Pemphigus vulgaris (PV) is a chronic, life-altering autoimmune disease due to the production of anti-desmoglein antibodies causing the loss of cell–cell adhesion in keratinocytes (acantholysis) and blister formation in both skin and mucous membranes. The dispase-based keratinocyte dissociation assay (DDA) is the method of choice to examine the pathogenic effect of antibodies and additional co-stimuli on cell adhesion in vitro. Despite its widespread use, there is a high variability of experimental conditions, leading to inconsistent results. In this paper, we identify and discuss pitfalls in the application of DDA, including generation of a monolayer with optimized density, appropriate culturing conditions to obtain said monolayer, application of mechanical stress in a standardized manner, and performing consistent data processing. Importantly, we describe a detailed protocol for a successful and reliable DDA and the respective ideal conditions for three different types of human keratinocytes: (1) primary keratinocytes, (2) the HaCaT spontaneously immortalized keratinocyte cell line, and (3) the recently characterized HaSKpw spontaneously immortalized keratinocyte cell line. Our study provides detailed protocols which guarantee intra- and inter-experimental comparability of DDA.

Pemphigus vulgaris is a chronic autoimmune blistering disease characterized by antibody production against Desmogleins 3 (Dsg3) and 1 (Dsg1) causing acantholysis. Increased levels of innate cytokines detected in the blister fluid suggest a role of innate immune system activity. To elucidate the discrepancy between binding of pathogenic antibodies and a lack of blister formation, we

Pemphigus vulgaris is a chronic, life-altering bullous autoimmune disease with production of antibodies against the desmosomal proteins Desmoglei | POSTER

Head and neck mucosal melanoma (HNMM) arises in the nasal and oral cavities and has the propensity to metastasize to local and distant body sites. HNMM is also notable for its resistance to available therapeutics. The rarity of this disease makes it difficult to conduct large-scale clinical studies to develop standard treatment protocols. In contrast to cutaneous melanoma, c-Kit-dependent pathways are well studied in HNNMM and provide a potential therapeutic target. We identified and isolated genetically distinct subpopulations with stem cell characteristics in HNMM samples bearing Kit wild-type and mutations. Functional analysis of these subpopulations reveals that, in addition to expressing the stem cell marker proteins CD20, CD117, CD133, and CD166, these subpopulations are characterized by self-renewal potential, migratory capacity, and resistance to Kit inhibitors such as Imatinib. Immunofluorescence staining and inhibition experiments demonstrate that the maintenance and resistance of HHMM subpopulations to Kit inhibitors is mediated by the Kit signal to the PI3K signaling pathway. The KIT signal to the PI3K signaling pathway does not result exclusively from a KIT mutation localized to Exon 17, but can also be triggered by mutations localized to Exons 11 and 13. In the present study, we identify and characterize an HNMM subpopulation with stemness properties in patients with c-Kit wild-type and mutation, and demonstrate for the first time the mechanisms by which the CD117⁺/CD133⁺ HNMM subpopulations survive and confer resistance to the specific inhibitor of c-Kit mutation.