The overall study population included 12 male SD subjects presenting face and chest manifestations. SD patients were recruited from the Dermatology Unit of the University of Naples Federico II, Italy. The inclusion and exclusion criteria of SD patients are summarized in Table 1.
Compliance with Ethics Guidelines
The experimental protocol was approved the March14, 2018 by the Ethics Committee for Biomedical Activities “Carlo Romano” of University of Naples Federico II and conformed to the principles outlined in the Declaration of Helsinki of 1964, as revised in 2013, concerning human and animal rights. Each subject gave written informed consent before entering the study. The patient gave permission for his photograph to be published in the manuscript.
This was an open-label, prospective and experimental research clinical trial. Patients were instructed to apply the non-steroidal cream commercialized as NUTRADEICA® (ISDIN Spain), containing piroctone olamine [% International Nomenclature of Cosmetic Ingredients (INCI), 0.45–0.55], zinc PCA, hydroxyphenyl propamidobenzoic acid (% INCI, 0.045–0.055), biosaccharide gum-2 (% INCI, 0.045–0.055), and stearyl glycyrrhetinate (% INCI, 0.27–0.33), twice daily on the face and on the chest, performing regular visits at baseline (W0), after 7 (W1) and 14 (W2) days of treatment. In addition, patients were instructed to not apply the cream the morning of the scheduled follow-up visits (W1, W2). Patients were evaluated through questionnaires filled out by both investigators and patients themselves. In particular, investigator’s assessments were represented by scoring index (SI) and investigator’s global assessment score (IGA). The SI ranking system, recommended by Koca et al. , was used at all visits (W0, W1 and W2). According to this system, erythema, scaling, itching and irritation of each area is ranked from 0 to 3 (non existence, 0; mild, 1; moderate, 2; severe, 3). The sum of these values is regarded as the SD rank: 0–4 (mild), 5–8 (moderate) and 9–12 (severe). IGA was performed at W1 and W2. According to this system, post-treatment rank was used to make a final evaluation of the recovery rate from 0 (no efficacy) to 10 (cured). In particular, the response was defined as excellent from 8 to 10, good from 5 to 7, mild lower than 5 and no response or worsening as 0. Regarding patients’ assessments, they were represented by visual analog scales (VAS), patient global assessment (PGA), and subjective questionnaire for cosmetic and efficacy properties. VAS measurement was assessed at W0, W1 and W2. This score, ranging from 0 to 100 mm, was used to assess each of the following symptoms (erythema, scaling, itching, hot sensation, pain and irritation) both for face and chest. PGA was assessed at W1 and W2. According to this system post-treatment rank was used to make a final subjective evaluation of the recovery rate from 0 (no efficacy) to 10 (cured). In addition, patients performed the subjective questionnaire for cosmetic and efficacy properties of the product at W1 and W2 (answers: completely disagree, disagree, agree, completely agree) composed by the following questions: (Q1, The product is well absorbed; Q2, The product has a pleasant texture; Q3, My skin tolerates the product well; Q4, The product acts quickly after being applied on my skin; Q5, The product applied is effective). Beyond characterizing patients from a clinical point of view in order to assess the efficacy of the product, patients underwent additional procedures to evaluate anti-fungal, anti-microbial and anti-inflammatory effects.
In order to assess NSC anti-fungal and anti-microbial effects, skin scale scrapings (50–100 mg) from the face (wings of the nose) and from the chest were collected at W0, W1 and W2 to be used for Malassezia furfur (MF) and Staphylococcus epidermidis (SE) colony-forming units. In parallel, in order to assess NSC anti-inflammatory effects, each recruited patient was biopsied (2 mm diameter) in the same area on the chest at W0 and W1 for gene expression analysis.
Malassezia furfur Colony-Forming Units
Skin scales (50–100 mg weight) were scraped from the face as well as the chest and cultured into Malassezia agar plates (Dixon’s, 14.0193 V) at 36 °C in the dark for 7 days in order to assess the MF growth. Affected sites were cleaned with 70% ethyl or isopropyl alcohol before removing skin scales.
Cultured samples were examined every 2 days for growth. Indeed, MF at 36 °C after 48 h of incubation initially presented smooth, creamy colonies, from yellow to brownish, which over time took on a rough appearance. MF colonies were counted and reported as total colony-forming units per dish (cfu/dish).
Staphylococcus epidermidis Colony-Forming Units
Skin scales (50–100 mg weight) were scraped from face as well as chest and cultured on Chapman-Mannitol salt agar plates (Scharlau, 064-PR0015) at 37 °C for 48 h in order to assess SE growth. This culture medium is specific for the isolation and enumeration of staphylococci. The selectivity of this medium is based on the presence of sodium chloride which inhibits the growth of most Gram (+) and Gram (−) bacteria. Differentiation of staphylococci is based on their capacity to ferment mannitol. Fermentation of mannitol induces acidification, which turns the medium yellow in the presence of phenol red (pH indicator). Strains of SE form small colonies which, in the majority of cases, grow without modifying the color of the medium. SE colonies were counted and reported as total colony-forming units per dish (cfu/dish).
Gene Expression Analysis
RNA was extracted from skin biopsies performed at W0 and W1 (RNeasy Mini Protocol Qiagen, Valencia, CA) and cDNA was prepared (Transcriptor High Fidelity cDNA Synthesis, Roche, Indianapolis, IN) according to the manufacturer’s instructions. qRT-PCR (LightCycler, Roche) was used to analyze the levels of expression of 18S (housekeeping gene) as well as all selected mediators (reported below). The PCR protocol and product quantification for all analyzed genes were performed as previously reported . PCR primers for the selected genes were designed based on published sequences, and their specificity was verified with a BLAST alignment search. A melting curve analysis was carried out after completion to confirm the presence of a single amplified species. The amount of mRNA for a given gene in each sample was normalized to the amount of mRNA of 18S reference gene in the same sample. Fold induction of gene expression was calculated using the ΔΔCT method as described previously . The analyzed genes were divided according to their function as follows, for anti-inflammatory effects: IL-1α, IL-1β, IL-6, IL-8, and TNF-α; for anti-microbial effects: HBD2 and HBD3, and for anti-pruritus effects: cathepsin S (CTS) and l-histidine decarboxylase (HDC).
Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). Data were analyzed with Wilcoxon matched-pairs tests to calculate statistical differences. Values of p < 0.05 were considered significant. Data are expressed as the mean ± SD.