Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells.

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Acute maculopapular eruption in Covid-19 patient: A case report.

We report the case of a positive COVID-19 patient who presented to our hospital for a maculopapular skin rash which appeared 7 days after the onset of COVID-19 symptoms. He was 34 years old and nothing relevant was recorded at his previous anamnesis. The patient was hospitalized for 3 days and received systemic therapy with steroid, antihistamines, tocilizumab, and hydroxicloroquine. On the third day of the hospitalization the cutaneous rash had almost completely disappeared.

Mesenchymal stem cells for the treatment of psoriasis: a comprehensive review.

Mesenchymal stem cells (MSCs) have recently been shown to have not only regenerative capabilities but also immunomodulating properties. For this reason, they are currently under investigation in clinical trials for the treatment of several autoimmune systemic disorders. Psoriasis is a systemic immune-mediated disease for which MSCs could have therapeutic potential. We analysed the existing literature with regard to MSC-based strategies for the treatment of psoriasis, using the MEDLINE, Embase, Scopus and Cochrane Library electronic databases from inception to the date of study. A number of studies confirm the involvement of MSCs in psoriasis pathogenesis and therefore designate MSCs as an important potential therapeutic tool in this setting. Preclinical data are mostly based on imiquimod-induced murine models of psoriasis, and confirm the anti-inflammatory and immunomodulatory action of MSCs in the setting of psoriasis. Six patients affected by psoriasis were described in four clinical studies. Despite significant differences in terms of therapeutic protocols and clinical outcomes, the MSC-based regimens were efficacious in 100% of the cases. Despite more data still being needed, MSCs could be a promising therapy for psoriasis.

Oral melanoma and other pigmentations: when to biopsy?

Oral pigmentations (OPs) are often neglected, although a meticulous examination of the oral cavity is important not only in the diagnosis of oral melanoma, but also for the detection of important clinical findings that may indicate the presence of a systemic disease. OPs may be classified into two major groups on the basis of their clinical appearance: focal and diffuse pigmentations, even though this distinction may not appear so limpid in some cases. The former include amalgam tattoo, melanocytic nevi, melanoacanthoma and melanosis, while the latter include physiological/racial pigmentations, smoker's melanosis, drug-induced hyperpigmentations, postinflammatory hyperpigmentations and OPs associated with systemic diseases. We will discuss the most frequent OPs and the differential diagnosis with oral mucosal melanoma (OMM), underlining the most frequent lesions that need to undergo a bioptic examination and lesions that could be proposed for a sequential follow-up.

The smart approach: feasibility of lentigo maligna superficial margin assessment with hand-held reflectance confocal microscopy technology.

BACKGROUND: Lentigo maligna may be challenging to clear surgically. OBJECTIVE: To evaluate feasibility of using superficial skin cuts as RCM imaging anchors for attaining negative surgical margins in lentigo maligna. METHODS: Included patients presented with lentigo maligna near cosmetically sensitive facial structures. We evaluated, with hand-held-RCM, microscopic clearance of melanoma beyond its dermoscopically detected edges. Evaluated margins were annotated using shallow skin cuts. If a margin was positive at 'first-step' RCM evaluation, we sequentially advanced the margin radially outward at that segment by 2-mm intervals until an RCM-negative margin was identified. Prior to final surgical excision, we placed sutures at the outmost skin cuts to allow comparison of RCM and histopathological margin assessments. Primary outcome measure was histopathological verification that RCM-negative margins were clear of melanoma. RESULTS: The study included 126 first-step margin evaluations in 23 patients, median age 70 years (range: 43-91). Seventeen patients (74%) had primary in-situ melanoma and six (26%) invasive melanoma, mean thickness 0.3 mm (range 0.2-0.4 mm). Six cases (26%) showed complete negative RCM margins on 'first-step', 11 (48%) were negative at 'second-step', and four (17%) at 'third-step'. In two additional cases (9%), margins clearance could not be determined via RCM due to widespread dendritic cells proliferation. The RCM-negative margins in all 21 cases proved clear of melanoma on histopathology. Of the 15 cases that returned at 1-year follow-up, none showed any residual melanoma on dermoscopic and RCM examinations. Interobserver reproducibility showed fair agreement between bedside RCM reader and blinded remote-site reader, with Spearman's rho of 0.48 and Cohen's kappa of 0.43; using bedside reader as reference, the remote reader's sensitivity was 92% and specificity 57% in positive margin detection. CONCLUSIONS: Margin mapping of lentigo maligna with hand-held-RCM, using superficial skin cuts, appears feasible. This approach needs validation by larger studies.

Dermoscopy of small melanomas: just miniaturized dermoscopy?

BACKGROUND: Small malignant melanomas (MMs) are usually MMs in an initial growth phase, deserving attention by the clinician aiming at an early diagnosis. OBJECTIVES: To identify clues for early diagnosis of small MMs, by comparing the dermoscopic features of MMs < 4 mm (micromelanomas) with those of larger MMs. METHODS: Our database consists of dermoscopic images of 482 MMs, which have been retrieved and measured digitally. The ABCD (asymmetry, border, colour, dimension) and 7-point criteria were evaluated for the whole database by three expert dermoscopists, whereas the main dermoscopic pattern was assessed only for micromelanomas. The dermoscopic aspects were correlated to clinical and histological features. RESULTS: Most 7-point and ABCD scores, and criteria referring to micromelanomas, differed from those of the MM database as a whole. Lesion asymmetry, number of colours, blue-whitish veil, atypical vessels, irregular globules/dots and regression increased according to MM diameter. An inverse trend was observed for atypical network and irregular pigmentation, which were more frequently observed in micromelanomas than in larger ones. Among the 22 micromelanomas, 12 lesions were in situ, whereas the other 10 were 0·2-2 mm thick. The clinical and dermoscopic characteristics of the two groups were similar. CONCLUSIONS: Micromelanomas are not a rarity. However, the clinician should be aware of the fact that the majority of them lack most of the dermoscopic features presented by larger lesions.

Dermoscopic yellow structures in basal cell carcinoma.

BACKGROUND: Yellowish structures in dermoscopy comprise milia-like cysts (MLCs) and yellow lobular-like structures. OBJECTIVE: This study aimed at establishing the frequency of these features in BCC and at describing their dermoscopic details. METHODS: A retrospective analysis of digital dermoscopic images referring to 400 BCCs was performed. Images were evaluated for the presence of starry and cloudy MLCs and yellow lobular-like structures. RESULTS: Among the 400 BCCs constituting our database, 40 presented yellowish structures (10%). "Yellow" BCCs were located more frequently on the head and were mainly of the nodular type. MLCs were observed in 7.75% of the cases (with a mean number of 4.9 MLCs per lesion), whereas yellow globules were noticed in 4.2% /ucodep of the lesions. CONCLUSION: In the presence of BCC specific dermoscopic criteria, the observation of MLCs and yellow lobular-like structures should not prompt the dermatologist to exclude the diagnosis of BCC.

Electrical stimulation as adjuvant treatment for chronic leg ulcers of different aetiology: an RCT.

OBJECTIVE: To investigate the effectiveness of an innovative electrical stimulation (ES) therapy as adjuvant treatment for chronic wounds of various aetiology, in terms of pain and ulcer healing. METHOD: Patients with chronic limb ulcers were enrolled for the study and randomised into the intervention or control group. The intervention group received conventional treatment plus ES therapy (FREMS; Lorenz Lifetech) while the control group received only conventional treatment. Each ES treatment cycle consisted of 12 sessions performed in 4 weeks (three sessions/week). All patients were treated until full wound healing occurred, or for a maximum of 9 ES cycles, with a 2-week rest between each cycle. RESULTS: A total of 60 patients were enrolled in the study and randomised into the two groups: the intervention group (n=30) and the control group (n=30). During follow-up, some patients terminated the protocol because they reached the ulcer closure before the maximum of 9 cycles. The analysis of the effect of ES on pain and ulcer healing was performed on all patients who underwent at least two consecutive clinical evaluations (two cycles), in order to reach a compatible sample size with the primary objective (one patient withdrew). In both groups, there was a significant reduction of pain compared with baseline (p < 0.05), starting from T6 visit in the first cycle. In particular, there was a significant reduction of pain in the intervention group compared with the control group after 14 days, and this reduction continued until the end of the second cycle. Similarly, there was a significant reduction of PUSH tool score in the intervention group compared with the control group after 14 days, and this reduction continued until the end of the second cycle. CONCLUSION: Data collected in this study support data in the literature. Analysis of longitudinal data analysed by simple models and complex models suggest that the ES therapy had a positive and significant effect on pain reduction (VAS) and on the improvement of ulcer healing process in terms of the PUSH tool total index compared with conventional treatment, and may have induced a significant acceleration of the wound-healing process.

Assessing physicians' preferences on skin cancer treatment in Europe.

BACKGROUND: A wide variety of both surgical and nonsurgical therapies is currently available for patients with skin cancer. OBJECTIVES: This part of the EPIDERM (European Prevention Initiative for Dermatological Malignancies) project is aimed at the evaluation of the treatment preferences for skin cancer in eight countries of the European Union. METHODS: A multicentre hospital-based case-control study was carried out at dermatology departments in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain. Patients with skin cancer (basal cell carcinoma, actinic keratosis, squamous cell carcinoma, cutaneous malignant melanoma and Bowen disease) were consecutively enrolled between July 2008 and July 2010. Information on the study variables (sex, age, country, tumour type, anatomical location and treatment) was obtained from questionnaires designed by the EPIDERM project. RESULTS: In total, 1708 patients with skin cancer were included. Surgery was the first treatment option in 76·5% of the patients (P = 0·001). Actinic keratosis was the only tumour type in which nonsurgical treatment was more frequent than surgery (91·4%). Tumours on the head were less likely to be surgically excised than those at other locations (odds ratio 0·25, P = 0·001). Simple excision or curettage was the most common surgical procedure (65·4%), followed by graft and flaps (22·4%). Cryotherapy was the most common nonsurgical option (52·4%), followed by imiquimod (18·0%), photodynamic therapy (PDT; 12·0%), 5-fluorouracil (5-FU; 5·7%), and diclofenac with hyaluronic acid (4·0%). CONCLUSIONS: Surgery remains the first-choice treatment of skin cancer. Regarding nonsurgical treatments, the conservative treatments available (imiquimod, 5-FU, PDT and diclofenac gel) have not yet exceeded the use of ablative options such as cryotherapy despite their accepted benefit of treating field cancerization.

Known and potential new risk factors for skin cancer in European populations: a multicentre case-control study.

BACKGROUND: During recent years numerous studies have suggested that personal and environmental factors might influence cancer development. OBJECTIVES: To investigate environmental and personal characteristics associated with skin cancer risk. METHODS: A multicentre hospital-based case-control study was performed in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain, including 409 patients with squamous cell carcinoma (SCC), 602 with basal cell carcinoma (BCC) and 360 with cutaneous malignant melanoma (CMM) and 1550 control persons. Exposures were assessed by questionnaires that were partly self-administered, partly completed by dermatologists. Unconditional logistic regression modelling was used to assess associations including the influence of certain drugs and food items on skin cancer risk. RESULTS: The usual associations were observed for sun exposure and pigmentation characteristics, with chronic sun exposure being most strongly associated with SCC risk, and naevi and atypical naevi with CMM risk. Use of ciprofloxacin was associated with a decreased risk of BCC [odds ratio (OR) 0·33] and use of thiazide diuretics was associated with an increased risk of SCC (OR 1·66). Ciprofloxacin was also associated with SCC (OR 0·34) and thiazines with BCC (OR 2·04), but these associations lost significance after correction for multiple testing. Consumption of pomegranate, rich in antioxidants, was associated with decreased BCC and SCC risk, also after correcting for multiple testing. Recent experience of stressful events was associated with increased risk, particularly of CMM. CONCLUSIONS: In this large case-control study from across Europe the expected associations were observed for known risk factors. Some new potential protective factors and potential risk factors were identified for consumption of certain food items, medication use and stress, which deserve further investigation in future studies.

Risk factors for actinic keratosis in eight European centres: a case-control study.

BACKGROUND: There are limited data regarding the association of actinic keratosis (AK) and other types of nonmelanoma skin cancer (NMSC); studies investigating possible correlation of AK with melanocytic naevi are even scarcer. To our knowledge, there are no data examining the risk of AK in people using specific medications. OBJECTIVE: To investigate constitutional and exposure risk factors leading to AK and the coexistence of AK with NMSC and melanoma. METHODS: A multicentre hospital-based case-control study was performed in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain, including 343 patients with actinic keratosis (AK), 409 with squamous cell carcinoma (SCC), 602 with basal cell carcinoma (BCC), 360 with invasive melanoma and 119 with in situ melanoma, and 686 control subjects. Exposures were assessed by questionnaires that were partly self-administered and partly filled out by dermatologists. Unconditional logistic regression modelling was used to assess associations including the influence of phenotypic characteristics, presence of naevi, sun-exposure habits and certain drugs on AK risk. RESULTS: Differences in hair and eye coloration variably influenced the risk for AK, with red hair signifying a seven times higher risk [odds ratio (OR) 6·9, 95% confidence interval (CI) 4·34-11·00), and brown - compared with blue - eyes, about a 40% reduced risk (OR 0·61, 95% CI 0·13-0·92). The darker the skin phototype, the lower the risk for AK, with phototype IV exhibiting nine times less risk of developing AK. Some and many freckles on the arms were associated with an OR of 1·8 (95% CI 1·08-2·81) and 3·0 (95% CI 1·10-3·54), respectively, while overall number of naevi and high educational level were inversely associated with AK. Sun exposure, thiazide diuretics and cardiac drugs had a higher risk for AK. SCC was the most frequent (58%) skin neoplasm coexisting with AKs, followed by BCC (30%), melanoma in situ (12%) and invasive melanoma (6%). CONCLUSION: In this large case-control study from across Europe the expected associations were confirmed for known risk factors. Some possible new risk factors, including cardiac and diuretic drugs, were identified, creating a new field for further investigation in future studies.

The patient journey: a report of skin cancer care across Europe.

BACKGROUND: There are poorly documented variations in the journey a skin cancer patient will follow from diagnosis to treatment in the European Union. OBJECTIVES: To investigate the possible difficulties or obstacles that a person with a skin malignancy in the European Union may have to overcome in order to receive adequate medical screening and care for his/her condition. In addition, we wished to explore differences in European health systems, which may lead to health inequalities and health inequities within Europe. METHODS: Ten European countries took part in this investigation (in alphabetical order): Finland, Germany, Greece, Italy, Malta, Poland, Romania, Spain, the Netherlands and the U.K. The individual participants undertook local and national enquiries within their own country and completed a questionnaire. RESULTS: This exercise has identified important differences in the management of a skin cancer patient, reflecting major disparities in health care between European countries. CONCLUSIONS: Further investigation of health disparities and efforts to address health inequalities should lead to improvements in European health care quality and reduction in morbidity from skin cancer.

Obesity and gastro-esophageal reflux disease: a 2009 update.

The reports describing that obesity per se predisposes to gastroesophageal reflux disease (GERD) have brought conflicting results. Establishing a causal link between these two conditions would be of major public health importance, because of their present epidemic proportions. To date, some large studies examining the relationship between obesity and GERD found a strongly positively relationship while others did not. The main cause of this discordance is the vast heterogeneity of such studies: sufficiently powerful design is found only in few investigations, GERD is defined with a low degree of homogeneity, biases are obvious in the choice of diagnostic methods, thus giving room for large variations in the adjustment of potential confounding factors. Future research should take three directions: 1) prospective population-based studies in which the incidence or recurrence of GERD should be evaluated in correlation with body mass index; 2) intervention trials, focusing on the benefit of weight loss in the prevention of GERD and its recurrence; 3) studies of physiopathology (both in the animal models and humans) to understand the potential biological plausibility.

Which are the main fluorophores in skin and oral mucosa? A review with emphasis on clinical applications of tissue autofluorescence.

OBJECTIVES: The present review provides information about which molecules appear to be the main fluorophores in skin and oral mucosa, together with their clinical applications. DESIGN: The MEDLINE database was searched, using "oral mucosa AND fluorophores", "skin AND fluorophores", "epidermal AND fluorophores", "dermal AND fluorophores" and "cutaneous AND fluorophores" as entry terms. We searched the literature following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The level of evidence in the studies was assessed using the Classification of the Oxford Centre for Evidence-based Medicine (CEBM) Levels for Diagnosis. RESULTS: Five papers and 17 were primarily focused on description of fluorophores in oral mucosa and skin Evidence exists that fluorophores of oral mucosa and skin are mainly proteins such as collagen, elastin, keratin and tryptophan. Other possible fluorophores identified are: porphyrins, advanced glycation end products, flavins, lipopigment, nicotinamide adenine dinucleotide, flavin adenine dinucleotide, pheomelanin, eumelanin and components of lipofuscin. Clinical applications of oral mucosal autofluorescence (AF) are related to management of malignant and potentially malignant lesions. In the skin, AF has been used for acne assessment, diagnosis of sweat-gland pathologies, glycemic control and management of malignant lesions and as a marker for skin aging. CONCLUSION: Fluorophores stimulated through AF devices are implied in different physiologic and pathologic processes. AF seems to be useful for several clinical applications, especially in skin department. Because most of the studies show a low level of evidence, further studies are necessary in such a promising and fascinating field.

Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for BCL-2.

Normal human keratinocytes synthesize and release nerve growth factor (NGF) and express both the low- and the high-affinity NGF receptor. Because NGF has been shown to rescue certain cell types from programmed cell death, we investigated the role of endogenous NGF in preventing keratinocyte apoptosis. We report here that apoptosis is induced in normal human keratinocytes in culture by blocking endogenous NGF signaling with either anti-NGF neutralizing antibody or K252, a specific inhibitor of the tyrosine kinase high-affinity NGF receptor. Apoptosis was assessed by DNA laddering, electron microscopy, and in situ nick end labeling technique. In anti-NGF-treated keratinocytes, the apoptotic process starts at 96 h, and is maximal at 120 h. After K252 treatment, apoptosis starts at 48 h and peaks at 120 h. Because the product of the bcl-2 proto-oncogene protects many cell types from apoptosis, we measured the levels of this protein in apoptotic keratinocytes. We found that both K252 and anti-NGF antibody strikingly downregulate bcl-2 expression, starting at 72 h. Furthermore, HaCat keratinocytes stably transfected with a plasmid containing bcl-2 cDNA fail to undergo apoptosis when treated with K252. These findings show that autocrine NGF acts as a survival factor for human keratinocytes in vitro through its high-affinity NGF receptor, possibly by maintaining constant levels of Bcl-2.

Ultraviolet B radiation induces activation of neutral and acidic sphingomyelinases and ceramide generation in cultured normal human keratinocytes.

The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which transduces an extracellular signal into the cell. During the past few years increasing evidence has shown that the sphingolipid ceramide may play a role as a second messenger in intracellular signal transduction. The ceramide generation via sphingomyelinase (SMase) is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of programmed cell death or apoptosis. The aim of this study is to investigate whether activation of SMases and generation of ceramide can be induced by UVB radiation in normal human keratinocytes. The present data show that exposure to UVB radiation results in rapid generation of ceramide. The ceramide accumulation starts 15 min after UV exposure and progressively increases up to 24 h. In vitro measurement of SMase activity following exposure to UVB evidences an activation of both neutral and acidic SMases. Moreover, UVB induces apoptosis in normal human keratinocytes as shown by TUNEL technique and FACS analysis. These data indicate that UVB induced ceramide generation and activation of both neutral and acidic SMases, suggesting that sphingolipids metabolism may be involved in the UVB signaling pathway.

Psoriasis and the nervous system.

Both clinical and experimental evidence is accumulating on the role of the nervous system in the pathogenesis of psoriasis. Sporadic reports as well as extensive studies indicate that emotional stress can act as an exacerbating event in psoriasis. Moreover, that neurogenic mechanisms are operating in psoriasis is suggested by clinical, pharmacologic and experimental data. We have focused our investigations on the role of vasoactive intestinal peptide (VIP) and substance P (SP) in psoriatic lesions using a variety of experimental approaches: 1) receptor autoradiography; 2) immunohistochemistry; 3) radio-immunoassay; 4) human keratinocytes cultures. Our results indicate that an imbalance of VIP and SP exists in psoriatic lesions, and that these neuropeptides exert different and specific effects on human keratinocytes. At present, however, the finding of psoriasis being exacerbated by psychological factors cannot be satisfactorily explained merely by alterations of neuropeptides in the skin.

[Effect of total++ time of extracorporeal circulation in in myocardial revascularization surgery on urinary urea nitrogen loss]. / Efeito do tempo total de circulação extracorpórea em cirurgia de revascularização miocárdica sobre a perda urinária de nitrogênio na forma de uréia.

Cardiopulmonary bypass probably should be an important factor increasing surgical stress when heart surgery is focused. This study was undertaken in order to evaluate the role of cardiopulmonary bypass in proteic catabolism. Study group consisted of patients who underwent cardiac aortic bypass graft as an isolated procedure. Inclusion criteria were elective surgery and absence of comorbidities after a rigorous preoperatory evaluation. One hundred and five patients were studied prospectively and urinary nitrogen loss was measured in the first 24 hour postoperative period. Operations performed were standard cardiopulmonary bypass procedures, under cardiopulmonary bypass, moderate hypothermia and hemodilution. Saphenous veins and mammary artery grafts were performed in all cases. Correlation and multiple linear regression were used. There was found no correlation between urinary nitrogen loss and age, gender and time under cardiopulmonary bypass. A positive correlation was found between number of grafts and increased urinary nitrogen loss. Further studies comparing cardiac aortic bypass graft with and without cardiopulmonary bypass are suggested.