RESUMEN
With the prevalence of allergic contact dermatitis (ACD) from the usage of skin-contact products, like wearable, skin care, and hair care products, screening their skin sensitizing potential is necessary, for the sake of alleviating the consequent public health impact. In the present study, a total of 77 skin-contact products classified by four categories, watch bands (WBs), skin care products (SCPs), hair care products (HCPs), and rubber gloves (RGs), were investigated, using an optimized in vitro assay of human cell line activation test (h-CLAT). Extracting the products using neutral artificial sweat simulated well the practical usage scenarios, and testing the extracts showed that 26 of them were allergy test positive, including nine WBs, six SCPs, two HCPs, and nine RGs. The allergenic response was mainly characterized by the induction of CD54 expression, and diverse paradigms of CD54 and CD86 levels were observed by analyzing dose-response curves, which could also be influenced by the compromised viability of the THP-1 cells. The data implicated the intricate regulation by different contributors to suspicious ingredients in the test samples. Altogether, a promising methodology for testing skin allergy potential was well established for commonly used commodities by neutral artificial sweat extraction coupled with h-CLAT screening. The findings would be of great help in tracing the potential allergens in practical products and improving their qualities.
Asunto(s)
Preparaciones para el Cabello , Hipersensibilidad , Humanos , Alérgenos/farmacología , Células THP-1 , PielRESUMEN
Type 2 inflammation in asthma develops with exposure to stimuli to include inhaled allergens from house dust mites (HDM). Features include mucus hypersecretion and the formation of pro-secretory ion transport characterised by elevated basal Cl- current. Studies using human sinonasal epithelial cells treated with HDM extract report a higher protease activated receptor-2 (PAR-2) agonist-induced calcium mobilisation that may be related to airway sensitisation by allergen-associated proteases. Herein, this study aimed to investigate the effect of HDM on Ca2+ signalling and inflammatory responses in asthmatic airway epithelial cells. Primary bronchial epithelial cells (hPBECs) from asthma donors cultured at air-liquid interface were used to assess electrophysiological, Ca2+ signalling and inflammatory responses. Differences were observed regarding Ca2+ signalling in response to PAR-2 agonist 2-Furoyl-LIGRLO-amide (2-FLI), and equivalent short-circuit current (Ieq) in response to trypsin and 2-FLI, in ALI-asthma and healthy hPBECs. HDM treatment led to increased levels of intracellular cations (Ca2+, Na+) and significantly reduced the 2-FLI-induced change of Ieq in asthma cells. Apical HDM-induced Ca2+ mobilisation was found to mainly involve the activation of PAR-2 and PAR-4-associated store-operated Ca2+ influx and TRPV1. In contrast, PAR-2, PAR-4 antagonists and TRPV1 antagonist only showed slight impact on basolateral HDM-induced Ca2+ mobilisation. HDM trypsin-like serine proteases were the main components leading to non-amiloride sensitive Ieq and also increased interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) from asthma hPBECs. These studies add further insight into the complex mechanisms associated with HDM-induced alterations in cell signalling and their relevance to pathological changes within asthma.
Asunto(s)
Alarminas , Asma , Humanos , Animales , Tripsina , Células Epiteliales , Alérgenos/farmacología , PyroglyphidaeRESUMEN
To obtain the recombinant group 2 allergen product of Dermatophagoides farinae (Der f 2), the Der f 2 gene was synthesized by RT-PCR. The full-length cDNA comprised 441 nucleotides and was 99.3 percent identical to the reference sequence (GenBank AB195580). The cDNA was bound to vector pET28a to construct plasmid pET28a(+)-Der f 2, which was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE showed a specific band of about 14kDa in the hole cell lysate. s estiated by chroatography, about 3.86 g of the recobinant product as obtained, which conjugated with serum IgE from asthmatic children. The protein had a signal peptide of 17 amino acids. Its secondary structure comprised an alpha helix (19.86 percent), an extended strand (30.82 percent), and a random coil (49.32 percent). The subcellular localization of this allergen was predicted to be at mitochondria. Furthermore, its function was shown to be associated with an MD-2-related lipid-recognition (ML) domain. The results of this study provide a solid foundation for large-scale production of the allergen for clinical diagnosis and treatent of allergic disorders.
Com a finalidade de obter o produto recombinante do alergeno grupo 2 do Dermatophagoides farinae (Der f2), o gene Der f2 foi sintetizado por RT-PCR. O cDNA continha 441 nucleotídeos e era idêntico em 99,3 por cento à sequência de referência (GenBank AB195580). O cDNA foi ligado ao vetor pET28a para construir o plasmídeo pET28a(+)-Der f2, o qual foi introduzido por transformação em E. coli BL21 e induzido por IPTG. Em SDS-PAGE foi vista mia banda específica de 14 kDa no lisado celular. Conforme estimado por cromatografia, cerca de 3,86 mg do produto recombinante foi obtido, que reagia com IgE sérica de crianças asmáticas. A proteína continha um peptídeo sinal de 17 amino ácidos. Sua estrutura secundária consistia de uma alfa hélice (19,86 por cento), uma fita estendida (30,82 por cento), e uma sequência randômica (49,32 por cento). A localização subcelular desse alergeno foi predita ocorrer nas mitocôndrias. Sua função foi associada com o domínio de reconhecimento lipídico (ML) relacionado a MD-2. Os resultados desse estudo permitem a produção em larga escala do alergeno para o diagnóstico clínico e tratamento das doenças alérgicas.
Asunto(s)
Animales , Preescolar , Humanos , Alérgenos/genética , Antígenos Dermatofagoides/genética , Dermatophagoides farinae/genética , Escherichia coli/genética , Secuencia de Aminoácidos , Alérgenos/química , Alérgenos/farmacología , Antígenos Dermatofagoides/química , Western Blotting , Clonación Molecular , ADN Complementario/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
A 1.9s albumin having allergenic activity and denoted Ric c III was isolated from an alcohol extract of defatted Ricinus communis seeds CB-1A, as a homogeneous protein by ion -exchange chromatography on SP-Sephadex, gel filtration on Sephadex G-75 and preparative polyacrylamide gel electrophoresis (6 mg Ric cIII/g CB-1A). The protein contained approximately 98 amino acid residues distributed in 2 chains of 67 anmd 34 residues, a molecular weight of 11.239 based on amino acid composition and pI=4.9 Ric c III can be aligned, on the basis of amino acid composition and partial amino acid sequence data, with residues 18 to 50 (51) and 66 to 130 of the 2S albumin precursor predicted by the cDNA data of S. D. Irwin, J. N. Ken, J. B. C. Findlary and J. M. Lord (Molecular and General Genetics, 222:400-408, 1990). The present data identify Ric c III as the second allergenic 2S storage albumin coded by this DNA