CD40–CD154 interaction is an important mediator of inflammation and has been implicated in T helper type 1-mediated autoimmune diseases including rheumatoid arthritis (RA). Linkage studies have shown association of markers in the proximity of the CD154 gene. In the present work we investigated whether specific allele variants of the microsatellite in the 3' UTR of the CD154 gene might modulate the risk of RA. The study, in a case-control setting, included 189 patients and 150 healthy controls from the Canary Islands, Spain. The 24CAs allele was less represented in female patients than in controls (0.444 in controls versus 0.307 in patients, P = 0.006, odds ratio (OR) 0.556, 95% confidence interval (CI) 0.372 to 0.831) but not in males (0.414 versus 0.408), and only when homozygous (P = 0.012; OR 0.35, 95% CI 0.16 to 0.77). We also verified that CD154 association with RA was independent of human leukocyte antigen (HLA) phenotype. A further functional study showed that after stimulation anti-CD3, CD154 mRNA was more stable in CD4⁺ T lymphocytes from patients with RA bearing the 24CAs allele (mRNA half-life 208 minutes) than in patients without the 24CAs allele (109 minutes, P = 0.009). However, a lower percentage of CD154⁺CD4⁺ T lymphocytes was seen in freshly isolated peripheral blood mononuclear cells from patients carrying 24CAs alleles (mean 4.28 versus 8.12; P = 0.033), and also in CD4⁺ T lymphocytes stimulated with anti-CD3 (median 29.40 versus 47.60; P = 0.025). These results were concordant with the smaller amounts of CD154 mRNA isolated from stimulated T lymphocytes with 24CAs alleles. The CD154 microsatellite therefore seems to affect the expression of the gene in a complex manner that implies not only mRNA stability. These data suggest that the CD154 microsatellite contributes to the regulation of mRNA and protein expression, although further studies will be necessary to elucidate its role in disease predisposition.

Allergic contact dermatitis is one of the most common adverse events associated with cosmetic use. Accordingly, assessment of skin sensitisation hazard is required for safety evaluation of cosmetic ingredients. The transition to the use of alternative methods for testing has made skin sensitisation an intense field in the past decades. The first alternative methods have been in place for almost a decade, but none as stand-alone replacement for the reference murine Local Lymph Node Assay (LLNA). While strategies to combine data from several methods are being evaluated and refined, individual methods face technical limitations. These include issues related to their applicability to highly lipophilic substances and the lack of reliable potency estimation, which remain important obstacles to their widespread adoption as replacement for animal methods. The unique characteristics of in vitro skin models represented an attractive alternative, potentially overcoming these limitations and offering a more physiologically relevant environment for the assessment of the response in keratinocytes and dendritic cells. In this review, we recapitulate how reconstructed human skin models have been used as platforms for skin sensitisation testing, including the latest approaches using organ-on-a-chip and microfluidic technologies, aimed to develop next-generation organotypic skin models with increased complexity and monitoring capabilities.

Allergic contact dermatitis is one of the most common adverse events associated with cosmetic use. Accordingly, assessment of skin sensitisation hazard is required for safety evaluation of cosmetic ingredients. Urged by the European cosmetics legislation ban on animal testing, skin sensitisation has been an intense field in the past two decades, focused on transitioning to the use of alternative methods for hazard prediction. The first alternative methods accepted for regulatory use have been in place for almost a decade, but none as stand-alone replacement for the reference animal method, the murine Local Lymph Node Assay (LLNA). While their strengths and limitations are being evaluated, and the search for new combinations of the adopted methods and other sources of information increase confidence, novel methods based on reconstructed human epidermis (RHE) have recently been validated and joined the available methods. Their unique characteristics provide important advantages that will potentially enhance non-animal testing for skin sensitisation in cosmetics and related industries. In this review, we recapitulate how reconstructed human skin models have been used as platforms for skin sensitisation testing, including the latest approaches using organ-on-a-chip and microfluidic technologies, aimed to develop next-generation organotypic skin models with increased complexity and monitoring capabilities.