Integrative genetic and genomic networks identify microRNA associated with COPD and ILD.

Chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) are clinically and molecularly heterogeneous diseases. We utilized clustering and integrative network analyses to elucidate roles for microRNAs (miRNAs) and miRNA isoforms (isomiRs) in COPD and ILD pathogenesis. Short RNA sequencing was performed on 351 lung tissue samples of COPD (n = 145), ILD (n = 144) and controls (n = 64). Five distinct subclusters of samples were identified including 1 COPD-predominant cluster and 2 ILD-predominant clusters which associated with different clinical measurements of disease severity. Utilizing 262 samples with gene expression and SNP microarrays, we built disease-specific genetic and expression networks to predict key miRNA regulators of gene expression. Members of miR-449/34 family, known to promote airway differentiation by repressing the Notch pathway, were among the top connected miRNAs in both COPD and ILD networks. Genes associated with miR-449/34 members in the disease networks were enriched among genes that increase in expression with airway differentiation at an air-liquid interface. A highly expressed isomiR containing a novel seed sequence was identified at the miR-34c-5p locus. 47% of the anticorrelated predicted targets for this isomiR were distinct from the canonical seed sequence for miR-34c-5p. Overexpression of the canonical miR-34c-5p and the miR-34c-5p isomiR with an alternative seed sequence down-regulated NOTCH1 and NOTCH4. However, only overexpression of the isomiR down-regulated genes involved in Ras signaling such as CRKL and GRB2. Overall, these findings elucidate molecular heterogeneity inherent across COPD and ILD patients and further suggest roles for miR-34c in regulating disease-associated gene-expression.

Age of onset defines two distinct profiles of atopic dermatitis in adults.

BACKGROUND: The incidence of adult-onset atopic dermatitis (AOAD) is increasing. However, the unique characteristics of AOAD compared to pediatric-onset AD persisting into adulthood (POAD) are underexplored, hampering the development of targeted-therapeutics for this growing population. We thus assessed the profile of AOAD in skin and blood compared to that of POAD. METHODS: We collected skin biopsies and blood from adults with AOAD, POAD, and healthy controls (n = 15 in each group). Skin samples were analyzed by RNA sequencing, qRT-PCR, and immunohistochemistry, and Olink Proseek multiplex assay was used to identify the serum proteomic profile. RESULTS: Compared to healthy controls, both AOAD and POAD showed cutaneous immune and barrier dysregulations with a shared Th2/Th22 hyperactivation. Overall, POAD showed greater inflammation in lesional skin, with more prominent expression of Th2/Th17/Th22 markers (CCL17/22, S100A8/9, IL-36A, PI3/Elafin, DEFB4) in POAD compared to AOAD (p-value < .05). In contrast, higher Th1-(IFN-γ, IL-2, IL-15, CCL5) upregulation and Th1-skewing were seen in AOAD. The epidermal barrier was also more compromised in POAD, with greater epidermal hyperplasia and lower expression of markers related to terminal differentiation, lipids, and cell adhesion. In parallel with increased rates of cardiovascular comorbidities, AOAD demonstrated many more significantly dysregulated proteins in serum (n = 148) compared to POAD (n = 86), including pro-inflammatory and cardiovascular-risk markers. Th1-related products showed significant correlations between their skin and blood expressions only in AOAD subjects. CONCLUSION: Age-of-onset delineates two distinct endophenotypes in adult AD potentially suggesting the need for broader (beyond Th2) therapeutic targeting in AOAD.

Crisaborole reverses dysregulation of the mild to moderate atopic dermatitis proteome toward nonlesional and normal skin.

BACKGROUND: Safe and effective long-term topical treatments for atopic dermatitis (AD) remain limited. OBJECTIVE: In this phase 2a, single-center, intrapatient, and vehicle-controlled study, we examine the mechanism of action of crisaborole 2% ointment, a topical nonsteroidal PDE4 (phosphodiesterase-4) inhibitor, in a proteomic analysis of 40 adults with mild to moderate AD and 20 healthy subjects. METHODS: Within the AD cohort, 2 target lesions were randomized in an intrapatient (1:1) manner to double-blind crisaborole/vehicle applied twice daily for 14 days. Punch biopsy specimens were collected for biomarker analysis at baseline from all participants, then from AD patients only at day 8 (optional) and day 15. RESULTS: Compared to the vehicle, crisaborole significantly reversed dysregulation of the overall lesional proteome and of key markers and pathways (eg, Th2, Th17/Th22, and T-cell activation) associated with AD pathogenesis toward both nonlesional and normal skin. Significant clinical correlations were observed with markers associated with nociception and Th2, Th17, and neutrophilic activation. LIMITATIONS: Study limitations include predominance of white patients in the cohort, relatively short treatment time, and regimented administration of crisaborole. CONCLUSION: Our results demonstrate crisaborole-induced normalization of the AD proteome toward a nonlesional molecular phenotype and further support topical PDE4 inhibition in the treatment of mild to moderate AD.

Proteomic characterization of atopic dermatitis blood from infancy to adulthood.

BACKGROUND: Patients with atopic dermatitis (AD) have systemic biomarker dysregulation that differs by age group; however, the proteomic characteristics of these age-based changes are unknown. OBJECTIVE: To profile blood proteins of patients with AD across different age groups versus age-appropriate controls. METHODS: Using the Olink high-throughput proteomic platform, we profiled 375 serum proteins of 20 infants (age, 0-5 years), 39 children (age, 6-11 years), 21 adolescents (age, 12-17 years), and 20 adults (age, ≥18 years) with moderate-to-severe AD and 83 age-appropriate controls. RESULTS: Each group presented a distinct systemic proteomic signature. Th2-related proteins were increased in infant AD and further intensified with age through adolescence and adulthood (interleukin 4/CCL13/CCL17). In contrast, Th1 axis down-regulation was detected in infants with AD and gradually reversed to increased Th1 products (interferon γ/CXCL9/CXCL10/CCL2) in patients with AD from childhood to adulthood. Despite their short disease duration, infants already had evidence of systemic inflammation, with significant upregulation of innate immunity (interleukin 17C/ interleukin-1RN), T-cell activation/migration (CCL19), Th2 (CCL13/CCL17), and Th17 (PI3) proteins. Adults with AD present unique upregulation of cardiovascular proteins related to coagulation and diabetes. LIMITATIONS: Cross-sectional observational study with a single time point. CONCLUSION: Systemic immune signatures of AD are age-specific beyond the shared Th2 immune activation. These data advocate for precision medicine approaches based on age-specific AD profiles.

Secukinumab responses vary across the spectrum of congenital ichthyosis in adults.

IMPORTANCE: Treatment of congenital ichthyoses primarily focuses on reversing skin scaling and is not pathogenesis based. Recent studies showed Th17 immune skewing, as in psoriasis, across the spectrum of ichthyosis, suggesting that targeting this pathway might broadly reduce disease severity. OBJECTIVE: To determine whether secukinumab, an IL-17A inhibitor, can improve ichthyosis across several congenital ichthyosis subtypes. DESIGN: Exploratory 16-week double-blind, randomized, placebo-controlled trial comparing secukinumab 300 mg every 4wks to placebo (1:1 randomization) in adults with the four major congenital ichthyosis subtypes (NCT03041038), followed by a 16-week open-label phase to evaluate response of the placebo-first group and a 20-week extension for safety. Significant differences in secukinumab- vs. placebo-treated subjects at Wk16 in the Ichthyosis Area Severity Index (IASI) score and lack of increased mucocutaneous bacterial and/or fungal infections were the co-primary efficacy and safety endpoints, respectively. SETTING: Two tertiary referral centers: Northwestern University Feinberg School of Medicine, Chicago, and Mount Sinai Icahn School of Medicine, New York. PARTICIPANTS: Twenty subjects ≥ 18 yo with genotype-confirmed epidermolytic ichthyosis, Netherton syndrome, lamellar ichthyosis, or congenital ichthyosiform erythroderma with at least moderate erythroderma. RESULTS: IL-17A inhibition did not significantly reduce severity or increase mucocutaneous infections among the 18 who completed the 16-week double-blind phase. Five patients with 29-50% clinical improvement at Wk32 requested drug continuation. Th17-related biomarkers were not significantly reduced vs. baseline or placebo-treated levels. LIMITATIONS: Small sample size; heterogeneous ichthyosis subsets. CONCLUSION: IL-17 inhibition with secukinumab is safe, but not efficacious across the spectrum of adult ichthyoses. GOV REGISTRATION NUMBER: NCT03041038; first posted on 02/02/2017.

Apremilast and narrowband ultraviolet B combination therapy suppresses Th17 axis and promotes melanogenesis in vitiligo skin: a randomized, split-body, pilot study in skin types IV-VI.

Improved repigmentation of generalized vitiligo in skin types IV-VI has been reported in clinical response to combined therapy with apremilast and narrowband (NB)-UVB; however, tissue responses to combined therapy versus NB-UVB monotherapy have not been elucidated. We compared the change from baseline in cellular and molecular markers in vitiligo skin after combined therapy versus NB-UVB monotherapy. We assessed lesional and nonlesional skin samples from enrolled subjects and evaluated for immune infiltrates, inflammatory, and melanogenesis-related markers which were compared across different treatment groups. Combined therapy resulted in significant reduction of CD8+T cells and CD11c+ dendritic cells, downregulation of PDE4B and Th17-related markers, and upregulation of melanogenesis markers. This study was limited to small sample size, skin types IV-VI, and high dropout rate. Our molecular findings support the clinical analysis that apremilast may potentiate NB-UVB in repigmentation of generalized vitiligo in skin types IV-VI.

Scalp biomarkers during dupilumab treatment support Th2 pathway pathogenicity in alopecia areata.

BACKGROUND: The mechanisms driving alopecia areata (AA) are still unclear, hindering development of targeted therapeutics. Specific Th2 targeting with dupilumab in AA provides a unique opportunity to dissect its pathogenesis and explore the role of Th2 pathway. METHODS: We evaluated changes in scalp biomarkers in AA patients (with and without concomitant atopy) randomized to weekly dupilumab or placebo for 24 weeks, followed by open-label dupilumab for 24 weeks. Changes in biomarker levels were measured at weeks 12, 24, and 48 and were also correlated with clinical hair regrowth. RESULTS: At week 24, preceding clinical hair regrowth outcomes, only dupilumab-treated patients presented significant suppression of cellular infiltrates, and multiple Th2-related, markers (CCL13/MCP-4, CCL18/PARC, CCL26/eotaxin-3, CCL24/Eotaxin-2), coupled with significant upregulation in the hair keratins. Th1-related suppression was evident later (week 48) when all patients received open-label dupilumab. Results were more pronounced in atopic AA patients, that showed 48% and 97% improvements in the lesional AA scalp profile at weeks 24 and 48, respectively, while 2% worsening was seen in the placebo arm at week 24. Moreover, placebo-treated patients presented 54% worsening in hair keratins when compared with baseline at week 24. At week 24, increases in hair keratins showed significant correlations only with decreases in Th2-related markers. CONCLUSIONS: Scalp biomarkers provide evidence of dupilumab efficacy in AA, detected even prior to clinical response, with exclusive correlations between early suppression of Th2 markers and increased hair keratins. These findings strengthen previous reports suggesting a possible role for Th2 cytokines as AA drivers.

Delayed type hypersensitivity reactions to various allergens may differently model inflammatory skin diseases.

BACKGROUND: Treatment of inflammatory skin diseases, including atopic dermatitis (AD) and psoriasis, is undergoing transformative changes, highlighting the need to develop experimental models of skin inflammation in humans to predict treatment responses. METHODS: We topically or intradermally administered four common sensitizers (dust mite (DM), diphencyprone (DPCP), nickel (Ni), and purified protein derivative (PPD)) to the backs of 40 healthy patients and the skin hypersensitivity response was biopsied and evaluated using immunohistochemistry, RNA-seq, and RT-PCR. RESULTS: All agents induced strong increases in cellular infiltrates (T-cells and dendritic cells) as compared to untreated skin (p < .05), with variable T helper polarization. Overall, DPCP induced the strongest immune responses across all pathways, including innate immunity (IL-1α, IL-8), Th1 (IFNγ, CXCL10), Th2 (IL-5, CCL11), and Th17 (CAMP/LL37) products, as well as the highest regulatory tone (FOXP3, IL-34, IL-37) (FDR <0.01). Nickel induced Th17 (IL-17A), Th1 (CXCL10) and Th2 (IL-4R) immune responses to a lesser extent than DPCP (p < .05). PPD induced predominantly Th1 (IFNγ, CXCL10, STAT1) and Th17 inflammation (IL-17A) (p < .05). DM induced modulation of Th2 (IL-13, CCL17, CCL18), Th22 (IL-22), and Th17/Th22 (S100A7/9/12) pathways (p < .05). Barrier defects that characterize both AD and psoriasis were best modeled by DPCP and Ni, followed by PPD, including downregulation of terminal differentiation (FLG, FLG2, LOR, LCEs), tight junction (CLDN1/CLDN8), and lipid metabolism (FA2H, FABP7)-related markers. CONCLUSION: Our data imply that DPCP induced the strongest immune response across all pathways, and barrier defects characteristic of AD and psoriasis.

Analysis of alopecia areata surveys suggests a threshold for improved patient-reported outcomes.

BACKGROUND: Although alopecia areata (AA) greatly impacts patients' quality of life (QoL), there is no adequate validation of AA-targeted QoL surveys in clinical trials, hindering sufficient representation of patient-reported outcomes. OBJECTIVES: Better understanding of patient-reported outcomes may guide treatment goals and future clinical trials. METHODS: In a recent randomized controlled trial testing dupilumab in AA, patients were administered the Alopecia Areata Quality of Life Index (AA-QLI) and the Alopecia Areata Symptom Impact Scale (AASIS) surveys, specifically evaluating QoL in patients with AA. An in-depth analysis was performed to assess the utility of these questionnaires in this patient population, both at baseline and after treatment, and to determine a threshold for improved patient-reported outcomes. RESULTS: While AASIS correlated with baseline Severity of Alopecia Tool (SALT) scores and with therapeutic response, AA-QLI showed no correlation with AA severity before or after treatment. Itch strongly correlated with serum IgE levels across both surveys. Using various approaches to estimate a discriminative threshold for decreased impact of AA on QoL (by AASIS) following treatment, a SALT score of 20 points or less post-treatment was associated with improved patient-reported outcomes, including both AA-related symptoms and items within the daily activities/feelings domain such as 'feeling sad' and 'feeling anxious or worry'. CONCLUSIONS: AASIS is better than AA-QLI to assess patient-reported outcomes. SALT ≤ 20 following treatment should be considered as a threshold for meaningful therapeutic outcome and as a clinical endpoint in future clinical trials for AA. What is already known about this topic? Alopecia areata greatly compromises quality of life, and affected patients have increased prevalences of depression, anxiety and social phobia. Despite the significant negative impact of the disease on patients' wellbeing, validation of targeted questionnaires in alopecia areata is lacking, and a therapeutic response threshold for improved patient-reported outcomes is unknown. What does this study add? This study investigated the utility of two different alopecia areata-targeted questionnaires - Alopecia Areata Quality of Life Index and Alopecia Areata Symptom Impact Scale (AASIS) - in a clinical trial setting. AASIS was found to correlate strongly with alopecia areata severity and clinical response. What are the clinical implications of this work? Patients with ≤ 20% scalp hair loss after treatment reported improvement in multiple quality-of-life items, suggesting this as a meaningful therapeutic outcome that may guide clinicians and improve the development of future clinical trials.

Transcriptomic Analysis of the Major Orphan Ichthyosis Subtypes Reveals Shared Immune and Barrier Signatures.

Preliminary work suggested upregulation of inflammatory pathways in patients with common forms of ichthyosis. However, a comprehensive characterization of skin from various ichthyosis subtypes is unavailable, precluding the development of targeted treatments. Thus, we sought to characterize the immune and barrier profiles of common and subtype-specific skin transcriptomes in a large group of patients with ichthyosis. We performed a global RNA-sequencing analysis in 54 patients with ichthyosis (7 with Netherton syndrome, 13 with epidermolytic ichthyosis, 16 with lamellar ichthyosis, and 18 with congenital ichthyosiform erythroderma) and 40 healthy controls. Differentially expressed genes were defined on the basis of fold changes > 2 and false discovery rate < 0.05 criteria. We found robust and significant T helper (Th) 22/Th17 skewing in all subtypes (e.g., IL-17A/C/F, S100A7/8/9/12; P < 0.001) with modest changes in Th2 pathway, primarily in Netherton syndrome, and Th1 skewing in congenital ichthyosiform erythroderma. Across all subtypes (less evident in epidermolytic ichthyosis), lipid metabolism and barrier junction markers were downregulated (e.g., FA2H, CDH10/11/12/2; P < 0.05), whereas epidermal cornification and proliferation measures were upregulated (e.g., SPRR1A/1B/2C/2G, EREG; P < 0.05). Our findings suggest that the common ichthyosis variants share aberrations in Th17/Th22 and barrier function, with minimal Th2 modulation. This may help to elucidate the pathogeneses of these subtypes and inform the development of subtype-specific treatments.

Th1, Th2 and Th17 inflammatory pathways synergistically predict cardiometabolic protein expression in serum of COVID-19 patients.

A predominant source of complication in SARS-CoV-2 patients arises from a severe systemic inflammation that can lead to tissue damage and organ failure. The high inflammatory burden of this viral infection often results in cardiovascular comorbidities. A better understanding of the interaction between immune pathways and cardiovascular proteins might inform medical decisions and therapeutic approaches. In this study we hypothesized that helper T-cell inflammatory pathways (Th1, Th2 and Th17) synergistically correlate with cardiometabolic proteins in serum of COVID-19 patients. We found that Th1, Th2 and Th17 cytokines and chemokines are able to predict expression of 186 cardiometabolic proteins profiled by Olink proteomics.

Ritlecitinib and brepocitinib demonstrate significant improvement in scalp alopecia areata biomarkers.

BACKGROUND: Janus kinase (JAK) inhibitors have shown encouraging results in the treatment of alopecia areata (AA), an autoimmune form of hair loss, in small, uncontrolled studies and case reports. OBJECTIVE: We conducted a biopsy substudy during the randomized, double-blind, placebo-controlled first 24 weeks of a phase 2a clinical trial that evaluated the efficacy and safety of ritlecitinib, an inhibitor of JAK3 and the tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase family, and brepocitinib, an inhibitor of tyrosine kinase 2 (TYK2)/JAK1 in the treatment of AA. METHODS: Change in biomarkers in lesional scalp biopsy samples between baseline and weeks 12 and 24 was an exploratory end point, and 46 patients participated from the ritlecitinib (n = 18), brepocitinib (n = 16), and placebo (n = 12) groups. Correlations of biomarkers with hair regrowth, measured using the Severity of Alopecia Tool (SALT) score, were also evaluated. CLINICAL TRIAL REGISTRATION: NCT02974868. RESULTS: At week 24, both ritlecitinib and brepocitinib demonstrated improvement exceeding 100% in the lesional scalp transcriptome toward a nonlesional profile. At week 12, the improvements in scalp tissue were greater with brepocitinib than ritlecitinib; however, at week 24, the improvements were greater with ritlecitinib. CONCLUSIONS: For both ritlecitinib and brepocitinib, improvement in the SALT scores was positively associated with expression of TH1 markers and negatively associated with expression of hair keratins. Larger, long-term clinical trials are warranted.

COVID-19 Symptoms Are Attenuated in Moderate-to-Severe Atopic Dermatitis Patients Treated with Dupilumab.

BACKGROUND: In the SARS-CoV-2/COVID-19 pandemic, we need to understand the impact of immunomodulatory medications on COVID-19 symptom severity in patients with inflammatory diseases, including the type 2/Th2 polarized skin disease, atopic dermatitis (AD). OBJECTIVE: Because it is believed that type 1/Th1 immunity controls viral infections and that there is a Th1/Th2 counter-regulation, we hypothesized that Th2 targeting with the IL-4Rα-antagonist, dupilumab, in patients with moderate-to-severe AD would rebalance the Th1/Th2 axis, potentially leading to attenuated COVID-19 symptoms. METHODS: A total of 1237 patients with moderate-to-severe AD in the Icahn School of Medicine at Mount Sinai Department of Dermatology were enrolled in a registry. Patients were screened for COVID-19-related symptoms and assigned a severity score (asymptomatic [0]-fatal [5]). Scores were compared among 3 treatment groups: dupilumab (n = 632), other systemic treatments (n = 107), and limited/no treatment (n = 498). Demographic and comorbid covariates were adjusted by multivariate generalized logistic regression models. RESULTS: The dupilumab-treated group showed reduced incidence and severity of COVID-19 symptoms versus other treatment groups. Dupilumab-treated patients were less likely to experience moderate-to-severe symptoms versus patients on other systemics (P = .01) and on limited/no treatment (P = .04), and less likely to experience any symptoms versus patients on other systemics (P = .01). This effect was seen in our entire cohort and in the subgroup of patients with verified COVID-19 or high-risk exposure. CONCLUSIONS: Patients on dupilumab experienced less severe COVID-19 manifestations and lesser symptoms compared with patients on other systemics and on limited/no treatment. These results suggest that Th2 modulation with dupilumab may have a protective effect on anti-viral immune response in patients with AD.

Scalp and serum profiling of frontal fibrosing alopecia reveals scalp immune and fibrosis dysregulation with no systemic involvement.

BACKGROUND: Frontal fibrosing alopecia (FFA) is a progressive, scarring alopecia of the frontotemporal scalp that poses a substantial burden on quality of life. Large-scale global profiling of FFA is lacking, preventing the development of effective therapeutics. OBJECTIVE: To characterize FFA compared to normal and alopecia areata using broad molecular profiling and to identify biomarkers linked to disease severity. METHODS: This cross-sectional study assessed 33,118 genes in scalp using RNA sequencing and 350 proteins in serum using OLINK high-throughput proteomics. Disease biomarkers were also correlated with clinical severity and a fibrosis gene set. RESULTS: Genes differentially expressed in lesional FFA included markers related to Th1 (IFNγ/CXCL9/CXCL10), T-cell activation (CD2/CD3/CCL19/ICOS), fibrosis (CXCR3/FGF14/FGF22/VIM/FN1), T-regulatory (FOXP3/TGFB1/TGFB3), and Janus kinase/JAK (JAK3/STAT1/STAT4) (Fold changes [FCH]>1.5, FDR<.05 for all). Only one protein, ADM, was differentially expressed in FFA serum compared to normal (FCH>1.3, FDR<.05). Significant correlations were found between scalp biomarkers (IL-36RN/IL-25) and FFA severity, as well as between JAK/STAT and fibrosis gene-sets (r>.6; P <.05). LIMITATIONS: This study was limited by a small sample size and predominantly female FFA patients. CONCLUSION: Our data characterize FFA as an inflammatory condition limited to scalp, involving Th1/JAK skewing, with associated fibrosis and elevated T-regulatory markers, suggesting the potential for disease reversibility with JAK/STAT inhibition.

Mapping atopic dermatitis and anti-IL-22 response signatures to type 2-low severe neutrophilic asthma.

BACKGROUND: Transcriptomic changes in patients who respond clinically to biological therapies may identify responses in other tissues or diseases. OBJECTIVE: We sought to determine whether a disease signature identified in atopic dermatitis (AD) is seen in adults with severe asthma and whether a transcriptomic signature for patients with AD who respond clinically to anti-IL-22 (fezakinumab [FZ]) is enriched in severe asthma. METHODS: An AD disease signature was obtained from analysis of differentially expressed genes between AD lesional and nonlesional skin biopsies. Differentially expressed genes from lesional skin from therapeutic superresponders before and after 12 weeks of FZ treatment defined the FZ-response signature. Gene set variation analysis was used to produce enrichment scores of AD and FZ-response signatures in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes asthma cohort. RESULTS: The AD disease signature (112 upregulated genes) encompassing inflammatory, T-cell, TH2, and TH17/TH22 pathways was enriched in the blood and sputum of patients with asthma with increasing severity. Patients with asthma with sputum neutrophilia and mixed granulocyte phenotypes were the most enriched (P < .05). The FZ-response signature (296 downregulated genes) was enriched in asthmatic blood (P < .05) and particularly in neutrophilic and mixed granulocytic sputum (P < .05). These data were confirmed in sputum of the Airway Disease Endotyping for Personalized Therapeutics cohort. IL-22 mRNA across tissues did not correlate with FZ-response enrichment scores, but this response signature correlated with TH22/IL-22 pathways. CONCLUSIONS: The FZ-response signature in AD identifies severe neutrophilic asthmatic patients as potential responders to FZ therapy. This approach will help identify patients for future asthma clinical trials of drugs used successfully in other chronic diseases.