Atopic dermatitis (AD) is a common pruritic dermatitis with macroscopically non-lesional skin that is often abnormal. Therefore, we used high-density oligonucleotide arrays to identify cutaneous gene transcription changes associated with early AD inflammation as potential disease control targets. Skin biopsy specimens analysed included normal skin from five healthy non-atopic adults and both minimally lesional skin and nearby or contralateral non-lesional skin from six adult AD patients. Data were analysed on an individual gene basis and to identify biologically relevant gene networks. Transcription levels of selected genes were also analysed by quantitative PCR. Differential transcription occurring early in AD skin was indicated for (i) individual genes such as C-C chemokine ligand (CCL)18, CCL13, and interferon-alpha2 (IFNalpha2), (ii) genes associated with peroxisome proliferator-activated receptor (PPAR)alpha- and PPARgamma-regulated transcription, and possibly for (iii) immunoglobulin J-chain and heavy chain isotype transcripts. These data suggest that local changes in immunoglobulin-associated transcription may favour IgE over secretory immunoglobulin (multimeric IgM and IgA) expression in AD skin. Decreased PPAR activity appears common to both AD and psoriasis, and reduced cutaneous IFNalpha2 transcription also appears characteristic of AD. Identification of these genes and pathways will direct future research towards controlling AD.

Meckel syndrome (MKS) is an embryonic lethal, autosomal recessive disorder characterized by polycystic kidney disease, central nervous system defects, polydactyly and liver fibrosis. This disorder is thought to be associated with defects in primary cilia; therefore, it is classed as a ciliopathy. To date, six genes have been commonly associated with MKS (MKS1, TMEM67, TMEM216, CEP290, CC2D2A and RPGRIP1L). However, mutation screening of these genes revealed two mutated alleles in only just over half of our MKS cohort (46 families), suggesting an even greater level of genetic heterogeneity. To explore the full genetic complexity of MKS, we performed exon-enriched next-generation sequencing of 31 ciliopathy genes in 12 MKS pedigrees using RainDance microdroplet-PCR enrichment and IlluminaGAIIx next-generation sequencing. In family M456, we detected a splice-donor site change in a novel MKS gene, B9D1. The B9D1 protein is structurally similar to MKS1 and has been shown to be of importance for ciliogenesis in Caenorhabditis elegans. Reverse transcriptase-PCR analysis of fetal RNA revealed, hemizygously, a single smaller mRNA product with a frameshifting exclusion of B9D1 exon 4. ArrayCGH showed that the second mutation was a 1.713 Mb de novo deletion completely deleting the B9D1 allele. Immunofluorescence analysis highlighted a significantly lower level of ciliated patient cells compared to controls, confirming a role for B9D1 in ciliogenesis. The fetus inherited an additional likely pathogenic novel missense change to a second MKS gene, CEP290; p.R2210C, suggesting oligogenic inheritance in this disorder.

Background: Pimecrolimus is a calcineurin inhibitor that inhibits T cell and mast cell activation and effectively treats atopic dermatitis. However, its effects on eosinophils, a cell type implicated in allergic disease pathology, are unknown. Therefore, we examined the effects of pimecrolimus on eosinophil superoxide anion production, degranulation and survival. Methods: Purified eosinophils from normal or atopic donors were incubated with serial dilutions of pimecrolimus (μM to nM) and then stimulated with platelet activating factor (PAF), interleukin 5 (IL5), secretory immunoglobulin A (sIgA) or Alternaria alternata (Alt) fungus extract. Eosinophil activation was monitored by cytochrome c reduction resulting from superoxide anion production and by a 2-site immunoassay for eosinophil-derived neurotoxin (EDN) in cellular supernatants, as a marker of degranulation. Eosinophil survival was measured by propidium iodide exclusion using flow cytometry after 4 days in culture. Results: Normal and atopic eosinophil superoxide anion production induced by PAF, and associated with increased intracellular calcium, was inhibited up to 37% with 1 μM pimecrolimus. However, superoxide anion production induced by IL5 and sIgA was not consistently inhibited. EDN release, which ultimately depends on calcium, was inhibited about 30% with PAF, IL5 and sIgA stimulation for normal and atopic donor eosinophils. Furthermore, calcium-dependent Alt-induced EDN release was inhibited up to 49% with nanomolar pimecrolimus. Finally, increased eosinophil survival promoted by IL5 and sIgA was not influenced by pimecrolimus. Conclusion: Pimecrolimus moderately inhibits eosinophil superoxide anion production and EDN release associated with calcium mobilization, which may contribute to its efficacy in treating atopic dermatitis.