Adone Baroni

Three-point checklist of dermoscopy

Background: Dermoscopy used by experts has been demonstrated to improve the diagnostic accuracy for melanoma. However, little is known about the diagnostic validity of dermoscopy when used by nonexperts. Objective: To evaluate the diagnostic performance of nonexperts using a new 3-point checklist based on a simplified dermoscopic pattern analysis. Methods: Clinical and dermoscopic images of 231 clinically equivocal and histopathologically proven pigmented skin lesions were examined by 6 nonexperts and 1 expert in dermoscopy. For each lesion the nonexperts assessed 3 dermoscopic criteria (asymmetry, atypical network and blue-white structures) constituting the 3-point method. In addition, all examiners made an overall diagnosis by using standard pattern analysis of dermoscopy. Results: Asymmetry, atypical network and blue-white structures were shown to be reproducible dermoscopic criteria, with a kappa value ranging from 0.52 to 0.55. When making the overall diagnosis, the expert had 89.6% sensitivity for malignant lesions (tested on 68 melanomas and 9 pigmented basal cell carcinomas), compared to 69.7% sensitivity achieved by the nonexperts. Remarkably, the sensitivity of the nonexperts using the 3-point checklist reached 96.3%. The specificity of the expert using overall diagnosis was 94.2% compared to 82.8 and 32.8% achieved by the nonexperts using overall diagnosis and 3-point checklist, respectively. Conclusion: The 3-point checklist is a valid and reproducible dermoscopic algorithm with high sensitivity for the diagnosis of melanoma in the hands of non-experts. Thus it may be applied as a screening procedure for the early detection of melanoma.

Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur.

Toll-like receptors (TLRs) are crucial players in the innate immune response to microbial invaders. The lipophilic yeast Malassezia furfur has been implicated in the triggering of scalp lesions in psoriasis. The aim of the present study was to assess the role of TLRs in the defence against M. furfur infection. The expression of the myeloid differentiation factor 88 (MyD88) gene, which is involved in the signalling pathway of many TLRs, was also analysed. In addition, a possible correlation of antimicrobial peptides of the β-defensin family to TLRs was tested. Human keratinocytes infected with M. furfur and a variety of M. furfur-positive psoriatic skin biopsies were analysed by RT-PCR, for TLRs, MyD88, human β-defensin 2 (HBD-2), HBD-3 and interleukin-8 (IL-8) mRNA expression. When keratinocytes were infected with M. furfur, an up-regulation for TLR2, MyD88, HBD-2, HBD-3 and IL-8 mRNA was demonstrated, compared to the untreated cells. The same results were obtained when psoriatic skin biopsies were analysed. The M. furfur-induced increase in HBD-2 and IL-8 gene expression is inhibited by anti-TLR2 neutralising antibodies, suggesting that TLR2 is involved in the M. furfur-induced expression of these molecules. These findings suggest the importance of TLRs in skin protection against fungi and the importance of keratinocytes as a component of innate immunity.

Antimicrobial human beta-defensin-2 stimulates migration, proliferation and tube formation of human umbilical vein endothelial cells

Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which is released upon microbial invasion and contributes to mucosal and epithelial defense modulating both innate and adaptive immunity. We found that hBD-2 stimulates chemotaxis of human endothelial cells with an extent similar to that exerted by the vascular endothelial growth factor (VEGF). The hBD-2-dependent chemotaxis is dose-dependent, maximal effect being reached at 500 ng/ml concentration. In the absence of any growth factor, hBD-2 favors wound healing of endothelial cells, causing an about 2-fold increase in the speed of wound closure with respect to the control. Furthermore, hBD-2 promotes endothelial cell proliferation, although at a minor extent as compared to VEGF. When plated on matrigel enriched with angiogenic factors, endothelial cells form a three-dimensional network of tubes that gives rise to capillary-like structures. Similarly to VEGF, hBD-2 promotes capillary-like tube formation of human endothelial cells. Pro-angiogenic effect promoted by hBD-2 is dose-dependent, peaks at a 500 ng/ml hBD-2 concentration and is prevented by blocking anti-alphavbeta3 monoclonal antibody. However, hBD-2-induced pro-angiogenic activity is not due to endogenously produced VEGF because it is not prevented by neutralizing anti-VEGF antibodies. Overall, our findings suggest that hBD-2 could link inflammation and the host defense through its pro-angiogenic activity.

Malassezia furfur induces the expression of β-defensin-2 in human keratinocytes in a protein kinase C-dependent manner

Antimicrobial peptides of the β-defensin family are expressed in all human epithelial tissues tested to date and have recently been the subject of vigorous investigation. Their localization and characteristics support the hypothesis that these peptides play a role in mucosal and skin defense. The lipophilic yeast Malassezia furfur is a saprophyte found in normal human cutaneous flora. Malassezia furfur is not only a saprophyte, but is also associated with several diseases such as Malassezia folliculitis, seborrheic dermatitis and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. Little is known about the mechanism by which M. furfur overcomes the natural barrier of the skin. To further define the role of the β-defensins in the innate human skin immune response, we analyzed the mRNA expression of two human β-defensins HBD-1 and HBD-2 in human keratinocytes treated with M. furfur. In addition, we looked into how M. furfur of TGF-β1 and IL-10, cytokines that interfere with the development of protective cell immunity, regulate their expression. Finally, we examined the signal transduction mechanisms involved during M. furfur uptake. Cultured human keratinocytes were treated with M. furfur. The mRNA and protein expression were analyzed, respectively, by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Our data demonstrate that M. furfur does not modify HBD-1 expression, whereas it up-regulates, via protein kinase C (PKC), the expression of HBD-2, TGFβ-1 and IL-10 48 h after treatment. Our results suggest that β-defensins are integral components of innate host defenses. They play an essential part in the resistance of the human skin surfaces against M. furfur uptake and other microbial invasion.

Peripheral Blood T Lymphocytes from Systemic Sclerosis Patients Show Both Th1 and Th2 Activation

Our objective was to investigate the phenotype of helper T cells in the peripheral blood of patients with systemic sclerosis (SSc). PBMC from 15 patients with SSc and 15 sex- and age-matched controls were investigated for lymphocyte subpopulations (CD3, CD4, CD8, CD19, CD16/CD56, CD3-DR); IL-2, IL-4, and IFN-γ mRNAs; and the relative cytokines in their cytoplasm. The last assay was carried out both in unstimulated and in PMA-activated PBMC. SSc patients presented a higher percentage of activated T cells, CD3+ DR+ (19.7 ± 9.9 vs 5.1 ± 2.5%; P < 0.0001); 12 of them presented IFN-γ mRNA-positive cells; and none IL-2 or IL-4 mRNAs. Under basal conditions, PBMC from six SSc patients contained IL-2, IL-4, and IFN-γ (i.e., they showed both Th1 and Th2 activation), and 1 IFN-γ only. PMA-stimulated PBMC of patients differed from those of controls only in the increased percentage of IFN-γ positive cells (52 ± 12 vs 37 ± 11%; P < 0.01). Our study demonstrates that Th1 activation occurs in the peripheral blood of SSc patients. This evidence must be faced with from both a pathogenetic and a therapeutical point of view.

Detection of herpesvirus DNA in peripheral blood mononuclear cells and skin lesions of patients with pemphigus by polymerase chain reaction

Pemphigus is an autoimmune disease where both endogenous (genetic) and exogenous (environmental) factors play a part. Viral infections, in particular herpesvirus infections, have been identified as a possible triggering factor for pemphigus. In this study, using the polymerase chain reaction, we studied peripheral blood mononuclear cells (PBMC) and skin biopsies from patients with pemphigus, and in some of these were able to demonstrate the presence of DNA sequences of herpes simplex virus 1/2 (50% in PBMC and 71% in skin biopsies), Epstein–Barr virus (15% in PBMC and 5% in skin biopsies) and human herpesvirus 6 (20% in PBMC only). However, the inability to detect herpesvirus DNA consistently in these cases suggests that viral infection may only be an occasional factor triggering the outbreak or exacerbation of the disease. The possible role of interferons and interleukins in the pathogenesis of virus‐induced pemphigus is discussed.

Oral pigmented lesions

Melanocytes have their embryologic origin in the neural crest and, during fetal life, migrate to the basal layer of the skin and mucosa through the peripheral nerves. They exhibit dendritic processes that extend to adjacent keratinocytes, where they transfer pigment. Melanocytes synthesize melanin in specialized granules, that is, the melanosomes, which deliver the pigment to the surrounding keratinocytes and sometimes to the macrophages. Genetic constitution, hormone production, and sun exposure usually influence the amount of pigment production. Oral pigmentations represent a group of several entities ranging from physiologic pigmentation to malignant melanoma. The clinical appearance of oral pigmentation ranges from brown to black, depending on the amount of melanin production and depth of the pigment location (usually brown lesions are related to a superficial pigment location and black/gray lesions to deep pigment location). Oral pigmented lesions are due to:

Possible role of Malassezia furfur in psoriasis: modulation of TGF‐β1, integrin, and HSP70 expression in human keratinocytes and in the skin of psoriasis‐affected patients

Background:  Psoriasis is a disease characterized by an abnormal pattern of keratinocyte growth and differentiation. Malassezia furfur forms part of the normal human skin flora. It may also be involved in the pathogenesis of psoriasis. To define the role of M. furfur in the pathogenesis of psoriasis, we investigated how M. furfur regulates molecules involved in cell migration and proliferation. The experiments were performed using human keratinocytes and skin biopsies from M. furfur‐positive and ‐negative psoriasis‐affected patients. In addition, we examined the signal transduction mechanisms involved.

Malassezia furfur invasiveness in a keratinocyte cell line (HaCat): effects on cytoskeleton and on adhesion molecule and cytokine expression

Abstract The lipophilic yeast Malassezia furfur is a member of the cutaneous microbiota, also associated with several chronic diseases such as pityriasis versicolor, folliculitis, seborrhoeic dermatitis, and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. In this study we determined the immunomodulatory and invasive capacity of M. furfur in a human keratinocyte cell culture, HaCat. At a yeast cell to HaCat ratio of 30 : 1, M. furfur penetration was only 30% with poor phagolysosome fusion and with cytoskeleton modification. Transglutaminase I gene expression was also inhibited, supporting the hypothesis that M. furfur causes an initial break in the barrier function of the epidermis. Moreover, we demonstrated that M. furfur modulates proinflammatory and immunomodulatory cytokine synthesis by downregulating IL-1α and by inhibiting IL-6 and TNF-α and by upregulating IL-10 and TGF-β1. The suppressed inflammatory response induced by M. furfur may play a role in chronic disease.

Artemisinin reduces human melanoma cell migration by down-regulating αVβ3 integrin and reducing metalloproteinase 2 production

SummaryArtemisinin and its derivatives are well known antimalarial drugs, particularly useful after resistance to traditional antimalarial pharmaceuticals has started to occur in Plasmodium falciparum. In recent years, anticancer activity of artemisinin has been reported both in vitro and in vivo. Artemisinin has inhibitory effects on cancer cell growth and anti-angiogenetic activity. In the present investigation, we analyzed the inhibitory effects of artemisinin on migratory ability of melanoma cell lines (A375P and A375M, low and medium metastatic properties, respectively). We demonstrate that artemisinin induces cell growth arrest in A375M, and affects A375P cells viability with cytotoxic and growth inhibitory effects, while it was not effective in contrasting proliferation of other tumor cell lines (MCF7 and MKN). In addition, artemisinin affected the migratory ability of A375M cells by reducing metalloproteinase 2 (MMP-2) production and down-regulating αvβ3 integrin expression. These findings introduce a potential of artemisinin as a chemotherapeutic agent in melanoma treatment.