| 1. |
- Kack, Ulrika, et al.
(author)
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Nasal upregulation of CST1 in dog-sensitised children with severe allergic airway disease
- 2021
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In: ERJ Open Research. - : European Respiratory Society (ERS). - 2312-0541. ; 7:2
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Journal article (peer-reviewed)abstract
- Background: The clinical presentation of children sensitised to dog dander varies from asymptomatic to severe allergic airway disease, but the genetic mechanisms underlying these differences are not clear. The objective of the present study was to investigate nasal transcriptomic profiles associated with dog dander sensitisation in school children and to reveal clinical symptoms related with these profiles. Methods: RNA was extracted from nasal epithelial cell brushings of children sensitised to dog dander and healthy controls. Blood sample analyses included IgE against dog dander, dog allergen molecules, other airborne and food allergens, basophil activation and white blood cell counts. Clinical history of asthma and rhinitis was recorded, and lung function was assessed (spirometry, methacholine provocation and exhaled nitric oxide fraction). Results: The most overexpressed gene in children sensitised to dog dander compared to healthy controls was CST1, coding for Cystatin 1. A cluster of these children with enhanced CST1 expression showed lower forced expiratory volume in 1 s, increased bronchial hyperreactivity, pronounced eosinophilia and higher basophil allergen threshold sensitivity compared with other children sensitised to dog dander. In addition, multi-sensitisation to lipocalins was more common in this group. Conclusions: Overexpression of CST1 is associated with more severe allergic airway disease in children sensitised to dog dander. CST1 is thus a possible biomarker of the severity of allergic airway disease and a possible therapeutic target for the future treatment of airborne allergy.
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| 2. |
- Røisgård, Solveig, et al.
(author)
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Basophil allergen threshold sensitivity to casein (casein-specific CD-sens) predicts allergic reactions at a milk challenge in most but not all patients
- 2024
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In: Immunity, Inflammation and Disease. - : John Wiley & Sons. - 2050-4527. ; 12:5
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Journal article (peer-reviewed)abstract
- Background: The basophil activation test is an emerging clinical tool in the diagnosis of cow's milk allergy (CMA). The aim was to assess the association between the basophil allergen threshold sensitivity to the major milk protein casein (casein-specific CD-sens), the levels of milk- and casein-specific Immunoglobulin E antibodies (IgE-ab), and the severity of allergic reactions at milk challenges.Methods: We enrolled 34 patients aged 5–15 (median 9) years who underwent a double-blind placebo-controlled milk-challenge (DBPCMC) as screening before inclusion in an oral immunotherapy study for CMA. The severity of the allergic reaction at the DBPCMC was graded using Sampson's severity score. Venous blood was drawn before the DBPCMC. Milk- and casein-specific IgE-ab were analyzed. Following in vitro stimulation of basophils with casein, casein-specific CD-sens, was determined.Results: Thirty-three patients completed the DBPCMC. There were strong correlations between casein-specific CD-sens and IgE-ab to milk (rs = 0.682, p <.001), and between casein-specific CD-sens and IgE-ab to casein (rs = 0.823, p <.001). There was a correlation between the severity of the allergic reaction and casein-specific CD-sens level (rs = 0.395, p =.041) and an inverse correlation between casein-specific CD-sens level and the cumulative dose of milk protein to which the patient reacted at the DBPCMC (rs = −0.418, p =.027). Among the 30 patients with an allergic reaction at the DBPCMC, 67% had positive casein-specific CD-sens, 23% had negative casein-specific CD-sens, and 10% were declared non-responders.Conclusion: Two thirds of those reacting at the DBPMC had positive casein-specific CD-sens, but reactions also occurred despite negative casein-specific CD-sens. The association between casein-specific CD-sens and the severity of the allergic reaction and cumulative dose of milk protein, respectively, was moderate.
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| 3. |
- Aljadi, Zenib, et al.
(author)
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A novel tool for clinical diagnosis of allergy operating a microfluidic immunoaffinity basophil activation test technique
- 2019
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In: Clinical Immunology. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1521-6616 .- 1521-7035. ; 209
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Journal article (peer-reviewed)abstract
- The Basophil Activation Test (BAT) is a valuable allergy diagnostic tool but is time-consuming and requires skilled personnel and cumbersome processing, which has limited its clinical use. We therefore investigated if a microfluidic immunoaffinity BAT (miBAT) technique can be a reliable diagnostic method. Blood was collected from allergic patients and healthy controls. Basophils were challenged with negative control, positive control (anti-FccRI), and two concentrations of a relevant and non-relevant allergen. CD203c and CD63 expression was detected by fluorescent microscopy and flow cytometry. In basophils from allergic patients the CD63% was significantly higher after allergen activation as compared to the negative control (p < .0001-p = .0004). Activation with non-relevant allergen showed equivalent CD63% expression as the negative control. Further, the miBAT data were comparable to flow cytometry. Our results demonstrate the capacity of the miBAT technology to measure different degrees of basophil allergen activation by quantifying the CD63% expression on captured basophils.
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| 4. |
- Aljadi, Zenib, et al.
(author)
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Activation of Basophils Is a New and Sensitive Marker of Biocompatibility in Hemodialysis
- 2014
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In: Artificial Organs. - : Wiley. - 0160-564X .- 1525-1594. ; 38:11, s. 945-953
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Journal article (peer-reviewed)abstract
- The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood-membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fc epsilon receptor I (Fc epsilon RI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-Fc epsilon RI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis.
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| 5. |
- Aljadi, Zenib, et al.
(author)
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Altered basophil function in patients with chronic kidney disease on hemodialysis
- 2017
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In: Clinical Nephrology. - : DUSTRI-VERLAG DR KARL FEISTLE. - 0301-0430. ; 88:2, s. 86-96
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Journal article (peer-reviewed)abstract
- Aims: Chronic kidney disease (CKD) leads to impairment of immune cell function. Given the potential role of basophils in the pathogenesis of CKD, we aimed to study the basophil responsiveness towards microbial antigen exposure, judged as adhesion molecule expression and degranulation, in CKD patients on hemodialysis. Materials and methods: We selected markers linked to two crucial biological phases: the transmigration and degranulation processes, respectively. For the transmigration process, we selected the adhesion molecules CD11b, active CD11b epitope, and CD62L and for the degranulation process CD203c (piecemeal degranulation marker), CD63 (degranulation marker), and CD300a (inhibitory marker of degranulation). We measured basophil responsiveness after stimulation of different activation pathways in basophils using lipopolysaccharide (LPS), peptidoglycan (PGN), formyl-methyinoyl-leucyl-phenylalanine (fMLP), and anti-FceRI-ab. Results: The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared to matched healthy controls, but no differences were observed after activation by anti-Fc.RI. CD300a expression was significantly higher in patients following activation by fMLP and anti-Fc.RI, and the active epitope CD11b expression was significantly higher in patients after LPS activation. In addition, we found that CD62L was not shed from the cell surface after activation with LPS and fMLP. A slight downregulation was noted after activation with anti-Fc.RI in healthy controls. Conclusion: Together, these data demonstrate that basophil functions related to adhesion and degranulation are altered in CKD patients on hemodialysis, which indicates a potential role for the basophil in the pathogenesis of complications related to infections.
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| 6. |
- Aljadi, Zenib, et al.
(author)
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Microfluidic Immunoaffinity Basophil Activation Test for Point-of-Care Allergy Diagnosis
- 2019
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In: Journal of Applied Laboratory Medicine (JALM). - : American Association for Clinical Chemistry. - 2475-7241 .- 2576-9456. ; 4:2, s. 152-163
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Journal article (peer-reviewed)abstract
- Background: The flow cytometry-based basophil activation test (BAT) is used for the diagnosis of allergic response. However, flow cytometry is time-consuming, requiring skilled personnel and cumbersome processing, which has limited its use in the clinic. Here, we introduce a novel microfluidic-based immunoaffinity BAT (miBAT) method. Methods: The microfluidic device, coated with anti-CD203c, was designed to capture basophils directly from whole blood. The captured basophils are activated by anti-FceRI antibody followed by optical detection of CD63 expression (degranulation marker). The device was first characterized using a basophil cell line followed by whole blood experiments. Weevaluated the device with ex vivo stimulation of basophils in whole blood from healthy controls and patients with allergies and compared it with flow cytometry. Results: The microfluidic device was capable of capturing basophils directly from whole blood followed by in vitro activation and quantification of CD63 expression. CD63 expression was significantly higher (P = 0.0002) in on-chip activated basophils compared with nonactivated cells. The difference in CD63 expression on anti-FceRI-activated captured basophils in microfluidic chip was significantly higher (P = 0.03) in patients with allergies compared with healthy controls, and the results were comparable with flow cytometry analysis (P = 0.04). Furthermore, there was no significant difference of CD63% expression in anti-FceRI-activated captured basophils in microfluidic chip compared with flow cytometry. Conclusions: We report on the miBAT. This device is capable of isolating basophils directly from whole blood for on-chip activation and detection. The new miBAT method awaits validation in larger patient populations to assess performance in diagnosis and monitoring of patients with allergies at the point of care.
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| 7. |
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| 8. |
- Brandström, Josef, et al.
(author)
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Individually dosed omalizumab facilitates peanut oral immunotherapy in peanut allergic adolescents
- 2019
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In: Clinical and Experimental Allergy. - : Wiley. - 0954-7894 .- 1365-2222. ; 49:10, s. 1328-1341
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Journal article (peer-reviewed)abstract
- Background: Peanut oral immunotherapy (pOIT) has showed good short-term outcomes, but allergic reactions may prevent effective up-dosing and is a major cause of stopping OIT. In placebo-controlled trials, omalizumab has been shown to facilitate allergen immunotherapy and increase tolerance to peanut.Objective: We hypothesized that by combining omalizumab with pOIT, and monitor treatment effects with basophil allergen threshold sensitivity tests (CD-sens), peanut allergic patients could safely initiate pOIT and thereafter slowly withdraw omalizumab.Methods: This is the 2nd part of a one-armed open phase-2 study where peanut allergic adolescents (n = 23) started pOIT after an individualized omalizumab treatment. The pOIT dose was increased from 280 to 2800 mg peanut protein in 8 weeks followed by an individualized step-wise withdrawal of omalizumab, based on clinical symptoms and CD-sens levels. pOIT continued for 12 weeks followed by an open peanut challenge. Peanut CD-sens and allergen-binding activity (ABA) and IgE-ab, IgG-ab and IgG4-ab to peanut and its components were measured during the study.Results: All 23 patients successfully reached the 2800 mg maintenance dose. Moderate/systemic allergic reactions were rare while receiving full-dose omalizumab. Eleven of 23 (48%) successfully continued with pOIT after omalizumab was stopped. Compared to treatment failures, median baseline IgE-ab to peanut components Ara h 1-3 and CD-sens to peanut were significantly lower among successfully treated patients and IgG4-ab to peanut, Ara h 2 and 6 increased significantly more during treatment.Conclusions and clinical relevance: This study indicates that omalizumab is an effective adjunctive therapy for initiation and rapid up-dosing of pOIT; however, adverse events from pOIT become more frequent as omalizumab doses are decreased.
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| 9. |
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| 10. |
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| 11. |
- Kalm, Frida, et al.
(author)
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Adhesion molecule cross-linking and cytokine exposure modulate IgE- and non-IgE-dependent basophil activation
- 2021
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In: Immunology. - : Wiley. - 0019-2805 .- 1365-2567. ; 162:1, s. 92-104
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Journal article (peer-reviewed)abstract
- Basophils are known for their role in allergic inflammation, which makes them suitable targets in allergy diagnostics such as the basophil activation test (BAT) and the microfluidic immunoaffinity basophil activation test (miBAT). Beside their role in allergy, basophils have an immune modulatory role in both innate immunity and adaptive immunity. To accomplish this mission, basophils depend on the capability to migrate from blood to extravascular tissues, which includes interactions with endothelial cells, extracellular matrix and soluble mediators. Their receptor repertoire is well known, but less is known how these receptor–ligand interactions impact the degranulation process and the responsiveness to subsequent activation. As the consequences of these interactions are crucial to fully appreciate the role of basophils in immune modulation and to enable optimization of the miBAT, we explored how basophil activation status is regulated by cytokines and cross-linking of adhesion molecules. The expression of adhesion molecules and activation markers on basophils from healthy blood donors was analysed by flow cytometry. Cross-linking of CD203c, CD62L, CD11b and CD49d induced a significant upregulation of CD63 and CD203c. To mimic in vivo conditions, valid also for miBAT, CD62L and CD49d were cross-linked followed by IgE-dependent activation (anti-IgE), which caused a reduced CD63 expression compared with anti-IgE activation only. IL-3 and IL-33 priming caused increased CD63 expression after IgE-independent activation (fMLP). Together, our data suggest that mechanisms operational both in the microfluidic chip and in vivo during basophil adhesion may impact basophil anaphylactic and piecemeal degranulation procedures and hence their immune regulatory function.
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| 12. |
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| 13. |
- Kalm, Frida, et al.
(author)
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Development and clinical testing of a microfluidic immunoaffinity basophil activation test for point-of-care allergy diagnosis
- 2019
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In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - : Chemical and Biological Microsystems Society. ; , s. 657-658
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Conference paper (peer-reviewed)abstract
- The Basophil Activation Test (BAT) is a valuable allergy diagnostic tool but is time-consuming and requires skilled personnel, which has limited its clinical use. We therefore developed and clinically tested a microfluidic immunoaffinity BAT (miBAT) technique where we captured basophils directly from whole blood followed by in vitro activation and quantification of activation markers. For the first time basophils captured from whole blood, from both allergic patients and healthy donors, have been activated using allergens.
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| 14. |
- Kalm, Frida
(author)
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Studies of cell-on-chip technology and basophil regulation for improved allergy diagnostics
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Allergic diseases affect approximately 30% of adults and has an impact on both the individual’s quality of life as well as an economic impact on society. Two effector cells involved in allergic disease are mast cells and basophils, where basophils are more readily available in blood and therefore of great interest when studying allergy. Basophils can be recruited into the tissue during inflammation originating from for example allergic reactions or parasite infections. Allergy diagnostics starts with evaluation of the patient’s medical history followed by in vivo and/or in vitro testing. All diagnostic tests have different advantages and disadvantages are chosen depending on the patient and the circumstances. In vivo tests include the gold standard of allergy diagnostics, which is the challenge tests, but also the commonly used skin prick test (SPT). Allergy diagnostics can also be done in vitro using allergen-specific IgE antibody assays and the basophil activation test (BAT). BAT is useful to study the cellular response to an allergen but is only available at university hospitals and therefore often require long transportations of blood samples. A research field that is growing fast is microfluidics which can miniaturize and improve existing methods and diagnostic tests. The aims of this thesis were to improve the existing BAT using microfluidic techniques to enable fast and cheap point-ofcare (POC) diagnostics as well as to further study basophil adhesion and activation for a better understanding of basophil behavior and regulation both in vivo and in vitro in a microfluidic chip. In paper I, we developed a novel microfluidic immunoaffinity-based basophil activation test (miBAT) assay to investigate whether it was possible to capture and activate basophils from whole blood in a microfluidic chip. The yield of captured basophils from whole blood was 64% at a capture flow rate of 3 µl/min. The captured basophils were activated using an anti-FceRI antibody and the basophil identification marker CD203c and the activation marker CD63 were detected using fluorescence microscopy. This was done using blood from both healthy donors and allergic patients and showed comparable results between BAT and miBAT. In paper II, we further investigated whether it was possible to detect a dose-dependent allergen activation for basophils captured in a microfluidic chip. We detected a significant difference in CD63-expression between the negative control and allergen-activated basophils from allergic patients but no difference between the negative control and the non-relevant allergen (an allergen to which the patient had no IgE antibodies). The healthy donors showed no significant difference in activation between the negative control and the allergens. The miBAT results were comparable to BAT. In paper III, we studied basophil adhesion and activation to better understand both basophil function as well as the effect that basophil capture and stimulation in a microfluidic chip has on the cell. The basophil capture in a microfluidic chip could potentially mimic basophil adhesion to the endothelium and was therefore of interest due to the elevated background activation seen in unstimulated basophils captured in a microfluidic chip, reported in paper I and II. Basophils did not upregulate CD63 after passage through a microfluidic chip, but there was a slight but significant activation after crosslinking of CD203c, which is the surface marker used for basophil-specific capture in miBAT, giving one potential factor for the background activation. IgE-dependent (anti-IgE) basophil degranulation after crosslinking of adhesion molecules, to mimic adhesion before transmigration into tissue, showed a significant decrease in CD63-expression compared to anti-IgE activation, which indicate a regulatory function. Cytokine stimulation followed by IgE-independent (fMLP) basophil degranulation on the other hand showed a significantly increased CD63-expression compared to non-primed fMLP activation. In conclusion, we have developed a novel microfluidic-based technique (miBAT) able to detect basophil activation (CD63-expression) using allergens in allergic patients. miBAT was also able to discriminate between relevant and non-relevant allergen activation as well as between allergic patients and healthy controls. miBAT has the potential to be used at POC for allergy diagnostics. We have also shown that crosslinking of CD203c is a potential contributor to the basophil activation seen in the negative controls in miBAT but also that IgE-dependent activation is downregulated from crosslinking of some adhesion molecules, which is of interest both in the microfluidic chip but also in vivo to better understand basophil functions
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| 15. |
- Nopp, Anna
(author)
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Characterisation of eosinophil activity markers : relation to allergic inflammation and apoptosis
- 2002
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Doctoral thesis (other academic/artistic)abstract
- An allergic inflammation is characterised by many different factors, among which eosinophils and the release of their granule content e.g. eosinophilic cationic protein (ECP) constitute main features. Activated eosinophils play an important role in both the early and late stages of the allergic inflammation and participate in the remodelling process in tissue, thus contributing to scarring and fibrosis development. Moreover, there are data showing that eosinophils in an activated state have prolonged survival, which could in part be explained by a decrease in apoptosis. This thesis focuses on identification of eosinophil activity markers of importance for the clinical evaluation of allergic inflammation, as well as the relation between activation and prolonged survival of the eosinophil granulocyte. We have shown that when analysing activated cells, primarily eosinophils, the choice of anticoagulant is of importance for the maintenance of cell survival and function, and that citrate as anticoagulant is preferable to EDTA. These studies were designed to allow detection of eosinophil activity markers, both in vitro (the intracellular expression of the EG2-epitope on ECP (EG2), ECP release as well as CD69 expression) and in vivo (eosinophil number, ECP, EG2 and CD69). When examining the kinetic relation between EG2 expression, ECP release, CD69 upregulation and apoptosis, we found that EG2 expression and ECP release precede CD69 upregulation and apoptosis, but we were not able to establish any relation between CD69 upregulation and level apoptosis susceptibility. However, one must take in account that the presence of the natural ligand for CD69, which was still unknown at the time of the investigation, may play a role in induction of apoptosis. To further examine the apoptosis process in eosinophils, we analysed eosinophils from allergic and non-allergic individuals, and we found an increased survival in cells from allergic individuals as compared to non-allergic individuals. In addition, we examined the possible role of the mitochondrial membrane potential, release of cytochrome c and caspase 3 activation. When we used the apoptosis inducer tributyltin (TBT), an extremely fast induction of apoptosis was seen (measured by caspase 3 activity and Annexin binding) without the prior mitochondrial changes generally associated with apoptosis (dissipation of the mitochondrial membrane potential and release of cytochrome c). In conclusion, we have identified different markers, which can be used for detection of activated eosinophils in relation to the allergic inflammation. We have also described the kinetic relation between markers for activation, degranulation and apoptosis m eosinophils. Our hope is that these results may eventually contribute to the identification of a drug that can selectively induce apoptosis in eosinophils without causing activation and degranulation, thus decreasing the pathophysiological effect of eosinophils both in the acute inflammation, as well as in the process of tissue remodelling after injury.
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| 16. |
- van der Heiden, Marieke, et al.
(author)
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A pilot study towards the immunological effects of omalizumab treatment used to facilitate oral immunotherapy in peanut-allergic adolescents
- 2021
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In: Scandinavian Journal of Immunology. - : Wiley. - 0300-9475 .- 1365-3083. ; 93:4
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Journal article (peer-reviewed)abstract
- Anti-IgE treatments, such as omalizumab, have shown promising effects in allergy treatment. Our previous work has shown that individualized omalizumab treatment (OT) allows a safe initiation and rapid up-dosing of peanut oral immunotherapy (OIT) in peanut-allergic adolescents. However, the broader immunological effects of this OT are incompletely understood. In this pilot study, we longitudinally followed the total B- and T-cell immunity during OT, using flow cytometry, ELISpot and ELISA. Peripheral blood mononuclear cells (PBMCs) and plasma were collected from participants (n = 17) at several timepoints during treatment, before starting OT (baseline), prior to starting OIT during OT (start OIT) and at maintenance dose OIT prior to OT reduction (maintenance). OT did not affect the total B-cell compartment over treatment time, but our results suggest an association between the OT dosage scheme and the B-cell compartment. Further, in vitro polyclonal T-cell activation at the different timepoints suggests a cytokine skewing towards the Th1 phenotype at the expense of Th2- and Th9-related cytokines during treatment. No differences in the frequencies or phenotype of regulatory T cells (Tregs) over treatment time were observed. Finally, plasma chemokine levels were stable over treatment time, but suggest elevated gut homing immune responses in treatment successes during the treatment as compared to treatment failures. The novel and explorative results of this pilot study help to improve our understanding on the immunological effects of OT used to facilitate OIT and provide guidance for future immunological investigation in large clinical trials.
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