SummaryImmune defence against microbes depends in part on the production of antimicrobial peptides, a process that occurs in a variety of cell types but is incompletely understood. In this study, the mechanisms responsible for the induction of cathelicidin and b-defensin antimicrobial peptides were found to be independent and specific to the cell type and stimulus. Vitamin D 3 induced cathelicidin expression in keratinocytes and monocytes but not in colonic epithelial cells. Conversely, butyrate induced cathelicidin in colonic epithelia but not in keratinocytes or monocytes. Distinct factors induced b-defensin expression. In all cell types, vitamin D 3 activated the cathelicidin promoter and was dependent on a functional vitamin D responsive element. However, in colonic epithelia butyrate induced cathelicidin expression without increasing promoter activity and vitamin D 3 activated the cathelicidin promoter without a subsequent increase in transcript accumulation. Induction of cathelicidin transcript correlated with increased processed mature peptide and enhanced antimicrobial activity against Staphylococcus aureus. However, induction of b-defensin-2 expression did not alter the innate antimicrobial capacity of cells in culture. These data suggest that antimicrobial peptide expression is regulated in a tissue-specific manner at transcriptional, post-transcriptional and post-translational levels. Furthermore, these data show for the first time that innate antimicrobial activity can be triggered independently of the release of other pro-inflammatory molecules, and suggest strategies for augmenting innate immune defence without increasing inflammation.

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Background: Management of pigmented lesions currently relies on visual assessment with surgical biopsy and histopathologic examination for those lesions suspicious for melanoma. A non-invasive genomic assay that detects two melanoma-associated biomarkers (PLA, 2-GEP) has recently been validated as an adjunct to visual assessment for distinguishing high-risk pigmented lesions appropriate for biopsy from those that can be safely monitored via clinical surveillance. Objectives: To calculate NPV and PPV of the PLA in real-world use and determine the distribution of PLA-positive lesions among categories in the MPATH-Dx classification scheme for melanocytic neoplasms. Methods: Real-world NPV was determined by following a cohort of 1,233 PLA-negative pigmented lesions for evidence of malignancy for up to 36 months and by re-testing a separate prospective cohort of 302 PLA-negative lesions up to 2 years after initial testing. Real-world PPV was determined by identifying melanoma diagnoses among PLA-positive lesions within a US-based registry of 3,418 PLA-tested cases. Results: Ten early-stage melanomas (4 in situ and 6 pT1a) were identified among 1,233 PLA-negative lesions (0.8%), corresponding to a real-world NPV of 99.2% (CI 95% = 98.5 - 99.6). Of 302 initially PLA-negative lesions subjected to repeat testing an average of 15 months later, 34 were PLA-positive. Biopsy revealed 3 melanomas (all in situ), further confirming an NPV of > 99%. Among 316 PLA-positive cases, 59 were diagnosed as melanoma by histopathology, corresponding to a PPV of 18.7%. Of all PLA-positive lesions, 30.5% had histopathologic diagnoses corresponding to high-risk MPATH-Dx categories (Classes III-V). Conclusions and Relevance: The PLA has an NPV of >99% within the real-world intended use population.  The PLA has a PPV of 18.7% for melanoma and also detects high-risk lesions such as dysplastic nevi with severe / high-grade atypia that are generally targeted for complete excision.

Apoptosis resistance in melanoma is a primary cause of treatment failure. Apoptotic pathways in melanocytes, from which melanoma arises, are poorly characterized. Human melanocytes were susceptible to apoptosis following exposure to UV radiation (UVB, 24-48 hours), 4-tert-butylphenol (4-TBP, 1-4 hours), and cisplatin (24-48 hours). These responses were associated with Bid cleavage, caspase activation (caspases 3, 8, and 9), mitochondrial depolarization and release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor (AIF), but not endonuclease G. The apoptotic responses and AIF release were caspase-independent, as they were not blocked by zVal-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk). While RNA interference-mediated knockdown of AIF protected melanocytes against apoptosis induced by serum withdrawal, apoptotic responses to UVB, cisplatin, and 4-TBP were not compromised by AIF knockdown, even in the presence of zVAD-fmk. Finally, adenoviral-mediated expression of Survivin, an inhibitor of apoptosis expressed in melanoma but not melanocytes, protected melanocytes against UVB-induced apoptosis. Survivin expression in melanocytes partially blocked caspase activation and release of mitochondrial release of AIF, cytochrome c, and Smac induced by UVB. These data indicate that multiple stimuli can activate both caspase-dependent and caspase-independent apoptotic pathways in melanocytes, and that endogenous expression of Survivin in melanoma may contribute to apoptosis resistance by multiple mechanisms.