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Phototherapy with narrow‐band Ultraviolet B (nb‐UVB) is a major therapeutic option in atopic dermatitis (AD), yet knowledge of the early molecular responses to this treatment is lacking. The objective of this study was to map the early transcriptional changes in AD skin in response to nb‐UVB treatment. Adult patients (n = 16) with AD were included in the study and scored with validated scoring tools. AD skin was irradiated with local nb‐UVB on day 0, 2 and 4. Skin biopsies were taken before and after treatment (day 0 and 7) and analysed for genome‐wide modulation of transcription. When examining the early response after three local UVB treatments, gene expression analysis revealed 77 significantly modulated transcripts (30 down‐ and 47 upregulated). Among them were transcripts related to the inflammatory response, melanin synthesis, keratinization and epidermal structure. Interestingly, the pro‐inflammatory cytokine IL‐36γ was reduced after treatment, while the anti‐inflammatory cytokine IL‐37 increased after treatment with nb‐UVB. There was also a modulation of several other mediators involved in inflammation, among them defensins and S100 proteins. This is the first study of early transcriptomic changes in AD skin in response to nb‐UVB. We reveal robust modulation of a small group of inflammatory and anti‐inflammatory targets, including the IL‐1 family members IL36γ and IL‐37, which is evident before any detectable changes in skin morphology or immune cell infiltrates. These findings provide important clues to the molecular mechanisms behind the treatment response and shed light on new potential treatment targets.

Background Loss-of-function mutations in the skin barrier gene filaggrin (FLG) increase the risk of atopic dermatitis (AD), but their role in skin barrier function, dry skin and eczema in infancy is unclear. Objectives To determine the role of FLG mutations in impaired skin barrier function, dry skin, eczema and AD at 3 months of age and throughout infancy. Methods FLG mutations were analysed in 1836 infants in the Scandinavian population-based PreventADALL study. Transepidermal water loss (TEWL), dry skin, eczema and AD were assessed at 3, 6 and 12 months of age. Results FLG mutations were observed in 166 (9%) infants. At 3 months, carrying FLG mutations was not associated with impaired skin barrier function (TEWL > 11 center dot 3 g m(-2) h(-1)) or dry skin, but was associated with eczema [odds ratio (OR) 2 center dot 89, 95% confidence interval (CI) 1 center dot 95-4 center dot 28; P < 0 center dot 001]. At 6 months, mutation carriers had significantly higher TEWL than nonmutation carriers [mean 9 center dot 68 (95% CI 8 center dot 69-10 center dot 68) vs. 8 center dot 24 (95% CI 7 center dot 97-8 center dot 15), P < 0 center dot 01], and at 3 and 6 months mutation carriers had an increased risk of dry skin on the trunk (OR 1 center dot 87, 95% CI 1 center dot 25-2 center dot 80; P = 0 center dot 002 and OR 2 center dot 44, 95% CI 1 center dot 51-3 center dot 95; P < 0 center dot 001) or extensor limb surfaces (OR 1 center dot 52, 95% CI 1 center dot 04-2 center dot 22; P = 0 center dot 028 and OR 1 center dot 74, 95% CI 1 center dot 17-2 center dot 57; P = 0 center dot 005). FLG mutations were associated with eczema and AD in infancy. Conclusions FLG mutations were not associated with impaired skin barrier function or dry skin in general at 3 months of age, but increased the risk for eczema, and for dry skin on the trunk and extensor limb surfaces at 3 and 6 months.

BackgroundThe kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropica’s digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss.Methodology/Principal FindingsThe genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007-2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units.Conclusion/SignificanceThese large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East.Author SummaryCutaneous leishmaniasis is mainly caused by Leishmania tropica in the Middle East, where it is known for treatment failure and a need for prolonged and/or multiple treatments. Several factors affect the clinical presentation and treatment outcome, such as host genetic variability and specific immune response, as well as environmental factors and the vector species. Little is known about the parasite genome and its influence on treatment response. By analysing the genome of 22 isolates of L. tropica, we have revealed extensive genomic variation and a complex population structure with evidence of genetic exchange within and among the isolates, indicating a possible presence of sexual or parasexual mechanisms. Understanding the Leishmania genome better may improve future treatment and better understanding of treatment failure and relapse.

The associations between atopic dermatitis (AD) and cardiovascular disease (CVD) are debated. The aim of this study was to investigate the association between AD and coronary artery disease or ischaemic stroke in a nationwide, register-based, case-control study (104,832 AD cases, 1,022,435 controls) based on linkage of Swedish national register data between 1968 and 2016. Patients were classified as having severe AD if they had received systemic pharmacotherapy for AD or had been treated in a dermatological ward with AD as the main diagnosis. Other AD was classified as non-severe. After multivariable adjustments for comorbidities and socioeconomic status, overall AD was associated with angina pectoris (adjusted odds ratio (aOR) 1.13, 95% confidence interval (CI) 1.08-1.19), but among males with severe AD this association was not found, compared with the general population. Male non-severe AD was associated with myocardial infarction (OR 1.15, 95% CI 1.07-1.23). Severe AD was associated with ischaemic stroke, with similar estimates in men and women (aOR 1.19, 95% CI 1.07-1.33). Subgroup analyses among women indicated smoking as an important risk factor among severe cases. Dia-betes mellitus, hyperlipidaemia, and hypertension were more prevalent in severe AD than in controls, and hyper-lipidaemia and hypertension were also more prevalent in non-severe AD than in controls. In conclusion, in this study, AD was associated with CVD, and this should be kept in mind, especially when managing patients with severe AD.

IMPORTANCE: Atopic dermatitis is a common and debilitating skin condition characterized by intense itching and chronic inflammation. Research on behavioral treatments with high accessibility is needed. OBJECTIVE: To investigate the efficacy of a highly scalable internet-delivered cognitive behavior therapy (CBT) for adults with atopic dermatitis. DESIGN, SETTING, AND PARTICIPANTS: This randomized clinical trial from a medical university in Stockholm, Sweden, included 102 adults with atopic dermatitis, recruited from across Sweden, who received 12 weeks of internet-delivered CBT (March 29, 2017, to February 16, 2018). The first participant provided screening data on November 27, 2016, and the last 1-year follow-up assessment was conducted on June 28, 2019. INTERVENTIONS: Participants were randomized in a 1:1 ratio to 12 weeks of therapist-guided internet-delivered CBT (n = 51) or a control condition (n = 51) that gave instructions about standard care. MAIN OUTCOMES AND MEASURES: The primary outcome was the between-group difference in mean reduction of atopic dermatitis symptoms as measured by the Patient-Oriented Eczema Measure and modeled intention to treat during the 12-week treatment period. RESULTS: A total of 102 participants (mean [SD] age, 37 [11] years; 83 [81%] female) were recruited and randomized. The primary analysis indicated that participants receiving internet-delivered CBT, relative to the controls, had a significantly larger mean weekly reduction in symptoms of atopic dermatitis as measured with the Patient-Oriented Eczema Measure (B = 0.32; 95% CI, 0.14-0.49; P

Background Hundreds of variants associated with atopic dermatitis (AD) and psoriasis, 2 common inflammatory skin disorders, have previously been discovered through genome-wide association studies (GWASs). The majority of these variants are in noncoding regions, and their target genes remain largely unclear. Objective We sought to understand the effects of these noncoding variants on the development of AD and psoriasis by linking them to the genes that they regulate. Methods We constructed genomic 3-dimensional maps of human keratinocytes during differentiation by using targeted chromosome conformation capture (Capture Hi-C) targeting more than 20,000 promoters and 214 GWAS variants and combined these data with transcriptome and epigenomic data sets. We validated our results with reporter assays, clustered regularly interspaced short palindromic repeats activation, and examination of patient gene expression from previous studies. Results We identified 118 target genes of 82 AD and psoriasis GWAS variants. Differential expression of 58 of the 118 target genes (49%) occurred in either AD or psoriatic lesions, many of which were not previously linked to any skin disease. We highlighted the genes AFG1L, CLINT1, ADO, LINC00302, and RP1-140J1.1 and provided further evidence for their potential roles in AD and psoriasis. Conclusions Our work focused on skin barrier pathology through investigation of the interaction profile of GWAS variants during keratinocyte differentiation. We have provided a catalogue of candidate genes that could modulate the risk of AD and psoriasis. Given that only 35% of the target genes are the gene nearest to the known GWAS variants, we expect that our work will contribute to the discovery of novel pathways involved in AD and psoriasis.

Background: The pathophysiology in atopic dermatitis (AD) is not fully understood, but immune dysfunction, skin barrier defects, and alterations of the skin microbiota are thought to play important roles. AD skin is frequently colonized with Staphylococcus aureus (S. aureus) and microbial diversity on lesional skin (LS) is reduced compared to on healthy skin. Treatment with narrow-band ultraviolet B (nb-UVB) leads to clinical improvement of the eczema and reduced abundance of S. aureus. However, in-depth knowledge of the temporal dynamics of the skin microbiota in AD in response to nb-UVB treatment is lacking and could provide important clues to decipher whether the microbial changes are primary drivers of the disease, or secondary to the inflammatory process. Objectives: To map the temporal shifts in the microbiota of the skin, nose, and throat in adult AD patients after nb-UVB treatment. Methods: Skin swabs were taken from lesional AD skin (n = 16) before and after 3 treatments of nb-UVB, and after 6–8 weeks of full-body treatment. We also obtained samples from non-lesional skin (NLS) and from the nose and throat. All samples were characterized by 16S rRNA gene sequencing. Results: We observed shifts towards higher diversity in the microbiota of lesional AD skin after 6–8 weeks of treatment, while the microbiota of NLS and of the nose/throat remained unchanged. After only 3 treatments with nb-UVB, there were no significant changes in the microbiota. Conclusion: Nb-UVB induces changes in the skin microbiota towards higher diversity, but the microbiota of the nose and throat are not altered.

Background Atopic dermatitis (AD) is a common chronic skin disorder and is well known to be associated with other atopic conditions. There is increasing evidence for an association also with nonatopic conditions, including autoimmune diseases, but data are limited about several autoimmune diagnoses. Objectives To investigate the association between AD and autoimmune diseases. Methods This case-control study used Swedish national healthcare registers. The source population comprised the entire Swedish population aged ≥ 15 years from 1968 to 2016. Cases, including all those with an inpatient diagnosis of AD (from 1968) and/or a specialist outpatient diagnosis of AD (from 2001), were matched by sex and age to healthy controls (104 832 cases of AD, 1 022 435 controls). Results AD was significantly associated with one or more autoimmune diseases compared with controls - adjusted odds ratio (aOR) 1·97, 95% confidence interval (CI) 1·93-2·01 - and this association was significantly stronger in the presence of multiple autoimmune diseases compared with only one. The association was strongest for autoimmune disorders involving the skin (aOR 3·10, 95% CI 3·02-3·18), the gastrointestinal tract (aOR 1·75, 95% CI 1·69-1·82) or connective tissue (aOR 1·50, 95% CI 1·42-1·58). In the overall analysis, men with AD had a stronger association with rheumatoid arthritis and coeliac disease than did women with AD. In subanalyses, the findings remained stable in multivariable analyses after adjustment for smoking and parental autoimmune disease. Conclusions This large population-based study indicates significant autoimmune comorbidity of adults with AD, especially between AD and autoimmune dermatological, gastrointestinal and rheumatological diseases. Having multiple autoimmune diseases resulted in a stronger association with AD than having only one autoimmune disease.

There is a need for unified guidance on the management of ocular manifestations of atopic dermatitis and ocular manifestations associated with dupilumab in the Nordic region (Denmark, Finland, Norway and Sweden). This initiative gathered Nordic dermatologists and ophthalmologists to identify consensus in this area using a modified Delphi process. The initiative was led by a Nordic expert panel who developed a questionnaire that was circulated to a wider group. The results informed an agenda consisting of 24 statements to be voted on using a 5-point Likert scale at a meeting in Copenhagen on 24 April 2019. A facilitator moderated discussion and revised statements according to expert feedback for a second vote when required to reach consensus. Consensus was reached for 23 statements regarding the diagnosis, treatment and referral of these patients, which we hope will improve patient management in the Nordic region.

Background Dupilumab, targeting the interleukin-4α receptor and inhibiting the action of interleukin-4 and interleukin-13, was recently approved for treatment of moderate to severe atopic dermatitis. There is limited data on long-term effects and safety among patients with severe atopic dermatitis treated with dupilumab. Weight gain was observed among patients treated with dupilumab in our clinic. The aim was to describe weight change in a cohort study of patients with severe atopic dermatitis treated with dupilumab from baseline to follow-up after 12 months, and to analyze if weight change was associated with effect of treatment, reported appetite, and/or disturbed night sleep due to itching. Methods All patients with atopic dermatitis receiving systemic treatment at the Unit of Dermatology, Karolinska University Hospital, have been registered and monitored consecutively since January 2017. This cohort constituted all patients who started treatment on dupilumab or methotrexate between 10 January 2017 and 30 June 2019 with at least 6 months of follow-up within the study period. The following variables were monitored at start of and during treatment: Eczema Severity Score Index, Patient-Oriented Eczema Measure, visual analogue scale for pruritus 10 cm, Montgomery-Åsberg Depression Rating Scale, Dermatology Life Quality Index, and weight. Data analyses were performed using two-sample Wilcoxon-Mann-Whitney rank-sum test, or the Wilcoxon matched-pairs sign-rank test with a p-value Results Patients treated with dupilumab (n = 12) gained weight (mean 6.1 kg, range [0.1–18.0], p = 0.002) after 1 year on treatment. The majority of patients showed a good response to treatment with dupilumab (n = 11); at follow-up at 6, 9, or 12 months, they reached EASI-90 (n = 6), EASI-75 (n = 4), or EASI-50 (n = 1). There was no significant association between weight gain and treatment response, reported appetite, or disturbed night-sleep due to itch. Patients treated with methotrexate showed no significant weight change (n = 8). Conclusions To our knowledge, this is the first report on a possible association between weight gain and dupilumab treatment; the extent of the association is yet to be seen, as is the mechanism behind this finding.

Atopic dermatitis (AD) is a common, complex trait, arising from the interplay of multiple genetic and environmental factors. This review provides an overview of developments in the field of AD genetics. AD shows high heritability; strategies to investigate genetic risk include linkage, candidate gene studies, genome-wide association and animal modelling. Loss-of-function mutations in FLG, encoding the skin barrier protein filaggrin, remain the strongest genetic risk factor identified for AD, but variants influencing skin and systemic immune function are also important. AD is at the forefront of genetic research, from large-scale population studies to in vitro models and detailed molecular analyses. An understanding of genetic risk factors has considerably improved knowledge of mechanisms leading to atopic skin inflammation. Together this work has identified avenues for therapeutic intervention, but further research is needed to fully realise the opportunities of personalised medicine for this complex disease, to optimise patient benefit.

Background Leishmaniasis is a neglected and poorly reported parasitic infection transmitted by sand flies in tropical and subtropical regions. Knowledge about leishmaniasis has become important in non-endemic countries due to increased migration and travel. Few studies of the clinical management of cutaneous, mucocutaneous and visceral leishmaniasis in non-endemic regions have been published to date. In this study, we aimed to evaluate patient characteristics, clinical manifestations and treatments of leishmaniasis in Sweden, over a 20-year period. Methods A retrospective observational nationwide study was performed using medical records of patients diagnosed with leishmaniasis in Sweden from 1996 to 2016. Cases with culture and polymerase chain reaction verified leishmaniasis were identified at the Public Health Agency of Sweden. Results In total, 165 cases of leishmaniasis were diagnosed from 1996 to 2016. Medical records from 156 patients (95%) were available for review and included in the study. Cutaneous leishmaniasis was the dominant manifestation (n = 149, 96%), and in 66 patients (44%) cutaneous leishmaniasis was due to Leishmania tropica. Other manifestations were mucocutaneous (n = 4, 3%), visceral (n = 2, 1%) and post-kala-azar dermal leishmaniasis (n = 1, 1%). During this time period, the number of cases increased, especially after 2013. Most patients (n = 81, 52%) were migrants who were infected in their countries of origin (from 2013 to 2016, mainly Syria or Afghanistan). Other groups were Swedish tourists (25%) and returning workers (13%). The time from collection of the diagnostic sample to the start of treatment was less than one month in 81 (66%) patients and under three months in 124 patients (96%). Among the 149 patients with cutaneous leishmaniasis, 125 patients received antileishmanial treatment, and in 88 of these patients (70%) cure was achieved, regardless of treatment. Conclusions The number of leishmaniasis cases diagnosed in Sweden increased between 1996 and 2016, mainly in migrants from endemic countries. Although leishmaniasis is a rare disease in Sweden, patients appear to be diagnosed early and treated according to current European guidelines, resulting in an overall high cure rate. Electronic supplementary material The online version of this article (10.1186/s12879-018-3539-1) contains supplementary material, which is available to authorized users.

Information on factors of importance for remission of eczema is scarce. This study explored factors related to the remission and course of preschool eczema (PSE) (eczema at 1, 2 and/or 4 years of age) to 16 years of age (n = 889) in a Swedish cohort. Half of the children were in complete remission by school age (at age 8, 12, and 16 years). In multivariate prognostic models, persistent PSE (eczema at 1, 2 and 4 years of age) (odds ratio 0.27 (95% confidence interval 0.18-0.41)), PSE with sleep disturbance (due to itch at least once a week at 1, 2 and/or 4 years of age) (0.59 (0.43-0.81)), parental allergy (0.73 (0.55-0.96)), parental smoking at child's birth (0.70 (0.50-0.99)) and filaggrin mutation (R501X, R2447X, 2282del4) (0.47 (0.26-0.85)) were inversely associated with complete remission by school age. Male sex (1.37 (1.03-1.82)) and exclusive breastfeeding ≥4 months (1.44 (1.01-2.05)) were positively associated with complete remission by school age. In conclusion, half of the children with PSE were in complete remission by school age. The most important prognostic factors were persistent PSE and PSE with sleep disturbance due to itch.

Similarities and differences in the everyday clinical management of moderate-to-severe atopic dermatitis in Nordic countries are unknown. Using a modified Delphi approach, 15 dermatologists from Denmark, Finland, Norway and Sweden completed face-to-face and online questionnaires and participated in summary discussions to map expert opinion on the clinical management of moderate-to-severe atopic dermatitis in these Nordic countries. Through discussions, 6 adult patient profiles, reflecting common disease presentations of atopic dermatitis, were identified. Using these case profiles, diagnostic work-up, treatment goals, patient education and treatment approaches were discussed. Patient education was identified as essential for effective management. A treatment sequence of moderate-to-potent topical glucocorticosteroids and emollients, followed by systemic treatment, was recommended, allowing 3 months to ascertain systemic treatment response before switching, if necessary. Consensus was not reached on systemic treatment choice, reflecting differences in clinical practice and reimbursement between countries. Practical, case-based clinical recommendations were developed for optimal patient care.

Background Filaggrin is an important protein for structure and function of the skin barrier. Filaggrin gene (FLG) mutations are known to result in dry skin, impaired skin barrier, and increased risk for atopic dermatitis. However, it is not clear whether these mutations are associated with contact allergy or hand eczema in adolescence. Objectives The purpose of this study was to investigate whether FLG mutations are associated with contact allergy, self-reported hand eczema, or dry skin in adolescence. Methods We used data from the 16-year follow-up in the BAMSE cohort, information obtained from a Web-based questionnaire including questions on hand eczema and dry skin, from FLG mutation analysis (R501X, R2447X, 2282del4), and patch testing (n = 1822). Results Logistic regression analyses showed no statistically significant associations between FLG mutations and contact allergy (any contact allergy, nickel allergy, or fragrance allergy) according to patch test, or self-reported hand eczema at 16 years, or hand eczema ever. However, FLG mutations were associated with self-reported dry skin at 16 years. Conclusions FLG mutations are associated with self-reported dry skin at 16 years. However, in this study no consistent associations were found between FLG mutations and contact allergy or hand eczema at 16.

Background The introduction of unclassified new psychoactive substances (NPS) on the recreational drugs market through open online sale ('legal highs' or 'Internet drugs') continues unabated and represents a growing health hazard. The use of NPS has resulted in numerous, severe, adverse events and fatalities, due to unintended overdose or unknown toxic side-effects. Objectives To try to find a possible common underlying cause for the skin-hair-eye symptoms complex observed in three men. Methods From late 2013 to mid-2014, three Swedish men aged 23-34 years with a history of recreational drug use independently presented with similar and very remarkable clinical signs, requiring extensive examination and prolonged treatment. Results Common clinical signs included hair depigmentation, hair loss, widespread folliculitis and dermatitis, painful intertriginous dermatitis, dry eyes, and elevated liver enzymes. Two of them also showed transverse white Mees' lines (leukonychia striata) on the fingernails and toenails, suggesting a temporary, drug-induced, disorganized keratinization. The clinical signs gradually disappeared over time. However, later on, two developed severe bilateral secondary cataracts requiring surgery. Because drug tests within the Swedish STRIDA project had demonstrated intake of the NPS opioid MT-45 in all patients, this was suspected to be the common causative agent. Conclusions These cases highlight the importance for physicians and health professionals to consider the increasing number of novel, untested recreational drugs, as a potential cause of unusual and otherwise unrecognized clinical signs and symptoms.

Interactions between the epidermis and the immune system govern epidermal tissue homeostasis. These epidermis-immune interactions are altered in the inflammatory disease psoriasis; however, the pathways that underlie this aberrant immune response are not well understood. Here, we determined that Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key mediator of epidermal dysfunction. RAC1 activation was consistently elevated in psoriatic epidermis and primary psoriatic human keratinocytes (PHKCs) exposed to psoriasis-related stimuli, but not in skin from patients with basal or squamous cell carcinoma. Expression of a constitutively active form of RAC1 (RACV12) in mice resulted in the development of lesions similar to those of human psoriasis that required the presence of an intact immune system. RAC1V12-expressing mice and human psoriatic skin showed similar RAC1-dependent signaling as well as transcriptional overlap of differentially expressed epidermal and immune pathways. Coculture of PHKCs with immunocytes resulted in the upregulation of RAC1-dependent proinflammatory cytokines, an effect that was reproduced by overexpressing RAC1 in normal human keratinocytes. In keratinocytes, modulating RAC1 activity altered differentiation, proliferation, and inflammatory pathways, including STAT3, NFκB, and zinc finger protein 750 (ZNF750). Finally, RAC1 inhibition in xenografts composed of human PHKCs and immunocytes abolished psoriasiform hyperplasia and inflammation in vivo. These studies implicate RAC1 as a potential therapeutic target for psoriasis and as a key orchestrator of pathologic epidermis-immune interactions.

SummaryBackground Epidemiological data and the effect of sun exposure on atopic eczema (AE) suggest that vitamin D (vitD) may be involved in the pathogenesis. Objectives To investigate if vitD levels were associated with the presence or severity of AE in the first 2 years of life in children living in south-east Norway. Methods Infants, recruited to a clinical trial on acute bronchiolitis (n = 404) and from the general population (n = 240), were examined at 1–13 months (first visit) and at 2 years of age (second visit). Caregivers were interviewed using a structured questionnaire. AE was diagnosed clinically, based on well-established criteria. Disease severity was assessed using the SCORing Atopic Dermatitis index. Blood samples were taken for vitD measurements, using liquid chromatography–tandem mass spectrometry and for common filaggrin mutation analyses. Complete data on AE and vitD were available in 596 and 449 children at the first and second visit, respectively. Results Atopic eczema was diagnosed in 67 children (11%) at the first visit and in 103 children (23%) at the second. Mean vitD levels were 58·2 nmol L−1 at the first visit and 66·9 nmol L−1 at the second. Using vitD level tertiles in multivariate regression analysis, there was no association between vitD levels and AE at either visit, regardless of filaggrin mutation. In children without AE at the first visit, vitD levels did not predict AE at the second. Conclusions In this cohort of young children in Norway, we found no association between vitD levels and the presence or severity of AE.

Majocchi granuloma is an uncommon deep follicular inflammation caused by dermatophytes and affects immunocompetent and immunocompromised patients. The clinical findings overlap with other skin conditions such bacterial infections and inflammatory skin diseases, thereby delaying correct diagnosis. We describe 2 cases in immunocompetent patients.

Atopic dermatitis (AD) is a common and debilitating inflammatory dermatological disorder and is marked by itch and inflamed skin. Scratching, sleep loss, and avoidance of situations associated with more AD symptoms are central hypothesized mechanisms that perpetuate the disorder and cause reduced quality of life. We developed an exposure-based cognitive behavioral treatment (CBT) that entailed mindfulness practice as a means to increase tolerance for aversive experiences during exposure. The aim of the present study was to test the treatment's acceptability and preliminary efficacy in adults with AD. We used an uncontrolled pretest-posttest design and recruited participants (

Background The strong association between epidermal barrier gene variants and Atopic Dermatitis (AD) highlights that impaired skin barrier is a key feature in the pathogenesis of AD. Although the filaggrin (FLG) gene is the major AD risk gene in European and Asian populations, disease-associated variants remain elusive in African populations. Objective A previous study has reported that variants in the tight junction gene CLDN1 have been associated with AD susceptibility and disease severity in African-Americans. Our aim was therefore to investigate the association of CLDN1 with AD in the Ethiopian population. Methods To investigate how CLDN1 variants may be involved in increasing the risk of AD in the Ethiopian population, we analysed whole exome sequencing (WES) data for all exons in CLDN1, and in addition, assayed four SNPs (rs17501010, rs9290927, rs9290929 and rs893051) which had previously showed association in African-American AD patients. Results No damaging variants were detected through WES in 22 Ethiopian samples. Genotyping of disease-associated CLDN1 SNPs in Ethiopian cases and control material showed no overall association. However, significant association was seen for rs893051 in patients who developed AD before the age of 5 years (P < 0.03). Conclusion Taken together, we demonstrate that tight junction genes and, in particular, CLDN1 rather than variants in FLG may be involved in the susceptibility of AD in the Ethiopian population.

Atopic dermatitis (AD) is a chronic, itchy, inflammatory skin disorder that may worsen due to stress and anxiety. Tachykinins have been suggested to be involved in the inflammation in AD, as well as pruritus. Aprepitant is a NK-1 receptor antagonist. This open randomized trial evaluated the effect of aprepitant added to topical treatment in adult patients with moderate-severe AD. The treatment group (n = 19) received 80 mg/day aprepitant for 7 days as a supplement to standardized topical treatment with a moderately strong steroid and a moisturizer. The control group (n = 20) received topical treatment alone. Patients were monitored for the extent of the disease (using SCORing of Atopic Dermatitis; SCORAD), pruritus, and scratching movements. In both the aprepitant-treated and the control groups there was a decrease in SCORAD, pruritus and scratching movements. How-ever, there was no significant additional improvement in any of these parameters in the aprepitant-treated group compared with the control group.

Background Overweight and atopic dermatitis (AD) are major health problems in most industrialised countries, but the relationship between overweight and AD in infants and young children is unclear. We investigated if weight-for-length at birth, in infancy and at two years, as well as early weight-gain velocity, are associated with the development of AD in early life. Methods Cohort study of infants (n = 642), all living in south-east Norway, hospitalized with acute bronchiolitis (n = 404) or recruited from the general population (n = 238), examined at mean age 5.1 months (enrolment) and at a two-year follow-up visit (n = 499; 78%) at mean age 24.6 months. Exposures were weight-for-length (g/cm) at birth, enrolment and two-year follow-up, and early weight-gain velocity (gram/month from birth to enrolment). Excessive weight-for-length was defined as weight-for-length >95th percentile of WHO child-growth standards. Data on weight-for-length at the three time points were obtained for 435, 428 and 473 children. AD was diagnosed according to the Hanifin & Rajka criteria or from a history of physician-diagnosed AD. We performed multivariate analyses with weight-for-length at birth, at enrolment and at the two-year follow-up visit and with early weight gain velocity for the endpoint AD at each visit. Results In adjusted analyses, excessive weight-for-length at enrolment was associated with concurrent AD (OR 3.03; 95% CI 1.23–7.50) and with AD at two years (OR 2.40; 1.11–5.17). In infants without AD, weight-for-length at enrolment increased the risk of AD at two years, with OR being 1.02 (95% CI 1.00–1.04) per increased gram/cm. AD at two years was not associated with concurrent excessive weight-for-length, nor was AD at any time associated with weight-for-length at birth or with early weight-gain velocity. Conclusions The results suggest that overweight in infancy may contribute to the development of AD in early life, highlighting the need for child health-care professionals to address potential overweight and atopic disease when advising infants’ caregivers. Trial registration ClinicalTrials.gov number, NCT00817466 , EudraCT number, 2009–012667-34.

Atopic dermatitis (AD), ichthyosis vulgaris (IV), and X-linked recessive ichthyosis (XLRI) are characterized by dry skin and impaired skin barrier. AD and IV are related to loss-of-function mutations in FLG (encoding filaggrin), whereas XLRI is caused by deletions or inactivating mutations in the steroid sulphatase gene (STS). Patients regularly use moisturizing creams, but little is known about the creams’ effects on the skin barrier.The present work combines objective scorings, non-invasive techniques, and molecular analyses of skin biopsies to characterize the skin in 57 patients with AD, IV, or XLRI, and in 14 healthy controls. Patients were classified according to their FLG and STS mutation status: AD with FLG+/+ (n = 14), AD with FLG+/– (n = 14), AD/IV with FLG–/– (n = 15), and XLRI with STS– (n = 14), as well as one man with a novel point mutation. Assessments were conducted at baseline and after four weeks of treatment with three different moisturizers applied to volar forearm skin.At baseline, dryness scoring and non-invasive assessments verified impaired skin barrier function in all patients. In patients with AD/IV, microarray analysis identified 300–3000 up- or downregulated mRNA transcripts involved in signalling pathways important for inflammation and barrier repair. The skin phenotype and number of altered transcripts were correlated with the FLG mutation status, with FLG–/– patients displaying the highest transepidermal water loss (TEWL) and the most altered transcript levels. In contrast, despite an equally dysfunctional skin barrier, only limited changes in mRNA transcripts occurred in XLRI patients. Treatment with moisturizers improved skin dryness similarly in all groups, but TEWL behaved differently: it decreased slightly in the AD/IV group and increased in the XLRI group, especially after urea treatment. Only minute effects on skin pH and mRNA expression were observed.In conclusion, FLG mutations elicit pro-inflammatory mechanisms probably aimed at restoring barrier competence. This does not occur in patients with XLRI, presumably because STS deficiency automatically increases the barrier thickness. Moisturizing treatment improves skin dryness in patients with AD, IV, or XLRI, but does not seem to normalize the altered epidermal gene expression profile in AD/IV patients.

Atopic dermatitis (AD), ichthyosis vulgaris (IV), and X-linked recessive ichthyosis (XLRI) are characterized by dry skin and impaired skin barrier. AD and IV are related to loss-of-function mutations in FLG (encoding filaggrin), whereas XLRI is caused by deletions or inactivating mutations in the steroid sulphatase gene (STS). Patients regularly use moisturizing creams, but little is known about the creams’ effects on the skin barrier.The present work combines objective scorings, non-invasive techniques, and molecular analyses of skin biopsies to characterize the skin in 57 patients with AD, IV, or XLRI, and in 14 healthy controls. Patients were classified according to their FLG and STS mutation status: AD with FLG+/+ (n = 14), AD with FLG+/– (n = 14), AD/IV with FLG–/– (n = 15), and XLRI with STS– (n = 14), as well as one man with a novel point mutation. Assessments were conducted at baseline and after four weeks of treatment with three different moisturizers applied to volar forearm skin.At baseline, dryness scoring and non-invasive assessments verified impaired skin barrier function in all patients. In patients with AD/IV, microarray analysis identified 300–3000 up- or downregulated mRNA transcripts involved in signalling pathways important for inflammation and barrier repair. The skin phenotype and number of altered transcripts were correlated with the FLG mutation status, with FLG–/– patients displaying the highest transepidermal water loss (TEWL) and the most altered transcript levels. In contrast, despite an equally dysfunctional skin barrier, only limited changes in mRNA transcripts occurred in XLRI patients. Treatment with moisturizers improved skin dryness similarly in all groups, but TEWL behaved differently: it decreased slightly in the AD/IV group and increased in the XLRI group, especially after urea treatment. Only minute effects on skin pH and mRNA expression were observed.In conclusion, FLG mutations elicit pro-inflammatory mechanisms probably aimed at restoring barrier competence. This does not occur in patients with XLRI, presumably because STS deficiency automatically increases the barrier thickness. Moisturizing treatment improves skin dryness in patients with AD, IV, or XLRI, but does not seem to normalize the altered epidermal gene expression profile in AD/IV patients.

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases in industrialized countries. To identify candidate genes involved in the pathogenesis of AD, we previously undertook a genome-wide approach using DNA microarrays. A transcript encoding the epidermal growth factor receptor (EGFR) was found to be among the down-regulated transcripts in AD skin. Here, we further investigated the expression pattern of two EGFR family members (EGFR and ErbB2) in AD skin on a protein level. Immunohistochemical (IHC) analysis of EGFR and ErbB2 showed decreased expression of EGFR and ErbB2 proteins in AD lesional skin as compared to skin from healthy individuals. Interestingly, we found that EGFR and ErbB2 were reciprocally expressed in an in vitro model of keratinocyte proliferation and differentiation, paralleling the expression patterns found in epidermis of healthy skin. The highest levels of EGFR transcripts were found in proliferating cells, while ErbB2 was found in differentiated cells. We show that blocking EGFR activity combined with co-stimulation of the Th2-cytokine IL4 in keratinocytes leads to induction of the inflammatory chemokine CCL26/eotaxin-3 in vitro. Accordingly, increased CCL26 transcriptional levels were observed in AD lesional skin. Taken together, suppression of EGFR may contribute to the pathogenesis of AD via the regulation of inflammatory chemokines.

In a commentary to our publication entitled “Acute skin and hair symptoms followed by severe, delayed eye complications in subjects using the synthetic opioid MT-45”,[1] Yip and Deng [2] argue that the lack of opioid symptoms would indicate involvement of another drug class, possibly arsenic or 2,4-dinitrophenol (DNP). In this respect, it should be emphasized that the patients did not seek hospital care for acute opioid overdose symptoms but for unexpected, late adverse side effects involving the skin, hair, and eyes. This article is protected by copyright. All rights reserved.

Genetic variants in filaggrin (FLG) involving truncating mutations or intragenic copy number variation are strongly associated with the risk of developing atopic dermatitis (AD) in European and Asian populations. Few loss-of-function mutations have been identified in Africans, although an association between FLG copy number variation and AD severity in a small African American cohort has been proposed. We studied the association between FLG copy number and AD in 132 Ethiopians and found no association between AD severity and FLG copy number, suggesting that other, still unidentified genetic factors are of more importance in predisposing Ethiopians to AD.

Eczema is often associated with development of allergic asthma. The Neuropeptide S Receptor 1 (NPSR1) gene has previously been associated with asthma and elevated serum IgE levels. The aim of this study was to investigate a potential association between the NPSR1 gene and eczema in patients and healthy individuals from five different populations in Western Europe, in total 6275 individuals. Seven single nucleotide polymorphisms previously associated with allergic asthma were genotyped. The protein expression of NPSR1 in the skin was studied using immunohistochemistry in six eczema patients and eight healthy individuals. No association was found be tween eczema and the seven single nucleotide polymor phisms in NPSR1 in any of the populations, either independently or in combinations. In addition, no difference was detected in epidermal NPSR1 expression between eczema patients and healthy individuals. These results strongly suggest that NPSR1 is not involved in the pathogenesis of eczema.

Recent studies have identified 2 loss-of-function variants, R501X and 2282del4, in the filaggrin gene as predisposing factors in the development of eczema. In this study, representing the first analysis of the variants in a Swedish population, we analysed transmission in 406 multiplex eczema families with mainly adult patients. In accordance with previous studies we found association between the filaggrin gene variants and atopic eczema (p=9.5 x 10(-8)). The highest odds ratio for the combined allele, 4.73 (1.98-11.29), p=3.6 x 10(-8), was found for the subgroup with a severe eczema phenotype, and association was also found with raised allergen-specific IgE, allergic asthma and allergic rhinoconjunctivitis occurring in the context of eczema. Our results support an important role for the filaggrin gene variants R501X and 2282del4 in the development and severity of atopic eczema and indicate a possible role for the subsequent progression into eczema-associated phenotypes.

Summary Hallucinogens, including mescaline, psilocybin, and lysergic acid diethylamide (LSD), profoundly affect perception, cognition, and mood. All known drugs of this class are 5-HT 2A receptor (2AR) agonists, yet closely related 2AR agonists such as lisuride lack comparable psychoactive properties. Why only certain 2AR agonists are hallucinogens and which neural circuits mediate their effects are poorly understood. By genetically expressing 2AR only in cortex, we show that 2AR-regulated pathways on cortical neurons are sufficient to mediate the signaling pattern and behavioral response to hallucinogens. Hallucinogenic and nonhallucinogenic 2AR agonists both regulate signaling in the same 2AR-expressing cortical neurons. However, the signaling and behavioral responses to the hallucinogens are distinct. While lisuride and LSD both act at 2AR expressed by cortex neurons to regulate phospholipase C, LSD responses also involve pertussis toxin-sensitive heterotrimeric G i/o proteins and Src. These studies identify the long-elusive neural and signaling mechanisms responsible for the unique effects of hallucinogens.

Moisturizers are widely used for atopic dermatitis (AD) and related conditions, but available evidence of their effectiveness has not been reviewed in a systematic fashion. Our objective was to investigate the effectiveness of emollients, as a group and individually, in the treatment of AD and related conditions, by means of a systematic review. Studies indexed in MEDLINE and/or Embase before 16 January 2015. Controlled clinical studies comparing the clinical effect of a moisturizer against its vehicle, another moisturizer, or no treatment were eligible. For the outcomes transepidermal water loss (TEWL) and stratum corneum hydration, uncontrolled before–after designs were also eligible. Participants were patients with AD, irritant hand dermatitis, and/or ichthyosis vulgaris. Out of the 595 publications initially identified, 45 (48 studies, 3262 patients) were eligible for inclusion. A vast majority of studies indicate that moisturizers have beneficial effects on clinical symptoms [SCORAD (SCORing Atopic Dermatitis) reductions ranging from 0 to 2.7 points], TEWL (range 0 to −12.2 g/m2h) and stratum corneum hydration (range +8 to +100 %). Direct comparisons between individual moisturizers are still scarce, but the clinical effect appears to be much more well-documented for urea and glycerin than, for example, propylene glycol, lactate, ceramide, and aluminum chlorohydrate. Compared with urea studies, glycerin studies were more often associated with a high risk of bias. Due to differences in study designs and outcome measures, a quantitative meta-analytic approach was not deemed feasible, and formal indicators of publication bias such as funnel plots could not be used. However, a large number of moderately sized studies with positive outcomes could be compatible with selective publishing of favourable results. The clinical effect of moisturizers is well-documented. Urea-based preparations may be preferable as a first-line treatment, but there is an unmet need for well-powered comparisons between individual moisturizers.

Background and Purpose— This work was undertaken to determine to what degree long-term neurofunctional outcome of neonatal hypoxic–ischemic (HI) brain injury in mice correlates with anatomical extent of cerebral damage assessed by magnetic resonance imaging (MRI) and histopathology. Methods— On postnatal day 7, mice were subjected to HI. At 7 to 9 weeks after HI neurofunctional outcome was assessed by water-maze, rota-rod, and open-field test performance, followed by cerebral MRI and histopathology evaluation. Results— At 10 weeks after HI, MRI revealed ipsilateral brain atrophy alone or with porencephalic cyst formation and contralateral ventriculomegaly. Adult HI-affected mice, especially those that developed a porencephalic cyst, demonstrated significant neurofunctional deficit compared with age-matched naïve mice. HI-affected mice with ipsilateral cerebral atrophy but without porencephaly demonstrated no or an intermediate level of neurofunctional deficit. Neurobehavioral assessment of mice subjected to HI insult revealed a strong correlation between degree of brain injury and functional neurohandicap. Conclusions— This is the first study to demonstrate that long-term neurofunctional outcome in mice after a neonatal HI correlates tightly with anatomical pattern/extent of cerebral damage, defined by MRI and histopathology.

International audience; Background As a key regulator of serotonergic activity and target of many antidepressant treatments, the serotonin transporter (SERT) represents a potential mediator of anxiety- and depression-related behaviors. Using mice lacking the SERT (SERT KO), we examined the role of SERT function in anxiety- and depression-related behaviors and serotonergic neuron function. Methods Serotonin transporter knockout mice were evaluated in paradigms designed to assess anxiety-, depression-, and stress-related behaviors. Dorsal raphe nucleus (DRN) function was assessed by quantitative serotonergic cell counting and extracellular electrical recording of neuronal firing properties. Results Serotonin transporter knockout mice showed an increase in latency to feed in a novel situation, more immobility in a forced swim, increased escape latency in a shock escape paradigm, and decreased immobility in tail suspension. No differences in anxiety-related behaviors were seen in the open field and the elevated plus maze. Serotonin transporter knockout mice exhibit a 50% reduction in serotonergic cell number and a fourfold decrease in firing rate in the DRN. Conclusions Developmental loss of SERT produces altered behaviors in models of depression that are generally opposite to those produced by antidepressant treatment. The reduced serotonergic cell number and firing rate in the DRN of adult SERT KO mice suggest a mechanism for these altered behaviors.

Birth asphyxia accounts for the majority of developmental motor and cognitive deficits. Studies were undertaken to develop a reproducible murine model of perinatal hypoxic-ischemic encephalopathy (HIE) which would permit both anatomic and neurofunctional quantification of injury. Short-term neurofunctional outcomes consisted of three developmental reflexes (righting, cliff aversion and geotaxis) assessed in 7-day-old mouse pups 24 h after unilateral carotid artery ligation followed by inhalation of 8% oxygen. Cerebral infarct volume was dependent on duration of hypoxia, being approximately 2.5-fold greater with longer (60 min) versus shorter (30 min) hypoxia exposure (P=0.001). All three sensorimotor neonatal reflexes assessed at 24 h after HIE injury correlated significantly with long-term neurofunction evaluated using a water-maze test of navigational learning and memory assessed 8 weeks later in the same animals. Cerebral atrophy, a delayed consequence of HIE in this model, also correlated strongly with water-maze performance (r=0.86, P=0.002). These data demonstrate for the first time that murine neonatal sensorimotor reflex performance can be rigorously quantified in the acute phase of perinatal HIE and has strong predictive value not only for anatomic extent of cerebral injury, but also for long-term neurofunctional outcome.

The role of the extracellular matrix in cutaneous morphogenesis is poorly understood. Here, we describe the essential role of laminin-10 (alpha5beta1gamma1) in hair follicle development. Laminin-10 was present in the basement membrane of elongating hair germs, when other laminins were downregulated, suggesting a role for laminin-10 in hair development. Treatment of human scalp xenografts with antibodies to laminin-10, or its receptor beta1 integrin, produced alopecia. E16.5 Lama5 -/- mouse skin, lacking laminin-10, contained fewer hair germs compared with controls, and after transplantation, Lama5 -/- skin showed a failure of hair germ elongation followed by complete hair follicle regression. Lama5 -/- skin showed defective basement membrane assembly, without measurable increases in anoikis. Instead, Lama5 -/- skin showed decreased expression of early hair markers including sonic hedgehog and Gli1, implicating laminin-10 in developmental signaling. Intriguingly, treatment of Lama5 -/- skin with purified laminin-10 corrected basement membrane defects and restored hair follicle development. We conclude that laminin-10 is required for hair follicle development and report the first use of exogenous protein to correct a cutaneous developmental defect.

Summary Background Atopic dermatitis (AD) is caused by genetic and environmental factors that interact to determine disease susceptibility and severity. Several lines of evidence suggest that the IL-4 gene and the IL-4-receptor alpha (IL-4Rα) gene are involved in the development of atopic diseases. Objective The objective of this study was to evaluate the possible involvement of the chromosomal regions 5q31 and 16p12, which include the genes coding for the IL-4 and the IL-4Rα in AD. Methods We conducted linkage analysis and association studies using the microsatellite markers D16S298 and D16S403 and a single nucleotide polymorphism in the promoter region of the IL-4 gene (− 590C/T) in 406 Swedish families with at least two siblings affected with AD, in total 1514 individuals. Results and Conclusion We report linkage (P

Atopic dermatitis (AD), ichthyosis vulgaris (IV), and X-linked recessive ichthyosis (XLRI) are characterized by dry skin and impaired skin barrier. AD and IV are related to loss-of-function mutations in FLG (encoding filaggrin), whereas XLRI is caused by deletions or inactivating mutations in the steroid sulphatase gene (STS). Patients regularly use moisturizing creams, but little is known about the creams’ effects on the skin barrier.The present work combines objective scorings, non-invasive techniques, and molecular analyses of skin biopsies to characterize the skin in 57 patients with AD, IV, or XLRI, and in 14 healthy controls. Patients were classified according to their FLG and STS mutation status: AD with FLG+/+ (n = 14), AD with FLG+/– (n = 14), AD/IV with FLG–/– (n = 15), and XLRI with STS– (n = 14), as well as one man with a novel point mutation. Assessments were conducted at baseline and after four weeks of treatment with three different moisturizers applied to volar forearm skin.At baseline, dryness scoring and non-invasive assessments verified impaired skin barrier function in all patients. In patients with AD/IV, microarray analysis identified 300–3000 up- or downregulated mRNA transcripts involved in signalling pathways important for inflammation and barrier repair. The skin phenotype and number of altered transcripts were correlated with the FLG mutation status, with FLG–/– patients displaying the highest transepidermal water loss (TEWL) and the most altered transcript levels. In contrast, despite an equally dysfunctional skin barrier, only limited changes in mRNA transcripts occurred in XLRI patients. Treatment with moisturizers improved skin dryness similarly in all groups, but TEWL behaved differently: it decreased slightly in the AD/IV group and increased in the XLRI group, especially after urea treatment. Only minute effects on skin pH and mRNA expression were observed.In conclusion, FLG mutations elicit pro-inflammatory mechanisms probably aimed at restoring barrier competence. This does not occur in patients with XLRI, presumably because STS deficiency automatically increases the barrier thickness. Moisturizing treatment improves skin dryness in patients with AD, IV, or XLRI, but does not seem to normalize the altered epidermal gene expression profile in AD/IV patients.

The prevalence of filaggrin gene (FLG) mutations varies between different populations. The initial association studies revealed that FLG mutations were causative of ichthyosis vulgaris and strongly associated with atopic dermatitis (AD) in patients of Irish, Scottish, and Danish descent. However, early prevalence data from additional populations indicated that subjects from a North African population lacked the most common FLG mutations (R501X, 2282del4) found in the European populations and were rare or absent in Asian populations. Since then, a high prevalence of FLG loss-of-function variants in the general population and a strong association to disease have been detected. All associated variants have in common that they are either nonsense or frameshift in a coding exon, leading to a loss of filaggrin expression. A high prevalence of FLG mutations is evident in Northern Europe, North America, and parts of Asia. However, the prevalence and association to AD and IV seem low in populations studied from Mediterranean or African countries; also, the prevalence of FLG mutations seems to be lower in Americans of African compared to people of European descent. The prevalence in many other populations, such as those in South America or Oceania, remains to be elucidated. Interestingly, although FLG mutations are prevalent and associated with disease in several Asian populations, the specific loss-of-function variants within the FLG gene vary from those prevalent in Europe and North America. Altogether, FLG displays a remarkable variation in both the type of loss-of-function variants and mutation prevalence between different global populations. Seemingly, there is a north-south gradient of association of those studied to date. The explanation for and significance of these variations may involve an evolutionary role of carrying FLG mutations and will be further discussed.

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