Bruce J. Goldberg

Superoxide generation and its modulation by adenosine in the neutrophils of subjects with asthma

Airway inflammation with neutrophil infiltration may play a role in airway hyperreactivity. Neutrophils may exert their effects through the generation of superoxide O2- anion and other oxygen-derived free radicals. O2- generation by neutrophils has been demonstrated to be modulated by adenosine at physiologic concentrations. Therefore, we have investigated the function of peripheral blood neutrophils with respect to O2- anion generation and its regulation by adenosine in both subjects with asthma and normal subjects and also the relationship between O2- anion generation and airway hyperresponsiveness in subjects with asthma. Purified neutrophils were obtained from eight subjects with stable asthma and seven normal control subjects not taking chronic medications. O2- anion generation in subjects with asthma was significantly higher compared with that of normal subjects after stimulation with either N-formyl-methionyl-leucyl-phenylalanine (mean, 14.8 nmol/10(6) cells for subjects with asthma versus mean, 9.6 nmol/10(6) cells for normal subjects; p less than 0.01) or phorbol myristate acetate (mean, 13.6 nmol/10(6) cells versus mean, 8.1 nmol/10(6) cells; p less than 0.05). Adenosine inhibited N-formyl-methionyl-leucyl-phenylalanine-stimulated O2- anion generation in a dose-related fashion in subjects with asthma and normal subjects to a similar degree. Adenosine had no effect on O2- anion generation after phorbol myristate acetate stimulation. These results indicate that neutrophils from subjects with asthma produce more O2- anion when they are stimulated than do neutrophils from normal subjects and that this difference is not due to adenosine modulation. In subjects with asthma, O2- anion generation correlated with the degree of airway hyperresponsiveness to inhaled methacholine.

IgE antibody response to vertebrate meat proteins including tropomyosin

BACKGROUND Although meat is a main source of proteins in western diets, little information is available regarding allergy to vertebrate meats or the allergens implicated in these reactions. OBJECTIVE To evaluate the in vitro IgE antibody response to different vertebrate meats in suspected meat-allergic subjects, as well as the possible role of tropomyosin in meat allergy and to analyze the cross-reactivity between vertebrate meats and the effect of heating on the IgE-binding to meat proteins. METHODS Fifty-seven sera from suspected meat-allergic subjects were tested by grid blot to extracts of beef, lamb, pork, venison, chicken, and turkey and to four mammalian tropomyosins of different origins. RESULTS Meat-allergic subjects have IgE antibodies to proteins in different mammalian meats (43/57 subjects); cross-reactivity with avian meat was limited: less than 50% (19/43) of meat positive sera reacted to chicken. In contrast, most of the poultry-positive sera also reacted to different mammalian meats. In general, there was stronger IgE reactivity to raw meats in comparison to cooked meats; an exception was six cases in which IgE reactivity to cooked poultry was stronger. Weak IgE reactivity to tropomyosin was detected in only 2/57 sera tested. CONCLUSIONS Suspected meat-allergic subjects have serum IgE directed to meat proteins. In vitro cross-reactivity among mammalian meats appears to be important, while cross-reactivity to poultry is limited indicating mammalian-specific proteins. Although cooking in general denatures meat proteins rendering them less allergenic, in some cases the process of cooking may result in the formation of new allergenic moieties. The muscle protein tropomyosin is not an important vertebrate meat allergen.

Receptor-specific threshold effects of cyclic AMP are involved in the regulation of enzyme release and superoxide production from human neutrophils

We have investigated the sequence of events leading from the activation of adenylate cyclase and increases in intracellular cyclic AMP to the modulation of enzyme release and superoxide production in human neutrophils. In the isolated plasma membrane, adenylate cyclase is activated by both prostaglandin E1 and isoproterenol. In the whole cell only a small increase in cyclic AMP is observed, though in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine a substantial amplification in intracellular cyclic AMP is observed with both isoproterenol and prostaglandin E1. These conditions are relevant to the regulation of cell function, since fMet-Leu-Phe-stimulated superoxide production is inhibited by either prostaglandin E1 or isoproterenol in the absence of methylisobutylxanthine, while enzyme release is inhibited only via the prostaglandin E1 receptor and then only in the presence of methylisobutylxanthine. For enzyme release and superoxide production, the order of potency for three prostaglandins tested was prostaglandin E1 greater than prostaglandin D2 much greater than prostaglandin F2 alpha. Our results suggest that (a) superoxide production is more sensitive to regulation by cyclic AMP than enzyme release, (b) the type of receptor occupied as well as the threshold level of cyclic AMP attained are important to the regulation of enzyme release, and (c) although elevation in cyclic AMP is inhibitory to neutrophil function, phosphodiesterase inhibition is required in addition to adenylate cyclase activation to effect maximal inhibition.

Use of commercial anti–penicillin IgE fluorometric enzyme immunoassays to diagnose penicillin allergy

BACKGROUND The intermittent unavailability of penicilloyl-polylysine since September 2000 has focused interest on commercial anti-penicillin IgE fluorometric enzyme immunoassay (FEIA) tests to evaluate penicillin allergy. There has been no published comparison of commercial anti-penicillin IgE FEIAs and penicillin skin testing performed in the United States. OBJECTIVE To determine whether the current commercial anti-penicillin IgE FEIAs can replace or augment penicillin skin testing and oral challenges when evaluating individuals with a history of penicillin allergy for future therapeutic penicillin tolerance. METHODS A prospective convenience sample of 150 individuals with a history of penicillin allergy were evaluated between January 23, 2007, and August 4, 2009, with both penicillin skin tests and commercial anti-penicillin IgE FEIAs to penicillin G, penicillin V, and amoxicillin. All individuals with a negative penicillin skin test result underwent oral penicillin class antibiotic challenges. All individuals with a positive anti-penicillin IgE FEIA result also underwent oral penicillin class antibiotic challenges. RESULTS Six individuals (4.0%; 95% confidence interval [CI], 0.9% to 7.1%) had positive penicillin skin test results, and none had positive FEIA results. Four individuals (2.7%; 95% CI, 0.1% to 5.3%) had positive FEIA results, and none had positive penicillin skin test results. Three individuals (2.0%; 95% CI, -0.2% to 4.2%) had positive oral challenge results, 1 with hives at 6 hours after challenge and 2 with delayed-onset (at >24 hours) nonurticarial rashes, and none had positive FEIA results. CONCLUSIONS The current commercial anti-penicillin IgE FEIAs are not useful in diagnosing penicillin allergy in patients with remote histories of penicillin allergy. Penicillin skin testing and, if the results are negative, an oral challenge remain the criterion standard tests to determine therapeutic penicillin tolerance.

Categorization of allergic disorders in the new World Health Organization International Classification of Diseases

BackgroundAlthough efforts to improve the classification of hypersensitivity/allergic diseases have been made, they have not been considered a top-level category in the International Classification of Diseases (ICD)-10 and still are not in the ICD-11 beta phase linearization. ICD-10 is the most used classification system by the allergy community worldwide but it is not considered as appropriate for clinical practice. The Systematized Nomenclature of Medicine Clinical Terms (SNOMED CT) on the other hand contains a tightly integrated classification of hypersensitivity/allergic disorders based on the EAACI/WAO nomenclature and the World Health Organization (WHO) may plan to align ICD-11 with SNOMED CT so that they share a common ontological basis.MethodsWith the aim of actively supporting the ongoing ICD-11 revision and the optimal practice of Allergology, we performed a careful comparison of ICD-10 and 11 beta phase linearization codes to identify gaps, areas of regression in allergy coding and possibly reach solutions, in collaboration with committees in charge of the ICD-11 revision.ResultsWe have found a significant degree of misclassification of terms in the allergy-related hierarchies. This stems not only from unclear definitions of these conditions but also the use of common names that falsely imply allergy. The lack of understanding of the immune mechanisms underlying some of the conditions contributes to the difficulty in classification.ConclusionsMore than providing data to support specific changes into the ongoing linearization, these results highlight the need for either a new chapter entitled Hypersensitivity/Allergic Disorders as in SNOMED CT or a high level structure in the Immunology chapter in order to make classification more appropriate and usable.

Constructing a classification of hypersensitivity/allergic diseases for ICD-11 by crowdsourcing the allergist community.

The global allergy community strongly believes that the 11th revision of the International Classification of Diseases (ICD‐11) offers a unique opportunity to improve the classification and coding of hypersensitivity/allergic diseases via inclusion of a specific chapter dedicated to this disease area to facilitate epidemiological studies, as well as to evaluate the true size of the allergy epidemic. In this context, an international collaboration has decided to revise the classification of hypersensitivity/allergic diseases and to validate it for ICD‐11 by crowdsourcing the allergist community. After careful comparison between ICD‐10 and 11 beta phase linearization codes, we identified gaps and trade‐offs allowing us to construct a classification proposal, which was sent to the European Academy of Allergy and Clinical Immunology (EAACI) sections, interest groups, executive committee as well as the World Allergy Organization (WAO), and American Academy of Allergy Asthma and Immunology (AAAAI) leaderships. The crowdsourcing process produced comments from 50 of 171 members contacted by e‐mail. The classification proposal has also been discussed at face‐to‐face meetings with experts of EAACI sections and interest groups and presented in a number of business meetings during the 2014 EAACI annual congress in Copenhagen. As a result, a high‐level complex structure of classification for hypersensitivity/allergic diseases has been constructed. The model proposed has been presented to the WHO groups in charge of the ICD revision. The international collaboration of allergy experts appreciates bilateral discussion and aims to get endorsement of their proposals for the final ICD‐11.

Optimal skin prick wheal size for diagnosis of cat allergy

BACKGROUND The skin prick test is the diagnostic procedure of choice for determination of immediate hypersensitivity. A wheal diameter of 3 mm or larger is generally accepted as the cutoff for a positive test result, although the validity of this assumption has not been rigorously demonstrated. OBJECTIVE To determine the skin prick wheal size that best identifies clinical allergy to cat. METHODS Forty-five patients referred for evaluation of rhinoconjunctivitis underwent determination of atopic status by skin testing using the Greer Dermapik device and a combination of other modalities, including history, in vitro determination of specific IgE level, and nasal challenge with standardized cat pelt extract. Parameters evaluated before and after nasal challenge included symptom score and nasal lavage tryptase and prostaglandin D (PGD2) levels. RESULTS The widely accepted 3-mm wheal for a positive skin test result to cat is highly sensitive but only moderately specific for diagnosis of cat allergy as evaluated by history, specific IgE level, postchallenge symptom score, and tryptase and PGD2 levels. Optimal cutoffs for a positive skin test result to cat based on receiver operating characteristic analysis and 95% positive predictive value were 5.5 mm or greater for each of these parameters. When a true-positive result for cat allergy was defined as a combination of positive history, specific IgE level, postchallenge symptom score, and tryptase and PGD2 levels and a true-negative result as all of these parameters being negative, a 6-mm cutoff was able to distinguish cat allergic from cat nonallergic individuals. CONCLUSION In a potentially allergic population undergoing skin prick testing with the Greer Dermapik using standardized extracts, a 3-mm skin prick wheal will overestimate the presence of cat allergy. A 6-mm wheal appears to distinguish those individuals who are cat allergic from those who are not.

Non-asthmatic respiratory symptomatology.

Because of the common nature of asthma and the attention this disease has been given in recent years, it is important to consider the possibility of other disorders that may present in a similar manner to asthma. Failure to consider alternative diagnoses often leads to inappropriate treatment with medications such as systemic corticosteroids that result in untoward morbidity. This review will discuss several diseases originating not only in the tracheobronchial tree but also in extrapulmonary sites, such as the gastrointestinal tract, that may be mistakenly diagnosed as asthma. Newly described entities such as irritant vocal cord dysfunction and exercise-induced hyperventilation syndrome are discussed in this article, as is new information pertaining to well-established diseases such as Churg-Strauss syndrome and sarcoidosis.

Detection of IgE antibodies to bacitracin using a commercially available streptavidin-linked solid phase in a patient with anaphylaxis to triple antibiotic ointment

BACKGROUND Bacitracin is a commonly used topical antibiotic that has on occasion been reported to cause anaphylaxis. Evidence of the role of bacitracin specific IgE in such reactions has been demonstrated by skin testing. Because of the potential for provoking a systemic reaction by skin testing, it would be advantageous to develop an in vitro test for bacitracin specific IgE. OBJECTIVE To report our experience coupling bacitracin to a solid phase and using it to detect specific IgE to bacitracin by fluorescent enzyme immunoassay. METHODS A patient with a history of recurrent anaphylaxis that developed after application of triple antibiotic ointment to an open wound underwent skin testing with triple antibiotic ointment. Bacitracin was biotinylated and coupled to streptavidin ImmunoCAPs. IgE against bacitracin in the patients serum was detected by fluorescent enzyme immunoassay. RESULTS Topical application of triple antibiotic ointment to intact skin produced a 7 X 8-mm wheal with pseudopods. IgE against bacitracin was detected using biotinylated bacitracin-streptavidin ImmunoCAPs at a level of approximately 0.6 KUA/L and confirmed with ImmunoCAPs using direct coupling of bacitracin to the solid phase. CONCLUSIONS We demonstrate the presence of IgE antibodies to bacitracin in a patient with anaphylaxis to triple antibiotic ointment using a recently described procedure for producing custom allergen solid phases for immunoassay.

A method for the preparation of mononuclear cells devoid of platelet contamination and its application to the evaluation of putative α-receptors in normal and asthmatic subjects

Receptor studies of human mononuclear leukocytes (MNLs) are complicated by the presence of contaminating platelets which have common receptors. A method was devised to produce MNLs free of platelets (less than 1%) and consists of sequential Ficoll-Hypaque gradients, a BSA gradient and a washing step. Lack of platelet contamination was confirmed by the following criteria: (a) microscopic evaluation using fluorescent dyes showed less than 1% platelets; (b) PGE1 stimulation of the leukocyte membrane adenylate cyclase required addition of exogenous GTP while the platelet cyclase did not; (c) immunoblots of the cells and membranes using antibodies strongly reactive against platelet membranes showed no reactivity against MNL membranes; (d) [3H]yohimbine showed no binding in MNL membranes under conditions where substantial binding to platelets was detected. MNLs were viable as judged by dye exclusion. PHA stimulation of lymphocytes was unimpaired. Plasma membranes of MNLs were prepared by brief sonication and fractionation on a sucrose step gradient. Binding studies using 3H-DHE, an alpha-receptor ligand, revealed no binding in MNLs from normal subjects (n = 6). By contrast, studies on cells from subjects with mild asthma with medication appropriately withheld (n = 8) showed low levels of binding (60-300 fmol/10(6) cells). The subtype and functionality of the putative alpha-receptors are being further evaluated.