Sarita U. Patil

The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease

The basophil activation test (BAT) has become a pervasive test for allergic response through the development of flow cytometry, discovery of activation markers such as CD63 and unique markers identifying basophil granulocytes. Basophil activation test measures basophil response to allergen cross‐linking IgE on between 150 and 2000 basophil granulocytes in <0.1 ml fresh blood. Dichotomous activation is assessed as the fraction of reacting basophils. In addition to clinical history, skin prick test, and specific IgE determination, BAT can be a part of the diagnostic evaluation of patients with food‐, insect venom‐, and drug allergy and chronic urticaria. It may be helpful in determining the clinically relevant allergen. Basophil sensitivity may be used to monitor patients on allergen immunotherapy, anti‐IgE treatment or in the natural resolution of allergy. Basophil activation test may use fewer resources and be more reproducible than challenge testing. As it is less stressful for the patient and avoids severe allergic reactions, BAT ought to precede challenge testing. An important next step is to standardize BAT and make it available in diagnostic laboratories. The nature of basophil activation as an ex vivo challenge makes it a multifaceted and promising tool for the allergist. In this EAACI task force position paper, we provide an overview of the practical and technical details as well as the clinical utility of BAT in diagnosis and management of allergic diseases.

Peanut oral immunotherapy transiently expands circulating Ara h 2-specific B cells with a homologous repertoire in unrelated subjects.

BACKGROUND Peanut oral immunotherapy (PNOIT) induces persistent tolerance to peanut in a subset of patients and induces specific antibodies that might play a role in clinical protection. However, the contribution of induced antibody clones to clinical tolerance in PNOIT is unknown. OBJECTIVE We hypothesized that PNOIT induces a clonal, allergen-specific B-cell response that could serve as a surrogate for clinical outcomes. METHODS We used a fluorescent Ara h 2 multimer for affinity selection of Ara h 2-specific B cells and subsequent single-cell immunoglobulin amplification. The diversity of related clones was evaluated by means of next-generation sequencing of immunoglobulin heavy chains from circulating memory B cells with 2x250 paired-end sequencing on the Illumina MiSeq platform. RESULTS Expression of class-switched antibodies from Ara h 2-positive cells confirms enrichment for Ara h 2 specificity. PNOIT induces an early and transient expansion of circulating Ara h 2-specific memory B cells that peaks at week 7. Ara h 2-specific sequences from memory cells have rates of nonsilent mutations consistent with affinity maturation. The repertoire of Ara h 2-specific antibodies is oligoclonal. Next-generation sequencing-based repertoire analysis of circulating memory B cells reveals evidence for convergent selection of related sequences in 3 unrelated subjects, suggesting the presence of similar Ara h 2-specific B-cell clones. CONCLUSIONS Using a novel affinity selection approach to identify antigen-specific B cells, we demonstrate that the early PNOIT-induced Ara h 2-specific B-cell receptor repertoire is oligoclonal and somatically hypermutated and shares similar clonal groups among unrelated subjects consistent with convergent selection.

Immunology in the Clinic Review Series; focus on allergies: basophils as biomarkers for assessing immune modulation

OTHER THEMES PUBLISHED IN THIS IMMUNOLOGY IN THE CLINIC REVIEW SERIES

The importance of vancomycin in drug rash with eosinophilia and systemic symptoms (DRESS) syndrome.

Drug rash with eosinophilia and systemic symptoms (DRESS) syndrome characterized by fever, rash, eosinophilia, atypical lymphocytes, and multiorgan involvement has a significant mortality. Inpatient vancomycin use is increasing and appears to be emerging as an important etiology of DRESS syndrome. This study highlights the importance of vancomycin as a cause of DRESS syndrome. We reviewed all cases of DRESS syndrome among inpatients consulted by the Allergy & Immunology service at Massachusetts General Hospital (MGH) from July 2009 through December 2010. We also reviewed the use of inpatient parenteral vancomycin over the past 4 years at MGH. Six patients fulfilled clinical criteria for DRESS syndrome, including rash, fever, eosinophilia, and hepatitis, with five (83%) having vancomycin as the attributable cause. Onset of symptoms varied from 12 days to 4 weeks after start of vancomycin treatment. Systemic findings included atypical lymphocytes, lymphadenopathy, nephritis, hypotension, tachycardia, and pharyngitis. Treatment with corticosteroids was required in three cases. Recurrence of peripheral eosinophilia was a marker of disease relapse. In three of the five patients (60%), elevated human herpesvirus 6 (HHV6) IgG titers correlated with greater systemic involvement and prolonged time to resolution. MGH pharmacy records indicate a progressive increase in the number of patients treated with parenteral vancomycin over the last 4 years. Causative agents for DRESS syndrome in an inpatient setting is likely different from that seen in the general population. With increasing use of vancomycin, we are likely to see more cases of DRESS syndrome caused by vancomycin. Recognition of vancomycin as a common cause of inpatient DRESS syndrome is important.

Vitamins A and D have antagonistic effects on expression of effector cytokines and gut-homing integrin in human innate lymphoid cells

Retinoic acid (RA), the main biologically active metabolite of vitamin A, is known to promote gut homing of lymphocytes, as well as various regulatory and effector immune responses. In contrast, the active form of vitamin D, 1,25‐dihydroxyvitamin D3 (1,25D3), is predominantly immunosuppressive. Little is known about the direct effects of these vitamins on the recently identified innate lymphoid cells (ILCs).

The usefulness of biomarkers of airway inflammation in managing asthma.

The goal of managing asthma is to maintain disease control. Current approaches to assessment of control do not include measurement of airway inflammation. This study was designed to assess the usefulness of biomarkers of airway inflammation in guiding asthma management decisions. A literature review was performed. Bronchial biopsy is a direct measure of airway inflammation but not practical for routine use. Enumeration of sputum eosinophils is very useful in guiding changes in controller medication to decrease asthma exacerbations, whereas measurement of exhaled nitric oxide has not proven to be useful in this regard. Serial measurement of airway hyperreactivity as a guide to asthma management yields inconclusive results. Use of indirect stimuli for bronchial challenge offers both practical and theoretical advantages in the assessment of airway hyperreactivity. Data on the analysis of exhaled breath condensate have not yet been studied adequately in guiding management decisions. Enumeration of sputum cell counts appears to be the most useful biomarker of airway inflammation in guiding asthma management decisions. Combined approaches using simple methods of measuring airway hyperreactivity and obtaining sputum samples hold promise for the future, particularly if rapid analysis of cellular products in sputum can be developed.

BATting above average: basophil activation testing for peanut allergy.

It could be argued that IgE-mediated peanut allergy has become a significant public health issue over the past decade as much because of the growing public awareness of food allergy as because of its true increased prevalence. This awareness and the broad availability of serum IgE testing have created the present situation, in which a pediatric specialist in the United States is more likely to be consulted on the question of peanut allergy for a patient with no convincing history of ingestion than one with a clinical history of an IgE-mediated reaction. In those cases for which the pretest probability is reduced to the relevant population prevalence, conventional testing often leaves the patient at an equivocal diagnostic point. In these patients accurate diagnosis requires an oral food challenge (OFC). The consequences of this heightened public awareness are significant to individual patients and their families, who bear the brunt of a potential misdiagnosis and its attendant anxiety, degradation of quality of life, unnecessary medical expenses, and avoidance measures. The costs are also significant for the health care system. The demand for OFCs, which are necessary to make an accurate diagnosis in the face of equivocal test results, greatly exceeds the capacity of specialists who are prepared to offer them. Part of the solution to this problem must be more judicious use of serum IgE testing. The current guidelines specifically recommend that children in the general population should not be routinely tested for food allergy, including to the most common allergens, such as peanut, milk, or egg, and that even when there is a higher likelihood of allergy (eg, sibling of child with peanut allergy), the evidence is not strong enough to recommend testing for IgE. When such testing is done, it should be with a plan to confirm equivocal results with OFCs. From a practical perspective, differentiation of peanut sensitization and allergy will remain an issue, even if testing is used less broadly. Nonetheless, there will still be many equivocal outcomes. For example, as a history of reactivity becomes more

Pathogen induced chemo-attractant hepoxilin A3 drives neutrophils, but not eosinophils across epithelial barriers

Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator, hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces, eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids, however, it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study, neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient, whereas only eosinophils migrated toward a prostaglandin D2 (PGD2) gradient. Interestingly, while pathogen induced neutrophil trans-epithelial migration was substantial, pathogen induced eosinophil trans-epithelial migration was not observed. Further, gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers, whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection, HXA3 does not appear to exhibit chemotactic activity toward eosinophils.

Data-driven programmatic approach to analysis of basophil activation tests

Conventional data analysis of flow cytometry‐based basophil activation testing requires repetitive, labor‐intensive analysis that hampers efforts to standardize testing for clinical applications. Using an open‐source platform, we developed and implemented a programmatic approach to the analysis of the basophil activation test (BAT) by flow cytometry.

Basophil activation testing in diagnosis and monitoring of allergic disease – an overview

The nature of basophil activation as an ex vivo challenge makes it a multifaceted and promising tool for the allergist. Through the development of flow cytometry, discovery of activation markers such as CD63 and markers identifying basophil granulocytes, the basophil activation test (BAT) has become a pervasive test. BAT measures basophil response to allergen crosslinking IgE on between 150 and 2,000 basophil granulocytes with remarkable analytical sensitivity in < 0.1 ml fresh blood. Dichotomous activation is assessed as the fraction of reacting basophils. In patients with food-, insect venom-, and drug allergy and patients with chronic urticaria BAT can be part of the diagnostic evaluation in addition to history, skin prick testing, and specific IgE determination. BAT may also be helpful in determining the clinically relevant allergen. Basophil sensitivity may be used to monitor patients on allergen immunotherapy, anti-IgE treatment, or in the natural resolution of allergy. The test may use fewer resources and be more reproducible than oral, sting, nasal or bronchial challenge testing. BAT may be useful before challenge testing as it is less stressful for the patient and avoids severe allergic reactions. It may be useful before challenge testing. An important next step is to standardize BAT and make it available in diagnostic laboratories. This article provides an overview of the practical and technical details as well as the utility of BAT in diagnosis and management of allergic diseases.