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.

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.

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.

Feasibility and diagnostic accuracy of a low radiation exposure protocol for prospective ECG-triggering coronary MDCT angiography.

AIM: To compare the feasibility, accuracy, and effective radiation dose (ED) of multidetector computed tomography (MDCT) in the detection of coronary artery disease using a combined ED-saving strategy including prospective electrocardiogram (ECG) triggering with a short x-ray window and a body mass index (BMI)-adapted imaging protocol using adaptive statistical iterative reconstruction (ASIR; group 1), in comparison with a prospective ECG triggering strategy alone (group 2). MATERIALS AND METHODS: One hundred and seventy patients scheduled for invasive coronary angiography (ICA) were evaluated. Fourteen patients were not eligible for MDCT. The remaining 156 patients were randomized to group 1 (78 patients) and group 2 (78 patients). Eight and 11 patients in groups 1 and 2, respectively, were excluded after randomization because the patients' heart rates were >65 beats/min. MDCT images were assessed for feasibility, signal-to-noise ration (SNR), and contrast-to-noise ratio (CNR), accuracy in detection of coronary stenoses >50% versus ICA and for ED. RESULTS: The feasibility, SNR, CNR, accuracy in a segment-based and patient-based model were similar in both groups (97 versus 95%, 14.5 ± 3.9 versus 14.2 ± 4.1, 16 ± 4.6 versus 16.5 ± 4.4, 95 versus 94% and 97 versus 99%, respectively). The ED in group 1 was 72% lower than in group 2 (2.1 ± 1.2 versus 7.5 ± 1.8 mSv, respectively; p<0.01). CONCLUSIONS: The use of a multi-parametric ED saving protocol results in a significant reduction in ED without a negative impact on accuracy.

Risk of acquiring tick-borne infections in forestry workers from Lazio, Italy.

The seroprevalence of antibodies to Borrelia burgdorferi and tick-borne encephalitis (TBE) virus was evaluated in a group of forestry rangers in the Lazio region of Italy. One hundred and forty-five forestry rangers and 282 blood donors were examined by two-tiered serological tests for B. burgdorferi and TBE virus. Information on occupation, residence, tick bites, outdoor leisure activities and other risk factors was obtained. The prevalence of IgG/IgM antibodies to B. burgdorferi showed no statistical difference between the two groups, but there was a higher occurrence of IgM antibodies. There were significant differences between indoor and outdoor, urban and rural workplaces among the 145 exposed workers (χ² test: p < 0.001), and a higher risk for outdoor rural than urban tasks was detected among the ten Western blot-tested forestry rangers positive to B. burgdorferi (χ² test: p < 0.1). No seropositivity was observed for the TBE virus. Forestry rangers from the Lazio region did not have a higher risk of Borrelia infection than the blood donors, though an increase in the risk for outdoor tasks in a rural environment was observed.

Intracardiac echocardiography in electrophysiology.

Intracardiac echocardiography (ICE) is a recent, invaluable tool which can provide real-time anatomical guidance in electrophysiological procedures. By inserting intravenously an ultrasound probe and advancing it into the heart, various different views can be obtained which allow to better visualize patient anatomy, to guide the placement of electrophysiological catheters, and to detect immediately procedural complications as they occur. In atrial fibrillation ablation, ICE proves particularly useful to achieve a safer trans-septal puncture (especially in the presence of anatomical anomalies of the interatrial septum) and to help to monitor the visualization of the mapping catheters (circular, high density), or the monitoring of the balloons catheter (Cryo, Laser) position. In ventricular tachycardia ablation, on the other hand, ICE allows for continuous correlation between electrophysiological and structural findings (such as wall motion anomalies or changes in echodensity), and helps to ensure correct catheter contact and to position it, particularly around delicate structures such as the aortic cusps. In any procedure, ICE is also useful to immediately detect procedural complications, such as thrombus formation along catheters, or pericardial effusion. Thanks to its real-time morphological information, ICE provides an ideal complement to simple fluoroscopy or to more complex electroanatomic mapping techniques and is set to gain a wider role in a broad range of electrophysiological procedures.

Markers of inflammation, thrombosis and endothelial activation correlate with carotid IMT regression in stable coronary disease after atorvastatin treatment.

BACKGROUND AND AIMS: MIAMI is a prospective multicenter clinical study designed to investigate the relationship between changes in carotid intima-media thickness (C-IMT) and changes in circulating markers of inflammation, thrombosis and endothelial activation in stable coronary patients treated for 20+/-3.7 months with 20mg/day atorvastatin. METHODS AND RESULTS: Eighty-five subjects had their C-IMT, blood lipids and soluble markers measured at baseline, at the 12th month and at the end of the study. Almost all soluble markers decreased upon treatment except for high-sensitivity C-reactive protein (hs-CRP), interleukin-18 (IL-18), tissue factor pathway inhibitor-free (TFPI-free) and soluble vascular cell adhesion molecules-1 (sVCAM-1) which did not change significantly, and interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and soluble CD40 ligand (sCD40L) which increased. sCD40L, fibrinogen, tissue factor pathway inhibitor-total (TFPI-total), soluble intercellular adhesion molecules-1 (sICAM-1), sE-selectin, interleukin-8 (IL-8) and von Willebrand factor (vWF) changed significantly even after application of the Bonferroni correction for multiple comparisons. Changes in lipids did not correlate with C-IMT regression either when considered singly or when combined in a lipid score. Changes in soluble markers correlated poorly with C-IMT regression when analyzed singly, but strongly when combined in relevant composite scores (inflammation/coagulation score, endothelial activation score, soluble markers score and total score). CONCLUSION: In patients with stable coronary artery disease treated with moderate doses of atorvastatin, carotid IMT regression correlated with changes of inflammation, thrombosis and endothelial activation profiles.

Bacterial protein toxins: current and potential clinical use.

Natural toxins are the product of a long-term evolution, and act on essential mechanisms in the most crucial and vital processes of living organisms. They can attack components of the protein synthesis machinery, actin polymerization, signal transduction pathways, intracellular trafficking of vesicles as well as immune and inflammatory responses. For this reason, toxins have increasingly being used as valuable tools for analysis of cellular physiology, and in the recent years, some of them are used medicinally for the treatment of human diseases. This review is devoted to protein toxins of bacterial origin, specifically those toxins that are currently used in therapy or those under study for their potential clinical applications. Bacterial protein toxins are all characterized by a specific mechanism of action that involves the central molecular pathways in the eukaryotic cell. Knowledge of their properties could be used for medical purposes.

E-selectin and TFPI are associated with carotid intima-media thickness in stable IHD patients: the baseline findings of the MIAMI study.

OBJECTIVE: MIAMI was a prospective multicenter clinical study designed to investigate the relationship between changes in carotid intima-media thickness (C-IMT) and those in the levels of circulating markers of inflammation, thrombosis and endothelial dysfunction. The study was performed in a group of stable coronary patients treated for two years with a moderate dosage of atorvastatin (20mg/day). In this paper the cross-sectional relationship between C-IMT and the same circulating markers of inflammation, thrombosis and endothelial dysfunction measured at baseline was investigated. METHODS: Eighty-five subjects that had not used statins for at least two months were enrolled in the study. At time of enrollment, the levels of vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin, interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-alpha, high-sensitivity C-reactive protein (hs-CRP), tissue factor (TF), tissue factor pathway inhibitor (TFPI), von Willebrand factor (vWF), fibrinogen, total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL), and triglycerides were measured, in parallel with C-IMT assessment. RESULTS: In cross-sectional analyses, markers of endothelial perturbation (i.e. E-selectin) and TFPI were more strongly correlated with arherosclerotic burden than markers of inflammation. The baseline picture in this study indicates that E-selectin and TFPI are linked with atherosclerotic burden.

Arboviruses in Italy.

A brief review of the arboviruses isolated In Italy is presented and discussed. Some of the Arboviruses considered in this paper are endemic in the country and are transmitted by arthropods that play actually a role of vectors, (sand flyes, hard ticks and mosquitoes); other arboviruses, sporadically isolated, are potential agents of emerging human or zoonotic diseases.

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function.

Potential of ultrasonography for epidemiological study of work-related wrist tenosynovitis.

OBJECTIVE: To explore the possible role of ultrasonography in case definitions for epidemiological studies of work-related wrist tenosynovitis. METHODS: Clinical and ultrasonography (7.5 MHz linear probe) data systematically collected from meat workers (n = 128) with biomechanical exposure characterisation were analysed. The diagnostic accuracy of different combinations of potentially relevant ultrasonography findings (nonhomogeneity, thickening and anechoic halo) was evaluated using symptomatology as a reference standard. The concordance between ultrasonography findings and symptoms was then analysed. RESULTS: Analysis of wrist biomechanical exposure was suggestive of increased prevalence of musculoskeletal disorders. Using symptoms as a reference standard, each of the three ultrasonography findings (and their combinations) showed good specificity (> or =85%) accompanied by low sensitivity (<60%); the positive likelihood ratio for at least two findings was 4.1. kappa Values (95% confidence intervals) between symptoms and different sets of ultrasonography findings were as follows: for non-homogeneity, kappa = 0.31 (0.19 to 0.43); at least one finding, kappa = 0.28 (0.16 to 0.40); at least two findings, kappa = 0.32 (0.20 to 0.44); all p<0.01. CONCLUSION: The use of ultrasonography in symptomatic subjects could contribute to a more specific epidemiological case definition of wrist tenosynovitis. The results of this study could help orient future research in this direction.

Rho-dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22.

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid-induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

Activation of rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells.

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases.

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.

Multinucleation and pro-inflammatory cytokine release promoted by fibrous fluoro-edenite in lung epithelial A549 cells.

An unusual cluster of malignant mesothelioma was evidenced in Biancavilla, a Sicily village where no inhabitant had been significantly and professionally exposed to asbestos. Mineralogical and environmental studies led to the identification of a new prismatic amphibole, named fluoro-edenite. We previously reported, by using the human lung epithelial A549 cells, that prismatic fluoro-edenite was unable to induce changes that could be somehow related to cellular transformation, and this was in accordance with studies carried out in vivo. More recently, a fibrous amphibole with a composition very similar to that of prismatic fluoro-edenite, was identified in Biancavilla. This fibrous fluoro-edenite was shown to induce mesothelioma in rats. In keeping with this effect in vivo, in the present work we observed multinucleation and spreading, common features of transformed cells, as well as pro-inflammatory cytokine release in A549 cells. Such cell changes occurred without interfering with the passage of the resulting multinucleated cells through the cell cycle and without condemning cells to death. Hence, in lung epithelial cells, fibrous fluoro-edenite behaved similarly to the unrelated asbestos type crocidolite, whose connection with severe inflammation and cancer of the lung is renowned.

Toxins from anaerobic bacteria: specificity and molecular mechanisms of action.

Major advances have been made in the past five years in the identification of cellular targets of toxins produced by anaerobic bacteria. These targets include the vesicular membrane docking and fusion apparatus, the actin cytoskeleton, the signal transduction machinery and the cell membrane. The recent discovery that large clostridial toxins (Clostridium difficile A and B toxins, C. sordellii lethal and hemorrhagic toxins, and alpha C. novyi toxin) are monoglucosyltransferases, together with the establishment of the perfringolysin crystal structure, has led to new insights in the field of toxins from anaerobic bacteria.