with NSAID-exacerbated respiratory disease (NERD). A set of tagging single-nucleotide polymorphisms (tagSNPs) in PTGS1, PTGS2, ALOX5 and LTC4S was genotyped using mass spectrometry coupled with endpoint polymerase chain reaction. RESULTS: The study included 1272 individuals. Thirty-five tagSNPs were successfully genotyped in the discovery cohort, with three being significantly associated after Bonferroni correction (rs10306194 and rs1330344 in PTGS1; rs28395868 in ALOX5). These polymorphisms were genotyped in the replication cohort: rs10306194 and rs28395868 remained associated with NIUA, and rs28395868 was marginally associated with NERD. Odds ratios (ORs) in the combined analysis (discovery and replication NIUA populations) were 1·7 for rs10306194 [95% confidence interval (CI) 1·34-2·14; Pcorrected = 2·83 × 10-4 ) and 2·19 for rs28395868 (95% CI 1·43-3·36; Pcorrected = 0·002). CONCLUSIONS: Variants of PTGS1 and ALOX5 may play a role in NIUA and NERD, supporting the proposed mechanisms of NSAID-hypersensitivity and shedding light on their genetic basis.

Natural evolution in patients with nonsteroidal anti-inflammatory drug-induced urticaria/angioedema.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most frequent triggers of drug hypersensitivity with NSAIDs-induced urticaria/angioedema (NIUA) the most common phenotype. Loss of hypersensitivity has been reported for IgE-mediated reactions; however, it has not been assessed in nonimmunological reactions such as NIUA. We evaluated NSAID-hypersensitivity over time in NIUA patients. METHODS: Patients confirmed as NIUA by positive drug provocation test (DPT) with acetylsalicylic acid (ASA) during 2005-2012 (V1) were included (n=38). Subjects were prospectively re-evaluated by DPT with ASA/other NSAIDs at two time points between 2013 and 2015 (V2 and V3). Atopy was assessed by skin prick test (SPT) using inhalant and food allergens. RESULTS: Patients were evaluated at V1 and re-evaluated after 60 months (V2; IR:48-81) and a further 18 months (V3; IR:14-24). At V2, the majority (24; 63.15%) tolerated ASA and other NSAIDs (Group A) while 14 (36.84%) still reacted (Group B). At V3, all Group A patients remained tolerant; all Group B patients remained hypersensitive. The number of previous episodes reported at V1 and the percentage of reactions induced by ASA/ibuprofen were significantly lower in Group A (P=.005 and P=.006, respectively). Group A patients developed tolerance 72 months (IR:45-87) after their last evaluated reaction (V1); this interval was shorter in nonatopics (P=.003), patients who experienced reactions over 1 hour after NSAIDs administration (P=.001), and those who experienced isolated urticaria after NSAID intake (P=.024). CONCLUSIONS: NIUA patients may develop tolerance to NSAIDs over time, a process that seems to be influenced by atopy and type of clinical reaction.

Update on the Genetic Basis of Drug Hypersensitivity Reactions.

Drug hypersensitivity reactions (DHRs) are unpredictable, complex responses to medicines in predisposed individuals. They represent a major health problem owing to the number of patients affected and the severity of the clinical conditions they can induce. In addition to environmental factors, the underlying mechanisms of DHRs are also influenced by genetic factors, although considerable gaps remain in our knowledge. Therefore, further study of the genetics of DHRs is necessary to shed light on their underlying mechanisms. In this manuscript, we provide an update on the genetic basis of the most frequent types of DHRs, including those mediated by immunological and nonimmunological mechanisms. For the first group, we will focus on immediate reactions to ß-lactam antibiotics, which are associated mainly with the IgE pathway (IL13, IL4R, LGALS3, and NOD2) and antigen presentation (HLA-DRA), and nonimmediate reactions to allopurinol, anticonvulsants, antibiotics, and antiretrovirals, which are often associated with polymorphisms in the HLA system. For the second group, we will focus on nonsteroidal anti-inflammatory drugs, which are mostly associated with genetic variants in enzymes and receptors from the arachidonic acid pathway (eg, ALOX5, ALOX5AP, PTGDR, and CYSLTR1). The information provided here will be of interest for medical practitioners from a range of disciplines who come across these reactions in their clinical practice, as well as for allergologists.

The Genetics of Drug Hypersensitivity Reactions.

DHRs are induced by various mechanisms and encompass a heterogeneous set of potentially life-threatening clinical entities. In addition to environmental effects, individual factors play a key role in this intricate puzzle. However, despite commendable efforts in recent years to identify individual predisposing factors, our knowledge of the genetic basis of these reactions remains incomplete. In this manuscript, we summarize current research on the genetics of DHRs, focusing on specific immune-mediated reactions (immediate and nonimmediate) and on pharmacologically mediated reactions (cross-intolerance to nonsteroidal anti-inflammatory drugs). We also provide some thoughts on potential technological approaches that would help us to decipher the molecular mechanisms underlying DHRs. We believe this manuscript will be of interest not only for allergists and basic researchers in the field, but also for clinicians from various areas of expertise who manage these reactions in their clinical practice.

Initial immunological changes as predictors for house dust mite immunotherapy response.

BACKGROUND: Although specific immunotherapy is the only aetiological treatment for allergic disorders, the underlying mechanisms are not fully understood. Specific immunotherapy induces changes in lymphocyte Th subsets from Th2 to Th1/Treg. Whether differences in immunological patterns underlie patient response to immunotherapy has not yet been established. OBJECTIVES: We studied the immunological changes occurring during a 1-year period of Dermatophagoides pteronyssinus (DP) immunotherapy and their relation with clinical outcome. METHODS: We included 34 patients with DP allergy who received subcutaneous specific immunotherapy (SCIT) for 1 year. Following treatment, patients were classified as responders or non-responders. Fourteen allergic subjects who did not receive SCIT were included as controls. Peripheral blood was obtained at 0, 1, 3, 6 and 12 months and cultured with nDer p 1. Phenotypic changes, cytokine production and basophil response were analysed by flow cytometry; transcription factors were measured by mRNA quantification. Serum immunoglobulin levels were also measured. RESULTS: After 1 year of SCIT, 82% of cases showed improved symptoms (responders). Although increases in sIgG4 were observed, BAT reactivity was not modified in these patients. Increases in T-BET/FOXP3 as well as nDer p 1-specific Th1/Treg frequencies were also observed, along with a decrease in Th2, Th9 and Th17. These changes corresponded to changes in cytokine levels. CONCLUSION: Patients who respond well to DP-SCIT show immunological differences compared to non-responders. In responders, basal differences include a lower frequency of Th1 and higher frequencies of Th2, Th9 and Th17 cells. After 1 year of treatment, an increased production of sIgG4 was observed in responders, along with a change in Th2 response towards Th1/Treg.

Influence of age on IgE response in peanut-allergic children and adolescents from the Mediterranean area.

BACKGROUND: Peanut allergens are common triggers of food allergy. Analyses of sensitization patterns, relationships with other allergens, clinical symptoms, and variation with age are needed. We studied sensitization to Ara h 2, Ara h 9, and Pru p 3 in a peanut allergic children/adolescents and the relationship with peach and pollen. METHODS: Peanut allergic patients aged between 1 and 20 years old were classified into two groups: A) allergic to peanut only and B) allergic to peach and peanut. The IgE response was measured to Ara h 2, Ara h 9, and Pru p 3. RESULTS: Of 964 subjects evaluated, 28% were allergic to peanut. From this group, 68% were also sensitized to pollen. Urticaria was the most frequent entity followed by anaphylaxis and OAS. Fifty-eight percent had Ara h 2- and/or Ara h 9-specific IgE. More than half reported symptoms with peanut alone (Group A) and 35% to peanut and peach (Group B). We observed significant differences in sex, age, onset of symptoms, and sensitization to Artemisia between groups. IgE response to Ara h 2 was more frequent in Group A, and Ara h 9 and Pru p 3 in Group B. We observed a decrease in sensitization to Ara h 2 and an increase to Ara h 9 and Pru p 3 with increasing age. CONCLUSION: Peanut allergy is frequent in subjects with allergy to plant foods, with Ara h 2 and Ara h 9 being two important allergens. In younger patients, Ara h 2 predominates over Ara h 9. The reverse was observed in older patients.

Hypersensitivity to Nonsteroidal Anti-inflammatory Drugs in Children and Adolescents: Cross-Intolerance Reactions.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used worldwide and are responsible for several types of drug hypersensitivity reactions (DHRs) in all age groups. The 2 major groups of DHRs to NSAIDs are those induced by immunological mechanisms (selective reactions) and those where inflammatory mediators are released through activation of the prostaglandin-leukotriene pathway without specific immunological recognition (cross-intolerance). In the present review, we focus on cross-intolerance reactions, which are the most frequent DHRs and are becoming a topic of major interest in children and adolescents. Paracetamol and ibuprofen are the drugs that most frequently cause DHRs in children; other NSAIDs are responsible for reactions in adolescents. In vivo and in vitro tests are of limited diagnostic value, with some exceptions for the less common selective reactions. In cross-intolerance, the clinical history and controlled administration are in many instances the only way to establish a diagnosis and look for alternatives. The clinical history is diagnostic when consistent symptoms occur repeatedly after exposure to NSAIDs with different chemical structures. Cutaneous and respiratory symptoms often co-occur in young children. The natural history of these reactions in children is unknown, and some patients can develop tolerance over time. Atopy remains a major risk factor for cross-intolerant reactions. The increasing interest in hypersensitivity to NSAIDs with improvements in patient phenotyping and the information provided by pharmacogenetics will improve our understanding and management of these reactions in the near future.

Hypersensitivity Reactions to Nonsteroidal Anti-inflammatory Drugs in Children and Adolescents: Selective Reactions.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used throughout the world to treat pain and inflammation; however, they can trigger several types of drug hypersensitivity reactions (DHRs) in all age groups. Although most such reactions occur through activation of the leukotriene pathway without specific immunological recognition (cross-intolerance), a significant number of DHRs to NSAIDs are due to immunological mechanisms (selective reactions [SRs]). SRs are thought to be induced by specific IgE antibodies or by T cells. In this manuscript, we focus on SRs, which are of great concern in children and adolescents and comprise a heterogeneous set of clinical pictures ranging from mild entities such as urticaria/angioedema to potentially life-threatening conditions such as Stevens-Johnson syndrome/toxic epidermal necrolysis. Paracetamol and ibuprofen are the most frequent elicitors of IgE-mediated SRs, although pyrazolones have also been implicated. T cell-mediated reactions are infrequent in children but have been associated with ibuprofen, naproxen, and dipyrone. In this review, we analyze the available literature on SRs in children and adolescents, with emphasis on epidemiological data, mechanisms, and drugs involved, as well as on diagnostic procedures.

Systems biology approaches to enhance our understanding of drug hypersensitivity reactions.

Hypersensitivity drug reactions (HDRs) encompass a wide spectrum of unpredictable clinical entities. They represent an important health problem, affecting people of all ages, and lead to a large strain on the public health system. Here, we summarize experiments that use high-throughput genomics technologies to investigate HDRs. We also introduce the field of systems biology as a relatively recent discipline concerned with the integration and analysis of high-throughput data sets such as DNA microarrays and next-generation sequencing data. We describe previous studies that have applied systems biology techniques to related fields such as allergy and asthma. Finally, we present a number of potential applications of systems biology to the study of HDRs, in order to make the reader aware of the types of analyses that can be performed and the insights that can be gained through their application.

Dermatophagoides pteronyssinus immunotherapy changes the T-regulatory cell activity.

Subcutaneous specific immunotherapy (SCIT) has been shown to modify the Dermatophagoides pteronissinus (DP) allergic response, characterized by generation of Treg cells. However, studies have reported no changes in the proportion of Treg cells after immunotherapy, indicating that the effects may be due to modifications in their regulatory activities. We aimed to determine whether Tregs generated by DP-SCIT can switch the allergic response to tolerant and study the involvement of suppressive cytokines on it. Twenty-four DP-allergic rhinitis patients were recruited, 16 treated with DP-SCIT and 8 untreated. Treg and T effector cells were isolated before and after DP-SCIT, and cocultured in different combinations with α-IL-10, α-TGF-ß blocking antibodies and nDer p 1. Treg cells after DP-SCIT increased Th1 and decreased Th2 and Th9 proliferation. Similarly, they increased IL-10 and decreased IL-4 and IL-9-producing cells. α-IL-10 affected the activity of Treg cells obtained after DP-SCIT only. Finally, DP-specific IgG4 levels, Treg percentage and IL-10 production were correlated after DP-SCIT. These results demonstrate that DP-SCIT induces Treg cells with different suppressive activities. These changes could be mediated by IL-10 production and appear to play an important role in the induction of the tolerance response leading to a clinical improvement of symptoms.

Non-insulin-dependent diabetes: 10-year outcome in relation to initial response to diet and subsequent sulfonylurea therapy.

Thirty-eight non-insulin-dependent diabetic patients within 130% of desirable body weight were given a 100-g oral glucose tolerance test (OGTT) at diagnosis and after at least 1 mo of dietary treatment with energy and carbohydrate restriction. Thirteen "responders" showed an improvement in fasting blood glucose, glucose tolerance, and insulin secretion, with near-normalization of plasma, lactate, pyruvate, free fatty acids, glycerol, and ketone bodies. There were no significant changes in the 25 "non-responders." The responders were, at diagnosis, heavier than the non-responders (75.5 versus 64.3 kg, P less than 0.01). Twenty non-responders subsequently completed a prospective controlled study of glibenclamide, chlorpropamide, and placebo lasting for 16 mo with OGTTs at the end of each 4-mo treatment phase. The results showed that there were no significant differences between the metabolic effects of glibenclamide and chlorpropamide. On active treatment, insulin levels rose coincident with a fall in fasting blood glucose and an improvement in glucose tolerance and near-normalization of plasma lactate, pyruvate, free fatty acids, glycerol, and ketone bodies, all of which relapsed to initial values after placebo. Ten years after the initial study, 29 of the original patients were traced and reviewed and the outcome related to their earlier tests. Twenty-two percent of the responders and 70% of the non-responders were now on insulin (P less than 0.02); the initial insulin response to OGTT at the end of the diet treatment was significantly lower in those subsequently treated with insulin (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Nanoscale separations combined with electrospray ionization mass spectrometry: Sulfonamide determination.

Nanoscale capillary liquid chromatography (nCLC) and capillary zone electrophoresis (CZE) have been combined with quadrupole mass spectrometry via an electrospray ionization (ESI) interface. These methodologies have been applied to the separation and determination of a variety of sulfonamides. CZE/ESI/MS is the more rapid and sensitive technique, but nCLC/ESI/MS shows promise for the analysis of dilute samples. Ultimately, the two techniques provide complementary methods of analysis. The detection limits of these techniques in the full-scan mode are in the low picomole range. Dissociation of the sulfonamides can be induced by increasing the skimmer voltage. This provides a limited means of discriminating between compounds of identical molecular weight but, more important, provides fragments that could be used to confirm the presence of analyte within a sample.

Application of nanoscale packed capillary liquid chromatography (75 µm id) and capillary zone electrophoresis/electrospray ionization mass spectrometry to the analysis of macrolide antibiotics.

Nanoscale separation techniques, nanoscale packed capillary columns (75 µm id), and capillary zone electrophoresis (CZE), on-line with electrospray mass spectrometry (ESI/MS), were applied to the separation of a series of ten macrolide antibiotics. Both techniques use sub-microliter-per-minute flow rates through the analytical column and therefore require an electrospray probe that incorporates coaxial sheath flow. Positive ion electrospray mass spectra of these compounds yielded mainly protonated molecules. Fragmentation to yield structurally significant fragment ions was achieved by collision-induced dissociation (CID) at increased skimmer voltages. Separations were achieved using both techniques, with CZE/ESI/MS showing improved peak shapes and detection limits combined with faster analysis times. Nanoscale packed capillary columns provided better chromatographic resolution and was less susceptible to peak broadening caused by overloading of the analytes.

Application of electrospray mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for molecular weight assignment of peptides in complex mixtures.

Electrospray mass spectrometry (ES/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) were used to provide mass spectra from seven elapid snake venoms. Spectral interpretation was much simpler for MALDI/TOF/MS. ES/MS proved more useful for the provision of molecular weight data for very closely related peptides, but suppression of higher molecular weight compounds was seen to occur during flow injection analysis. MALDI/TOF/MS proved useful for providing a complete picture of the venom, but the low resolution led to obscuring of major ions, and the mass accuracy was poorer for known peptides. Suppression also occurred during MALDI/TOF/MS but could be overcome using alternative matrices because the spectra were very dependent on the choice of matrix. ES/MS and MALDI/TOF/MS provide complementary and confirmatory information such that for the anal sis of complex peptide mixtures (snake venoms), the use of both techniques is desirable.

Characterization of the lower-molecular-mass fraction of venoms from Dendroaspis jamesoni kaimosae and Micrurus fulvius using capillary-electrophoresis electrospray mass spectrometry.

Capillary electrophoresis (CE) with electrospray ionization (ESI) and selected ion-monitoring mass-spectrometric (SIM-MS) detection has been used to provide as much information as possible about the lower molecular-mass fraction (peptides of molecular masses up to 8500 Da) of the venoms of Dendroaspis jamesoni kaimosae (Jameson's Mamba) and Micrurus fulvius (Eastern Coral Snake). Method development was based on the venom of D. jamesoni kaimosae, which contains some previously described peptides, with subsequent application to the completely unknown venom of M. fulvius. CE-ESI-SIM-MS provides a rapid and extremely sensitive method for the detection and molecular-mass determination of peptides present in venoms. It has been utilized to provide molecular masses and thus, by inference, confirmation of the peptide compositions for those toxins which have been previously described in the literature. Our methodology indicates the presence of 83 peptides in the venom of D. jamesoni kaimosae and 49 peptides in the venom of M. fulvius in the molecular-mass range 6000-8500 Da.

The physiological role of liver alcohol dehydrogenase.

1. Yeast alcohol dehydrogenase was used to determine ethanol in the portal and hepatic veins and in the contents of the alimentary canal of rats given a diet free from ethanol. Measurable amounts of a substance behaving like ethanol were found. Its rate of interaction with yeast alcohol dehydrogenase and its volatility indicate that the substance measured was in fact ethanol. 2. The mean alcohol concentration in the portal blood of normal rats was 0.045mm. In the hepatic vein, inferior vena cava and aorta it was about 15 times lower. 3. The contents of all sections of the alimentary canal contained measurable amounts of ethanol. The highest values (average 3.7mm) were found in the stomach. 4. Infusion of pyrazole (an inhibitor of alcohol dehydrogenase) raised the alcohol concentration in the portal vein 10-fold and almost removed the difference between portal and hepatic venous blood. 5. Addition of antibiotics to the food diminished the ethanol concentration of the portal blood to less than one-quarter and that of the stomach contents to less than one-fortieth. 6. The concentration of alcohol in the alimentary canal and in the portal blood of germ-free rats was much decreased, to less than one-tenth in the alimentary canal and to one-third in the portal blood, but detectable quantities remained. These are likely to arise from acetaldehyde formed by the normal pathways of degradation of threonine, deoxyribose phosphate and beta-alanine. 7. The results indicate that significant amounts of alcohol are normally formed in the gastro-intestinal tract. The alcohol is absorbed into the circulation and almost quantitatively removed by the liver. Thus the function, or a major function, of liver alcohol dehydrogenase is the detoxication of ethanol normally present. 8. The alcohol concentration in the stomach of alloxan-diabetic rats was increased about 8-fold. 9. The activity of liver alcohol dehydrogenase is generally lower in carnivores than in herbivores and omnivores, but there is no strict parallelism between the capacity of liver alcohol dehydrogenase and dietary habit. 10. The activity of alcohol dehydrogenase of gastric mucosa was much decreased in two out of the three germ-free rats tested. This is taken to indicate that the enzyme, like gastric urease, may be of microbial origin. 11. When the body was flooded with ethanol by the addition of 10% ethanol to the drinking water the alcohol concentration in the portal vein rose to 15mm and only a few percent of the incoming ethanol was cleared by the liver.

The characterization of snake venoms using capillary electrophoresis in conjunction with electrospray mass spectrometry: Black Mambas.

Capillary electrophoresis has been used in conjunction with electrospray mass spectrometry using both full-scan and selected ion monitoring modes to supply as much information as possible about the venom of Dendroaspis polylepis polylepis (Black Mamba). As an example of the application of capillary electrophoresis/electrospray mass spectrometry (CE/ESI/MS) to the analysis of a complex mixture of small proteins, we have analyzed the venom of Dendroaspis polylepis polylepis using the combined techniques. Both full-scan and selected ion monitoring modes were employed. CE/ESI/MS provides a rapid and extremely sensitive method for molecular weight determination, particularly when selected ion monitoring is employed. It has been utilized to provide sequence confirmation for those toxins which have already been described in the literature. Our methodology indicates the presence of at least 70 peptides in the molecular weight range 6000-9000.

Analysis of sulphonamides using supercritical fluid chromatography and supercritical fluid chromatography-mass spectrometry.

Packed-column supercritical fluid chromatography has been used for the separation of mixtures of sulphonamides on silica and amino-bonded stationary phases utilizing carbon dioxide with methanol modifier as the mobile phase. The effect of modifier concentration, column pressure and modifier identity on retention was also studied. Packed-column supercritical fluid chromatography-mass spectrometry (SFC-MS) of these mixtures utilizing both moving-belt and modified thermospray interfaces was also studied. The identification of sulphamethazine in a spiked porcine kidney extract was performed by SFC-MS using the moving-belt interface.