Healthy Control Skin Research Articles

P168 Modelling macrophage-fibroblast interaction in scleroderma to evaluate new treatments

Abstract Background Alternatively-activated M2-like macrophages are known to express CD206 and are thought to have an important role in pathological fibrosis in scleroderma through stimulation of fibroblasts. RP peptides are 10-12 mer synthetic peptides which have been developed to engage with human macrophages via CD206, but the selectivity of this receptor for macrophages requires clarification. In this study, we investigate the relative expression levels of CD206 in SSc macrophages and fibroblasts, and explore two approaches in modelling macrophage-fibroblast interactions to evaluate the efficacy of the RP peptides. Methods Macrophages were derived from peripheral blood mononuclear cells by culture of buffy layer cells in RPMI supplemented by M-CSF (4ng/ml) for 7 days. Skin fibroblasts were obtained by explant culture of 4 mm punch biospies from the involved forearm skin of SSc patients and healthy controls, maintained in DMEM with 10-% FCS and studied at passage 3-5. Stimulation with TGFβ (4ng/ml) was used to further enhance the activation state of fibroblasts. Macrophages and skin fibroblast lysates were evaluated using qPCR to determine the level of expression of CD206. Macrophages were co-cultured with fibroblasts within respective monolayers on 50kPa gels (models stiff scleroderma skin) for four days with or without the peptide inhibitors. Collagen mRNA expression from the cellular monolayers were quantified by qPCR and Western blot. In the second approach, macrophages and fibroblasts were co-cultured together with or without the peptide inhibitors in a collagen gel contraction assay. The gel was weighed and imaged after 48 hours of incubation and the collagen gel contraction was then quantified as a measure of fibrotic activity. Results Expression of CD206 was specific to macrophages and not seen in fibroblast cultures (relative expression level in macrophages 26.63 versus 0.004 in unstimulated fibroblasts versus 0.04 in TGFβ-stimulated fibroblasts. Fibroblasts co-cultured with macrophages in monolayers show increased CTGF and collagen mRNA by qPCR. There was a dose response reduction in CTGF and collagen mRNA in assays cultured with the RP peptide inhibitor. CTGF mRNA was positively correlated with CD206 expression (r2=0.81). Co-culture of SSc macrophages led to enhanced contraction of collagen gels (0.1g in macrophages, 0.08g in fibroblasts, 0.062 for fibroblasts co-cultured with macrophages) which was fully reversed by the RP peptide treatment (0.1g). Conclusion SSc macrophages express CD206 which has potential as a biomarker of ongoing fibrotic activity whereas fibroblasts express very minimal levels of CD206 even when stimulated by the pro-fibrotic TGFβ. RP peptides that are specific for CD206 on macrophages led to a suppression of the macrophage signature and inhibition of the pro-fibrotic cross talk in macrophages and fibroblasts in both monolayer and 3D collagen gels. Both approaches of evaluating macrophage-fibroblast interactions in scleroderma are viable approaches to evaluate new treatments. Disclosures L. Lei None. B. Abdi None. A. Mujkavnovic None. X. Shi Wen None. H. Lopez Shareholder/stock ownership; Scientific director at Riptide Biosciences. R. Stratton None.

Synthesis and Bioactivity Assessment of Novel Spiro Pyrazole-Oxindole Congeners Exhibiting Potent and Selective in vitro Anticancer Effects.

The present work aims to design and synthesize novel series of spiro pyrazole-3,3’-oxindoles analogues and investigate their bioactivity as antioxidant and antimicrobial agents, as well as antiproliferative potency against selected human cancerous cell lines (i.e., breast, MCF-7; colon, HCT-116 and liver, HepG-2) relative to healthy noncancerous control skin fibroblast cells (BJ-1). The mechanism of their cytotoxic activity has been also examined by immunoassaying the levels of key anti- and proapoptotic protein markers. The analytical and spectral data of the all synthesized target congeners were compatible with their structures. Synthesized compounds showed diverse moderate to powerful antimicrobial and antioxidant activities. Results of MTT assay revealed that seven synthesized compounds (i.e., 11a, 11b, 12a, 12b, 13b, 13c and 13h) particularly exhibited significant cytotoxicity against the three cancerous cell lines under investigation. Ranges of IC50 values obtained were 5.7–21.3 and 5.8–37.4 µg/mL against HCT-116 and MCF-7, respectively; which is 3.8 and 6.5-fold (based on the least IC50 values) more significant relative to the reference chemotherapeutic drug doxorubicin. In HepG-2 cells, the analogue 13h exhibited the highest cytotoxicity with IC50 value of 19.2µg/mL relative to doxorubicin (IC50 = 21.6µg/mL). The observed cytotoxicity was specific to cancerous cells, as evidenced by the minimal toxicity in the noncancerous control skin-fibroblast cells. ELISA results indicated that the observed antiproliferative effect against examined cancer cell lines is mediated via engaging the activation of apoptosis as illustrated by the significant increase in proapoptotic protein markers (p53, bax and caspase-3) and reduction in the antiapoptotic marker bcl-2. Taken together, results of the present study emphasize the potential of spiro pyrazole-oxindole analogues as valuable candidate anticancer agents against human cancer cells.

Decreased CCN3 in Systemic Sclerosis Endothelial Cells Contributes to Impaired Angiogenesis

Systemic sclerosis (SSc) is a rare and severe connective tissue disease combining autoimmune and vasculopathy features, ultimately leading to organ fibrosis. Impaired angiogenesis is an often silent and life-threatening complication of the disease. We hypothesize that CCN3, a member of the CCN family of extracellular matrix proteins, which is an antagonist of the profibrotic protein CCN2 as well as a proangiogenic factor, is implicated in SSc pathophysiology. We performed skin biopsies on 26 patients with SSc, both in fibrotic and nonfibrotic areas for 17 patients, and collected 18 healthy control skin specimens for immunohistochemistry and cell culture. Histological analysis of nonfibrotic and fibrotic SSc skin shows a systemic decrease of papillary dermis surface as well as disappearance of capillaries. CCN3 expression is systematically decreased in the dermis of patients with SSc compared with healthy controls, particularly in dermal blood vessels. Moreover, CCN3 is decreased invitro in endothelial cells from patients with SSc. We show that CCN3 is essential for endothelial cell migration and angiogenesis invitro. In conclusion, CCN3 may represent a promising therapeutic target for patients with SSc presenting with vascular involvement.

Expression of microRNA-31 and microRNA-205 in basal cell carcinoma, squamous cell carcinoma, and healthy controls: a comparative study

Background MicroRNAs (miRNAs) are small nonprotein coding RNAs that play an important role in physiological cellular processes. MicroRNA (miR)-205 and miR-31 are among the most frequently altered miRNAs in human malignancies. Objective To measure miR-205 and miR-31 expression in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) skin biopsies in comparison with sun-exposed and nonexposed skin of healthy controls in order to evaluate their possible role in the pathogenesis. Patients and methods This study included 15 BCC patients, 15 SCC patients and 20 age-matched and sex-matched healthy controls (10 with biopsies from sun-exposed areas and 10 with biopsies from nonexposed areas). Skin biopsies were taken from all participants for assessment of miR-205 and miR-31 levels by real-time PCR. Results MiR-205 levels were significantly lower in BCC and in SCC than in controls, whether sun-exposed or nonexposed. MiR-31 levels were significantly higher in BCC and in SCC than in controls, whether sun-exposed or nonexposed. There was no significant relation between miR-205 or miR-31 and age, sex, duration, site, shape, or size in all groups. There was no significant correlation between miR-205 and miR-31 in BCC, SCC, sun-exposed controls or nonexposed controls. Conclusion The study revealed lower miR-205 and higher miR-31 levels in both BCC and SCC patients than in controls, suggesting a possible role for these miRNAs in the pathogenesis and management of BCC and SCC.

Fli1 deficiency induces endothelial adipsin expression, contributing to the onset of pulmonary arterial hypertension in systemic sclerosis.

Adipsin, or complement factor D, is a serine proteinase catalysing complement factor C3 breakdown, leading to the production of opsonin (C3b), membrane attack complex (C5b-C9) and anaphylatoxins (C3a and C5a). Since adipsin is potentially associated with pulmonary arterial hypertension in SSc, we investigated adipsin expression in dermal small vessels of SSc-involved skin, the mechanism regulating adipsin expression in endothelial cells, and the correlation of serum adipsin levels with SSc clinical symptoms. Adipsin expression was assessed by immunohistochemistry with skin sections of SSc and healthy subjects. mRNA levels of target genes and transcription factor binding to the ADIPSIN promoter were evaluated by quantitative reverse transcription PCR and chromatin immunoprecipitation, respectively. Serum adipsin levels were determined by enzyme-linked immunosorbent assay. Adipsin expression was remarkably increased in dermal small vessels of SSc-involved skin as compared with those of healthy control skin. Consistent with the notion that Fli1 deficiency induces SSc-like phenotypes in various types of cells, FLI1 siRNA enhanced adipsin expression at protein and mRNA levels and Fli1 bound to the ADIPSIN promoter in human dermal microvascular endothelial cells. Serum adipsin levels were significantly lower in diffuse cutaneous SSc patients than in limited cutaneous SSc patients and healthy controls, and were associated positively with elevated right ventricular systolic pressure and inversely with interstitial lung disease by multivariate regression analysis. Adipsin is up-regulated at least partially by Fli1 deficiency in endothelial cells, potentially contributing to the development of pulmonary vascular involvement in SSc.

Regulation of Fibroblast Apoptosis and Proliferation by MicroRNA-125b in Systemic Sclerosis.

To analyze the expression, regulation, and role of microRNA-125b (miR-125b) in systemic sclerosis (SSc). MiR-125b expression was assessed by quantitative polymerase chain reaction (qPCR) of RNA from dermal fibroblasts and whole skin biopsy specimens from healthy controls and SSc patients. To identify downstream effectors, RNA from healthy control fibroblasts was sequenced after miR-125b knockdown and further validated using qPCR and Western blotting. Fibrosis, apoptosis, and proliferation were assessed by Caspase-Glo 3/7 assay, Western blotting, immunofluorescence staining for cleaved caspase 3, and annexin V real-time assay in dermal fibroblasts. Expression of miR-125b was significantly down-regulated in SSc skin biopsy specimens by 53% (median fold change 0.47 [interquartile range 0.35-0.69]; P < 0.001) and in SSc dermal fibroblasts by 47% (median fold change 0.53 [interquartile range 0.36-0.58]; P < 0.001) compared to healthy control skin biopsy specimens and fibroblasts, respectively (n = 10 samples per group). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin A significantly decreased the expression of miR-125b in dermal fibroblasts. MiR-125b knockdown significantly reduced cell proliferation and α-smooth muscle actin (α-SMA) expression at the messenger RNA (mRNA) and protein levels. RNA-Seq identified BAK1, BMF, and BBC3 as potential targets of miR-125b. Quantitative PCR confirmed that knockdown of miR-125b up-regulated these genes (P < 0.01; n = 12). Bcl-2 homologous antagonist killer 1 showed the strongest induction confirmed at the protein level (P < 0.01; n = 10). Consequently, miR-125b knockdown increased apoptosis compared to scrambled control. Accordingly, miR-125b overexpression decreased apoptosis. Our findings indicate that miR-125b is down-regulated in SSc skin and primary dermal fibroblasts. MiR-125b down-regulation increases apoptosis and decreases proliferation and α-SMA expression in dermal fibroblasts, indicating that its compensatory, antifibrotic mechanism may be a potential novel therapeutic option.

Short communication: Metagenomic evaluation of skin biopsies of udder sores in dairy cows

The objectives of this study were (a) to evaluate skin biopsies of udder sores and negative control cows for the presence of mange and nonbacterial pathogens via histopathology and (b) to identify and compare bacterial abundance in the lesions of cows with udder sores and from the skin of healthy controls from the same farms. Cows from 3 dairy farms with (n = 23) and without (n = 12) udder sore lesions were enrolled, and punch biopsies (23 lesions, 23 negative control samples of cows with lesions, and 12 control samples of cows without lesions) were collected. The biopsies were evaluated histopathologically, and their 16S metagenome was analyzed. No signs of mange or viral or fungal infections were detected histopathologically in any samples. The α diversity of microbial populations decreased in lesions, across all farms, and the abundance of spirochaetes did not notably change, compared with controls. However, compared with control samples, the microbial fractions of Fusobacterium, Helcococcus spp., Anaerococcus spp., Porphyromonas spp., Prevotella spp., and Trueperella spp. increased several-fold in lesions. In summary, our results suggest that spirochaetes, viruses, and mange are unlikely to cause udder sores. Instead, sores were associated with a marked increase in the abundance of Fusobacterium, Helcococcus, Anaerococcus, Porphyromonas, Prevotella, and Trueperella. Future studies are needed to determine which of these bacteria initiates this polymicrobial infection.

Construction of a lncRNA-miRNA-mRNA network to determine the regulatory roles of lncRNAs in psoriasis.

Psoriasis is a chronic inflammatory skin disorder that impairs the quality of life of affected patients. Emerging studies indicate that certain long non-coding RNAs (lncRNAs) have important roles in psoriasis. However, the exact functions of lncRNAs and their regulatory mechanisms as competitive endogenous RNAs (ceRNAs) in psoriasis have remained to be fully elucidated. In the present study, differentially expressed lncRNAs, microRNAs (miRNAs) and mRNAs were identified by analyzing public datasets, and a psoriasis-associated lncRNA-miRNA-mRNA network was constructed based on the ceRNA theory. Furthermore, previously validated abnormally expressed miRNAs in psoriasis were identified by a systematic literature search in the PubMed and Web of Science databases, and a specific miRNA-associated lncRNA-miRNA-mRNA sub-network was extracted. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using DAVID 6.8. A total of 253 lncRNAs, 106 miRNAs and 1,156 mRNAs were identified as being differentially expressed between psoriasis skin and healthy control skin. The present study identified two key lncRNAs that may potentially have a role in the pathogenesis of psoriasis: AL035425.3 and Prader Willi/Angelman region RNA 6. This integrative analysis enhances the understanding of the molecular mechanism of psoriasis and may provide novel therapeutic targets for the treatment of psoriasis.

435 Human group 2 innate lymphoid cells differentiate into interleukin-17A producing cells in psoriasis

The IL-23/IL-17A axis is critical in the defense against extracellular pathogens as well as the development of several autoimmune diseases including psoriasis. Here we focused on the capability of human innate lymphoid cell (ILC) subsets to produce IL-17A. Initial experiments revealed unexpectedly that purified dermis-derived group 2 ILCs (ILC2s), but not ILC3s, were induced to produce this cytokine after co-culture with Candida albicans-exposed undivided dermal cells. The CRTH2+ ILC2s transdifferentiated towards IL-17A producing NKp44– ILC3-like cells. This feature was also found in ILC2s derived from peripheral blood or cord blood and appeared to be driven by IL-1β, IL-23 and TGF-β and coincided with up-regulation of RORγt and reciprocal down-regulation of GATA3. Flow cytometry, RNA expression microarray, and unbiased single-cell RNA analyses disclosed a c-Kit (CD117)+ CCR6+ ILC2 subpopulation having some features in common with ILC3s, including expression of RORγt which accommodates IL-17A production in response to IL-1β and IL-23. This ILC2 subset also expressed the skin homing receptor CCR10. The c-Kit– ILC2s were also able to produce IL-17A but required during stimulation with IL-1β and IL-23 the additional presence of TGF-β, which promoted the upregulation of IL23R, CCR6 and c-Kit in these cells. The switch into IL-17A producing ILC3-like cells could be reversed by IL-4. The plasticity process we describe here has clinical relevance as we show that the proportion of IL-17A producing ILC3s is increased at the expense of ILC2s within the lesional skin of psoriatic patients as compared to healthy control skin. We identify ILC2s as a novel cellular source of IL-17A, providing a novel therapeutic target to ameliorate IL-17A pathogenicity.

The P2X7 Receptor Is Overexpressed in the Lesional Skin of Subjects Affected by Hidradenitis Suppurativa: A Preliminary Study

Background: P2X receptors (P2XRs) are plasma membrane channels involved in the modulation of immune responses. The role of the P2X7 receptor (P2X7R) has never been investigated in hidradenitis suppurativa (HS), which is a recurrent skin disease characterized by inflammatory nodules, scarring, and suppuration. Objective: Our aim was to investigate by immunohistochemistry (IHC) P2X7R, NLRP3 (NOD-like receptor family, pyrin domain-containing 3), and interleukin-1β (IL-1β) expression in HS lesions compared to healthy control (HC) skin. Method: The intensity of IHC immunostaining was semi-quantitatively graded for keratinocytes, neutrophils, lymphocytes, and monocytes. Statistical significance was assessed by the Mann-Whitney U test, Cohen’s κ coefficient, and χ² test. Results: A total of 59 samples, 31 from HS and 28 from HC, were collected and analysed. In skin keratinocytes, lymphocytes, and monocytes, but not in neutrophils, P2X7R and NLRP3 protein expression was significantly increased in HS versus the HC group. IL-1β protein expression was also higher in HS versus the HC group both in skin keratinocytes and in the inflammatory infiltrate. Cohen’s κ correlation coefficients for the expression of P2X7R versus NLRP3 or IL-1β in skin keratinocytes were significant (κ = 0.43 and 0.34, respectively). The same association between P2X7R and NLRP3 or IL-1β was confirmed by χ² tests. Conclusion: P2X7R, NLRP3, and IL-1β are overexpressed, and therefore the entire P2X7R/NLRP3/IL-1β pro-inflammatory axis is likely overactive in the skin of HS patients. This observation might provide clues to the pathogenesis of this disease and suggest novel therapies and markers of disease activity.

Interferon regulatory factor 7 (IRF7) represents a link between inflammation and fibrosis in the pathogenesis of systemic sclerosis

ObjectivesThere is considerable evidence that implicates dysregulation of type I interferon signalling (or type I IFN signature) in the pathogenesis of systemic sclerosis (SSc). Interferon regulatory factor 7 (IRF7) has...

Combined culture and metagenomic analyses reveal significant shifts in the composition of the cutaneous microbiome in psoriasis.

The treatment of psoriasis has been revolutionized by the development of biologic therapies. However, the pathogenesis of psoriasis, in particular the role of the cutaneous microbiome, remains incompletely understood. Moreover, skin microbiome studies have relied heavily on 16S rRNA sequencing data in the absence of bacterial culture. To characterize and compare the cutaneous microbiome in 20 healthy controls and 23 patients with psoriasis using metagenomic analyses and to determine changes in the microbiome during treatment. Swabs from lesional and nonlesional skin from patients with psoriasis, and from controls matched for site and skin microenvironment, were analysed using both 16S rRNA sequencing and traditional culture combined with mass spectrometry (MALDI-TOF) in a prospective study. Psoriasis was associated with an increased abundance of Firmicutes and a corresponding reduction in Actinobacteria, most marked in lesional skin, and at least partially reversed during systemic treatment. Shifts in bacterial community composition in lesional sites were reflected in similar changes in culturable bacteria, although changes in the microbiota over repeated swabbing were detectable only with sequencing. The composition of the microbial communities varied by skin site and microenvironment. Prevotella and Staphylococcus were significantly associated with lesional skin, and Anaerococcus and Propionibacterium with nonlesional skin. There were no significant differences in the amount of bacteria cultured from the skin of healthy controls and patients with psoriasis. Shifts in the cutaneous microbiome in psoriasis, particularly during treatment, may shed new light on the pathogenesis of the disease and may be clinically exploited to predict treatment response. What's already known about this topic? Alterations in the composition of the cutaneous microbiome have been described in psoriasis, although methodological differences in study design prevent direct comparison of results. To date, most cutaneous microbiome studies have focused on 16S rRNA sequencing data, including both living and dead bacteria. What does this study add? This prospective observational study confirms that changes in the composition of the cutaneous microbiome, detected by 16S rRNA sequencing, are consistent with those identified by bacterial culture and mass spectrometry. The changes in the microbiome during antipsoriasis therapy should be further investigated to determine whether these represent potential novel biomarkers of treatment response. What is the translational message? Characterization of cutaneous microbiota may ultimately move into the clinic to help facilitate treatment selection, not only by optimizing currently available treatments, but also by identifying new therapeutic targets.

A potential contribution of decreased galectin-7 expression in stratified epithelia to the development of cutaneous and oesophageal manifestations in systemic sclerosis.

Stratified epithelia have caught much attention as potential contributors to the development of dermal and oesophageal fibrosis in systemic sclerosis (SSc). Galectin-7 is a marker of all types of stratified epithelia, which is involved in the maintenance of epidermal homeostasis. So far, the role of galectin-7 has not been studied in SSc. To investigate the potential contribution of galectin-7 to the development of clinical manifestations in SSc. Galectin-7 expression was examined in skin samples and cultured keratinocytes by immunostaining and/or quantitative reverse transcription PCR. Serum galectin-7 levels were determined by enzyme-linked immunosorbent assay in 63 SSc and 20 healthy subjects. Galectin-7 expression was markedly decreased in the epidermis of SSc lesional skin compared with that of healthy control skin. Serum galectin-7 levels were significantly lower in SSc patients than in healthy controls and inversely correlated with skin score. In addition, SSc patients with diffuse pigmentation and those with oesophageal dysfunction had significantly decreased serum galectin-7 levels as compared to those without each symptom. Importantly, endothelin-1 stimulation suppressed galectin-7 expression in normal human keratinocytes, and bosentan, a dual endothelin receptor antagonist, reversed circulating galectin-7 levels and epidermal galectin-7 expression in SSc patients. Galectin-7 downregulation in stratified epithelia, which is mediated at least partially by autocrine endothelin stimulation, may contribute to the development of cutaneous manifestations and oesophageal dysfunction in SSc patients.

The anti-inflammatory potency of biologics targeting tumour necrosis factor-α, interleukin (IL)-17A, IL-12/23 and CD20 in hidradenitis suppurativa: an ex vivo study.

SummaryBackgroundBiologics targeting inflammatory mediators can achieve clinical improvements in hidradenitis suppurativa (HS). However, their clinical efficacy shows great interpatient variability in daily practice.ObjectivesTo investigate the anti‐inflammatory potency of a selection of currently available biologics and prednisolone for the treatment of HS in an ex vivo skin culture system using lesional HS biopsies.MethodsLesional skin samples from 10 patients with HS and skin samples from five healthy controls were cultured ex vivo and exposed to prednisolone or biologics targeting tumour necrosis factor (TNF)‐α, interleukin (IL)‐17A, IL‐12/23p40 or CD20 (adalimumab, infliximab, secukinumab, ustekinumab and rituximab, respectively). Real‐time quantitative polymerase chain reaction and cytokine bead arrays were used to measure the inhibitory effect of the biologics on cytokines and antimicrobial peptides (AMPs).ResultsThe relative mRNA expression of all tested cytokines and AMPs was significantly downregulated by all anti‐inflammatory agents (P < 0·001). The protein production of the proinflammatory cytokines TNF‐α, interferon γ, IL‐1β, IL‐6 and IL‐17A was significantly inhibited by adalimumab, infliximab, ustekinumab, prednisolone (all P < 0·001) and rituximab (P = 0·0071), but not by secukinumab (P = 0·0663). On both mRNA and protein levels, adalimumab, infliximab and prednisolone reduced the levels of a broader mix of individual cytokines than secukinumab, ustekinumab and rituximab. Moreover, a significant inhibitory effect on mRNA expression levels of inflammatory markers in healthy control skin was observed only for TNF‐α inhibitors (P < 0·001) and prednisolone (P = 0·0015).ConclusionsThis ex vivo study suggests that TNF‐α inhibitors and prednisolone are the most powerful inhibitors of proinflammatory cytokines and AMPs in HS lesional skin, which concurs with our clinical experience in patients with HS.

Abstract 85: Jun Drives A Fibrogenic Cutaneous Wound Healing Response Through Differential Action On Distinct Fibroblast Subpopulations

PURPOSE: Skin fibrosis and scarring can result in devastating disfigurement and permanent functional loss. Currently, there are no treatment modalities able to prevent or reverse this fibrotic process, and scars and their consequences result in an enormous medico-economic burden. Thus, it is of paramount importance to better understand the key pathogenic mechanisms driving this pathological fibrotic process. We recently described a mouse model in which overexpression of JUN, an AP-1 transcription factor, can be induced to produce global tissue fibrosis. Here, we explored the effects of JUN overexpression in skin scarring and fibrosis and the ability of our mouse to model hypertrophic scarring and excessive skin fibrosis in response to wounding. METHODS: Stented excisional dorsal wounds were created in transgenic JUN (c-JuntetO R26-M2rtTA) and Rosa (Rosa26-rtTA) control mice. Doxycycline (2mg/ml) was used to induce JUN overexpression in the wound beds on the day of surgery (POD 0) and every other day until the wounds healed (postoperative day 14, POD 14). On alternate days throughout the healing response, wounds were harvested and analyzed histologically for thickness and collagen deposition, and by fluorescent activated cell sorting (FACS) to compare the relative percentages of fibroblast subpopulations. To study the role of JUN in human scars, dermal fibroblasts were isolated from hypertrophic scars (HTS) and healthy control skin (NS) and transduced to knock out JUN using a CRISPR-Cas 9 method. Proliferation and apoptosis were compared in the knock-out (KO) and non-KO human dermal fibroblasts. RESULTS: The wounds of JUN mice healed at a significantly accelerated rate between POD5 and POD14 (*p<0.05). Compared to the wounds of Rosa control mice, the scars of JUN mice on POD14 were significantly thicker, and although collagen content was not different, it was more disordered on Hematoxylin and Eosin staining, and brighter and more branched upon computational assessment of Picrosirius stained wounds (*p<0.05). JUN overexpression resulted in a significant expansion of reticular fibroblasts at the expense of lipofibroblasts, evident on POD 7 (*p<0.05). Translating these results to human scars; JUN CRISPR-Cas 9 deletion increased apoptosis and decreased proliferation of primary cultures of HTS and NS fibroblasts. CONCLUSION: JUN overexpression increases the fibrotic cutaneous wound healing response by significantly expanding reticular dermal fibroblasts at the expense of the dermal lipofibroblasts. In addition, assessment of HTS and NS fibroblasts isolated from human skin indicate that JUN also mediates the fibrotic response in human disease by inhibiting apoptosis and driving proliferation of the dermal fibroblast subsets. Thus, our novel inducible JUN mouse model can be used to explore the mechanisms driving HTS and other pathological skin fibrosis and facilitate targeted identification of new treatments.

The Effect of Potato (Solanum tuberosum L.) Skin Extract on Alkaline Phosphatase Level in Periodontitis

Periodontitis is a chronic inflammation on tooth supporting tissues leads to alveolar bone resorption and tooth loss. Increase of alkaline phosphatase (ALP) level is found in periodontitis condition. Potato skin extract is known for its antioxidant and antiinflammation compounds therefore presumably potential as adjuvant therapy for periodontitis. This study aim was to know the effect of oral intake of potato skin extract on alveolar bone ALP and serum ALP level in model periodontitis Sprague dawley rats. Type of the study was experimental with posttest-only control group design. Thirty-five male Sprague dawley rats were divided into five groups: healthy control, negative control (aquadest-treated periodontitis), and potato skin extract- treated periodontitis in three different concentrations (25%, 50%, 75%). After 14-day treatments, ALP level on alveolar bone and blood serum samples from each group were measured using UV-Vis spectrophotometer. The results showed both alveolar bone ALP and serum ALP level were lower in potato skin extract-treated periodontitis groups in comparison to negative control group. Statistic analysis using One-way Anova showed significant difference of ALP level amongst groups (p<0.05). Post-hoc LSD test showed significant differences of ALP level between each treated and control groups (p<0.05). This study concluded potato skin extract decreased ALP level, with concentration 75% showed lowest both ALP levels of alveolar bone and serum samples, indicated alveolar bone remodeling. Moreover, it is suggested that ALP level in serum can represent ALP level on alveolar bone tissues in periodontitis condition.

Skin and Systemic Inflammation in Schnitzler's Syndrome Are Associated With Neutrophil Extracellular Trap Formation.

Schnitzler's syndrome is a rare autoinflammatory disorder characterized by interleukin-1ß-mediated and neutrophil-dominated inflammation. Neutrophil extracellular traps (NETs) are web-like structures of decondensed chromatin, histones, and antimicrobial peptides released by neutrophils. NETs were initially described in the context of pathogen defense but are also involved in autoimmune-mediated skin diseases. Here, we assessed the role of neutrophil extracellular trap formation (NETosis) in Schnitzler's syndrome. Immunofluorescence co-staining of myeloperoxidase and subnucleosomal complex was performed on lesional skin samples from patients with Schnitzler's syndrome, other neutrophilic dermatoses (cryopyrin-associated periodic syndrome, Sweet syndrome, and pyoderma gangrenosum), urticarial vasculitis and chronic spontaneous urticaria as well as healthy control skin. Blood neutrophils from patients with Schnitzler's syndrome and controls were isolated, and NETosis was induced by phorbol 12-myristate 13-acetate (PMA). Also, NETosis of control neutrophils induced by symptomatic Schnitzler's syndrome sera, cytokines and sub-threshold PMA doses was studied. Immunofluorescence co-staining revealed widespread and substantial NET formation in lesional skin of Schnitzler's syndrome patients but absence of NETs in chronic spontaneous urticaria and control skin. Neutrophils undergoing NETosis were observed in the skin of other neutrophilic diseases too. Correspondingly, blood neutrophils from Schnitzler's syndrome patients showed significantly elevated NETosis rates compared to control neutrophils following stimulation with PMA. Increased NETosis correlated well with high levels of C-reactive protein (CRP). SchS patients with the lowest NETosis rates had persistent joint and bone pain despite IL-1 blockade. Stimulation of control neutrophils and sub-threshold PMA with sera of symptomatic Schnitzler's syndrome patients disclosed enhanced NETosis as compared to control sera. Our results suggest that the induction of NET formation by neutrophils contributes to skin and systemic inflammation and may support the resolution of local inflammation in Schnitzler's syndrome.

Dysbiosis of Skin Microbiota in Psoriatic Patients: Co-occurrence of Fungal and Bacterial Communities.

Psoriasis is a chronic inflammatory skin disease, whose pathogenesis involves dysregulated interplay among immune cells, keratinocytes and environmental triggers, including microbiota. Bacterial and fungal dysbiosis has been recently associated with several chronic immune-mediated diseases including psoriasis. In this comprehensive study, we investigated how different sampling sites and methods reflect the uncovered skin microbiota composition. After establishing the most suitable approach, we further examined correlations between bacteria and fungi on the psoriatic skin. We compared microbiota composition determined in the same sample by sequencing two distinct hypervariable regions of the 16S rRNA gene. We showed that using the V3V4 region led to higher species richness and evenness than using the V1V2 region. In particular, genera, such as Staphylococcus and Micrococcus were more abundant when using the V3V4 region, while Planococcaceae, on the other hand, were detected only by the V1V2 region. We performed a detailed analysis of skin microbiota composition of psoriatic lesions, unaffected psoriatic skin, and healthy control skin from the back and elbow. Only a few discriminative features were uncovered, mostly specific for the sampling site or method (swab, scraping, or biopsy). Swabs from psoriatic lesions on the back and the elbow were associated with increased abundance of Brevibacterium and Kocuria palustris and Gordonia, respectively. In the same samples from psoriatic lesions, we found a significantly higher abundance of the fungus Malassezia restricta on the back, while Malassezia sympodialis dominated the elbow mycobiota. In psoriatic elbow skin, we found significant correlation between occurrence of Kocuria, Lactobacillus, and Streptococcus with Saccharomyces, which was not observed in healthy skin. For the first time, we showed here a psoriasis-specific correlation between fungal and bacterial species, suggesting a link between competition for niche occupancy and psoriasis. However, it still remains to be elucidated whether observed microbial shift and specific inter-kingdom relationship pattern are of primary etiological significance or secondary to the disease.

Assessment of telomerase activity in nonsegmental vitiligo tissue: apilot study.

Vitiligo is characterized by loss of melanocytes; therefore, an increased risk of photoageing and cancer are expected. However, a low incidence of cancer and sun damage in vitiliginous skin has been reported. Telomerase is a specialized cellular enzyme catalysing the synthesis of telomeres, and an increased level of the telomerase activity has been highlighted in most of human cancer cells and cancer cell lines. To assess relative telomerase activity (RTA) among patients with nonsegmental vitiligo. In this case-control study, skin biopsy specimens were taken from 20 patients (one from lesional and another from nonlesional skin) and from sun-protectedskin from 10 healthy age-, sex- and skin phototype-matched healthy controls. PCR ELISA was performed for assessment of RTA. RTA in lesional skin biopsies from patients with nonsegmental vitiligo was significantly decreased compared with nonlesional skin and healthy control skin samples, with no significant difference between the latter two. RTA in lesional skin was negatively correlated with Vitiligo Area Scoring Index but not correlated with Vitiligo Disease Activity score or RTA of nonlesional skin. Neither lesional nor nonlesional RTA levels showed any correlation with patient sex, age, skin phototype or with disease duration. Low levels of RTA in vitiliginous skin may help to explain the lower chance of developing skin cancer and decreased incidence of actinic damage in vitiliginous skin.

Cellular retinol-binding protein-1 expression increases with increasing clinical severity of alopecia areata.

Alopecia areata (AA) is multifactorial disease mostly autoimmune affecting anagen hair follicles. Many researchers hypothesize that adequate retinoic acid (RA) levels are important for proper hair follicle behavior. Previous animal studies revealed increase in RA synthesis proteins and decrease in RA degradation proteins in AA patients when compared with controls. To evaluate cellular retinol-binding protein-1 expression in lesional skin of alopecia areata in comparison with controls, in an attempt to know its role in the pathogenesis of alopecia areata . Immunohistochemical expression of cellular retinol-binding protein-1 CRBP1 was evaluated in skin biopsies taken from lesions of alopecia areata in 30 patients and 10 normal biopsy specimens taken from skin of healthy controls (HC) who were within the same age and sex. CRBP1 expression was significantly increased in lesional alopecia areata skin in comparison with normal skin of controls (P<0.001*). Significant positive correlation was found between expression of CRBP-1 and percentage of hair loss in the scalp (SALT score; r=0.840, P=<0.001). These results may enhance the idea of the possible role of CRBP1 in the pathogenesis of AA, and ensuring the importance of its level in AA treatment.