The University of Southampton
University of Southampton Institutional Repository

Measurement of mast cell and basophil activation in vitro as means for investigation of drug hypersensitivity

Measurement of mast cell and basophil activation in vitro as means for investigation of drug hypersensitivity
Measurement of mast cell and basophil activation in vitro as means for investigation of drug hypersensitivity
Allergic drug reactions to drugs are becoming more frequent throughout the world and are among the most common and serious form of immuno-pathological process in modern clinical medicine. These reactions can lead to life-threatening anaphylaxis which can occur within minutes of receiving the drug. Symptoms provoked by the explosive release of mediators from mast cells and basophils release may include itchiness, angioedema, difficulty breathing and circulatory collapse. There are few reliable laboratory tests available to identify those with drug hypersensitivity who may be at risk of anaphylactic shock, or even to establish if such a reaction has occurred. Confirmation of drug hypersensitivity by skin testing or oral challenge can put the patient at risk of serious reaction, and as such are expensive and performed at few centres.

The aim of the study was to seek to develop new tests for drug allergy based on assessment of mast cell or basophil activation either in vitro or in vivo. Patients were recruited who were suspected of having suffered an allergic reaction to medicine and who were undergoing skin testing or oral drug challenge. Blood was collected before and following the challenge, the nature of symptoms provoked were recorded, and clinical history and demographic data obtained. The activation of basophils in vitro in response to the drug was assessed by measurement of histamine release by immunoassay or up-regulation of CD63 by flow cytometry. In parallel studies, cells of a rat basophil leukaemia cell line (RBL-703-21) that had been transfected with the α chain of the human IgE receptor, were passively sensitised with serum and challenged with the drug implicated. The release of β-hexosaminidase in cell supernatants was measured to determine the degree of drug-induced cell activation. Mast cell tryptase was successfully purified from human lung tissue, and preliminary attempts made to optimise the detection of the basophil product basogranulin by dot blotting with specific antibodies BB1 and BB5, but time did not allow measurement of either of these markers of allergic reaction in the patients studied.

Skin weals were provoked in some 40% of patients following skin testing, though other symptoms were frequently recorded in the presence or even absence of a weal. These included hives, post-nasal drip, abdominal pain, vomiting, cough, shortness of breath, wheeze and stridor, and there was considerable heterogeneity in clinical responses. In patients with a positive test outcome on skin or oral challenge, raised levels of serum IgE specific to that drug were commonly observed (where the test was available). Increased expression of CD63 was provoked by in vitro challenge of basophils in a minority of patients who responded on in vivo challenge, but no histamine release into cell supernatants was detected where this was examined. Drug challenge of RBL-703-21 cells passively sensitised with patient serum resulted in the release of only a small quantities of β-hexosaminidase, and no evidence was found for direct activation of cells by the drugs themselves. There was, however, significantly higher net release of β-hexosaminidase from patients with positive skin reactions to the neuromuscular blocking agent rocuronium than in those who did not respond on skin challenge.

The reliable diagnosis of drug hypersensitivity remains a formidable challenge, and there is no ready replacement of skin or oral challenge of patients with drugs suspected of causing a reaction. The present study indicates some of the difficulties as well as ways forward in developing new laboratory tests. In vitro challenge of blood basophils or passively sensitised cells of mast cell or basophil-like cell lines hold some promise, but it will be crucial for the sensitivity of such procedures to be increased and optimised with a range of drugs. Assays for mast cell or basophil products in the circulation of patients experiencing reactions should also be valuable as diagnostic tools.
University of Southampton
Ludwig, Desiree
6b5252b2-c65c-4909-9c83-371a11001250
Ludwig, Desiree
6b5252b2-c65c-4909-9c83-371a11001250
Walls, Andrew
aaa7e455-0562-4b4c-94f5-ec29c74b1bfe
Zhou, Xiaoying
84558a96-3129-44de-b295-869d9ee4d19f
Eren, Efrem
ac449fc8-4ae2-4efd-ad91-9dcea3f355e2

Ludwig, Desiree (2015) Measurement of mast cell and basophil activation in vitro as means for investigation of drug hypersensitivity. University of Southampton, Doctoral Thesis, 141pp.

Record type: Thesis (Doctoral)

Abstract

Allergic drug reactions to drugs are becoming more frequent throughout the world and are among the most common and serious form of immuno-pathological process in modern clinical medicine. These reactions can lead to life-threatening anaphylaxis which can occur within minutes of receiving the drug. Symptoms provoked by the explosive release of mediators from mast cells and basophils release may include itchiness, angioedema, difficulty breathing and circulatory collapse. There are few reliable laboratory tests available to identify those with drug hypersensitivity who may be at risk of anaphylactic shock, or even to establish if such a reaction has occurred. Confirmation of drug hypersensitivity by skin testing or oral challenge can put the patient at risk of serious reaction, and as such are expensive and performed at few centres.

The aim of the study was to seek to develop new tests for drug allergy based on assessment of mast cell or basophil activation either in vitro or in vivo. Patients were recruited who were suspected of having suffered an allergic reaction to medicine and who were undergoing skin testing or oral drug challenge. Blood was collected before and following the challenge, the nature of symptoms provoked were recorded, and clinical history and demographic data obtained. The activation of basophils in vitro in response to the drug was assessed by measurement of histamine release by immunoassay or up-regulation of CD63 by flow cytometry. In parallel studies, cells of a rat basophil leukaemia cell line (RBL-703-21) that had been transfected with the α chain of the human IgE receptor, were passively sensitised with serum and challenged with the drug implicated. The release of β-hexosaminidase in cell supernatants was measured to determine the degree of drug-induced cell activation. Mast cell tryptase was successfully purified from human lung tissue, and preliminary attempts made to optimise the detection of the basophil product basogranulin by dot blotting with specific antibodies BB1 and BB5, but time did not allow measurement of either of these markers of allergic reaction in the patients studied.

Skin weals were provoked in some 40% of patients following skin testing, though other symptoms were frequently recorded in the presence or even absence of a weal. These included hives, post-nasal drip, abdominal pain, vomiting, cough, shortness of breath, wheeze and stridor, and there was considerable heterogeneity in clinical responses. In patients with a positive test outcome on skin or oral challenge, raised levels of serum IgE specific to that drug were commonly observed (where the test was available). Increased expression of CD63 was provoked by in vitro challenge of basophils in a minority of patients who responded on in vivo challenge, but no histamine release into cell supernatants was detected where this was examined. Drug challenge of RBL-703-21 cells passively sensitised with patient serum resulted in the release of only a small quantities of β-hexosaminidase, and no evidence was found for direct activation of cells by the drugs themselves. There was, however, significantly higher net release of β-hexosaminidase from patients with positive skin reactions to the neuromuscular blocking agent rocuronium than in those who did not respond on skin challenge.

The reliable diagnosis of drug hypersensitivity remains a formidable challenge, and there is no ready replacement of skin or oral challenge of patients with drugs suspected of causing a reaction. The present study indicates some of the difficulties as well as ways forward in developing new laboratory tests. In vitro challenge of blood basophils or passively sensitised cells of mast cell or basophil-like cell lines hold some promise, but it will be crucial for the sensitivity of such procedures to be increased and optimised with a range of drugs. Assays for mast cell or basophil products in the circulation of patients experiencing reactions should also be valuable as diagnostic tools.

Text
Masterthesis - Measurement of mast cell and basophil activation in vitro as means for investigation of drug hypersensitivity - Desiree Ludwig - Version of Record
Available under License University of Southampton Thesis Licence.
Download (2MB)

More information

Submitted date: May 2015
Published date: May 2015

Identifiers

Local EPrints ID: 416617
URI: https://eprints.soton.ac.uk/id/eprint/416617
PURE UUID: 3789966e-27c2-4da1-95bf-528a17f922d8

Catalogue record

Date deposited: 03 Jan 2018 17:30
Last modified: 13 Mar 2019 19:06

Export record

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×