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BACKGROUND: Food challenges carry a burden of safety, effort and resources. Clinical reactivity and presentation, such as thresholds and symptoms, are considered challenging to predict ex vivo. AIMS: To identify changes of peripheral immune signatures during oral food challenges (OFC) that correlate with the clinical outcome in patients with peanut allergy (PA). METHODS: Children with a positive (OFC+ , n = 16) or a negative (OFC- , n = 10) OFC-outcome were included (controls, n = 7). Single-cell mass cytometry/unsupervised analysis allowed unbiased immunophenotyping during OFC. RESULTS: Peripheral immune profiles correlated with OFC outcome. OFC+ -profiles revealed mainly decreased Th2 cells, memory Treg and activated NK cells, which had an increased homing marker expression signifying immune cell migration into effector tissues along with symptom onset. OFC- -profiles had also signs of ongoing inflammation, but with a signature of a controlled response, lacking homing marker expression and featuring a concomitant increase of Th2-shifted CD4+ T cells and Treg cells. Low versus high threshold reactivity-groups had differential frequencies of intermediate monocytes and myeloid dendritic cells at baseline. Low threshold was associated with increased CD8+ T cells and reduced memory cells (central memory [CM] CD4+ [Th2] T cells, CM CD8+ T cells, Treg). Immune signatures also discriminated patients with preferential skin versus gastrointestinal symptoms, whereby skin signs correlated with increased expression of CCR4, a molecule enabling skin trafficking, on various immune cell types. CONCLUSION: We showed that peripheral immune signatures reflected dynamics of clinical outcome during OFC with peanut. Those immune alterations hold promise as a basis for predictive OFC biomarker discovery to monitor disease outcome and therapy of PA.
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Hipersensibilidad al Cacahuete , Linfocitos T CD8-positivos , Linfocitos T Reguladores , Fenotipo , Alérgenos , Arachis/efectos adversosRESUMEN
INTRODUCTION: Detection of specific food allergens brought into circulation following ingestion is complicated by the minute amounts present in serum. We therefore aimed to assess the utility of selective removal of IgE against peanut components using ImmunoCAP combined with the basophil histamine release (HR) assay (BHRA) to identify the absorbed allergen components after ingestion of peanut. METHODS: Serum from six healthy individuals was drawn before and 1 h after ingestion of 100 g peanut. Serum from two peanut allergic patients was depleted for IgE against single allergen components by incubation with ImmunoCAPs coupled with either Ara h 1; 2; 3 or 6 before donor basophils were passively sensitized with the serum preparations. The sensitized cells were challenged with serum obtained from the six individuals before and after ingestion of peanut, and HR was measured after serum provocation. RESULTS: Ara h 2 and Ara h 6 were detected in serum 1 h after ingestion in 6/6 individuals by negative selection. Depletion of specific IgE against Ara h 2 or Ara h 6 almost completely abolished the response to serum provocation, indicating a sequence homology between the two allergen components in serum. Ara h 1 was demonstrated in 5/6 sera and Ara h 3 in 1/6 sera. CONCLUSION: This study is a proof of concept showing that passive sensitization of basophils with sera depleted of component-specific IgE can be used to identify food allergen components present in serum after ingestion.
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Hipersensibilidad a los Alimentos , Hipersensibilidad al Cacahuete , Albuminas 2S de Plantas , Alérgenos , Antígenos de Plantas , Arachis , Ingestión de Alimentos , Glicoproteínas , Humanos , Inmunoglobulina E , Hipersensibilidad al Cacahuete/diagnóstico , Proteínas de PlantasRESUMEN
BACKGROUND: Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide can provoke cluster headache attacks in up to half of cluster headache patients in their active phase. At present, it is unknown whether provoked attacks are mediated via calcitonin gene-related peptide or mast cell activation. METHODS: All enrolled patients with cluster headache were randomly allocated to receive a continuous infusion of either PACAP38 (10 pmol/kg/min) or vasoactive intestinal polypeptide (8 pmol/kg/min) over 20 min. We collected clinical data and measured plasma levels of calcitonin gene-related peptide and markers of mast cell activation (tryptase and histamine) at fixed time points: at baseline (T0), at the end of the infusion (T20), 10 min after the infusion (T30), and 70 min after the infusion (T90). RESULTS: Blood was collected from episodic cluster headache patients in active phase (n = 14), episodic cluster headache patients in remission (n = 15), and chronic cluster headache patients (n = 15). At baseline, plasma levels of calcitonin gene-related peptide, tryptase, and histamine were not different among the three study groups. Plasma levels of calcitonin gene-related peptide (p = 0.7074), tryptase (p = 0.6673), or histamine (p = 0.4792) remained unchanged during provoked attacks compared to attack-free patients. CONCLUSION: Cluster headache attacks provoked by either PACAP38 or vasoactive intestinal polypeptide were not accompanied by alterations of plasma calcitonin gene-related peptide, tryptase or histamine. The provoked attacks may not be mediated by calcitonin gene-related peptide or mast cell activation.Trial Registration: The study is registered at ClinicalTrials.gov (NCT03814226).
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Péptido Relacionado con Gen de Calcitonina , Cefalalgia Histamínica , Mastocitos , Péptido Relacionado con Gen de Calcitonina/sangre , Histamina , Humanos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Triptasas , Péptido Intestinal VasoactivoRESUMEN
Chronic spontaneous urticaria (CSU) is considered to be primarily a mast cell-driven disease. However, recent evidence suggests that eosinophils may also have an axial role in symptomology. Histologic studies have demonstrated the presence of both eosinophils and eosinophil granules, indicative of activation, in CSU lesions. Although many allergic and inflammatory conditions are associated with a peripheral blood eosinophilia, the converse appears to be the case in CSU, with a peripheral blood eosinopenia being observed in many patients. Possible mechanisms include the depletion of blood eosinophils by recruitment into the skin during active disease and immunologic destruction in the blood. We also address in some detail the interactions between eosinophils and mast cells, particularly the cytokine cross-talk of these cells and mediator release possibly leading to clinical symptoms. Also, activation by eosinophil proteins of the coagulation pathway leads to the generation of thrombin and increased mast cell degranulation. Finally, treatments aimed at reducing eosinophil accumulation and activation, such as the anti-IL-5 antibodies mepolizumab, reslizumab, and benralizumab, have been reported to reduce CSU symptoms. Clearly, a new picture of an important role of eosinophils in the pathogenesis of CSU is emerging.
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Urticaria Crónica/inmunología , Eosinófilos/inmunología , Anticuerpos Monoclonales/inmunología , Enfermedad Crónica , Citocinas/inmunología , Humanos , Mastocitos/inmunología , Piel/inmunologíaRESUMEN
Microdialysis is a well-established technique for sampling of small molecules from the human skin, but larger molecules are more difficult to recover. Consequently, sampling feasibility must be evaluated before microdialysis is used in vivo. This report presents a tool for estimating the recovery of large biomarkers from human skin by microdialysis, using previously frozen human skin specimens as reservoirs for biomarker reference solutions. Recovery of the following 17 biomarkers was assessed: CCL27/CTACK, CXCL1/GROα, CXCL7/NAP-2, CXCL10/IP-10, EGF, GM-CSF, IFN-γ, IL-1α, IL-6, IL-8, IL-17, IL-22, IL-23, MIF, TNF-α, TSLP and VEGF. The relative skin recoveries of 13/17 biomarkers were successfully determined in the range 4.0-18.4%. Sampling in the skin reservoir model was not associated with probe leakage, as fluid recovery was stable, at between 80% and 110%. Furthermore, the skin reservoir model enabled studies and optimization of different parameters known to affect biomarker recovery, including flow rate and perfusate composition.
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Biomarcadores/metabolismo , Microdiálisis/métodos , Piel/patología , HumanosRESUMEN
BACKGROUND: Autoimmune chronic spontaneous urticaria (aiCSU) is an important subtype of chronic spontaneous urticaria (CSU) in which functional IgG autoantibodies to IgE or its high-affinity receptor (FcεRI) induces mast cell degranulation and subsequent symptom development. However, it has not been tightly characterized. This study aimed to better define the clinical and immunological features and to explore potential biomarkers of aiCSU. METHODS: This was a multinational, multicenter study of 182 CSU patients. The clinical features studied included: urticaria activity and impact (UAS7 and quality of life); autologous serum skin test (ASST); IgG anti-FcεRI and IgG anti-IgE; IgG-anti-thyroperoxidase (IgG anti-TPO); total serum IgE; and basophil reactivity (BASO) using the basophil activation test (BAT) and basophil histamine release assay (BHRA). RESULTS: Of the 182 patients, 107 (59%) were ASST+, 46 (25%) were BASO+, and 105 (58%) were IgG anti-FcεRI+/IgE+. Fifteen patients (8%) fulfilled all three criteria of aiCSU. aiCSU patients appeared more severe (UAS7 21 vs 9 P < 0.016) but showed no other clinical or demographic differences from non-aiCSU patients. aiCSU patients also had markedly lower total IgE levels (P < 0.0001) and higher IgG anti-TPO levels (P < 0.001). Of biomarkers, positive BAT and BHRA tests were 69% and 88% predictive of aiCSU, respectively. CONCLUSIONS: aiCSU is a relatively small but immunologically distinct subtype of CSU that cannot be identified by routine clinical parameters. Inclusion of BHRA or BAT in the diagnostic workup of CSU patients may aid identification of aiCSU patients, who may have a different prognosis and benefit from specific management.
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Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Biomarcadores , Urticaria Crónica/inmunología , Urticaria Crónica/metabolismo , Adolescente , Adulto , Anciano , Anticuerpos Antiidiotipos/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes/diagnóstico , Basófilos/inmunología , Basófilos/metabolismo , Urticaria Crónica/diagnóstico , Femenino , Liberación de Histamina , Humanos , Inmunoglobulina G/inmunología , Yoduro Peroxidasa/inmunología , Proteínas de Unión a Hierro/inmunología , Masculino , Persona de Mediana Edad , Fenotipo , Receptores de IgE/metabolismo , Evaluación de Síntomas , Adulto JovenAsunto(s)
Antialérgicos/uso terapéutico , Autoanticuerpos/inmunología , Omalizumab/uso terapéutico , Urticaria/tratamiento farmacológico , Urticaria/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antiidiotipos/sangre , Anticuerpos Antiidiotipos/inmunología , Autoanticuerpos/sangre , Enfermedad Crónica , Femenino , Humanos , Inmunoglobulina E/inmunología , Masculino , Persona de Mediana Edad , Urticaria/sangre , Adulto JovenRESUMEN
Many patients experience reactions during penicillin treatment. The diagnosis may be difficult and is mainly based on short-term tests. The European Network for Drug Allergy (ENDA) guidelines proposed for diagnosing penicillin allergy do not include long-term challenge. In this study a total of 405 patients were evaluated. The ENDA guidelines were extended, to include a 7-day oral treatment (p.o.7) with penicillin for all patients who were negative in the ENDA programme. Among the 405 patients; 85 had an immediate reaction to penicillin, and a further 13 reacted during p.o.7. Among the 307 patients with a negative outcome, 88 had a case history of reaction to other ß-lactam antibiotics and were subsequently tested with the culprit drug. Thirteen patients had a positive outcome: 3 on single-dose challenge and 10 during p.o.7. The extended penicillin diagnostic work-up was positive in 111 patients, 30.0% showed immediate reactions and 5.7% reacted during p.o.7. Approximately 20% of all patients with positive outcome during penicillin challenge are detected by adding p.o.7 with penicillin to the original ENDA guidelines.
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Antibacterianos/inmunología , Erupciones por Medicamentos/diagnóstico , Hipersensibilidad Tardía/diagnóstico , Hipersensibilidad Inmediata/diagnóstico , Pruebas Inmunológicas/métodos , Penicilinas/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Angioedema/inducido químicamente , Antibacterianos/administración & dosificación , Antibacterianos/efectos adversos , Femenino , Humanos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Inmediata/inducido químicamente , Inmunoglobulina E , Masculino , Persona de Mediana Edad , Penicilinas/administración & dosificación , Penicilinas/efectos adversos , Guías de Práctica Clínica como Asunto , Factores de Tiempo , Adulto Joven , beta-Lactamas/administración & dosificación , beta-Lactamas/efectos adversos , beta-Lactamas/inmunologíaRESUMEN
Basophil testing is the most effective single approach for diagnosing type-IIb autoimmune chronic spontaneous urticaria (TIIbaiCSU). A positive basophil test has been linked to long disease duration, higher disease activity, a poor response to antihistamines and omalizumab, and a better response to cyclosporine and fenebrutinib. As of now it is unclear what other features are connected to a positive basophil test in chronic spontaneous urticaria (CSU). We aimed to identify features of basophil test-positive CSU patients. We performed a cross-sectional study of 85 CSU patients. Basophil testing was done with the basophil activation test (BAT) and the basophil histamine release assay (BHRA). Data were analysed using SPSS: Student's t-test, Chi-square test, Odds Ratio, Spearman's correlation test. Of 85 CSU patients, 44% and 28% tested positive with the BAT and BHRA, respectively. These patients showed higher disease activity and impact, lower levels of disease control and total serum IgE, as well as higher rates of having a positive autologous serum skin test (ASST), angioedema, nocturnal symptoms, symptoms for >5 days/week, and thyroid autoantibodies. The ASST, by itself, was not a good predictor of basophil test results, but it predicted a positive basophil test in up to 100% of cases when combined with angioedema, thyroid autoantibodies or low IgE. In conclusion, a positive basophil test is linked to known features of TIIbaiCSU and novel characteristics including nocturnal symptoms. Further studies on basophil test-positive and -negative CSU patients can help to better understand CSU endotypes and to develop better management approaches.
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Prueba de Desgranulación de los Basófilos/métodos , Urticaria Crónica/diagnóstico , Urticaria Crónica/inmunología , Adulto , Basófilos/inmunología , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: Patients with chronic spontaneous urticaria (CSU) have an increased risk for comorbid autoimmune diseases. In this retrospective multicenter study of CSU patients, we evaluated clinical and laboratory features of CSU associated with a higher risk of comorbid autoimmune diseases. METHODS: We analyzed records of CSU patients (n = 1,199) for a history or presence of autoimmune diseases. Patients were diagnosed with type IIb autoimmune CSU (aiCSU) if all 3 tests were positive: autologous serum skin test (ASST), basophil histamine release assay (BHRA) and/or basophil activation test (BAT), and IgG autoantibodies against FcεRIα/IgE detected by immunoassay. RESULTS: Twenty-eight percent of CSU patients had at least 1 autoimmune disease. The most prevalent autoimmune diseases were Hashimoto's thyroiditis (HT) (≥ 21%) and vitiligo (2%). Two percent of CSU patients had ≥ 2 autoimmune diseases, most frequently HT plus vitiligo. Comorbid autoimmune diseases, in patients with CSU, were associated with female sex, a family history of autoimmune diseases, and higher rates of hypothyroidism and hyperthyroidism (P < 0.001). Presence of autoimmune diseases was linked to aiCSU (P = 0.02). The risks of having autoimmune diseases were 1.7, 2.9 and 3.3 times higher for CSU patients with a positive ASST, BHRA and BAT, respectively. In CSU patients, markers for autoimmune diseases, antinuclear antibodies and/or IgG anti-thyroid antibodies were associated with non-response to omalizumab treatment (P = 0.013). CONCLUSIONS: In CSU, autoimmune diseases are common and linked to type IIb autoimmune CSU. Our results suggest that physicians assess and monitor all adult patients with CSU for signs and symptoms of common autoimmune diseases, especially HT and vitiligo.
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BACKGROUND: Allergen-specific immunotherapy (SIT) leads to reduced symptoms upon allergen exposure through as yet unresolved mechanisms. Desensitization of basophils to specific allergens during the updosing phase of injection immunotherapy may contribute to the clinical effect of SIT. Here we report a protocol for efficient in vitro allergen-mediated desensitization of basophils in whole blood and the effect of desensitization on the expression of basophil activation markers (CD203c and CD63) as well as histamine release in response to allergen challenge. METHODS: Whole blood from grass pollen-allergic subjects was incubated with Phleum pratense extract by stepwise increase of the allergen concentration in the culture from well below to well above the allergen threshold concentration for activation of basophils. Desensitization was determined by measuring the expression of the basophil activation markers CD63 and CD203c by FACS following challenge with high allergen concentrations. RESULTS: The basophil desensitization protocol reported here affected both the expression of the cell-surface markers and the levels of histamine release. Following the stepwise desensitization procedure the whole-blood basophils were not activated when challenged with more than 10-fold increased allergen concentration. CONCLUSION: We have established a protocol for basophil desensitization. By mimicking the updosing phase of immunotherapy we raised the allergen threshold for basophil activation and obtained efficient desensitization for all donors. We showed that conditions leading to desensitization affect histamine release and expression of different basophil markers alike.
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Antígenos CD/metabolismo , Basófilos/metabolismo , Desensibilización Inmunológica , Hidrolasas Diéster Fosfóricas/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Pirofosfatasas/metabolismo , Rinitis Alérgica Estacional/inmunología , Alérgenos/inmunología , Alérgenos/metabolismo , Antígenos CD/inmunología , Prueba de Desgranulación de los Basófilos , Basófilos/inmunología , Basófilos/patología , Biomarcadores/metabolismo , Separación Celular , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Citometría de Flujo , Histamina/metabolismo , Humanos , Phleum , Hidrolasas Diéster Fosfóricas/inmunología , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Glicoproteínas de Membrana Plaquetaria/inmunología , Pirofosfatasas/inmunología , Rinitis Alérgica Estacional/diagnóstico , Rinitis Alérgica Estacional/tratamiento farmacológico , Tetraspanina 30RESUMEN
BACKGROUND: Chronic spontaneous urticaria (CSU) is characterized by the degranulation of skin mast cells and the influx of basophils and eosinophils to affected skin sites. Blood basopenia has been linked to severe antihistamine-resistant CSU and type IIb autoimmunity, whereas the role of eosinophils in CSU is largely unknown. OBJECTIVE: To analyze data from 1613 patients with CSU from 2 centers to study the prevalence, role, and relevance of eosinopenia in CSU. METHODS: Peripheral blood eosinophil and basophil counts were measured by automated hematology analyzers. Patient files were screened for clinical characteristics, results of laboratory tests, the autologous serum skin test, the serum-induced basophil histamine release assay, and response to second-generation H1-antihistamines and omalizumab. RESULTS: Ten percent of patients with CSU had eosinopenia. Eosinopenia was associated with the female sex, high disease activity, autologous serum skin test and basophil histamine release assay positivity, low total IgE, and high levels of C-reactive protein and IgG-antithyroperoxidase (P ≤ .007). Nonresponders to second-generation H1-antihistamines or omalizumab had significantly lower eosinophils as compared with responders (P < .05 and P < .01, respectively). Blood eosinophil counts correlated with basophil counts (r = 0.396; P < .001), and 81% of patients with CSU with undetectable eosinophils had basopenia. The combination of eosinopenia and basopenia is a better predictor of nonresponse to second-generation H1-antihistamines than eosinopenia alone (odds ratio of 9.5 vs 4.8). CONCLUSIONS: Eosinopenia in patients with CSU is associated with type IIb autoimmunity, high disease activity, and poor response to treatment. Eosinophils should be explored as biomarkers and investigated for their contribution to the pathogenesis of CSU.
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Urticaria Crónica , Urticaria , Autoinmunidad , Enfermedad Crónica , Femenino , Humanos , Omalizumab , Urticaria/tratamiento farmacológico , Urticaria/epidemiologíaRESUMEN
Food allergy is a collective term for several immune-mediated responses to food. IgE-mediated food allergy is the best-known subtype. The patients present with a marked diversity of clinical profiles including symptomatic manifestations, threshold reactivity and reaction kinetics. In-vitro predictors of these clinical phenotypes are evasive and considered as knowledge gaps in food allergy diagnosis and risk management. Peanut allergy is a relevant disease model where pioneer discoveries were made in diagnosis, immunotherapy and prevention. This review provides an overview on the immune basis for phenotype variations in peanut-allergic individuals, in the light of future patient stratification along emerging omic-areas. Beyond specific IgE-signatures and basophil reactivity profiles with established correlation to clinical outcome, allergenomics, mass spectrometric resolution of peripheral allergen tracing, might be a fundamental approach to understand disease pathophysiology underlying biomarker discovery. Deep immune phenotyping is thought to reveal differential cell responses but also, gene expression and gene methylation profiles (eg, peanut severity genes) are promising areas for biomarker research. Finally, the study of microbiome-host interactions with a focus on the immune system modulation might hold the key to understand tissue-specific responses and symptoms. The immune mechanism underlying acute food-allergic events remains elusive until today. Deciphering this immunological response shall enable to identify novel biomarker for stratification of patients into reaction endotypes. The availability of powerful multi-omics technologies, together with integrated data analysis, network-based approaches and unbiased machine learning holds out the prospect of providing clinically useful biomarkers or biomarker signatures being predictive for reaction phenotypes.
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Alérgenos/inmunología , Arachis/efectos adversos , Biomarcadores , Inmunoglobulina E/inmunología , Hipersensibilidad al Cacahuete/diagnóstico , Hipersensibilidad al Cacahuete/etiología , Fenotipo , Animales , Genómica/métodos , Humanos , Microbiota/inmunología , Hipersensibilidad al Cacahuete/metabolismo , Pronóstico , Proteómica/métodosRESUMEN
The aim of this study was to develop an ex vivo method that allows to quantify the transfollicular penetration of topically applied substances by combining microdialysis and selective follicular closure with varnish. An experimental setup with three skin areas on ex vivo intact porcine ear skin was designed (varnish on hair follicle, varnish next to hair follicle, no varnish). On each area, 10 µl/cm2 caffeine-hydroxyethyl-cellulose-gel was applied. Samples were collected for 22 h by microdialysis. After sampling, the skin layers were separated, homogenized and caffeine was quantified by high pressure liquid chromatography (HPLC) in all samples. Potential impact of the varnish placed next to the follicle by tension on the follicle during the drying process was monitored by a microscopic setup and could be excluded. The microdialysis and homogenization study showed a significantly reduced penetration of caffeine when the hair follicles were closed. In areas with open hair follicles caffeine was detected already in the first ten minutes after application. The reported novel combination of two methods is suitable to investigate ex vivo transfollicular penetration. Possible impact of the closure material in the control area can be ruled out by adjusting the design of the control area in future studies.
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Cafeína/metabolismo , Folículo Piloso/metabolismo , Microdiálisis , Absorción Cutánea , Administración Cutánea , Animales , Cafeína/administración & dosificación , Cromatografía Líquida de Alta Presión , Cinética , Permeabilidad , Sus scrofaAsunto(s)
Glicoles de Etileno/inmunología , Etilenos/inmunología , Liberación de Histamina/inmunología , Hipersensibilidad Inmediata/inmunología , Polietilenglicoles/efectos adversos , Adulto , Glicoles de Etileno/farmacología , Etilenos/farmacología , Femenino , Liberación de Histamina/efectos de los fármacos , Humanos , Hipersensibilidad Inmediata/diagnósticoRESUMEN
Skin microdialysis (SMD) is a versatile sampling technique that can be used to recover soluble endogenous and exogenous molecules from the extracellular compartment of human skin. Due to its minimally invasive character, SMD can be applied in both clinical and preclinical settings. Despite being available since the 1990s, the technique has still not reached its full potential use as a tool to explore pathophysiological mechanisms of allergic and inflammatory reactions in the skin. Therefore, an EAACI Task Force on SMD was formed to disseminate knowledge about the technique and its many applications. This position paper from the task force provides an overview of the current use of SMD in the investigation of the pathogenesis of chronic inflammatory skin diseases, such as atopic dermatitis, chronic urticaria, psoriasis, and in studies of cutaneous events during type 1 hypersensitivity reactions. Furthermore, this paper covers drug hypersensitivity, UVB-induced- and neurogenic inflammation, and drug penetration investigated by SMD. The aim of this paper is to encourage the use of SMD and to make the technique easily accessible by providing an overview of methodology and applications, supported by standardized operating procedures for SMD in vivo and ex vivo.
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The application of recombinant (His)(6)-tagged proteins in cell culture assays is associated with problems due to lipopolysaccharide (LPS) contamination. LPS stimulates cells of the immune system, thereby masking antigen-specific activation of T cells. Due to the affinity of LPS for histidine it is associated with difficulties to remove LPS from recombinant (His)(6)-tagged proteins. Here we describe that the Triton X-114 phase separation method can be used to remove LPS from (His)(6)-tagged proteins and that the recombinant proteins retain their biological activity.