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Papers by P. Skov

Journal of Immunological Methods, 1997

Journal of Allergy and Clinical Immunology, 1996

Transfusion, 1996

Background: The mechanisms of the detrimental effects of perioperative allogeneic blood transfusi... more Background: The mechanisms of the detrimental effects of perioperative allogeneic blood transfusion are still unclear. Previous studies have suggested a higher incidence of adverse effects after the use of blood stored for prolonged time. Therefore, a possible time-dependent release of various white cell-and platelet-derived bioactive substances in stored human red cell suspensions was studied. Study Design and Methods: Whole blood (6 units), plasma-reduced whole blood (6 units), and saline-adenine-glucose-mannitol blood (6 units) from 18 unpaid, normal blood donors were stored under standard blood bank conditions at 4°C for 35 days. After refrigeration, samples were collected from all blood bags on Days 0, 2, 5, 9, 14, 21, 28, and 35 of storage. Extracellular concentrations of eosinophil cationic protein, eosinophil protein X, plasminogen activator inhibitor 1, myeloperoxidase, and interleukin 6 were analyzed by enzyme-linked immunosorbent assay and radioimmunoassay. The total intracellular and donor plasma levels of these substances also were analyzed at the time of blood donation. Results: Eosinophil cationic protein, eosinophil protein X, and myeloperoxidase increased 10-to 25-fold (p<0.05) in a time-dependent manner in whole blood, plasmareduced whole blood, and saline-adenine-glucose-mannitol blood during storage for 35 days. Piasminogen activator inhibitor 1 increased threefold to sixfold (pc0.05) in whole blood and plasma-reduced whole blood, but not in saline-adenine-glucosemannitol blood. Interleukin 6 was not detected in either plasma or samples obtained from the blood bags. Conclusion: Stored whole blood, plasma-reduced whole blood, and saline-adenineglucose-mannitol blood may release white cell-and platelet-derived bioactive substances in a time-dependent manner, which may be related to the detrimental effects of perioperative blood transfusions. Therefore, prestorage white cell reduction should be considered for further improvement of red cell suspensions. Abbreviations: ECP = eosinophil cationic protein; EPX = eosinophil protein X; IL-6 = interleukin 6; MPO = myeloperoxidase; PAI-1 = plasminogen activator inhibitor 1 ; SAGM = saline-adenine-glucose-mannitol; WBC(s) = white ceii(s). PERIOPERATIVE TRANSFUSION OF allogeneic blood is reported to increase the risk of infectious complications and to reduce recurrence-free and long-term survival after potentially curative cancer operations.' Furthermore, both acute and delayed adverse transfusion reactions to whole blood, white cell (WBC)-reduced blood, and platelet and plasma components are frequently r e p~r t e d~-~ and may play a role in posttransfusion morbidity and These adverse reactions to blood transfusion may be mediated primarily by bioactive and immunomodulatory

Journal of Immunological Methods, 2002

We have developed a microtiter assay for evaluating basophil spontaneous adhesion to extracellula... more We have developed a microtiter assay for evaluating basophil spontaneous adhesion to extracellular matrix (ECM) proteins examplified by fibronectin and cytokine induced basophil adhesion to bovine serum albumin (BSA). The percentage of basophils adhering to either ECM or BSA was quantified by the histamine content of the adhering basophils. The spontaneous adhesion to fibronectin was higher than to laminin and collagen type I. Both spontaneous adhesion to fibronectin and interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte/macrophage colony stimulating factor (GM-CSF) induced adhesion to BSA increased with time between 5 and 45 min. The histamine release in both spontaneous and induced basophil adhesion was lower than 3.1%. This microtiter assay is simple and reproducible and can be applied for basic and clinical studies using a limited number of partially purified basophils.

Journal of Allergy and Clinical Immunology, 1996

International Archives of Allergy and Immunology, 1998

Background: A growing body of evidence suggests that proinflammatory cytokines play a role in all... more Background: A growing body of evidence suggests that proinflammatory cytokines play a role in allergic inflammation by attracting and activating inflammatory cells. In this study, we have investigated the relationship between interleukin-8 (IL-8) in nasal lavage fluid and the local activation of eosinophils and neutrophils following nasal allergen challenge of allergic patients. Methods: Nasal challenges were performed with grass pollen extract in 14 allergic patients and 5 nonallergic controls. Nasal lavage fluid was collected repeatedly for 10 h, and the levels of eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were used as markers of eosinophil and neutrophil activation, respectively. The levels of these molecules were compared with that of IL-8 in nasal lavage fluid. Results: Allergen challenge of allergic patients produced a significant late-phase increase in the levels of ECP and MPO. Furthermore, the level of MPO showed a highly significant correlation with the le...

Allergy, 1998

A simple microtiter assay for eosinophil activation is described. The assay used 1000-4000 eosino... more A simple microtiter assay for eosinophil activation is described. The assay used 1000-4000 eosinophils/microtiter well, and the design allows for a separate or simultaneous quantitative assessment of eosinophil adhesion to protein-coated microtiter wells and degranulation after stimulation with eosinophil-activating factors. The number of adherent eosinophils is quantified indirectly by expressing the amount of eosinophil cationic protein (ECP) extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the cells added to the wells. Degranulation is quantified in the same way by expressing the amount of ECP or eosinophil protein X (EPX) released to the supernatant in percentage of the total amount of ECP or EPX. Known eosinophil-activating agents such as PMA, interleukin (IL)-3, IL-5, GM-CSF, and platelet-activating factor (PAF) all induced a time- and dose-dependent adhesion to albumin-coated wells, whereas L-PAF did not. Kinetic experiments showed that most adhesion occurred within the first 15-30 min, reaching a plateau around 60 min. After prolonged incubation, a decline in adhesion was detected. GM-CSF-induced adhesion was completely inhibited by incubation with monoclonal antibodies directed against the common beta 2-chain (CD18) of the LFA-1, Mac-1, p150,95 complexes. Monoclonal antibodies against CD11a, CD11b, CD11c, VLA-4 ALFA, ICAM-1, VCAM-1, Sialyl-Le(x), ELAM-1, and LECAM had no inhibitory effect. Simultaneous monitoring of adhesion and degranulation after stimulation of eosinophils in albumin-coated wells with either PMA or GM-CSF showed that adhesion always preceded degranulation. Replacing the albumin coating of the microtiter wells with IgG or secretory IgA augmented both the spontaneous and the PMA- or GM-CSF-stimulated responses. In conclusion, the assay allows dynamic evaluation of eosinophil activation and can be used to assess soluble eosinophil-activating factors as well as to study eosinophil activation by solid-phase-bound proteins.

Journal of Allergy and Clinical Immunology, 2000

Background: The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive ... more Background: The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell-derived factor 1α (SDF-1α) is the only known ligand for CXCR4. To date, the CXCR4 expression and function of SDF-1α in basophils are unknown. Objective: The purpose of this study was to investigate the expression of CXCR4 and functions of SDF-1α in basophils and to characterize the role of the CXCR4-SDF-1α receptor ligand pair in the allergic inflammation. Methods: Basophil purification, flow cytometry, real-time quantitative RT-PCR assay, Northern blotting, intracellular free Ca 2+ change, chemotaxis assay, and histamine release assay were used. Results: CXCR4 is abundantly expressed on peripheral blood resting basophils (91%). Likewise, CXCR4 messenger (m)RNA is expressed in resting basophils (3.2 × 10 3 copies per 2 × 10 2 cells). The existence of CXCR4 mRNA was also confirmed in basophils by means of Northern blot analysis. SDF-1α induces an increase in intracellular free Ca 2+ in basophils. SDF-1α activates basophils to chemotaxis (chemotactic index = 3.8) and histamine release (36% of total content) through CXCR4 on the cells. The chemokines SDF-1α, eotaxin, RANTES, monocyte chemoattractant protein (MCP) 1, and macrophage inflammatory protein (MIP) 1α have been demonstrated at different potencies in induction of chemotaxis (eotaxin > SDF-1α > RANTES ≅ MCP-1 >> MIP-1α) and histamine release (MCP-1 ≅ SDF-1α > eotaxin > RANTES > MIP-1α). The optimal concentration seen for SDF-1α effects (chemotaxis and histamine release) on basophils was 100 ng/mL. Conclusion: These results indicate that the CXCR4-SDF-1α receptor ligand pair may be important for the recruitment and activation of the basophils, which is a characteristic effector cell of the allergic inflammation. (J Allergy Clin Immunol 2000;106:313-20.)

BMC immunology, Jan 21, 2004

Parasite-specific IgE levels correlate with human resistance to reinfection with Schistosoma spp.... more Parasite-specific IgE levels correlate with human resistance to reinfection with Schistosoma spp. after chemotherapy. Although the role of eosinophils in schistosomiasis has been the focus of a great deal of important research, the involvement of other Fcepsilon receptor-bearing cells, such as mast cells and basophils, has not been investigated in relation to human immunity to schistosomes. Chemotherapy with praziquantel (PZQ) kills schistosomes living in an in vivo blood environment rich in IgE, eosinophils and basophils. This releases parasite Ags that have the potential to cross-link cell-bound IgE. However, systemic hypersensitivity reactions are not induced by treatment. Here, we describe the effects of schistosomiasis, and its treatment, on human basophil function by following changes in total cellular histamine and in vitro histamine-release induced by schistosome Ags or anti-IgE, in blood samples from infected Ugandan fishermen, who are continuously exposed to S. mansoni inf...

Journal of the American Academy of Dermatology, 1990

Pityrosporum ovale is a lipophilic yeast commonlypresent in the seborrheic areas of the skin of a... more Pityrosporum ovale is a lipophilic yeast commonlypresent in the seborrheic areas of the skin of adults. Fifty-five young adult patients with atopic dermatitis, 19 patients with seborrheic dermatitis, and 19 healthy controlsubjects were examined for immune reactions to P. ovale, includingtests for specificIgE antibodies (prick test, histamine release), IgG antibodies and epicutaneoustesting. IgE antibodiesagainst P. ovalewere found in two thirds of the patients with atopic dermatitis and were more frequent in patients with lesions predominantly in the seborrheicareas. In addition,someatopic patients had positivereactionsto epicutaneous tests, which suggest that delayed allergic reactions to P. ovalemay also be important. In patients with seborrheic dermatitis, no evidenceof immediate or delayed hypersensitivity to P. ovale was found. IgG antibody levels were low in all groups. (JAM ACAD DERMATOL 1990;22: 739-42.)

The Journal of Immunology, 2003

Journal of Allergy and Clinical Immunology, 1996

Journal of Allergy and Clinical Immunology, 1996

Cress-reactivity between latex and a medicinal plant: Maradeaia conduinmgo. JL Justicia MD~ MA Mu... more Cress-reactivity between latex and a medicinal plant: Maradeaia conduinmgo. JL Justicia MD~ MA Mufioz MD, E Segurado MD, M Barceio MD, JJ Garcfa MD, M Blanca MD. Malaga, Spain. Marsdenia co,durango (MC) is a tree that belongs to the Ascleoadaceae family. Its berk is used in different countries as a medicinal plant. We describe a case of a subject sonsitised to latex that developed an anaphylaetic reaction after the first contact with an infusion that contained MC. A 32 yo woman had several episodes of nasal pruritus, sneezing, angioedema and urticaria after contact with latex gloves. She developed a severe anaphylactic reaction after taking an infusion containing camomile, marjoram, coriander, mint and MC. Skin test to 12 common inhalant allcrgons, marjoram, coriander, mint and camomile were negative and positive to latex, chesnut, banana and MC. Nine subjects allergic to latex were skin tested with MC and were positive in all cases. Skin tests were negative in a control group of ten subjects. Inhibition studies using RAST discs conjugated to latox showed cross-reactivity with MC. ELISA inhibition studies using rabbit antibodies to MC also showed cross-reactivity with latex. Allergy to latex is a growing phenomeno~ and cross-reactivity with allergcns of different nature has been repotted. The clinical symptoms of the subject after exposure to latex, the results of the skin tests, the immonochemical studies and the reaction to MC after first exposure indicate that the subject reacted to MC due to cross-resctivity with latex allergens. Since MC is a medicinal plant used for different purposes, subjects allergic to latex should he aware of the risks that may take after exposure to this plant.

Journal of Allergy and Clinical Immunology, 2007

Investigations at the Danish Anesthesia Allergy Centre have included testing for allergy to chlor... more Investigations at the Danish Anesthesia Allergy Centre have included testing for allergy to chlorhexidine since 1999. To investigate whether measurement of IgE and histamine release confirm an IgE-mediated mechanism for chlorhexidine allergy. Twenty-two patients with clinical history suggestive of chlorhexidine allergy were included. Skin tests with chlorhexidine and tryptase measurements were performed during initial investigations. Sera were analyzed retrospectively for IgE and histamine release (passive sensitization) to chlorhexidine. Twelve patients were skin test positive and 10 were skin test negative. Of the skin test-positive patients, 11 of 12 had IgE to chlorhexidine and 7 of 11 had a positive histamine release test. None of the skin test-negative patients had specific IgE or positive histamine release to chlorhexidine. Skin test-positive patients had higher median age (64 vs 49 y) and were mainly male (11/12 vs 6/10). In both groups, 8 patients had hypotension, but bronchospasm mainly appeared in skin test-negative patients (1/12 vs 6/10). Reactions occurred more often during urologic surgery in skin test-positive patients (5/12 vs 0/10). Baseline tryptase was higher in skin test-positive patients (median, 11.5 vs 3.7 microg/L), and 6 of 7 patients had elevated IgE to chlorhexidine in serum at the time of reaction. This study confirms that chlorhexidine allergy is IgE-mediated and that measurement of specific IgE and histamine release are good adjuncts to skin testing in patients with clinical history suggesting chlorhexidine allergy. IgE and histamine release can be used to support the diagnosis of allergy to chlorhexidine.

Journal of Allergy and Clinical Immunology, 1992

We have previously reported that a lipophilic yeast, Pityrosporum ovale (P. ovale) produced a hig... more We have previously reported that a lipophilic yeast, Pityrosporum ovale (P. ovale) produced a high frequency of positive skin prick tests and in vitro histamine-release (HR) tests in patients suffering from atopic dermatitis (AD) of the face, scalp, and neck. In the present study, our aim was to confirm the involvement of P. ovale-specific lgE and to produce a standardized extract for diagnostic tests; 7/20 sera from patients with a positive HR test were positive in RAST. Several lgE-binding proteins could be detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting. Comparison of different extraction methods demonstrated that allergens were not released from P. ovale until after mechanical destruction of the yeast cells. Extraction of cultured P. ovale, obtained from the skin of various individuals suffering from AD of the face, scalp, and neck, resulted in very heterogenous patterns of constituents in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis. Nevertheless, all extracts were recognized by patients' lgE. P. ovale may express varying protein patterns, depending on either source or stage of differentiation. Thus, at present, skin and HR tests may be superior to other available assays for diagnosing type I reactions to P. ovale in AD of the face, neck, and scalp. ( J ALLERGY CLIN IMMUNOL 1992;89: 44-51 .)

Journal of Allergy and Clinical Immunology, 1996

The chemokines monocyte chemoattractant factor-l, RANTES, and macrophage inflammatory protein-l a... more The chemokines monocyte chemoattractant factor-l, RANTES, and macrophage inflammatory protein-l a release histamine from human basophils, as well as rat and mouse mast cells. The purpose of this investigation was to determine whether these chemokines release histamine from human skin mast cells in situ. Methods: A microdialysis technique was used to measure histamine release in skin. First, the model was validated by using anti-IgE, codeine, and stem cell factor (SCF); then the histamine-releasing effects of the chemokines were investigated. A total of 47 skin specimens from 41 donors were investigated. Hollow microdialysis fibers were inserted intradermally, and each fiber was slowly perfused (3 txl/min). Anti-IgE, codeine, SCF, or chemokines were injected intradetrnally above individual fibers, and dialysate was collected at 2-minute intervals for 20 minutes. Each series of investigations comprised five to eight single experiments. Results: Anti-IgE (4 to 4000 U/ml), codeine (0.001 to 1 mg/ml), and SCF (5.4 × (10 -'-1 to 10 -s tool~L)) released histamine in a dose-dependent manner; maximum histamine release was 97.4, 116.3, and 9.5pmol/20 min, respectively. Monocyte chemoattractant factor-1, RANTES, and macrophage inflammatory protein-l a in concentrations of lO -9 to 10 -6 mol/L did not release histamine; histamine release by 10 6 mol/L chemokine was less than 0.2 pmol/20 rain. None of the chemokines modulated anti-IgE-induced histamine release. In contrast, SCF significantly potentiated anti-IgE-induced histamine release by 33%. All chemokines, but not SCF, released histamine from human basophils. Conclusion: We conclude that the chemokines monocyte chemoattractant factor-I, RANTES, and macrophage inflammatory protein-l a do not release histamine from human skin mast cells. (J Allergy Clin Immunol 1996;98:790-6.)

Journal of Allergy and Clinical Immunology, 1996

Background: The purposes of the study were to characterize allergen-induced histamine release in ... more Background: The purposes of the study were to characterize allergen-induced histamine release in intact human skin in vivo by using a novel microdialysis technique and to study covariates influencing histamine releasability. Methods: Hollow microdialysis fibers were inserted into the upper dermis in 15 timothysensitive subjects. Up to 12 fibers were inserted in each subject. Each fiber was perfused with Krebs-Ringer's solution at a rate of 3.0 ~l/min. Three to four serial dilutions of allergen were applied to the skin by intracutaneous injections or skin prick test above individual fibers. Samples were collected in two 2-minute fractions before skin challenge and in 10 consecutive samples for 20 minutes after skin challenge. Histamine was assayed spectrofluorometrically.

Clinical & Experimental Allergy, 2010

Purified allergens are required to detect cross-contamination with other allergenic foods and to ... more Purified allergens are required to detect cross-contamination with other allergenic foods and to understand allergen interaction with other components of the food matrix. Pure allergens are also used for the diagnosis and treatment of food allergies. For example, serological methods are being developed to improve the quality of diagnosis, and to reduce the need for food challenge tests. In addition, recombinant allergens are being evaluated as candidate vaccines for safe and efficacious specific immunotherapy. Pure allergens are indispensable as reference materials for the calibration and standardization of methods between different laboratories and operators for risk assessment in the food industry. Therefore, there is a need for well-defined purified food allergens. In this context, a panel of 46 food allergens from plant and animal sources has been purified, from either the food sources or as recombinant forms, within the EU-funded EuroPrevall project. These allergens have been characterized by a battery of diagnostic tests demonstrating that they constitute an authentic, well-defined library of comparable quality. The review summarizes the applications, potentials and limitations of key techniques used for the characterization and authentication of these allergen preparations, with a special emphasis on protein purity and identity, folding, post-translational modifications and immunochemical properties. One key area identified is the development of powerful analytical techniques, such as mass spectrometry and nuclear magnetic resonance, to improve the authentication of allergens for routine applications in allergy management.

Anesthesia & Analgesia, 2004

Activation of mast cells and the systemic release of histamine is a common side effect of opioids... more Activation of mast cells and the systemic release of histamine is a common side effect of opioids. Nevertheless, fentanyl and its derivatives show only a slight activation of mast cells with a subsequent liberation of histamine and tryptase. In this study, we used intradermal microdialysis to assess whether this stimulatory effect of opioids on mast cells depends on the activation of opioid receptors. This new approach allowed us to measure the dose-dependent release of histamine and tryptase from mast cells and the subsequent vascular and sensory effect without systemic side effects in volunteers. The opiate codeine and the synthetic opioids meperidine, fentanyl, alfentanil, sufentanil, remifentanil, buprenorphine, and the opioid antagonist naloxone were tested. Only codeine and meperidine induced mast cell activation with the release of tryptase and histamine, leading to protein extravasation, flare reactions, and itch sensations. Because naloxone did not attenuate these effects, it is unlikely that mu-opioid receptors are involved in the activation of mast cells. Opioid effects on mast cells were assessed using intradermal microdialysis. Mast cell activation was seen with codeine and meperidine; no other opioid induced degranulation. Therefore, histamine release seen at large concentrations of potent micro agonists is caused by an unspecific effect rather than an activation of opioid receptors.

Journal of Immunological Methods, 1997

Journal of Allergy and Clinical Immunology, 1996

Transfusion, 1996

Background: The mechanisms of the detrimental effects of perioperative allogeneic blood transfusi... more Background: The mechanisms of the detrimental effects of perioperative allogeneic blood transfusion are still unclear. Previous studies have suggested a higher incidence of adverse effects after the use of blood stored for prolonged time. Therefore, a possible time-dependent release of various white cell-and platelet-derived bioactive substances in stored human red cell suspensions was studied. Study Design and Methods: Whole blood (6 units), plasma-reduced whole blood (6 units), and saline-adenine-glucose-mannitol blood (6 units) from 18 unpaid, normal blood donors were stored under standard blood bank conditions at 4°C for 35 days. After refrigeration, samples were collected from all blood bags on Days 0, 2, 5, 9, 14, 21, 28, and 35 of storage. Extracellular concentrations of eosinophil cationic protein, eosinophil protein X, plasminogen activator inhibitor 1, myeloperoxidase, and interleukin 6 were analyzed by enzyme-linked immunosorbent assay and radioimmunoassay. The total intracellular and donor plasma levels of these substances also were analyzed at the time of blood donation. Results: Eosinophil cationic protein, eosinophil protein X, and myeloperoxidase increased 10-to 25-fold (p<0.05) in a time-dependent manner in whole blood, plasmareduced whole blood, and saline-adenine-glucose-mannitol blood during storage for 35 days. Piasminogen activator inhibitor 1 increased threefold to sixfold (pc0.05) in whole blood and plasma-reduced whole blood, but not in saline-adenine-glucosemannitol blood. Interleukin 6 was not detected in either plasma or samples obtained from the blood bags. Conclusion: Stored whole blood, plasma-reduced whole blood, and saline-adenineglucose-mannitol blood may release white cell-and platelet-derived bioactive substances in a time-dependent manner, which may be related to the detrimental effects of perioperative blood transfusions. Therefore, prestorage white cell reduction should be considered for further improvement of red cell suspensions. Abbreviations: ECP = eosinophil cationic protein; EPX = eosinophil protein X; IL-6 = interleukin 6; MPO = myeloperoxidase; PAI-1 = plasminogen activator inhibitor 1 ; SAGM = saline-adenine-glucose-mannitol; WBC(s) = white ceii(s). PERIOPERATIVE TRANSFUSION OF allogeneic blood is reported to increase the risk of infectious complications and to reduce recurrence-free and long-term survival after potentially curative cancer operations.' Furthermore, both acute and delayed adverse transfusion reactions to whole blood, white cell (WBC)-reduced blood, and platelet and plasma components are frequently r e p~r t e d~-~ and may play a role in posttransfusion morbidity and These adverse reactions to blood transfusion may be mediated primarily by bioactive and immunomodulatory

Journal of Immunological Methods, 2002

We have developed a microtiter assay for evaluating basophil spontaneous adhesion to extracellula... more We have developed a microtiter assay for evaluating basophil spontaneous adhesion to extracellular matrix (ECM) proteins examplified by fibronectin and cytokine induced basophil adhesion to bovine serum albumin (BSA). The percentage of basophils adhering to either ECM or BSA was quantified by the histamine content of the adhering basophils. The spontaneous adhesion to fibronectin was higher than to laminin and collagen type I. Both spontaneous adhesion to fibronectin and interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte/macrophage colony stimulating factor (GM-CSF) induced adhesion to BSA increased with time between 5 and 45 min. The histamine release in both spontaneous and induced basophil adhesion was lower than 3.1%. This microtiter assay is simple and reproducible and can be applied for basic and clinical studies using a limited number of partially purified basophils.

Journal of Allergy and Clinical Immunology, 1996

International Archives of Allergy and Immunology, 1998

Background: A growing body of evidence suggests that proinflammatory cytokines play a role in all... more Background: A growing body of evidence suggests that proinflammatory cytokines play a role in allergic inflammation by attracting and activating inflammatory cells. In this study, we have investigated the relationship between interleukin-8 (IL-8) in nasal lavage fluid and the local activation of eosinophils and neutrophils following nasal allergen challenge of allergic patients. Methods: Nasal challenges were performed with grass pollen extract in 14 allergic patients and 5 nonallergic controls. Nasal lavage fluid was collected repeatedly for 10 h, and the levels of eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were used as markers of eosinophil and neutrophil activation, respectively. The levels of these molecules were compared with that of IL-8 in nasal lavage fluid. Results: Allergen challenge of allergic patients produced a significant late-phase increase in the levels of ECP and MPO. Furthermore, the level of MPO showed a highly significant correlation with the le...

Allergy, 1998

A simple microtiter assay for eosinophil activation is described. The assay used 1000-4000 eosino... more A simple microtiter assay for eosinophil activation is described. The assay used 1000-4000 eosinophils/microtiter well, and the design allows for a separate or simultaneous quantitative assessment of eosinophil adhesion to protein-coated microtiter wells and degranulation after stimulation with eosinophil-activating factors. The number of adherent eosinophils is quantified indirectly by expressing the amount of eosinophil cationic protein (ECP) extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the cells added to the wells. Degranulation is quantified in the same way by expressing the amount of ECP or eosinophil protein X (EPX) released to the supernatant in percentage of the total amount of ECP or EPX. Known eosinophil-activating agents such as PMA, interleukin (IL)-3, IL-5, GM-CSF, and platelet-activating factor (PAF) all induced a time- and dose-dependent adhesion to albumin-coated wells, whereas L-PAF did not. Kinetic experiments showed that most adhesion occurred within the first 15-30 min, reaching a plateau around 60 min. After prolonged incubation, a decline in adhesion was detected. GM-CSF-induced adhesion was completely inhibited by incubation with monoclonal antibodies directed against the common beta 2-chain (CD18) of the LFA-1, Mac-1, p150,95 complexes. Monoclonal antibodies against CD11a, CD11b, CD11c, VLA-4 ALFA, ICAM-1, VCAM-1, Sialyl-Le(x), ELAM-1, and LECAM had no inhibitory effect. Simultaneous monitoring of adhesion and degranulation after stimulation of eosinophils in albumin-coated wells with either PMA or GM-CSF showed that adhesion always preceded degranulation. Replacing the albumin coating of the microtiter wells with IgG or secretory IgA augmented both the spontaneous and the PMA- or GM-CSF-stimulated responses. In conclusion, the assay allows dynamic evaluation of eosinophil activation and can be used to assess soluble eosinophil-activating factors as well as to study eosinophil activation by solid-phase-bound proteins.

Journal of Allergy and Clinical Immunology, 2000

Background: The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive ... more Background: The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell-derived factor 1α (SDF-1α) is the only known ligand for CXCR4. To date, the CXCR4 expression and function of SDF-1α in basophils are unknown. Objective: The purpose of this study was to investigate the expression of CXCR4 and functions of SDF-1α in basophils and to characterize the role of the CXCR4-SDF-1α receptor ligand pair in the allergic inflammation. Methods: Basophil purification, flow cytometry, real-time quantitative RT-PCR assay, Northern blotting, intracellular free Ca 2+ change, chemotaxis assay, and histamine release assay were used. Results: CXCR4 is abundantly expressed on peripheral blood resting basophils (91%). Likewise, CXCR4 messenger (m)RNA is expressed in resting basophils (3.2 × 10 3 copies per 2 × 10 2 cells). The existence of CXCR4 mRNA was also confirmed in basophils by means of Northern blot analysis. SDF-1α induces an increase in intracellular free Ca 2+ in basophils. SDF-1α activates basophils to chemotaxis (chemotactic index = 3.8) and histamine release (36% of total content) through CXCR4 on the cells. The chemokines SDF-1α, eotaxin, RANTES, monocyte chemoattractant protein (MCP) 1, and macrophage inflammatory protein (MIP) 1α have been demonstrated at different potencies in induction of chemotaxis (eotaxin > SDF-1α > RANTES ≅ MCP-1 >> MIP-1α) and histamine release (MCP-1 ≅ SDF-1α > eotaxin > RANTES > MIP-1α). The optimal concentration seen for SDF-1α effects (chemotaxis and histamine release) on basophils was 100 ng/mL. Conclusion: These results indicate that the CXCR4-SDF-1α receptor ligand pair may be important for the recruitment and activation of the basophils, which is a characteristic effector cell of the allergic inflammation. (J Allergy Clin Immunol 2000;106:313-20.)

BMC immunology, Jan 21, 2004

Parasite-specific IgE levels correlate with human resistance to reinfection with Schistosoma spp.... more Parasite-specific IgE levels correlate with human resistance to reinfection with Schistosoma spp. after chemotherapy. Although the role of eosinophils in schistosomiasis has been the focus of a great deal of important research, the involvement of other Fcepsilon receptor-bearing cells, such as mast cells and basophils, has not been investigated in relation to human immunity to schistosomes. Chemotherapy with praziquantel (PZQ) kills schistosomes living in an in vivo blood environment rich in IgE, eosinophils and basophils. This releases parasite Ags that have the potential to cross-link cell-bound IgE. However, systemic hypersensitivity reactions are not induced by treatment. Here, we describe the effects of schistosomiasis, and its treatment, on human basophil function by following changes in total cellular histamine and in vitro histamine-release induced by schistosome Ags or anti-IgE, in blood samples from infected Ugandan fishermen, who are continuously exposed to S. mansoni inf...

Journal of the American Academy of Dermatology, 1990

Pityrosporum ovale is a lipophilic yeast commonlypresent in the seborrheic areas of the skin of a... more Pityrosporum ovale is a lipophilic yeast commonlypresent in the seborrheic areas of the skin of adults. Fifty-five young adult patients with atopic dermatitis, 19 patients with seborrheic dermatitis, and 19 healthy controlsubjects were examined for immune reactions to P. ovale, includingtests for specificIgE antibodies (prick test, histamine release), IgG antibodies and epicutaneoustesting. IgE antibodiesagainst P. ovalewere found in two thirds of the patients with atopic dermatitis and were more frequent in patients with lesions predominantly in the seborrheicareas. In addition,someatopic patients had positivereactionsto epicutaneous tests, which suggest that delayed allergic reactions to P. ovalemay also be important. In patients with seborrheic dermatitis, no evidenceof immediate or delayed hypersensitivity to P. ovale was found. IgG antibody levels were low in all groups. (JAM ACAD DERMATOL 1990;22: 739-42.)

The Journal of Immunology, 2003

Journal of Allergy and Clinical Immunology, 1996

Journal of Allergy and Clinical Immunology, 1996

Cress-reactivity between latex and a medicinal plant: Maradeaia conduinmgo. JL Justicia MD~ MA Mu... more Cress-reactivity between latex and a medicinal plant: Maradeaia conduinmgo. JL Justicia MD~ MA Mufioz MD, E Segurado MD, M Barceio MD, JJ Garcfa MD, M Blanca MD. Malaga, Spain. Marsdenia co,durango (MC) is a tree that belongs to the Ascleoadaceae family. Its berk is used in different countries as a medicinal plant. We describe a case of a subject sonsitised to latex that developed an anaphylaetic reaction after the first contact with an infusion that contained MC. A 32 yo woman had several episodes of nasal pruritus, sneezing, angioedema and urticaria after contact with latex gloves. She developed a severe anaphylactic reaction after taking an infusion containing camomile, marjoram, coriander, mint and MC. Skin test to 12 common inhalant allcrgons, marjoram, coriander, mint and camomile were negative and positive to latex, chesnut, banana and MC. Nine subjects allergic to latex were skin tested with MC and were positive in all cases. Skin tests were negative in a control group of ten subjects. Inhibition studies using RAST discs conjugated to latox showed cross-reactivity with MC. ELISA inhibition studies using rabbit antibodies to MC also showed cross-reactivity with latex. Allergy to latex is a growing phenomeno~ and cross-reactivity with allergcns of different nature has been repotted. The clinical symptoms of the subject after exposure to latex, the results of the skin tests, the immonochemical studies and the reaction to MC after first exposure indicate that the subject reacted to MC due to cross-resctivity with latex allergens. Since MC is a medicinal plant used for different purposes, subjects allergic to latex should he aware of the risks that may take after exposure to this plant.

Journal of Allergy and Clinical Immunology, 2007

Investigations at the Danish Anesthesia Allergy Centre have included testing for allergy to chlor... more Investigations at the Danish Anesthesia Allergy Centre have included testing for allergy to chlorhexidine since 1999. To investigate whether measurement of IgE and histamine release confirm an IgE-mediated mechanism for chlorhexidine allergy. Twenty-two patients with clinical history suggestive of chlorhexidine allergy were included. Skin tests with chlorhexidine and tryptase measurements were performed during initial investigations. Sera were analyzed retrospectively for IgE and histamine release (passive sensitization) to chlorhexidine. Twelve patients were skin test positive and 10 were skin test negative. Of the skin test-positive patients, 11 of 12 had IgE to chlorhexidine and 7 of 11 had a positive histamine release test. None of the skin test-negative patients had specific IgE or positive histamine release to chlorhexidine. Skin test-positive patients had higher median age (64 vs 49 y) and were mainly male (11/12 vs 6/10). In both groups, 8 patients had hypotension, but bronchospasm mainly appeared in skin test-negative patients (1/12 vs 6/10). Reactions occurred more often during urologic surgery in skin test-positive patients (5/12 vs 0/10). Baseline tryptase was higher in skin test-positive patients (median, 11.5 vs 3.7 microg/L), and 6 of 7 patients had elevated IgE to chlorhexidine in serum at the time of reaction. This study confirms that chlorhexidine allergy is IgE-mediated and that measurement of specific IgE and histamine release are good adjuncts to skin testing in patients with clinical history suggesting chlorhexidine allergy. IgE and histamine release can be used to support the diagnosis of allergy to chlorhexidine.

Journal of Allergy and Clinical Immunology, 1992

We have previously reported that a lipophilic yeast, Pityrosporum ovale (P. ovale) produced a hig... more We have previously reported that a lipophilic yeast, Pityrosporum ovale (P. ovale) produced a high frequency of positive skin prick tests and in vitro histamine-release (HR) tests in patients suffering from atopic dermatitis (AD) of the face, scalp, and neck. In the present study, our aim was to confirm the involvement of P. ovale-specific lgE and to produce a standardized extract for diagnostic tests; 7/20 sera from patients with a positive HR test were positive in RAST. Several lgE-binding proteins could be detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting. Comparison of different extraction methods demonstrated that allergens were not released from P. ovale until after mechanical destruction of the yeast cells. Extraction of cultured P. ovale, obtained from the skin of various individuals suffering from AD of the face, scalp, and neck, resulted in very heterogenous patterns of constituents in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis. Nevertheless, all extracts were recognized by patients' lgE. P. ovale may express varying protein patterns, depending on either source or stage of differentiation. Thus, at present, skin and HR tests may be superior to other available assays for diagnosing type I reactions to P. ovale in AD of the face, neck, and scalp. ( J ALLERGY CLIN IMMUNOL 1992;89: 44-51 .)

Journal of Allergy and Clinical Immunology, 1996

The chemokines monocyte chemoattractant factor-l, RANTES, and macrophage inflammatory protein-l a... more The chemokines monocyte chemoattractant factor-l, RANTES, and macrophage inflammatory protein-l a release histamine from human basophils, as well as rat and mouse mast cells. The purpose of this investigation was to determine whether these chemokines release histamine from human skin mast cells in situ. Methods: A microdialysis technique was used to measure histamine release in skin. First, the model was validated by using anti-IgE, codeine, and stem cell factor (SCF); then the histamine-releasing effects of the chemokines were investigated. A total of 47 skin specimens from 41 donors were investigated. Hollow microdialysis fibers were inserted intradermally, and each fiber was slowly perfused (3 txl/min). Anti-IgE, codeine, SCF, or chemokines were injected intradetrnally above individual fibers, and dialysate was collected at 2-minute intervals for 20 minutes. Each series of investigations comprised five to eight single experiments. Results: Anti-IgE (4 to 4000 U/ml), codeine (0.001 to 1 mg/ml), and SCF (5.4 × (10 -'-1 to 10 -s tool~L)) released histamine in a dose-dependent manner; maximum histamine release was 97.4, 116.3, and 9.5pmol/20 min, respectively. Monocyte chemoattractant factor-1, RANTES, and macrophage inflammatory protein-l a in concentrations of lO -9 to 10 -6 mol/L did not release histamine; histamine release by 10 6 mol/L chemokine was less than 0.2 pmol/20 rain. None of the chemokines modulated anti-IgE-induced histamine release. In contrast, SCF significantly potentiated anti-IgE-induced histamine release by 33%. All chemokines, but not SCF, released histamine from human basophils. Conclusion: We conclude that the chemokines monocyte chemoattractant factor-I, RANTES, and macrophage inflammatory protein-l a do not release histamine from human skin mast cells. (J Allergy Clin Immunol 1996;98:790-6.)

Journal of Allergy and Clinical Immunology, 1996

Background: The purposes of the study were to characterize allergen-induced histamine release in ... more Background: The purposes of the study were to characterize allergen-induced histamine release in intact human skin in vivo by using a novel microdialysis technique and to study covariates influencing histamine releasability. Methods: Hollow microdialysis fibers were inserted into the upper dermis in 15 timothysensitive subjects. Up to 12 fibers were inserted in each subject. Each fiber was perfused with Krebs-Ringer's solution at a rate of 3.0 ~l/min. Three to four serial dilutions of allergen were applied to the skin by intracutaneous injections or skin prick test above individual fibers. Samples were collected in two 2-minute fractions before skin challenge and in 10 consecutive samples for 20 minutes after skin challenge. Histamine was assayed spectrofluorometrically.

Clinical & Experimental Allergy, 2010

Purified allergens are required to detect cross-contamination with other allergenic foods and to ... more Purified allergens are required to detect cross-contamination with other allergenic foods and to understand allergen interaction with other components of the food matrix. Pure allergens are also used for the diagnosis and treatment of food allergies. For example, serological methods are being developed to improve the quality of diagnosis, and to reduce the need for food challenge tests. In addition, recombinant allergens are being evaluated as candidate vaccines for safe and efficacious specific immunotherapy. Pure allergens are indispensable as reference materials for the calibration and standardization of methods between different laboratories and operators for risk assessment in the food industry. Therefore, there is a need for well-defined purified food allergens. In this context, a panel of 46 food allergens from plant and animal sources has been purified, from either the food sources or as recombinant forms, within the EU-funded EuroPrevall project. These allergens have been characterized by a battery of diagnostic tests demonstrating that they constitute an authentic, well-defined library of comparable quality. The review summarizes the applications, potentials and limitations of key techniques used for the characterization and authentication of these allergen preparations, with a special emphasis on protein purity and identity, folding, post-translational modifications and immunochemical properties. One key area identified is the development of powerful analytical techniques, such as mass spectrometry and nuclear magnetic resonance, to improve the authentication of allergens for routine applications in allergy management.

Anesthesia & Analgesia, 2004

Activation of mast cells and the systemic release of histamine is a common side effect of opioids... more Activation of mast cells and the systemic release of histamine is a common side effect of opioids. Nevertheless, fentanyl and its derivatives show only a slight activation of mast cells with a subsequent liberation of histamine and tryptase. In this study, we used intradermal microdialysis to assess whether this stimulatory effect of opioids on mast cells depends on the activation of opioid receptors. This new approach allowed us to measure the dose-dependent release of histamine and tryptase from mast cells and the subsequent vascular and sensory effect without systemic side effects in volunteers. The opiate codeine and the synthetic opioids meperidine, fentanyl, alfentanil, sufentanil, remifentanil, buprenorphine, and the opioid antagonist naloxone were tested. Only codeine and meperidine induced mast cell activation with the release of tryptase and histamine, leading to protein extravasation, flare reactions, and itch sensations. Because naloxone did not attenuate these effects, it is unlikely that mu-opioid receptors are involved in the activation of mast cells. Opioid effects on mast cells were assessed using intradermal microdialysis. Mast cell activation was seen with codeine and meperidine; no other opioid induced degranulation. Therefore, histamine release seen at large concentrations of potent micro agonists is caused by an unspecific effect rather than an activation of opioid receptors.