A. J. Schuerwegh

Flow-assisted allergy diagnosis: current applications and future perspectives

Physicians predominantly rely upon quantification of serum‐specific immunoglobulin E (IgE) and/or skin test to confirm clinically suspected IgE‐mediated allergy. However, for various reasons, identification of the offending allergen(s) and potentially cross‐reactive structures is not always straightforward. Flow‐assisted allergy diagnosis relies upon quantification of alterations in the expression of particular basophilic activation markers. Actually, upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. Currently, the technique has been applied in the investigation of IgE‐mediated allergy caused by classical inhalant allergens, food, Hevea latex, hymenoptera venoms and drugs. It is also appreciated; the technique proves valuable in the diagnosis of non‐IgE‐mediated (anaphylactoid) reactions such drug hypersensitivity and the detection of autoantibodies in certain forms of chronic urticaria. This review will not address immunologic features, characteristics and general pitfalls of flow‐assisted analysis of in vitro‐activated basophils as summarized elsewhere. After a recapitulation of the principles and some specific technical issues of flow‐assisted analysis of in vitro‐activated basophils, we principally focus on the current clinical and research applications of the basophil activation tests. Personal experience of both research groups is provided, where appropriate. Finally, a viewpoint on how the field might evolve in the following years is provided.

In vitro allergy diagnosis: should we follow the flow?

During the last 5 years, an increasing number of studies have demonstrated that flow cytometric quantification of in vitro basophil activation can be a quite performant and reliable tool to measure IgE‐dependent allergen‐specific responses in allergic patients. So far, most assays have used CD63 as a basophil activation marker and native allergen extracts for stimulation. However, other basophil markers and recombinant allergens have recently been introduced. The technique has been applied for the diagnosis of allergy to pollen, house dust mite, food, natural rubber latex, hymenoptera venom and drugs. In addition, the technique has proven to be useful in non‐IgE‐mediated reactions such as hypersensitivity to drugs as well as detection of auto‐antibodies in chronic urticaria. This review will focus on some specific issues: (1) principles of flow cytometric analysis of in vitro‐activated basophils, (2) general technical aspects of the technique (including passive sensitization), (3) clinical applications and (4) recommendations for further development and evaluation of the technique.

Influence of pro-inflammatory (IL-1α, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4) cytokines on chondrocyte function

Abstract Objective: Cytokines produced by inflammatory cells play a pivotal role in synovial inflammation and joint destruction in rheumatoid arthritis. To investigate the influence of pro-inflammatory cytokines (IL-1α, IL-6, TNF-α, IFN-γ) and subsequently the possible beneficial role of an anti-inflammatory cytokine (IL-4) on chondrocyte viability (necrosis/apoptosis), proliferation and nitric oxide (NO) production. Methods: Primary bovine chondrocytes were cultured until monolayers were obtained. Cells were incubated with cytokines (IL-1α, IFN-γ, TNF-α, IL-4, IL-6) at 0.1, 1, 10 and 100ng/mL. After 48h, the viability of the chondrocytes was measured flow cytometrically with propidium iodide. Proliferation was determined by the incorporation of tritiated thymidine. The morphology of the chondrocytes, including presence of apoptotic nuclei, was evaluated by a May-Grunwald–Giemsa staining. In addition, the number of apoptotic chondrocytes was detected flow cytometrically with a TUNEL technique and annexin-V/propidium iodide staining. NO production was evaluated using a spectrophotometric assay, based upon the Griess reaction. Results: The viability and proliferation of bovine chondrocytes decreased after incubation with 100ng/mL IL-1α, TNF-α or IFN-γ. In contrast, incubation of chondrocytes with IL-4 or IL-6 had no influence on the viability or the proliferation of cells. IL-1α was able to enhance NO production in a dose dependent manner. IFN-γ and TNF-α induced NO production only at the highest concentration (100ng/mL), whereas IL-4 and IL-6 did not. There was a dose dependent increase in apoptosis of bovine chondrocytes cultured in the presence of IL-1α and TNF-α. This effect could not be prevented by preincubation with IL-4. Preincubation with IL-4 diminished IL-1α and TNF-α induced NO production and increased proliferation of chondrocytes. In an additional experiment, incubation of human chondrocytes with anti-Fas did not induce apoptosis as measured by annexin-V/propidium iodide staining. Conclusions: Pro-inflammatory cytokines are able to induce apoptosis, whereas IL-4 as an anti-inflammatory cytokine can inhibit the effect of IL-1α and TNF-α on NO production and proliferation of bovine chondrocytes.

Differences in circulating dendritic cell subtypes in cord blood and peripheral blood of healthy and allergic children

Background Different types of circulating dendritic cells have been described. Dendritic cells influence differentiation of naive T lymphocytes into T helper type 1 (Th1) and Th2 effector cells.

Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy

Specific immunoglobulin E (IgE) and commercially available skin prick tests have been demonstrated to be unreliable methods to diagnose pollen‐associated food allergy. To evaluate the predictive value of the basophil activation test (BAT) in pollen‐associated food allergy, the apple‐mediated oral allergy syndrome (OAS) in patients with birch pollinosis was chosen as a representative model.

Flow-Assisted Quantification of In Vitro Activated Basophils in the Diagnosis of Wasp Venom Allergy and Follow-up of Wasp Venom Immunotherapy

Correct identification of the culprit venom is a prerequisite for specific venom immunotherapy (VIT). Despite the efficacy of VIT, issues as how to monitor treatment and when to discontinue maintenance therapy remain to be established.

Validation of a two-color flow cytometric assay detecting in vitro basophil activation for the diagnosis of IgE-mediated natural rubber latex allergy

Background:  IgE‐dependent triggering of basophils not only elicits the release of different mediators but also the up‐regulation of certain markers, e.g. CD63, which can be detected by flow cytometry. We intended to investigate if flow cytometric analysis of basophil activation could be a valuable tool in the diagnosis of latex allergy, and to evaluate if the basophil activation test (BAT) could be helpful in determining the clinical significance of a positive latex IgE in individuals with negative history and negative latex skin test. Additionally we aimed to determine the role of cross‐reactive carbohydrate determinants (CCDs) in causing positive latex IgE without apparent clinical significance.

Selective intestinal decontamination in advanced chronic heart failure: a pilot trial.

Endotoxin, derived from intestinal aerobic Gram‐negative bacilli (AGNB), could be an important monocyte activator in chronic heart failure (CHF). The effect of selective decontamination of the digestive tract (SDD) on intracellular monocyte cytokine production, monocyte CD14 expression, circulating endotoxin and cytokines, and flow‐mediated dilation (FMD) was studied in patients with severe CHF.

Effect of bisphosphonates on viability, proliferation, and dexamethasone-induced apoptosis of articular chondrocytes.

Background: Bisphosphonates (BP) increase bone mass in patients with rheumatoid arthritis and are effective in the prevention and treatment of steroid-induced osteoporosis. However, little is known about their direct effects on chondrocytes. Objectives: To study the influence of BP on articular chondrocytes in vitro and to investigate whether BP can prevent steroid-induced apoptosis of articular chondrocytes. Methods: Bovine articular chondrocytes were cultured and incubated with different concentrations of clodronate, pamidronate, risedronate, or dexamethasone. In the second part of the study, BP were added to the chondrocyte cultures one hour before co-incubation with dexamethasone. Viability and proliferation were evaluated using propidium iodide staining and tritium labelled thymidine incorporation. Apoptosis was measured with annexin V staining or the TUNEL method. Results: Only high concentrations (>10−6 mol/l) of clodronate, pamidronate, and risedronate induced a decrease in the viability and proliferation of chondrocytes. None of the BP at concentrations ranging from 10−12 to 10−3 mol/l induced apoptosis. Growth retardation and apoptosis induced by dexamethasone (10−7 mol/l) was prevented by addition of pamidronate (10−6 mol/l) or risedronate (10−8 or 10−6 mol/l). Conclusion: Bisphosphonates in therapeutic concentrations are safe for articular chondrocytes in vitro. Moreover, pamidronate and risedronate prevent dexamethasone-induced growth retardation and apoptosis of chondrocytes. These findings add evidence for a chondroprotective effect of nitrogen-containing BP, especially in patients treated with corticosteroids.

Anaphylaxis to starch.

24±36 h. We decided to resume low-dose oral prednisone (5 mg daily) together with cetirizine (10 mg daily). This treatment controlled symptoms, but after 1 month the patient stopped taking cetirizine, and urticaria returned. In the attempt to control the patients symptoms, we prescribed a combined therapy of low-dose prednisone, cetirizine, and the leukotriene-receptor antagonist montelukast (Singulair) ± 10 mg daily. The patient did not present any further urticarial rash, and after 1 month cetirizine and prednisone were stopped. Montelukast is now the only drug taken by the patient, and she has not presented any symptom in over 2 months. Recently, a few reports have shown that leukotriene antagonists (za®rlukast or montelukast) (4, 5) or leukotriene-synthesis inhibitors (zileuton) (6) may be effective in controlling CIU, particularly when associated with aspirin intolerance. Moreover, chronic autoreactive urticaria serum has been demonstrated to induce leukotriene production, in addition to histamine release, by basophils (7). Our patients urticaria was characterized by an immediate and prolonged response to intradermal autologous serum and therefore can be considered autoreactive. The favorable clinical response to montelukast provides an indirect demonstration that cysteinyl-leukotrienes play an important role in both early and late skin responses elicited by autologous serum. Leukotriene antagonists may have a role in the treatment of some patients with chronic autoreactive urticaria.