Mast Cell Mediator Release Research Articles

Novel Approaches in the Inhibition of IgE-Induced Mast Cell Reactivity in Food Allergy.

Allergy is an IgE-dependent type-I hypersensitivity reaction that can lead to life-threatening systemic symptoms such as anaphylaxis. In the pathogenesis of the allergic response, the common upstream event is the binding of allergens to specific IgE, inducing cross-linking of the high-affinity FcεRI on mast cells, triggering cellular degranulation and the release of histamine, proteases, lipids mediators, cytokines and chemokines with inflammatory activity. A number of novel therapeutic options to curb mast cell activation are in the pipeline for the treatment of severe allergies. In addition to anti-IgE therapy and allergen-specific immunotherapy, monoclonal antibodies targeted against several key Th2/alarmin cytokines (i.e. IL-4Rα, IL-33, TSLP), active modification of allergen-specific IgE (i.e. inhibitory compounds, monoclonal antibodies, de-sialylation), engagement of inhibitory receptors on mast cells and allergen-specific adjuvant vaccines, are new promising options to inhibit the uncontrolled release of mast cell mediators upon allergen exposure. In this review, we critically discuss the novel approaches targeting mast cells limiting allergic responses and the immunological mechanisms involved, with special interest on food allergy treatment.

Decoding the intricacies of the mast cell compartment.

Historically, understanding of the human mast cell (MC) compartment has lagged behind the appreciation of other cell lineages. MCs exist in vascularised tissues but do not under normal circumstances circulate in blood, and there has been no pharmacological agent identified that totally and selectively inhibits human MC function. There are no substantiated accounts of an apparently healthy individual who is severely lacking in MCs. Thus, some of the approaches employed to understand the function of a specific immune cell are not available to the MC biologist. The disease categories that have provided the greatest insight into MC biology have been monoclonal and IgE-mediated MC disorders. This has led to the categorisation of MC diseases as intrinsic or extrinsic to the MC compartment and to the recognition of the role of mediators in MC activation disorders. Mastocytosis as a clonal disorder not only impacts the MC compartment through changes intrinsic to the MC, but also by the effects of episodes of significant release of MC mediators. The availability of newer therapeutic approaches developed to treat monoclonal MC disorders offer insights into how tomore selectively approach management of MC centric diseases.

Detection of drug-specific immunoglobulin E (IgE) and acute mediator release for the diagnosis of immediate drug hypersensitivity reactions

The diagnosis of a drug hypersensitivity reaction (DHR) is complex. The first step after taking the clinical history is to look for a sensitization to confirm or exclude the diagnosis and to identify the culprit drug. Skin tests are the primary means of detecting sensitization in DHR, but are associated with a risk for a severe reaction and may be contraindicated. In vitro tests offer the potential to support or confirm a diagnosis of DHR and influence medical decision making. For immediate-type DHR, a few validated assays for measurement of specific IgE (sIgE) are commercially available to a limited number of drugs. In addition, several home-made sIgE radioimmunoassays have been used in other studies. The sensitivity of the sIgE assay is drug-dependant and generally low (0–85%) for betalactams and reported heterogeneous for other drugs ranging from 26% for chlorhexidine and 44% for suxamethonium to 92% for chlorhexidine. However, as all these studies included patients, in whom DHR was confirmed only by skin tests and not by provocation, the results have to be interpreted carefully and may be unreliable. Determination of mediators during an acute phase of a reaction may indirectly support the diagnosis of a DHR by demonstrating mast cell and basophil mediator release. Negative in vitro tests do not exclude a DHR or imputability of a drug, but a positive result may support causality and eliminate the necessity for a drug provocation test. Unfortunately, evidence is limited with a lack of well-controlled studies in larger numbers of well-phenotyped patients, which results in susceptibility for bias and a need for future multicenter studies.

Cytokine Storm Syndrome in SARS-CoV-2 Infections: A Functional Role of Mast Cells.

Cytokine storm syndrome is a cascade of escalated immune responses disposing the immune system to exhaustion, which might ultimately result in organ failure and fatal respiratory distress. Infection with severe acute respiratory syndrome-coronavirus-2 can result in uncontrolled production of cytokines and eventually the development of cytokine storm syndrome. Mast cells may react to viruses in collaboration with other cells and lung autopsy findings from patients that died from the coronavirus disease that emerged in 2019 (COVID-19) showed accumulation of mast cells in the lungs that was thought to be the cause of pulmonary edema, inflammation, and thrombosis. In this review, we present evidence that a cytokine response by mast cells may initiate inappropriate antiviral immune responses and cause the development of cytokine storm syndrome. We also explore the potential of mast cell activators as adjuvants for COVID-19 vaccines and discuss the medications that target the functions of mast cells and could be of value in the treatment of COVID-19. Recognition of the cytokine storm is crucial for proper treatment of patients and preventing the release of mast cell mediators, as impeding the impacts imposed by these mediators could reduce the severity of COVID-19.

Safety of BNT162b2 mRNA COVID-19 vaccine in patients with mast cell disorders

Mast cell (MC) disorders (MCDs) are characterized by the proliferation and accumulation of MCs in different tissues, including skin and bone marrow, and/or the inappropriate release of MC mediators, creating symptoms in multiple organ systems. The clinical presentation of MCD is heterogeneous, ranging from a typical rash (urticaria pigmentosa) to a more aggressive systemic variant.1 All MCD patients can experience symptoms of anaphylaxis owing to massive MC activation and the release of mediators.

Gastrointestinal Manifestations in Systemic Mastocytosis: The Need of a Multidisciplinary Approach.

Simple SummaryMastocytosis is a group of neoplastic mast cell disorders ranging from a skin-limited disease to a systemic form with multi-organ involvement, including gut involvement. Clinical manifestations and outcome of systemic mastocytosis are variable. Symptoms may result from either release of mast cell mediators or tissue infiltration by mast cell proliferation. Gastrointestinal symptoms are one of the major causes of morbidity in these patients. The diagnosis of gastrointestinal mastocytosis can be tricky, as symptoms often mimic other more common gastrointestinal diseases; the endoscopic appearance is often unremarkable or nonspecific and the infiltrate can be focal and subtle and easily missed unless special stains are used. This review aims to better define the gastrointestinal involvement in systemic mastocytosis, discussing potential diagnostic pitfalls and pointing out the importance of a multidisciplinary approach for a prompt diagnosis and treatment.Mastocytosis represents a heterogeneous group of neoplastic mast cell disorders. The basic classification into a skin-limited disease and a systemic form with multi-organ involvement remains valid. Systemic mastocytosis is a disease often hard to diagnose, characterized by different symptoms originating from either the release of mast cell mediators or organ damage due to mast cell infiltration. Gastrointestinal symptoms represent one of the major causes of morbidity, being present in 60–80% of patients. A high index of suspicion by clinicians and pathologists is required to reach the diagnosis. Gastrointestinal mastocytosis can be a challenging diagnosis, as symptoms simulate other more common gastrointestinal diseases. The endoscopic appearance is generally unremarkable or nonspecific and gastrointestinal mast cell infiltration can be focal and subtle, requiring an adequate sampling with multiple biopsies by the endoscopists. Special stains, such as CD117, tryptase, and CD25, should be performed in order not to miss the gastrointestinal mast cell infiltrate. A proper patient’s workup requires a multidisciplinary approach including gastroenterologists, endoscopists, hematologists, oncologists, and pathologists. The aim of this review is to analyze the clinicopathological features of gastrointestinal involvement in systemic mastocytosis, focusing on the relevance of a multidisciplinary approach.

Prostaglandin D2 inhibits mediator release and antigen induced bronchoconstriction in the Guinea pig trachea by activation of DP1 receptors

The mechanism by which cyclooxygenase (COX) inhibition increases antigen-induced responses in airways remains unknown.Male albino guinea pigs were sensitized to ovalbumin (OVA). Intact rings of the trachea were isolated and mounted in organ baths for either force measurements or lipid mediator release analysis by UPLC-MS/MS or EIA following relevant pharmacological interventions.First, challenge with OVA increased the release of all primary prostanoids (prostaglandin (PG) D2/E2/F2α/I2 and thromboxanes). This release was eliminated by unselective COX inhibition (indomethacin) whereas selective inhibition of COX-2 (lumiracoxib) did not inhibit release of PGD2 or thromboxanes. Additionally, the increased levels of leukotriene B4 and E4 after OVA were further amplified by unselective COX inhibition. Second, unselective inhibition of COX and selective inhibition of the prostaglandin D synthase (2-Phenyl-Pyrimidine-5-Carboxylic Acid (2,3-dihydro-indol-1-yl)-amide) amplified the antigen-induced bronchoconstriction which was reversed by exogenous PGD2. Third, a DP1 receptor agonist (BW 245c) concentration-dependently reduced the antigen-induced constriction as well as reducing released histamine and cysteinyl-leukotrienes, a response inhibited by the DP1 receptor antagonist (MK-524). In contrast, a DP2 receptor agonist (15(R)-15-methyl PGD2) failed to modulate the OVA-induced constriction.In the guinea pig trachea, endogenous PGD2 is generated via COX-1 and mediates an inhibitory effect of the antigen-induced bronchoconstriction via DP1 receptors inhibiting mast cell release of bronchoconstrictive mediators. Removal of this protective function by COX-inhibition results in increased release of mast cell mediators and enhanced bronchoconstriction.

Drug desensitization in the coronavirus disease 2019 pandemic era: Local success to widespread potential

Drug desensitization in the coronavirus disease 2019 pandemic era: Local success to widespread potential

Induction of the endogenous sialoglycan ligand for eosinophil death receptor Siglec-8 in chronic rhinosinusitis with hyperplastic nasal polyposis.

Siglec-8, an immune-inhibitory sialoglycan binding lectin (S8), is expressed on the surface of eosinophils and mast cells, which are potent mediators of allergic inflammation. When S8 engages endogenous sialoglycan ligands, eosinophils undergo apoptosis and mast cell mediator release is inhibited. In the human airway, Siglec-8 ligands (S8L) are sialylated keratan sulfate chains carried on isoforms of the protein Deleted in Malignant Brain Tumors-1 (DMBT1), an immunoregulatory protein that we recently identified as the endogenous ligand for S8, DMBT1S8. We herein report that S8L is overexpressed in chronic rhinosinusitis with nasal polyposis (CRSwNP), a prevalent eosinophilic laden airway disease. Quantification and comparison of the degree to which DMBT1 carries the S8L by immunoblot analysis and lectin blot overlay, respectively, from nasal lavage showed that the S8L/DMBT1 ratio was significantly increased in CRSwNP vs. control or CRS patients. We identified the histological sites of S8L and DMBT1 expression in fresh surgically resected human nasal polyps. Histochemistry of diseased polyps and adjacent nondiseased middle turbinate (MT) tissue from CRSwNP demonstrated colocalization of S8L and DMBT1 with highest staining in submucosal glands >> epithelium > stoma. S8L expression was specifically elevated in the submucosal glands and epithelium of polyp tissue compared to MT. We hypothesize that expression of the isoform of DMBT1 carrying the Siglec-8 binding sialoglycan, DMBT1S8, is induced in polyps of CRSwNP specifically at the site of disease, is produced in the submucosal glands of polyps and secreted into the lumen of the sinonasal cavity as a host response to mitigate eosinophil-mediated inflammation.

Genetic Regulation of Tryptase Production and Clinical Impact: Hereditary Alpha Tryptasemia, Mastocytosis and Beyond.

Tryptase is a serine protease that is predominantly produced by tissue mast cells (MCs) and stored in secretory granules together with other pre-formed mediators. MC activation, degranulation and mediator release contribute to various immunological processes, but also to several specific diseases, such as IgE-dependent allergies and clonal MC disorders. Biologically active tryptase tetramers primarily derive from the two genes TPSB2 (encoding β-tryptase) and TPSAB1 (encoding either α- or β-tryptase). Based on the most common gene copy numbers, three genotypes, 0α:4β, 1α:3β and 2α:2β, were defined as “canonical”. About 4–6% of the general population carry germline TPSAB1-α copy number gains (2α:3β, 3α:2β or more α-extra-copies), resulting in elevated basal serum tryptase levels. This condition has recently been termed hereditary alpha tryptasemia (HαT). Although many carriers of HαT appear to be asymptomatic, a number of more or less specific symptoms have been associated with HαT. Recent studies have revealed a significantly higher HαT prevalence in patients with systemic mastocytosis (SM) and an association with concomitant severe Hymenoptera venom-induced anaphylaxis. Moreover, HαT seems to be more common in idiopathic anaphylaxis and MC activation syndromes (MCAS). Therefore, TPSAB1 genotyping should be included in the diagnostic algorithm in patients with symptomatic SM, severe anaphylaxis or MCAS.

Fibroblasts Regulate Mast Cell Activation in Nasal Polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory sinus disease associated with mast cell (MC) expansion and activation. We find that MCs in the subepithelium of nasal polyps exhibit elevated CD69 expression, a known leukocyte activation marker. However, it is unclear which factors within the polyp drive this activation. As subepithelial MCs colocalize with fibroblasts, cells known to influence MC development and mediator release, we hypothesized that nasal polyp fibroblasts could directly drive MC activation.

English

BACKGROUND Allergic conjunctivitis is one of the commonly found clinical conditions in ophthalmology practice. Diagnostic features consist of itching, redness and swelling of conjunctiva. Cornea is rarely involved. The physiologic basis of allergy is mast cell degranulation releasing histamine and other pro-inflammatory mediators. Mast cells release histamine which is responsible for early acute phase of allergies and pro-inflammatory mediators like prostaglandins, leukotrienes etc. which are responsible for the late phase. Activation of H1 receptors on conjunctival neurons causes itching while that of H1 and H2 receptors on vascular endothelium is responsible for vasodilation (appearing as redness) and endothelial swelling. Itching and inflammation are caused by response of H4 receptors on immune and inflammatory cells. Olopatadine is an H1 receptor antagonist and mast cell mediator release inhibitor. It is available as 0.1 % formulation used twice daily, and 0.2 % and 0.7 % formulations, both used once daily. Alcaftadine has high affinity and specificity for H1 and H2 receptors, and moderate affinity for the H4 receptors. It is an inverse agonist of H1, H2 and H4 receptors and also acts as mast cell stabiliser. Thus, antihistaminic effect relieves the early phase and mast cell stabilisation relieves the late phase of ocular allergic response. Both have become the most important therapy for allergic conjunctivitis. METHODS This was a prospective, observer-masked, single centre clinical trial conducted at outpatient department of ophthalmology, S.N. Medical College, Agra, from February 2018 to July 2019. Subjects with allergic conjunctivitis (n = 136) were registered for a prospective study and followed up for two weeks. Subjects were randomised using computer generated random number tables into one of the two treatment groups: alcaftadine 0.25 % eye drop and olopatadine 0.2 % eye drop. In this study, efficacy of the drug was taken as mean reduction in severity score of the subjects evaluated at one week and two weeks follow up. RESULTS Patients treated with alcaftadine 0.25 % eye drop showed comparatively early alleviation of signs and symptoms in comparison to patients treated with olopatadine 0.2 % eye drop. Alcaftadine 0.25 % treated subjects experienced significantly higher mean reduction in severity score than olopatadine 0.2 % treated subjects at every follow up visit; after 1 week (p = 0.0205) and after 2 weeks (p = 0.0475). No adverse effects were reported with either drug. CONCLUSIONS Once daily alcaftadine 0.25 % eye drop showed higher efficacy than once daily olopatadine 0.2 % eye drop in relieving signs and symptoms of allergic conjunctivitis at both one week and two weeks follow up. Both treatment arms were found to be safe and effective. KEYWORDS Allergic conjunctivitis, Olopatadine, Alcaftadine

Criteria for the Regression of Pediatric Mastocytosis: A Long-Term Follow-Up

Mastocytosis is a neoplastic condition characterized by the accumulation of mast cells (MCs) in 1 or more organ. Adults tend to have persistent, systemic mastocytosis, whereas MC infiltration in children is usually limited to the skin and typically regresses after several years. Both adults and children could display mast cell activation symptoms (MCASs) due to MC mediator release. In more than 85% of both adult and pediatric cases, KIT mutations are present, with the KIT D816V mutation being present in most affected adults but in only half the affected children. To identify the clinical, biological, and molecular factors associated with the regression of cutaneous mastocytosis (CM) in children, and to assess the correlation between MCASs and CM regression. Patients having suffered from pediatric-onset mastocytosis for at least 8 years were included in a longitudinal cohort study. Clinical data, the baseline serum tryptase level, the KIT sequence, and the progression of MCASs and CM were recorded. CM regressed in 210 of the 272 included patients (77.2%; mean time to regression, 6.10 years). The rare cases of aggressive systemic mastocytosis were symptomatic from the outset. Congenital mastocytosis and the KIT D816V mutation were associated with CM regression (odds ratio, 0.48, P= .031, and 0.173, P= .031, respectively). Aggravation of MCASs over time was correlated with the persistence of skin lesions. However, the MCASs became more intense in 19% of the patients with MCASs at baseline and CM regression, justifying long-term follow-up in this setting. Our results open up new hypotheses with regard to the spontaneous regression of CM in pediatric patients.

Pioneer Part 2: A Randomized, Double-Blind, Placebo-Controlled, Phase 2 Study to Evaluate Safety and Efficacy of Avapritinib in Indolent Systemic Mastocytosis

Pioneer Part 2: A Randomized, Double-Blind, Placebo-Controlled, Phase 2 Study to Evaluate Safety and Efficacy of Avapritinib in Indolent Systemic Mastocytosis

M554 A RARE PATHOGENIC MUTATION ASSOCIATED WITH A PEDIATRIC CASE OF DIFFUSE CUTANEOUS MASTOCYTOSIS

Diffuse cutaneous mastocytosis (DCM) is a rare disease resulting from widespread mast cell accumulation within the dermis. Disease manifestations may include erythroderma and blistering skin rashes with systemic symptoms related to mast cell mediator release. DCM has commonly been associated with sporadic gain-of-function KIT D816V mutations. Familial mastocytosis cases from germline mutations are rare and not well described.

Diagnostyka i leczenie mastocytozy układowej — stanowisko ekspertów

Systemic mastocytosis (SM) is a rare hematological malignancy characterized by an abnormal expansion and accumulation of pathological mast cells in bone marrow and other organs including skin, liver, spleen and lymph nodes. The clinical manifestation can be extremely heterogeneous, from limited skin changes to multi-organ involvement or mast cell tumors. The median survival of patients diagnosed with indolent form is comparable to healthy population, while prognosis for patients with advanced disease is poor, with an estimated survival ranging from several months to several years. In most patients (> 90%), a somatic mutation in codon 816 of the c-KIT gene encoding tyrosine kinase receptor is detected. Additional molecular abnormalities and even coexistence of other hematological cancers, e.g . acute leukemia, are also observed. Regardless of the form of disease or serum tryptase concentration, patients are exposed to symptoms resulting from the release of mast cell mediators — most often itching, paroxysmal redness and blisters, and general mediator-induced symptoms — such as nausea, vomiting, diarrhea, abdominal pain, hypotensive episodes, fatigue, headache, fever, shortness of breath, osteopenia, osteoporosis and severe anaphylactic reactions. This paper presents current outlook on the diagnostic and treatment process of SM, taking into account the interdisciplinary aspects of the disease.

Increased group 2 innate lymphoid cells in peripheral blood of adults with mastocytosis

Systemic mastocytosis is a hematological disease in which aberrant mast cells accumulate because of gain-of-function mutations in the KIT receptor. Group 2 innate lymphoid cells (ILC2s) are effector cells of type 2 immune responses that also express KIT and colocalize with mast cells at barrier tissue sites. In mouse models, mast cell-ILC2 crosstalk can drive local inflammation. However, a possible role for ILC2s in the pathophysiology of mastocytosis remains unexplored. We sought to characterize circulating ILC2s in a clinically diverse cohort of patients with mastocytosis. We included 21 adults with systemic mastocytosis and 18 healthy controls. Peripheral blood ILC2 abundance and phenotype were analyzed by flow cytometry and correlated to clinical characteristics, including the presence of the D816V KIT mutation. ILC2 levels were significantly higher in D816V+ patients with mastocytosis compared with D816V- patients or healthy controls. We observed increased proportions of KIT+ ILC2s among patients with mastocytosis, regardless of D816V status. Patients with skin involvement and itch showed the highest levels of ILC2s, which was independent from atopy or serum tryptase levels. Allele-specific quantitative PCR showed that the vast majority of ILC2s did not carry the D816V mutation. Our findings suggest a role for ILC2s and pathogenic ILC2-mast cell crosstalk in mastocytosis. We hypothesize that a high cutaneous D816V+ mast cell burden alters the skin microenvironment to induce chronic local ILC2 activation and their dissemination into the circulation. Activated ILC2s could contribute to skin symptoms by producing inflammatory mediators and by further augmenting mast cell mediator release.

Laparoscopic procedures impact on mast cell mediators, extracellular matrix and adhesion scoring system in rats

BackgroundLaparoscopic procedures under certain pressure have the potential to cause intra-abdominal adhesions. However, the pathomechanism of this disorder is unknown. Release of mast cell mediators due to mast cell degranulation is thought to be the cause. Materials and methodsThirty male Sprague-Dawley rats were grouped into five groups (n = 6 per group): one control group and four intervention groups to which 60 min insufflation was performed using carbon dioxide at 5, 8, 10 and 12 mmHg. Seven days after laparoscopy, we euthanized and evaluated the levels of histamine, tryptase, and chymase of peritoneal fluid, the thickness of ECM of peritoneal tissue, and intraabdominal adhesion scoring system. ResultsHistamine and tryptase levels in peritoneal fluid were significantly higher at the 10- and 12 mm Hg intervention compared to control (histamine: 0.50 ± 0.35 vs. 0.41 ± 0.41 vs. 0.04 ± 0.02 ng/mL, respectively; and tryptase: 0.69 ± 0.11 vs. 0.65 ± 0.05 vs. 0.48 ± 0.02 ng/ml respectively). The ECM was significantly thicker in the intervention groups at 10- and 12-mm Hg compared to control (71.3 [66.7–85.2] vs. 48.4 [34.5–50.3] vs. 10.25 [8.7–12.1] μm, respectively). Moreover, the intra-abdominal scoring was also significantly higher in the intervention groups at 10- and 12 mm Hg compared to control (4 [0–4] vs. 4.5 [4–5], vs. 0, respectively). ConclusionsLaparoscopic procedures increase the release of mast cell mediators in peritoneal fluid, the thickness of ECM and intraabdominal adhesion scoring in rats, implying that it might increase the possibility of intrabdominal adhesion in humans.

The role of IL-17, IL-23 and IL-31, IL-33 in allergic skin diseases.

Allergic skin diseases such as urticaria, atopic dermatitis and allergic contact dermatitis are among the most common skin diseases with severe socioeconomic consequences. The pathogenesis of allergic skin diseases is complex. This review provides an overview of cytocines IL-17, IL-23, IL-31 and IL-33. Current research results show a variety of immunological processes in the pathogenesis of the allergic skin diseases, including the role of cytokines. In addition to the Th1 and Th2 immune response, the immune response via Th17 is becoming increasingly important in allergic skin diseases but also the cytokines IL-23, IL-31 and IL-33 have been discussed in the literature recently. Different cytokines promote in a kind of orchestra the different symptoms seen in the different allergic skin diseases, including pruritus, dermatitis, mast cell mediator release and inflammation. We are still in the early stages of understanding pathophysiology of allergic skin diseases and the role of various cytokines in the immune system. With the development of targeted antibodies against the proinflammatory cytokines, the variety of normal therapeutic options can be expected to evolve.

TPC1 deficiency or blockade augments systemic anaphylaxis and mast cell activity

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+ release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+ homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+ uptake and filling of endoplasmic reticulum Ca2+ stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.