Cellular Antigen Stimulation Test Research Articles

An atypical presentation of non-IgE-mediated cow’s milk allergy associated with Staphylococcus aureus: A case study

Cow’s milk allergy (CMA) is one of the most common food allergies in the first years of life. CMA is classified as immunoglobulin E (IgE)or non-IgE-mediated reactions. IgE-mediated reactions are typically of immediate onset, with symptoms manifesting within 2 hours after ingestion. However, non-IgE-mediated reactions are delayed and mostly occur between 2 and 48 hours post exposure. This report describes a 17-year-old who presented with a long-standing history of pustular lesions immediately after cow’s milk ingestion. Allergy tests, which included skin-prick test (SPT); specific IgE to cow’s milk; cow’s milk IgE components; and Cellular antigen stimulation test (CAST) yielded insignificant results. However, an oral food challenge resulted in pustular lesions and abdominal pain within 30 and 120 minutes, respectively. A swab from a pustule cultured Staphylococcus aureus. CMA was confirmed and managed with dietary restriction. It is uncommon for non- IgE reactions to occur with immediate symptoms. S. aureus may be associated with atypical skin manifestation of CMA.

Anaphylaxis to food additives

AbstractA variety of additives found in foodstuffs have been associated with triggering of a number of clinical pictures. Hypersensitivity reactions of allergic and non-allergic etiology have been widely described, but are reported to be rare overall. This review presents a selection of food additives for which anaphylactic reactions have been described. These include colorants (e.g., tartrazine, carmine, patent blue), preservatives (e.g., sulfites, sodium benzoate), and thickeners/gelling agents (e.g., carboxymethylcellulose, guar, gum arabic). Possible diagnostic procedures (skin test, specific IgE, cellular antigen stimulation test, oral provocation) are presented. For those affected, avoidance of consumption is facilitated by the mandatory declaration (class name, E‑number/designation) required in the European Union. Only sulfur dioxide and sulfites have to be highlighted as food allergens, depending on the concentration. Due to the limited diagnostic procedures available, it cannot be excluded that the frequency of hypersensitivity to food additives is underestimated.

ORAL ALLERGY SYNDROME (OAS) AKIBAT REAKSI ALERGI MAKANAN

Allergic condition such as itching of the skin, or runny nose and sneezing are widely known, but different case of allergic reaction in the mouth. Are still unknown, one of them is Oral Allergy Syndrome (OAS). Almost everyone knows food allergens such as shrimp, or marine fish, but actually fresh fruits and vegetables also can trigger allergic reactions like OAS in the form of itching or swelling of the lips, tongue, palate, and pharynx. Author intends to provide further information about the OAS in this paper. The mechanism of OAS is a type I, immunoglobulin E-mediated hypersensitivity reaction, common in atopic people with history of atopic rhinitis, bronchial asthma, or urticaria. Food allergen that causes OAS have a cross reaction/ homolog with pollen from protein pathogen respons / PR-10 family, such as Bet v 1 (Birch) and Bet v 2 (Birch). Some OAS allergens from vegetables that homolog with Bet v 1 includes celery (Api g 1) and soybean (Gly m 4), and other allergens from fruits homolog with Bet v 2, such as Ana c1 alergen pineapple. The diagnosis of OAS based on medical history, clinical examination and diagnostic tests includes skin prick test, specific IgE serum, or basophil activation test, and cellular antigen stimulation test with an enzyme-linked immunosorbent assay. Management of OAS consists of non-pharmacological includes information and education to avoid the causes, and give suggestion to eat cooked food. Pharmacologically are prescription of antihistamines or epinephrine injection in case of emergencies. Signs and symptoms of OAS in fact have long been found, but its diagnosis and management are still not widely known. Dentist should know about signs and symptoms of food allergy in the oral mucosa include OAS which caused by fruits and vegetables such as celery, soybean, carrot, apple, pineapple, and strawberry, so finally dentist will provide good management for the patient..

Assessment of Cellular Antigen Stimulation Test in Diagnosis of Drug Allergy

Assessment of Cellular Antigen Stimulation Test in Diagnosis of Drug Allergy

Assessment of sensitization to grape and wine allergens as possible causes of adverse reactions to wine: a pilot study.

BackgroundIn a recently performed survey with 4000 randomly selected persons, 68 (7.2 %) of 948 respondents reported intolerance and/or allergy-like symptoms to wine. The aim of this study was to analyze whether a real sensitization to wine proteins could be confirmed by diagnostic and/or immunological settings.FindingsFor this purpose, 19 subjects with self-reported intolerance to wine of the invited subjects and 10 controls without a history of intolerance participated in an allergological examination (skin prick test, ImmunoCAP for determination of specific IgE antibodies, CAST for testing basophil activation, ImmunoBlot for testing specificity of IgE-antibodies). For the allergological work-up red and white grapes, selected wines, and the purified lipid transfer protein (LTP), a known grape allergen, were used. 7 subjects showed evidence of IgE sensitization to wine or grape extracts, including one control. One participant with symptoms of intolerance showed a positive skin prick test to red grape, a positive ImmunoCAP to grape, a positive cellular antigen stimulation test (CAST) and inhibition of Western blot by removal of cross-reactive carbohydrate determinants (CCD).ConclusionThe presented study focused on the grape protein-related IgE-mediated cause of intolerance to wine (true allergy) and not on other wine components or fining agents (other forms of intolerance). A sensitization to grape and wine proteins was observed in our cohort. In one case, this reactivity could be explained by cross-reactivity to CCD. The results of this pilot study need to be validated in greater cohorts.

Diagnostic value of the ISAC® allergy chip in detecting latex sensitizations

Latex allergy can be diagnosed by different test methods such as IgE quantification, Western blot, cellular antigen stimulation test (CAST), and in vivo methods [e.g. skin prick test (SPT)]. Phadia provides two modern methods using recombinant latex allergens: ImmunoCAP(®) and the Immuno Solid-phase Allergen Chip (ISAC(®)), which enables simultaneous determination of specific IgE against five latex allergens. We compared the diagnostic sensitivity of the ISAC(®) test kit and the conventional Hev b 5-spiked ImmunoCAP(®) latex extract. Forty sera were sampled from subjects with suspected natural rubber latex (NRL) allergy. These patients had positive SPT to NRL extract, positive NRL Western blots, and positive results in the CAST. All sera were analysed using the ISAC(®) and recombinant NRL ImmunoCAP(®) allergens and compared to the results of 20 negative control sera. Only 22 of the 40 subjects (55%) showed positivity to at least one latex allergen on the ISAC(®) (sensitivity ISAC(®) 55%). The sensitivity of the ImmunoCAP(®) latex extract was 70%. The most frequently detected sensitization was against Hev b 6.01 (n=12). When the serum samples were tested with all recombinant ImmunoCAP(®) allergens, three additional sensitizations against latex could be detected compared to the ISAC(®). Microarrays do offer many potential benefits such as elegant simultaneous determination of sensitizations against different NRL allergens with minimal amounts of serum. However, a negative NRL test result should be regarded with caution and at least be confirmed by other in vitro methods.

Basophil activation test in the diagnosis of insect venom allergies

Basophil activation test in the diagnosis of insect venom allergies

Targeting the MHC class II pathway of antigen presentation enhances immunogenicity and safety of allergen immunotherapy

Current s.c. allergen-specific immunotherapy (SIT) leads to amelioration of IgE-mediated allergy, but it requires numerous allergen injections over several years and is frequently associated with severe side-effects. The aim of this study was to test whether modified recombinant allergens can improve therapeutic efficacy in SIT while reducing allergic side-effects. The major cat allergen Fel d 1 was fused to a TAT-derived protein translocation domain and to a truncated invariant chain for targeting the MHC class II pathway (MAT-Fel d 1). The immunogenicity was evaluated in mice, while potential safety issues were assessed by cellular antigen stimulation test (CAST) using basophils from cat-dander-allergic patients. MAT-Fel d 1 enhanced induction of Fel d 1-specific IgG2a antibody responses as well as the secretion of IFN-gamma and IL-2 from T cells. Subcutaneous allergen-specific immunotherapy of mice using the modified Fel d 1 provided stronger protection against anaphylaxis than SIT with unmodified Fel d 1, and MAT-Fel d 1 caused less degranulation of human basophils than native Fel d 1. MAT-Fel d 1 allergen enhanced protective antibody and Th1 responses in mice, while reducing human basophil degranulation. Immunotherapy using MAT-Fel d 1 allergen therefore has the potential to enhance SIT efficacy and safety, thus, shortening SIT. This should increase patient compliance and lower treatment costs.

Cellular Antigen Stimulation Test (CAST) as a Novel, Affordable Laboratory Technique to Assess Potential Hypersensitivity to All Available Antiretroviral Compounds

Cellular Antigen Stimulation Test (CAST) as a Novel, Affordable Laboratory Technique to Assess Potential Hypersensitivity to All Available Antiretroviral Compounds

Cellular Allergen Stimulation Test with Acetylsalicylic Acid-Lysine Is Not a Useful Test to Discriminate between Asthmatic Patients with and without Acetylsalicylic Acid Sensitivity

Background: The aspirin provocation test, considered to be the gold standard in the diagnosis of aspirin sensitivity, may be associated with severe adverse reactions; thus, alternative procedures with a higher safety profile are highly desirable. Although the cellular antigen stimulation test (CAST) has been proposed to be such an alternative, there is limited information about its clinical usefulness. Objective: It was the aim of our study to evaluate the clinical usefulness of CAST in the diagnosis of aspirin sensitivity. Material and Methods: Patients with aspirin-sensitive asthma and/or nasal polyps (n = 40), patients with aspirin-tolerant asthma and/or nasal polyps (n = 13) and healthy volunteers (n = 26) were included. A 2-day, single-blind placebo-controlled oral aspirin provocation test was performed. In vitro release of cysteinyl leukotrienes (Cys-LTs) by peripheral blood leukocytes was measured after stimulation with both stimulation buffer and lysine aspirin (2.5 mg/ml) by CAST. Results: Baseline Cys-LT levels were similar among the 3 groups. After lysine aspirin stimulation, the net increase in Cys-LTs was significantly higher in patients with aspirin sensitivity (median 91 pg/ml, interquartile range 22–206) compared with aspirin-tolerant patients (20 pg/ml, range 0–46) and healthy controls (23 pg/ml, range 0–71; p = 0.004). The assay had a sensitivity of 25%, a specificity of 92.3%, and positive and negative predictive values of 28.7 and 90.7%, respectively. Conclusion: Although the leukocytes of patients with aspirin sensitivity produce higher amounts of Cys-LTs as measured by CAST, the low sensitivity and predictive values limit the clinical usefulness of this test in the diagnosis of aspirin sensitivity.

Effects of a new nutraceutical ingredient on allergen-induced sulphidoleukotrienes production and CD63 expression in allergic subjects

Allergic diseases represent conditions affecting millions of individuals across the world. The objective of this study was to investigate the potential anti-allergic effects of a new nutraceutical ingredient, Pantescal® (Bionap, Italy), contained in different food supplements. Pantescal is a mixture of plant extracts, such as Capparis spinosa, Olea europaea, Panax Ginseng and Ribes nigrum.The study was a randomized, double-blind, placebo controlled design. 60 patients allergic to common aeroallergens were chosen. Allergic patients were divided into two groups: one group was supplemented by Pantescal and the other, using a placebo formulation. Two in vitro tests were performed on blood samples taken from patients before and at 2 h, 2, 3 and 10 days after supplementation: cellular antigen stimulation test (CAST) was used to analyze the amount of sulphidoleukotrienes (SLT) production and flow-cytometric antigen stimulation test (FAST) to measure expression of basophil degranulation marker (CD63) was also performed.CAST showed that after 2 and 3 days, a slight decrease of SLT production was evident but only after 10 days did it become significant with a percentage of inhibition (P.I)=43.3%. FAST revealed that there were no statistical differences for the first 2 days after supplementation although there was an inhibitory trend in the supplemented patients. CD63 expression was significantly reduced after 10 days (P.I.=64.8%).This study suggests that Pantescal is effective in reducing allergic biomarkers such as CD63 protein and SLT in atopic subjects. The higher inhibitory effect on CD63 expression compared to SLT production allows us to hypothesize cell membrane stabilization as the main potential mechanism to explain the observed Pantescal protective effects.

Predictive value of the sulfidoleukotriene release assay in oral allergy syndrome to celery, hazelnut, and carrot.

Patients sensitized to birch pollen frequently suffer from a food allergy to plant foods such as celery, carrots, or hazelnut. One of the main manifestations of birch pollen-related food allergy is the oral allergy syndrome. Skin tests and allergen-specific immunoglobulin (Ig) E determinations are poor predictors of such reactions when assessed by double-blind placebo-controlled food challenge (DBPCFC). To investigate whether a cellular test based on leukotriene release from basophils, the cellular antigen stimulation test in combination with enzyme-linked immunosorbent assay (CAST-ELISA), is predictive of pollen-related food allergy. Birch pollen-sensitized patients with positive DBPCFC to celery (n=21), hazelnut (n=15), and carrot (n=7) underwent skin tests along with determination of specific IgE and CAST-ELISA for the respective allergens. The results were compared with those of 24 birch pollen-sensitized patients with negative open food challenge to celery, hazelnut, and carrot. While skin prick tests had a sensitivity of 85%, 80%, and 29% for commercial extracts of celery, hazelnut, and carrot, respectively, prick testing with self-prepared extracts yielded sensitivities of 100%, 80%, and 100%, respectively. For specific IgE determinations, sensitivities were 71%, 73%, and 57%, respectively, and the respective specificities were 67%, 73%, and 60%. For CAST-ELISA with various sources and doses of allergens, the sensitivity varied from 71% to 95% for celery, 73% to 80% for hazelnut, and 43% to 86% for carrot. The respective specificities were 67% to 92%, 75% to 88%, and 77% to 91%. Analysis of the predictive value of CAST-ELISA with receiver operating characteristic curves showed that the results of the tests were more predictive of pollen-related food allergy than quantitative allergen-specific IgE determinations. CAST-ELISA is more specific than routine diagnostic tests for the diagnosis of pollen-related food allergy to celery, hazelnut, and carrot.

Clinical Ritonavir and Lopinavir Hypersensitivity Confirmed by a Specific In Vitro Cellular Allergen Stimulation Test

A HIV-infected patient treated since eight years with all antiretroviral classes save boosted protease inhibitors, at the time of changing therapy due to an emerging genotyping resistance to non-nucleoside reverse transcriptase inhibitors, experienced repeated episodes of hypersensitivity reactions to all available boosted protease inhibitors. After documenting a combined ritonavir and lopinavir hypersensitivity by means of a specific in vitro cellular antigen stimulation test (CAST), antiretroviral therapy was safely continued with unboosted atazanavir. According to our knowledge, we report the first case of application of the in vitro CAST assay to antiretroviral intolerance, and the subsequent, specific regimen selection in a HIV-infected subject who showed multiple allergy to all boosted protease inhibitors. Further, controlled investigation is strongly needed to implement in vitro allergometric testing in patients with HIV infection and related diseases, who are prone to show unpredictable drug intolerance reactions. In fact, HIV-infected patients may suffer from frequent allergic drug reactions which may be difficult to be systematically recognized (due to the frequent, multiple concurrent pharmacotherapy), while eventual drug rechallenges are expected to be potentially dangerous.

In-vitro-Testverfahren bei Analgetikaintoleranz

Acute intolerance reactions to aspirin or nonsteroidal antiinflammatory drugs are referred to as aspirin-exacerbated respiratory disease (AERD), a nonallergic hypersensitivity reaction with potentially anaphylactoid character. AERD is essentially based on a continuous and progressive subclinical airway inflammation, due to an altered arachidonic acid metabolism. Intake of COX-1 inhibitors induces an excessive production of leukotrienes due to an insufficient and dysbalanced production of prostaglandin E 2 . Clinically, the resulting reactions comprise attacks of rhinitis and asthma. Todays gold standard for confirming the diagnosis of AERD is provocation testing with COX-1 inhibitors. However, due to the potentially severe complications resulting from provocation testing, it should only be performed in centers with suitable emergency facilities. Based on the known pathophysiological mechanisms of the disease during the last decade, many attempts have been made to establish a reliable in vitro testing approach to substitute provocation testing. Current assays can be classified according to the underlying methodology: mediator release assays, tests based on flow cytometry, or combinations of these methods. Accordingly, the present article reviews existing mediator release assays: the cellular antigen stimulation test (CAST), the Analgetikaintoleranztest (AIT), and the aspirin-sensitive patients identification test (ASPItest) as well as tests based on flow-cytometric quantification of CD63 expression (CAST-Flow, Basotest), and the hybrid test system CAST-Combi. Based on the most recent data concerning their sensitivity and specificity, the review provides an overview on the current status of this challenging field of in vitro diagnostic testing.

Alergia a veneno de himenópteros. Novos métodos de diagnóstico.

During the past years, the number of patients with both anaphylaxis to hymenoptera sting and negative IgE detected by conventional methods has been increasing. To obviate this problem new assays have been tried, such as Western blot and in vitro challenge tests involving basophil activation, with either the measurement of histamine and sulphidoleukotrien released (CAST--Cellular Antigen Stimulation Test) or the detection of activation markers on the cell surface with flow cytometric analysis (Flow CAST). In this paper the principles of these assays as well as their diagnostic value in hymenoptera allergy are briefly reviewed.

Investigations of Interactions of Chlormezanone Racemate and Its Enantiomers on Human Keratinocytes and Human Leucoytes in vitro

Chlormezanone is a centrally acting muscle relaxant introduced in human therapy as a racemic substance. The following investigation was performed in order to investigate whether the racemate and both enantiomers differ in their potential cytotoxicty in vitro. We investigated antiproliferative effects and cytotoxicity (PicoGreen and ATP assay) for human HaCaT keratinocytes, production of oxygen radicals (ROS) by human interleukin-3-stimulated leukocytes (Lucigenin assay) and production of sulfoleukotrienes (Cellular Antigen Stimulation Test – CAST) by human leukocytes. In the dosage range of 0.001 to 0.1 mg/ ml chlormezanone, no antiproliferative effects were measured with the racemate and both enantiomers. At 1.0 mg/ml, a decrease of proliferative activity was seen after 48 h incubation time of about 50% for the enantiomers and of about 80% for the racemate (PicoGreen) and 50% (enantiomers) or 21% (racemate) in the ATP assay, respectively. ROS production was significantly inhibited at concentrations ≤0.01 mg/ ml by the racemate and the (+)-enantiomer, whereas the (–)-enantiomer was less effective. There was no stimulation of sulfidoleukotrienes in human leukocytes by chlormezanone. Present data argue for absence of significant cytotoxicity against human HaCaT keratinocytes and a dose-dependent suppression of ROS production by human leukocytes that is not uniform among the racemate and its enantiomers.

Anaphylaktischer Schock nach Ingestion von Pinienkernen

Pine nuts belong to the family of Pineaceae spp. and are widespread in the Mediterranean region. Allergy against pine nuts are rather rare up to now. We report a case of a male patient, showing a shock syndrom with mentalblackout after ingestion of pine nuts. The sensitization to pine nuts was investigated by means of the skin prick test (SPT), the determination of specific IgE antibodies, the cellular antigen stimulation test (CAST) and Westernblots. A self-produced pine nut extract and extracts of several nuts were used in the prick tests as well as in the CAP and CAST experiments. CAP inhibition assays were carried out using pine nut extracts with different protein concentrations. The determination of the characteristic proteins of the pine nut extract was performed by means of the SDS-Page/immunoblotting. The IgE-mediated sensitization to pine nuts was demonstrated clearly. Inhibition assay showed the homogenous inhibition of the specific IgE antibody up to 98% by the self-produced pine nut extract. An increased sulfidoleukotrien release (sLT) (2,400 -2,600 pg/ml) could be shown by the CAST experiments. The sLT values of the controls amounted under the background values. The characteristic pine nut allergen bands of 10, 30 and 47.5 kDa could be detected in the immunoblots. In conclusions, our patient demonstrated an isolated, IgE-mediated type C (according to Pichler's nomenclature) hypersensitivity to pine nuts. Because of the increasing use of pine nut in food, these proteins seem to become an important food allergen with increasing significance.

Assessment of determinants of optimum performance of the CAST®-2000 ELISA procedure

The Cellular Antigen Stimulation Test (CAST®), a recent innovation in the laboratory detection of allergen sensitivity, is a functional assay based on the release of sulphidoleukotrienes (LTs) from allergen-activated circulating basophils. The current study was undertaken to establish optimum determinants of this procedure. The results demonstrate that treatment of blood with preservative-free heparin in polypropylene containers, and removal of erythrocytes by selective lysis with 0.83% ammonium chloride are excellent alternatives to the use of CAST® venepuncture tubes and dextran, respectively. Similarly, decreasing and increasing the leucocyte concentration by 0.5- and 4-fold, respectively, as well as omission of interleukin-3 (IL-3) were also without significant effect. However, treatment of leucocyte suspensions with bacterial endotoxin (>1 μg/ml) activated the release of sulphidoleukotrienes from basophils and/or other types of leucocytes. The findings of this study are clearly relevant, both with respect to cost and performance, when implementing the CAST® in clinical immunology laboratories.

Ficus-benjamina-Allergie ohne Kreuzreaktivität zu Latex

Ficus benjamina is a plant very often used for indoor decorations that can generate allergies, e.g. rhinoconjunctivitis, urticaria and Quinke's edema as well as anaphylactic reactions. We report the case of a patient withhypersensitivity to Ficus benjamina without cross-reactivity to Natural Rubber Latex (NRL) or several fruits, like mango, banana and spinach, but with cross-reactivity to fig. The hypersensitivity to Ficus benjamina was investigated by means of the prick test, the determination of specific IgE antibodies, the cellular antigen stimulation test (CAST) and Westemblots. A self-produced Ficus benjamina extract, a Ficus benjamina extract from Allergopharma, extract of Hevea brasiliensis and extracts of several fruits were used in the prick tests as well as in the CAP and CAST experiments. CAP inhibition assays were carried out to study a suspected cross-reactivity of Ficus benjamina to NRL and several fruits, respectively. The hypersensitivity to Ficus benjamina was clearly demonstrated. Inhibition assay showed the inhibition of the specific IgE antibody up to 95%, and 82% by the self-produced Ficus benjamina extract and the commercial Ficus benjamina, respectively. Neither sensitization nor cross-reactivity to Hevea brasiliensis and several fruit proteins could be found, apart from cross-reactivity to fig. In our patient, an isolated type I hypersensitivity to Ficus benjamina with cross-reactivity to fig could be found, but without cross-reactivity to NRL. The Ficus benjamina protein seems to become an important indoor allergen with increasing significance.

In-vitro-Leukotrienproduktion gemessen im CAST-ELISA über einen Beobachtungszeitraum von 5 Monaten

Background: The Cellular Antigen Stimulation Test (CAST; Buhlmann Laboratory AG, CH) is a diagnostic tool to quantify the In-vitro-Cystinly-Leukotriene production (= CysLT: LTC 4 , LTD 4 , LTE 4 ) from isolated leukocytes.The leukotrienes are responsible for the early inflammatory response with mucosal edema and smooth muscle contraction, clinically resulting in allergic diseases such as asthma and rhinitis. Objective: To evaluate seasonal effects of the in-vitro CysLT release. Population and methods: Blood samples were taken monthly from 9 children and young adults (7 - 18 years; 3 without allergic sensitization, 2 with sensitization to house dust mite and 4 with grasspollen sensitization) from February to June. Leukocytes were separated using Dextran sedimentation and stimulated either with medium alone (negative control), or with an Anti-IgE-receptor-antibody (Anti-IgE-rAK; 0.5 μg/mL; positive control) or with grass-pollen allergen (Six-Gras-MIX, GX1, 20 ng/ml). The CysLT release (LTC 4 , LTD 4 , LTE 4 ) was quantified using ELISA technique (CAST-ELISA, Buhlmann Laboratory AG, Schonenbuch, Switzerland). Results: After stimulation with Anti-IgE-rAK in-vitro CysLT-production increased from March (range: 1163 und 6187 pg/ml) to May (range: 2048 and 7620 pg/ml; median increase 63%; range: 35% -114%; p = 0.012). In response to grasspollen allergen we observed a similar increase from March (158 und 208 pg/ml) to May (337 und 7057 pg/ml; median increase 49.9%; range: 38 - 133%; p = 0.004). The median in-vitro CysLT-concentrations after stimulation with grasspollen was 6137 pg/ml (range: 4249-7620) in grass-pollen sensitized persons and 2130 pg/ml (2048 - 5421) in persons, who are not allergic to grasspollen. Conclusion: We observed a significant higher in-vitro CysLT production in May compared to March. Seasonal effects of the CysLT release must be considered performing longitudinal studies.