Fluorescence microscopy for the evaluation of elastic tissue patterns within fibrous proliferations of the skin on hematoxylin-eosin-stained slides.

BACKGROUND: Diagnosis of fibrous tumors can be challenging and expensive due to the use of special stains. OBJECTIVE: Determine the usefulness of fluorescence microscopy in the evaluation of elastic tissue patterns on hematoxylin-eosin-stained slides. METHODS: In total, 228 slides representing different fibrous tumors were evaluated for their elastic tissue patterns by fluorescence microscopy, and sensitivity and specificity were determined for relevant comparisons. RESULTS: Fluorescence microscopy was found to be useful, especially for distinguishing dermatofibroma from dermatofibrosarcoma protuberans and dermatomyofibroma from other fibrous tumors. LIMITATIONS: In some cases, excessive background staining made patterns difficult to interpret. CONCLUSION: Evaluation of elastic tissue patterns by fluorescence microscopy in fibrous tumors is a cheap and efficient means to further delineate these often challenging tumors.

False-Negative Rate of Direct Immunofluorescence on Lower Extremities in Bullous Pemphigoid.

ABSTRACT: Bullous pemphigoid (BP) is the most common autoimmune blistering disorder of the skin. It is typified by tense blisters with a subepidermal split and mixed dermal inflammatory infiltrate on histology. Biopsy of the perilesional skin for direct immunofluorescence (DIF) has become the gold standard in the diagnosis of BP. Currently there is a pervasive clinical opinion that the lower extremity is a site with a high false-negative rate (FNR) for DIF in the diagnosis of BP. This notion is primarily based on 2 early studies from the 1980s without more recent confirmatory studies. To readdress this question regarding the lower extremities, a retrospective study from 2012 to 2018 was performed in our institution that evaluated the FNR of DIF by an anatomical site in the diagnosis of BP. Cases of BP were identified using standard criteria (clinical and histological data reviewed in cases with negative DIF), and overall, 79 patients were included in the study. A total of 4 false-negative DIF biopsies were verified. Two negative DIF were from the lower extremity yielding a FNR of 10% compared with 4% on the trunk and 3% from the upper extremity, with no statistically significant difference by anatomical sites. Our study fails to demonstrate a high FNR of DIF from the lower extremity in the diagnosis of BP.

Epidemiology of Keratinocyte Carcinoma.

PURPOSE OF THE REVIEW: To provide a synopsis of recent research advances in the epidemiology of keratinocyte carcinoma (KC), with a focus on indoor tanning and known risk factors for other forms of cancer such as cigarette smoking and alcohol drinking. RECENT FINDINGS: The evidence is strong enough to infer that use of UVR-emitting indoor tanning devices cause KC. Epidemiologic studies of cigarette smoking, alcohol drinking, and menopausal hormone therapy all show some suggestion for increased risk of KC but the evidence is not yet strong enough to determine if there is a true etiologic role. Body mass index is clearly inversely associated with KC risk but this is more likely to be due to lower UVR exposure in overweight and obese individuals than it is due to a true etiologic role. SUMMARY: The epidemic of KC continues unabated, and the causal role of indoor tanning is contributing to this unfavorable trend in KC incidence rates. Advances in understanding the etiology of KC should not divert attention away from the fact that the primary public health strategy to prevent KC is known: minimize population exposure to UVR from the sun and from UVR-emitting indoor tanning devices, particularly among those with sun-sensitive phenotypes.

Magnetic studies of mesoporous nanostructured iron oxide materials synthesized by one-step soft-templating.

A combined magnetization and (57)Fe spin-echo nuclear magnetic resonance (NMR) study has been carried out on mesoporous nanostructured materials consisting of the magnetite (Fe3O4) and maghemite (γ-Fe2O3) phases. Two series of samples were synthesized using a recently developed one-step soft-templating approach with systematic variations in calcination temperature and reaction atmosphere. Nuclear magnetic resonance has been shown to be a valuable tool for distinguishing between the two magnetic iron oxide spinel phases, Fe3O4 and γ-Fe2O3, on the nanoscale as well as monitoring phase transformation resulting from oxidation. For the Fe3O4 and γ-Fe2O3 phases, peaks in the NMR spectra are attributed to Fe in the tetrahedral (A) sites and octahedral (B) sites. The magnetic field dependence of the peaks was observed and confirmed the site assignments. Fe3O4 on a nanoscale readily oxidizes to form γ-Fe2O3 and this was clearly evident in the NMR spectra. As evidenced by transmission electron microscope (TEM) images, the porous mesostructure for the iron oxide materials is formed by a random close-packed aggregation of nanoparticles; correspondingly, superparamagnetic behavior was observed in the magnetic measurements. Although X-ray diffraction (XRD) shows the spinel structure for the Fe3O4 and γ-Fe2O3 phases, unlike NMR, it is difficult to distinguish between the two phases with XRD. Nitrogen sorption isotherms characterize the mesoporous structures of the materials, and yield BET surface area values and limited BJH pore size distribution curves.

Defining a role for acid sphingomyelinase in the p38/interleukin-6 pathway.

Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Previous literature has implicated sphingolipids in the regulation of cytokines such as interleukin-6 (IL-6), but the specific sphingolipid pathways and mechanisms involved in inflammatory signaling need to be further elucidated. In this work, we sought to define the role of ASM in IL-6 production because our previous work showed that a parallel pathway of ceramide metabolism, acid ß-glucosidase 1, negatively regulates IL-6. First, silencing ASM with siRNA abrogated IL-6 production in response to the tumor promoter, 4ß-phorbol 12-myristate 13-acetate (PMA), in MCF-7 cells, in distinction to acid ß-glucosidase 1 and acid ceramidase, suggesting specialization of the pathways. Moreover, treating cells with siRNA to ASM or with the indirect pharmacologic inhibitor desipramine resulted in significant inhibition of TNFα- and PMA-induced IL-6 production in MDA-MB-231 and HeLa cells. Knockdown of ASM was found to significantly inhibit PMA-dependent IL-6 induction at the mRNA level, probably ruling out mechanisms of translation or secretion of IL-6. Further, ASM knockdown or desipramine blunted p38 MAPK activation in response to TNFα, revealing a key role for ASM in activating p38, a signaling pathway known to regulate IL-6 induction. Last, knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel. Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology.

Acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages.

Periodontal disease is more prevalent and severe in patients with diabetes than in nondiabetic patients. In addition to diabetes, a large number of studies have demonstrated an association between obesity and chronic periodontal disease. However, the underlying mechanisms have not been well understood. Since plasma free fatty acids (FAs) are elevated in obese patients and saturated FAs such as palmitic acid (PA) have been shown to increase host inflammatory response, we sought to find out how PA interacts with lipopolysaccharide (LPS), an important pathological factor involved in periodontal disease, to enhance inflammation. We found that whereas low concentration of LPS (1 ng/ml) stimulated interleukin (IL)-6 expression in RAW 264.7 macrophages, PA further augmented it fourfold. Besides IL-6, PA amplified the stimulatory effect of LPS on a large amount of Toll-like receptor (TLR)4-mediated expression of proinflammatory signaling molecules such as IL-1 receptor-associated kinase-like 2 and proinflammatory molecules, including monocyte chemotactic protein-1 and colony-stimulating factor. We also observed that PA augmented TLR4 but not TLR2 signal, and the augmentation was mediated by nuclear factor-κB (NF-κB) pathways. To further elucidate the regulatory mechanism whereby PA amplifies LPS signal, our studies showed that PA and LPS synergistically increased hydrolysis of sphingomyelin by stimulating acid sphingomyelinase (ASMase) activity, which contributed to a marked increase in ceramide production and IL-6 upregulation. Taken together, this study has demonstrated that PA markedly augments TLR4-mediated proinflammatory signaling triggered by low concentration of LPS in macrophages, and ASMase plays a key role in the augmentation.

Identification and characterization of protein phosphatase 2C activation by ceramide.

Ceramide is a bioactive sphingolipid with many associated biological outcomes, yet there is a significant gap in our current understanding of how ceramide mediates these processes. Previously, ceramide has been shown to activate protein phosphatase (PP) 1 and 2A. While continuing this line of work, a late fraction from a Mono-Q column was consistently observed to be activated by ceramide, yet PP1 and PP2A were undetectable in this fraction. Proteomic analysis of this fraction revealed the identity of the phosphatase to be PP2Cγ/PPM1G. This was consistent with our findings that PP2Cγ 1-eluted in a high salt fraction due to its strongly acidic domain, and 2-was insensitive to okadaic acid. Further characterization was performed with PP2Cα, which showed robust activation by C(6)-ceramide. Activation was specific for the erythro conformation of ceramide and the presence of the acyl chain and hydroxyl group at the first carbon. In order to demonstrate more physiological activation of PP2Cα by ceramide, phospho-p38δ was utilized as substrate. Indeed, PP2Cα induced the dephosphorylation of p38δ only in the presence of C(16)-ceramide. Taken together, these results show that the PP2C family of phosphatases is activated by ceramide, which may have important consequences in mediating the biological effects of ceramide.

Drug targeting of sphingolipid metabolism: sphingomyelinases and ceramidases.

Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. Sphingomyelinases and ceramidases are key enzymes of sphingolipid metabolism that regulate the formation and degradation of ceramide, one of the most intensely studied classes of sphingolipids. Improved understanding of these enzymes that control not only the levels of ceramide but also the complex interconversion of sphingolipid metabolites has provided the foundation for the functional analysis of the roles of sphingolipids. Our current understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from loss-of-function/gain-of-function studies utilizing genetic deletion/downregulation/overexpression of enzymes of sphingolipid metabolism (e.g. knockout animals, RNA interference) and from the use of pharmacologic inhibitors of these same enzymes. While genetic approaches to evaluate the functional roles of sphingolipid enzymes have been instrumental in advancing the field, the use of pharmacologic inhibitors has been equally important in identifying new roles for sphingolipids in important cellular processes.The latter also promises the development of novel therapeutic targets with implications for cancer therapy, inflammation, diabetes, and neurodegeneration. In this review, we focus on the status and use of pharmacologic compounds that inhibit sphingomyelinases and ceramidases, and we will review the history, current uses and future directions for various small molecule inhibitors, and will highlight studies in which inhibitors of sphingolipid metabolizing enzymes have been used to effectively treat models of human disease.

Regulated secretion of acid sphingomyelinase: implications for selectivity of ceramide formation.

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase(WT) were treated with inflammatory cytokines. Interleukin-1ß and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C(16)-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase(WT) exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase(S508A) MCF7. Secretion of the S508A mutant was also defective in response to IL-1ß, as was the regulated generation of C(16)-ceramide. Taken together, these data support a crucial role for Ser(508) in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.