Reflectance confocal microscopy and optical coherence tomography for the diagnosis of bullous pemphigoid and pemphigus

Introduction & Objectives: Bullous pemphigoid (BP) and pemphigus (P) are autoimmune diseases characterized by the presence of blisters on the skin and/or the mucous membranes. The diagnosis of these bullous diseases is based on a combination of criteria encompassing clinical features, histology, immunofluorescence and laboratory data. The aim of this study was to evaluate features of BP and P at reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) in order to provide a rapid non-invasive bed-side diagnosis. Secondary objective was to evaluate the detectability of clinically non-visible lesions. Material & Methods: This was an observational, retrospective, multicentre study (University of Modena, Italy and University of Saint-Etienne, France) in which patients with suspicious lesions for BP or P underwent clinical assessment, RCM, OCT, blood tests and skin biopsy for histological and direct immunofluorescence examinations. A total of 72 lesions in 24 patients (16 with PB and 8 with P) were evaluated. Apparently unaffected skin was examined in order to test sub-clinical lesion detectability in all patients. Data analysis was performed from January 2014 to December 2015. Results: RCM was able to detect sub-epidermal and intra-epidermal blisters respectively in 75% and 50% of the patients affected by BP and P. At OCT the exact blister level was identified in all BP and P cases’. Acantholytic cells were observed only at RCM in P (62.5%). Fibrin deposition inside the blisters was only found in PB, evidenced both at RCM and OCT. Subclinical bullae were revealed on clinically healthy skin at OCT in some cases of BP and P. Conclusions: RCM and/or OCT can assist the clinician in providing rapid information through a non-invasive procedure for a rapid diagnosis of BP and P. Combined use of RCM and OCT for a real-time examination of the skin lesions associates the higher resolution of RCM with the greater penetration depth in cross-sectional view of OCT, providing in vivo quasi-histologic information

p75 neurotrophin receptor mediates apoptosis in transit-amplifying cells and its overexpression restores cell death in psoriatic keratinocytes.

p75 neurotrophin receptor (p75NTR) belongs to the TNF-receptor superfamily and signals apoptosis in many cell settings. In human epidermis, p75NTR is mostly confined to the transit-amplifying (TA) sub-population of basal keratinocytes. Brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), which signals through p75NTR, induces keratinocyte apoptosis, whereas β-amyloid, a ligand for p75NTR, triggers caspase-3 activation to a greater extent in p75NTR transfected cells. Moreover, p75NTR co-immunoprecipitates with NRAGE, induces the phosphorylation of c-Jun N-terminal kinase (JNK) and reduces nuclear factor kappa B (NF-κB) DNA-binding activity. p75NTR also mediates pro-NGF-induced keratinocyte apoptosis through its co-receptor sortilin. Furthermore, BDNF or β-amyloid cause cell death in TA, but not in keratinocyte stem cells (KSCs) or in p75NTR silenced TA cells. p75NTR is absent in lesional psoriatic skin and p75NTR levels are significantly lower in psoriatic than in normal TA keratinocytes. The rate of apoptosis in psoriatic TA cells is significantly lower than in normal TA cells. BDNF or β-amyloid fail to induce apoptosis in psoriatic TA cells, and p75NTR retroviral infection restores BDNF- or β-amyloid-induced apoptosis in psoriatic keratinocytes. These results demonstrate that p75NTR has a pro-apoptotic role in keratinocytes and is involved in the maintenance of epidermal homeostasis

In Vivo Melanoma Cell Morphology Reflects Molecular Signature and Tumor Aggressiveness

Melanoma is the deadliest type of skin cancer, characterized by high cellular heterogeneity which contributes to therapy resistance and unpredictable disease outcome. Recently, by correlating Reflectance-Confocal-Microscopy (RCM) morphology with histopathological type, we identified four distinct melanoma-subtypes: dendritic-cell (DC), round-cell (RC), dermal-nest (DN), and combined-type (CT) melanomas. In the present study, each RCM-melanoma subtype expressed a specific biomolecular profile and biological behavior in vitro. Markers of tumor aggressiveness, including Ki67, MERTK, nestin and stemness markers, were highest in the most invasive CT and DN melanomas, as compared to DC and RC. This was also confirmed in multicellular tumor spheroids. Transcriptomic analysis showed a modulation of cancer progression-associated genes from DC to CT melanomas. The switch from E- to N-cadherin expression proved the epithelial-to-mesenchymal transition from DC to CT subtypes. The DN melanoma was predominantly located in the dermis, as also shown in skin reconstructs. It displayed a unique behavior and a molecular profile associated with a high degree of aggressiveness. Altogether, our results demonstrate that each RCM-melanoma subtype has a distinct biological and gene expression profile, related to tumor aggressiveness, confirming that RCM can be a dependable tool for in vivo detecting different types of melanoma and for early diagnostic screening

Un inusuale pemfigo localizzato

Si presenta il caso di un uomo di 74 anni giunto alla nostra osservazione per la comparsa da circa 4 mesi di una lesione squamo-crostosa del dorso del naso suggestiva per un epitelioma e con pattern dermoscopico aspecifico. Veniva eseguita la microscopia laser confocale e la tomografia a coerenza ottica che mostravano inaspettate caratteristiche di pemfigo. Per confermare tale ipotesi venivano eseguiti ulteriori accertamenti. L’esame istologico evidenziava acantolisi a livello degli strati spinoso e granuloso, mentre l’immunofluorescenza diretta rivelava depositi di IgG e C3 in tutto lo spessore dell’epidermide. Anticorpi intercellulari soprabasali (1:40) sono stati evidenziati dall’immunofluorescenza indiretta. Infine, l’immunoblot mostrava la presenza nel siero di anticorpi diretti contro gli antigeni 130 kDa e 160 kDa, successivamente identificati come anticorpi diretti contro la desmogleina 1 e 3 mediante sistemi colorimetrici AP. Tali dati deponevano per una forma mista di pemfigo con caratteristiche sia della forma volgare che di quella foliacea

Uso della microscopia laser confocale e della tomografia a coerenza ottica nella diagnosi del pemfigoide bolloso e del pemfigo

Il pemfigoide bolloso (PB) ed il pemfigo (P) sono malattie autoimmuni caratterizzate dalla formazione di bolle sulla pelle e/o sulle mucose. La diagnosi di queste patologie viene posta sulla base della valutazione clinica, dei dati di laboratorio, dell\u2019istologia, dell\u2019immunofluorescenza diretta ed indiretta. Nel nostro studio osservazionale multicentrico sono stati valutati con la microscopia laser confocale (RCM) e con la tomografia a coerenza ottica (OCT) di 16 pazienti con PB e di 8 con P sia la cute apparentemente sana che tre lesioni bollose. L\u2019OCT \ue8 risultato essere pi\uf9 preciso del RCM nella definizione della localizzazione delle bolle e nell\u2019identificazione delle lesioni subcliniche. Grazie al potere risolutivo maggiore, l\u2019RCM \ue8 in grado di visualizzare strutture a risoluzione quasi istologica e pertanto \ue8 risultato essere capace di valutare pi\uf9 parametri rispetto all\u2019OCT, dimostrando di essere un ottimo strumento nella diagnosi differenziale con altre patologie bollose come ad esempio quelle di natura virale

Nerve Growth Factor Protects Human Keratinocytes from Ultraviolet-B-Induced Apoptosis

Ultraviolet radiation is a potent inducer of apoptosis, whereas autocrine nerve growth factor protects human keratinocytes from programmed cell death. To evaluate the role of nerve growth factor in the mechanisms of ultraviolet B-induced apoptosis, cultured human keratinocytes were ultraviolet B irradiated following pretreatment with K252, a specific inhibitor of the tyrosine kinase high-affinity nerve growth factor receptor. Here we report that the addition of K252 significantly enhanced keratinocyte apoptosis. We then transfected normal human keratinocytes with pNUT-hNGF. Nerve growth factor overexpressing keratinocytes secreted the highest amounts of nerve growth factor in culture supernatants, were more viable, and had a higher rate of proliferation than mock-transfected cells. Whereas ultraviolet B radiation downregulated nerve growth factor mRNA and protein as well as the tyrosine kinase high-affinity nerve growth factor receptor in normal keratinocytes, it failed to do so in nerve growth factor-transfected cells. Moreover, nerve growth factor overexpressing keratinocytes were partially resistant to apoptosis induced by increasing doses of ultraviolet B at 24 and 48 h. These results indicate that downregulation of nerve growth factor function plays an important part in the mechanisms of ultraviolet B-induced apoptosis in human keratinocytes. In addition, ultraviolet B caused a decrease in BCL-2 and BCL-xL expression in mock-transfected keratinocytes, but not in nerve growth factor overexpressing cells. Finally, nerve growth factor prevented the cleavage of the enzyme poly(ADP-ribose) polymerase induced in human keratinocytes by ultraviolet B. These results are consistent with a model whereby the autocrine nerve growth factor protects human keratinocytes from ultraviolet B-induced apoptosis by maintaining constant levels of BCL-2 and BCL-xL, which in turn might block caspase activation