BackgroundEarly detection and treatment of melanoma is important for optimal clinical outcome, leading to biopsy of pigmented lesions deemed suspicious for the disease. The vast majority of such lesions are benign. Thus, a more objective and accurate means for detection of melanoma is needed to identify lesions for excision.ObjectivesTo provide proof-of-principle that epidermal genetic information retrieval (EGIR™; DermTech International, La Jolla, CA, U.S.A.), a method that noninvasively samples cells from stratum corneum by means of adhesive tape stripping, can be used to discern melanomas from naevi.MethodsSkin overlying pigmented lesions clinically suspicious for melanoma was harvested using EGIR. RNA isolated from the tapes was amplified and gene expression profiled. All lesions were removed for histopathological evaluation.ResultsSupervised analysis of the microarray data identified 312 genes differentially expressed between melanomas, naevi and normal skin specimens (P<0·001, false discovery rate q<0·05). Surprisingly, many of these genes are known to have a role in melanocyte development and physiology, melanoma, cancer, and cell growth control. Subsequent class prediction modelling of a training dataset, consisting of 37 melanomas and 37 naevi, discovered a 17-gene classifier that discriminates these skin lesions. Upon testing with an independent dataset, this classifier discerned in situ and invasive melanomas from naevi with 100% sensitivity and 88% specificity, with an area under the curve for the receiver operating characteristic of 0·955.ConclusionsThese results demonstrate that EGIR-harvested specimens can be used to detect melanoma accurately by means of a 17-gene genomic biomarker.
Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-beta-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-alpha and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-alpha made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.
To determine which lymphocytes are required for vaccine-induced immunity to coccidioidomycosis, we used a temperature-sensitive mutant of Coccidioides immitis to immunize mice lacking subsets of lymphocytes or specific cytokines and infected the mice 4 weeks later with virulent C. immitis. After 2 weeks, we determined the number of fungi in their lungs and spleens. Vaccine-induced immunity required alpha beta T lymphocytes. beta -2 microglobulin knockout (KO) mice were protected by immunization, and we transferred protection using CD4+ T cells from immunized mice. However, vaccination also protected CD4+ KO mice, which suggests that CD8+ T cells played a role in vaccine-induced immunity, even though they were not required. We adaptively transferred protection using spleen cells from immunized CD4+ KO mice to nonimmune B6 mice, but CD8+ -depleted spleen cells did not protect against infection. Recipients of spleen cells from immunized CD4+ KO mice had 6 times more tumor necrosis factor (TNF)- alpha mRNA in their lungs than did mice that received nonimmune spleen cells, and TNF receptor-1 KO mice were not fully protected by immunization. These results show that both CD4+ and CD8+ T cells can protect against coccidioidomycosis and that TNF- alpha is a necessary component of the acquired immune response.
We have shown previously that there is a direct correlation between IL-10 levels and susceptibility to Coccidioides immitis peritonitis in C57BL/6 (B6), DBA/2, and BXD recombinant inbred mice. We now show that B6 mice are also more susceptible to C. immitis pneumonia and that interleukin-10 (IL-10)-deficient (IL-10 ؊/؊ ) B6 mice are more resistant to C. immitis pneumonia. In addition, we established that high levels of IL-10 are sufficient to make genetically resistant mice susceptible to both C. immitis peritonitis and pneumonia by infecting h.IL-10 transgenic mice. Infected h.IL-10 transgenic mice express lower levels of gamma interferon, IL-12 p40, and inducible nitric oxide synthetase 2 (NOS2) mRNA in their lungs, implicating inducible NOS as a defense mechanism in this disease. We treated DBA/2 mice with aminoguanidine, and they became more susceptible to C. immitis peritonitis and pneumonia. We conclude that high levels of IL-10 are both necessary and sufficient to make mice susceptible to C. immitis, regardless of the genetic background of the mice, and that IL-10 impairs resistance to C. immitis in part by suppressing NO synthesis.
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