The cAMP assay: A functional in vitro alternative to the in vivo Histamine Sensitization test

Hoonakker, Marieke; Ruiterkamp, Nicole; Hendriksen, Coenraad

doi:10.1016/j.vaccine.2009.11.009

Cited by 18 publications

(24 citation statements)

References 27 publications

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“…38 Briefly, cells were kept in serum-free medium for 24 h, exposed to stimulators and extracted with ethanol (98%, −20°C). Phosphodiesterase inhibitors were omitted from incubations unless stated otherwise.…”

Section: Methodsmentioning

confidence: 99%

Impact of human autoantibodies on β1-adrenergic receptor conformation, activity, and internalization

Bornholz

Weidtkamp‐Peters

Schmitmeier

et al. 2012

Cardiovascular Research

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AimsAutoantibodies against second extracellular loops of β1-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function.Methods and resultsIgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to β1-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed.Conclusions(i) Autoantibodies trigger conformation changes in the β1-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels.

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Section: Methodsmentioning

confidence: 99%

Impact of human autoantibodies on β1-adrenergic receptor conformation, activity, and internalization

Bornholz

Weidtkamp‐Peters

Schmitmeier

et al. 2012

Cardiovascular Research

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“…This approach might be of interest for pertussis vaccine safety testing since not all effector mechanisms of PTx are completely understood, nor are all of its clinical effects [17,18]. Current in vitro alternatives under development to replace the HIST focus mainly on the binding and entry of PTx and consequent ADP-ribosylation of G i proteins [14][15][16]. Although the enzymatic activity is a principal activity of PTx involved in many of its biological effects, over the past years several studies have described PTx effects independent of this enzymatic activity [21,22,27,28].…”

Section: Discussionmentioning

confidence: 99%

“…Several in vitro alternatives are currently under development, i.e. a cAMP assay in rat A10 cells [14], a fetuin binding assay [15,16] and an enzymatic HPLC method [15]. These assays reflect known activities of PTx, i.e.…”

Section: Introductionmentioning

confidence: 99%

Identification of biomarkers to detect residual pertussis toxin using microarray analysis of dendritic cells

Vaessen

Verkoeijen

Vandebriel

et al. 2013

Vaccine

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“…Several in vitro alternatives have been developed as possible replacements for the HIST or the CHO clustering assay. [11][12][13][14] They are based on the assumed mechanism underlying increased histamine sensitization and CHO cell clustering, i.e., PT-induced ADP-ribosylation of G proteins. However, these in vitro alternatives assume that this mechanism is the only effect of PT that should be taken into consideration.…”

Section: Clinical Effects Of Pertussis Toxin Should Be the Basis For mentioning

confidence: 99%

Toward a mechanism-based in vitro safety test for pertussis toxin

Vaessen¹,

Bruysters

Vandebriel

et al. 2014

Human Vaccines & Immunotherapeutics

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Pertussis vaccines are routinely administered to infants to protect them from whooping cough. Still, an adequate safety test for pertussis toxin (PT), one of the main antigens in these vaccines, is not available. The histamine sensitization test is currently the only assay accepted by regulatory authorities to test for the absence of active PT in vaccines. This is however, a lethal animal test with poor reproducibility. In addition, it is not clear whether the assumed underlying mechanism, i.e., ADP-ribosylation of G proteins, is the only effect that should be considered in safety evaluation of PT. The in vitro safety test for PT that we developed is based on the clinical effects of PT in humans. For this, human cell lines were chosen based on the cell types involved in the clinical effects of PT. These cell lines were exposed to PT and analyzed by microarray. In this review, we discuss the clinical effects of PT and the mechanisms that underlie them. The approach taken may provide as an example for other situations in which an in vitro assay based on clinical effects in humans is required.

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The cAMP assay: A functional in vitro alternative to the in vivo Histamine Sensitization test

Cited by 18 publications

References 27 publications

Impact of human autoantibodies on β1-adrenergic receptor conformation, activity, and internalization

Impact of human autoantibodies on β1-adrenergic receptor conformation, activity, and internalization

Identification of biomarkers to detect residual pertussis toxin using microarray analysis of dendritic cells

Toward a mechanism-based in vitro safety test for pertussis toxin

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