Evidence that keratinocyte microvesicle particles carrying Platelet-activating factor mediate the widespread multi-organ damage associated with intoxicated thermal burn injury.

Thermal burn injuries can result in significant morbidity and mortality. The combination of ethanol intoxication with thermal burn injury results in increased morbidity through an exaggerated inflammatory response involving many organs. Recent studies have linked involvement of the lipid mediator Platelet-activating factor (PAF) in the pathology associated with intoxicated thermal burn injury (ITBI). The present studies tested the roles of PAF and the elevated levels of subcellular microvesicle particles (MVP) generated in response to ITBI in the subsequent multi-organ toxicity. First, thermal burn injury of HaCaT keratinocytes preincubated with ethanol resulted in augmented MVP release, which was blocked by inhibiting the PAF-generating enzyme cytosolic phospholipase A2 and the PAF receptor (PAFR). Second, ITBI of mice resulted in increased pro-inflammatory cytokine production and neutrophilic inflammation in multiple organs which were not present in mice deficient in PAFRs nor the MVP-generating enzyme acid sphingomyelinase (aSMase). Moreover, the increased bacterial translocation from the gut to mesenteric lymph nodes previously reported in murine ITBI was also dependent upon PAFR and aSMase. MVP released from ITBI-treated keratinocytes contained high levels of PAFR agonistic activity. Finally, use of topical aSMase inhibitor imipramine following ITBI attenuated the widespread organ inflammatory response of ITBI, suggesting a potential therapeutic for this condition. These studies provide evidence for PAF-enriched MVP generated in skin, which then act upon the gut PAFR resulting in bacterial translocation as the mechanism for the multi-organ dysfunction associated with ITBI. Inasmuch as aSMase inhibitors are widely available, these studies could result in effective treatments for ITBI.

Topical Photodynamic Therapy Generates Bioactive Microvesicle Particles: Evidence for a Pathway Involved in Immunosuppressive Effects.

Although effective in treating actinic damage, topical photodynamic therapy (PDT) has been shown to be immunosuppressive through unknown mechanisms, which could potentially limit its effectiveness. Multiple types of environmental stressors, including PDT, can produce the immunosuppressive lipid mediator platelet-activating factor (PAF). Because PAF can produce subcellular microvesicle particles (MVPs), these studies tested whether PDT can generate PAF and MVP release and whether these are involved in PDT-induced immunosuppression. Previously, topical PDT using blue light and 5-aminolevulinic acid was found to be a potent stimulus for PAF production in mice and human skin explants and human patients, and we show that experimental PDT also generates high levels of MVP. PDT-generated MVPs were independent of the PAF receptor but were dependent on the MVP-generating enzyme acid sphingomyelinase. Patients undergoing topical PDT treatment to at least 10% of body surface area showed local and systemic immunosuppression as measured by inhibition of delayed-type hypersensitivity reactions. Finally, using a murine model of contact hypersensitivity, PDT immunosuppression was blocked by genetic and pharmacologic inhibition of acid sphingomyelinase and genetic inhibition of PAF receptor signaling. These studies describe a mechanism involving MVP through which PDT exerts immunomodulatory effects, providing a potential target to improve its effectiveness.

Platelet-activating factor and microvesicle particles as potential mediators for the toxicity associated with intoxicated thermal burn injury.

Thermal burn injuries (TBIs) in patients who are alcohol-intoxicated result in greater morbidity and mortality. The systemic toxicity found in human patients, which includes both immediate systemic cytokine generation with multiple organ failure and a delayed systemic immunosuppression, has previously been replicated in mouse models combining ethanol and localized TBI. Though considerable insights have been provided with these models, the exact mechanisms for these pathologic effects are unclear. In this review, we highlight the roles of the lipid mediator platelet-activating factor (PAF) and subcellular microvesicle particle (MVP) release in response to intoxicated thermal burn injury (ITBI) as effectors in the pathology. Particularly, MVP is released from keratinocytes in response to PAF receptor (PAFR) activation due to excess PAF produced by ITBI. These subcellular particles carry and thus protect the metabolically labile PAF which enable binding of this potent lipid mediator to several key sites. We hypothesize that PAF carried by MVP can bind to PAFR within the gut, activating myosin light chain kinase (MLCK). The subsequent gut barrier dysfunction in response to MLCK activation then allows bacteria to invade the lymphatic system and, eventually, the bloodstream, resulting in sepsis and resultant dysregulated inflammation in multiple organs. PAF in MVP also activate the skin mast cell PAFR resulting in migration of this key effector cell to the lymph nodes to induce immunosuppression. This review thus provides a mechanism and potential therapeutic approaches for the increased toxicity and immunosuppressive outcomes of TBI in the presence of acute ethanol exposure.

Acute ethanol exposure stimulates microvesicle particle generation in keratinocytes.

Ethanol has been demonstrated to exert profound effects upon cells and tissues via multiple mechanisms. One recently appreciated means by which cells can communicate with other cells is via the production and release of extracellular vesicles. Though smaller exosomes have been demonstrated to be released in response to ethanol exposure, the ability of ethanol to modulate the generation and release of larger microvesicle particles (MVP) is lesser studied. The present studies examined the ability of exogenous ethanol to generate MVP with a focus on skin cells. Acute ethanol exposure resulted in augmented MVP release in keratinocytes and in the skin and blood of mice. Unlike other stimuli such as ultraviolet B radiation or thermal burn injury, ethanol-mediated MVP release was independent of the Platelet-activating Factor receptor (PAFR). However, ethanol pretreatment was found to augment thermal burn injury-induced MVP in a PAFR-dependent manner. These studies provide a novel mechanism for ethanol-mediated effects, that could be relevant in the significant toxicity associated with thermal burn injury in the setting of alcohol intoxication.

A genomic survey of sarcomas on sun-exposed skin reveals distinctive candidate drivers and potentially targetable mutations.

Sarcomas on photodamaged skin vary in prognosis and management, but can display overlapping microscopic and immunophenotypic features. Improved understanding of molecular alterations in these tumors may provide diagnostic and therapeutic insights. We characterized 111 cutaneous sarcomatoid malignancies and their counterparts, including primary cutaneous angiosarcoma (n = 7), atypical fibroxanthoma (AFX) (n = 21), pleomorphic dermal sarcoma (PDS) (n = 17), extracutaneous undifferentiated pleomorphic sarcoma (n = 8), cutaneous leiomyosarcoma (LMS) (n = 5), extracutaneous LMS (n = 9), sarcomatoid squamous cell carcinoma (spindle cell squamous cell carcinoma) (S-SCC) (n = 24), and conventional cutaneous squamous cell carcinoma (SCC) (n = 20), by next-generation sequencing (NGS) using the StrataNGS panel for copy number variations, mutations, and/or fusions in more than 60 cancer-related genes. TP53 mutations were highly recurrent in most groups. Angiosarcoma displayed previously reported MYC amplifications, as well as CCND1 gains. RB1 mutations were relatively restricted to cutaneous LMS. As previously reported, PIK3CA mutations occurred in AFX, whereas RAS activation was more frequent in PDS. CDKN2A mutations were recurrent in AFX and S-SCC, whereas PDS displayed frequent CDKN2A deletion. S-SCC displayed mutational similarity to conventional SCC. BRCA1/2 mutations were specific to tumors with disease progression. In a subset, we detected potential driver events novel to these tumor types: activating mutations in IDH2 (PDS), MAP2K1 (angiosarcoma, PDS), and JAK1 (S-SCC) and copy gains in FGFR1 (angiosarcoma, S-SCC), KIT (AFX), MET (PDS), and PDGFRA (PDS). Our findings confirm and expand the spectrum of known genomic aberrations, including potential targetable drivers, in cutaneous sarcomatoid malignancies. In addition, certain events are relatively specific to particular tumors within this differential diagnosis and hence might be diagnostically informative.

Vitreal pathogenic role in optic pit foveolar retinoschisis and central serous retinopathy.

PURPOSE: To expand on current theories concerning the vitreal-induced mechanism underlying the development of foveolar retinoschisis and macular sensory detachments associated with optic nerve head pits. To propose the notion that vitreal traction may contribute to the pathogenesis of serous detachments in central serous chorioretinopathy (CSC). REPORTS: We describe two patients, one with macular retinoschisis and the other with central serous detachment. The first patient, a 45-year-old Hispanic female, presented with a temporally located optic nerve head pit, foveolar retinoschisis and schisis retinal spaces extending to the surrounding macula and to the disc. The second patient, a 43-year-old Haitian male, developed a central serous retinal detachment OS with decreased visual acuity one day following in-office administration of Apraclonidine (0.5 per cent Iopidine, Alcon) and Dorzolamide-Timolol Maleate (Cosopt, Merck) to lower elevated intraocular pressure (IOP). Macular retinal pigment mottling and epiretinal membrane sheen OU had been observed on his initial visit. Visual acuity improved within a three-day period with resolution of the serous detachment. CONCLUSION: We suggest that the persistence of Cloquet's canal may permit fluid leakage into the proximal vitreous in cases of congenital optic nerve head pits. Tangential vitreal traction may promote the opening of a fistula at the optic pit and additionally thrust vitreal fluid into the pit and retinal space inducing the formation of schisis spaces, foveolar-schisis and underlying sensory serous detachment. We question whether a reduction in vitreous volume, induced by initial administration of anti-glaucoma medications, may contribute to the development and/or recurrence of central serous choroidopathy in predisposed individuals.