The Impact of Infant Feeding Regimen on Cow's Milk Protein Allergy, Atopic Dermatitis and Growth in High-Risk Infants during the First 6 Months of Life: The Allergy Reduction Trial.

The development of early-onset cow's milk protein allergy and atopic dermatitis during the first months of life is multifactorial, including both genetic and nutritional aspects. This study aims to assess the impact of different feeding patterns on the incidence of cow's milk protein allergy, atopic dermatitis, and growth among infants with a family history of allergy. A total of 551 high-risk infants were randomly recruited from 3 European countries in three feeding regimens: exclusive breastfeeding, partially hydrolyzed formula, or standard formula with intact protein either exclusively or supplementary to breastfeeding. During the first 6 months of intervention, amongst infants with a family history of atopic dermatitis, 6.5% of partially hydrolyzed formula-fed infants and 22.7% of exclusively breastfed infants (p = 0.007) presented with atopic dermatitis respectively. Growth as assessed by weight increase did not differ between the aforementioned groups. Although cow's milk protein allergy was not related to the different milk feeding regimens in the whole cohort, when adjusting for high breast milk intake, the respective incident was significantly lower in the infants consuming partially hydrolyzed formula (p < 0.001). This data indicates that a specific partially hydrolyzed formula could serve as a more appropriate complement to breast milk compared to a standard intact protein formula in high-risk infants, to reduce the incidence of atopic dermatitis.

The Risk Reduction Effect of a Nutritional Intervention With a Partially Hydrolyzed Whey-Based Formula on Cow's Milk Protein Allergy and Atopic Dermatitis in High-Risk Infants Within the First 6 Months of Life: The Allergy Reduction Trial (A.R.T.), a Multicenter Double-Blinded Randomized Controlled Study.

Background: The role of partially hydrolyzed formulas (pHF) as part of nutritional interventions to prevent the development of allergic manifestations (AM) is questioned, and efficacy of each specific pHF should be substantiated. Objective: To investigate the risk-reduction effect of a whey-based pHF on the development of cow's milk protein allergy (CMPA) and atopic dermatitis (AD) in infants at high-risk for allergy within the first 6 months of life. Materials and Methods: In a multicenter double-blinded randomized controlled setting, healthy non-exclusively breastfed full-term infants, received either a specific whey-based pHF or a standard cow's milk-based formula (SF) and were clinically assessed for AM at 2, 4, and 6 months of age, supported by the objective scoring tools SCORAD and CoMiSS. CMPA was confirmed by open food challenge. Intention-to-Treat (ITT) and Per-Protocol (PP) analyses were performed. Results: Of 331 randomized subjects (ITT analysis set), 160 received the pHF and 171 the SF. Six (3.8%) infants in the pHF and 12 (7%) in the SF group developed CMPA (p = 0.186). AD incidence was significantly lower in those receiving pHF as compared to SF (10.6% vs. 18.7%, p = 0.024) with a relative risk (RR, 95% CI) of 0.54 (0.32, 0.92), in particular when adjusting for family history of AD [6.5% vs. 27.3%, RR 0.24 (0.07, 0.78), p = 0.018] representing a risk reduction of 76%. The PP analysis showed similar results. Conclusion: This specific whey-based pHF reduced the risk of AD development, particularly in those with a family history of AD, and tended to reduce the development of CMPA in non-exclusively breastfed infants at high-risk for allergy. The A.R.T. study suggests that this particular pHF may contribute to measures aimed at prevention of allergic manifestations. However, further studies are needed to confirm this risk-reduction effect.

Early-life predictors and risk factors of peanut allergy, and its association with asthma in later-life: Population-based birth cohort study.

BACKGROUND: Understanding risk factors for peanut allergy (PA) is essential to develop effective preventive measures. OBJECTIVE: The objective was to ascertain associates and predictors of PA, and the relationship between PA and asthma severity. METHODS: In a population-based birth cohort, we investigated the association between objectively confirmed PA with early-life environmental exposures, filaggrin (FLG)-loss-of-function mutations and other atopic disease. We then examined the association of PA with longitudinal trajectories of sensitization, wheeze and allergic comorbidities, which were previously derived using machine learning. Finally, we ascertained the relationship between PA and asthma severity. RESULTS: PA was confirmed in 30/959 participants with evaluable data. In the multivariate analysis, eczema in infancy (OR = 4.4, 95% CI 1.5-13.2, p = 0.007), egg sensitization at age 3 years (OR = 9.7, 95% CI 3.3-29.9, p < 0.001) and early-life cat ownership (OR = 3.0, 95% CI 1.1-8.4, p = 0.04) were independent associates of PA. In the stratified analysis among 700 participants with genetic information, in children with early-life eczema there was no difference in FLG mutations between children with and without PA (3/18 [16.7%] vs. 42/220 [19.1%], p = 1.00). In contrast, among children without eczema, those with PA were almost eight times more likely to have FLG mutations (2/6 [33.3%] vs. 27/456 [5.9%], p = 0.049). We observed associations between PA and multiple allergic sensitization profiles derived using machine learning, with ~60-fold increase in risk among individuals assigned to multiple early sensitization. PA was significantly associated with persistent wheeze (but not other wheeze phenotypes), and with trajectories of atopic disease characterized by co-morbid persistent eczema and wheeze (but not with transient phenotypes). Children with PA were more likely to have asthma, but among asthmatics we found no evidence of an association between PA and asthma severity. CONCLUSIONS: Peanut allergy is associated with multiple IgE sensitization and early-onset persistent eczema and wheeze. FLG loss-of-function mutations were associated with peanut allergy in children without eczema.

Strategies and Future Opportunities for the Prevention, Diagnosis, and Management of Cow Milk Allergy.

The prevalence of food allergy has increased over the last 20-30 years, including cow milk allergy (CMA) which is one of the most common causes of infant food allergy. International allergy experts met in 2019 to discuss broad topics in allergy prevention and management of CMA including current challenges and future opportunities. The highlights of the meeting combined with recently published developments are presented here. Primary prevention of CMA should start from pre-pregnancy with a focus on a healthy lifestyle and food diversity to ensure adequate transfer of inhibitory IgG- allergen immune complexes across the placenta especially in mothers with a history of allergic diseases and planned c-section delivery. For non-breastfed infants, there is controversy about the preventive role of partially hydrolyzed formulae (pHF) despite some evidence of health economic benefits among those with a family history of allergy. Clinical management of CMA consists of secondary prevention with a focus on the development of early oral tolerance. The use of extensive Hydrolysate Formulae (eHF) is the nutrition of choice for the majority of non-breastfed infants with CMA; potentially with pre-, probiotics and LCPUFA to support early oral tolerance induction. Future opportunities are, among others, pre- and probiotics supplementation for mothers and high-risk infants for the primary prevention of CMA. A controlled prospective study implementing a step-down milk formulae ladder with various degrees of hydrolysate is proposed for food challenges and early development of oral tolerance. This provides a more precise gradation of milk protein exposure than those currently recommended.

Nutritional Management With a Casein-Based Extensively Hydrolysed Formula in Infants With Clinical Manifestations of Non-IgE-Mediated CMPA Enteropathies and Constipation.

Background: The majority of mixed-fed infants with non-IgE-mediated cow's milk protein allergy (CMPA) enteropathies are managed with an extensively hydrolysed cow's milk based infant formula (eHF). Given the high variability in peptide distribution of available eHFs, it is important to understand the suitability of a specific product in the management of distinct phenotypes. Objective: To assess the symptom resolution of various phenotypes of clinical manifestations of CMPA enteropathies and constipation managed by a casein-based eHF. Methods: The data of 20 full-term infants (n = 15 with non-IgE-mediated CMPA and n = 5 with constipation) attending a paediatric allergy clinic in Cyprus and managed with a casein-based eHF were retrospectively analysed. Results: Based on the clinical symptoms and history, infants were classified into the following phenotypes: (a) 11/15 (73.3%) FPIAP, (b) 3/15 (20%) FPIES, and (c) 1/15 (6.7%) severe diarrhoea. Overall, 14 (93.3%) patients were successfully managed with the casein-based eHF and 1 (6.7%) required an AAF. This formula was effective in 91% of patients with FPIAP, in 100% with FPIES and with diarrhoea. Three (60%) patients with constipation responded to the eHF. Conclusion: This case-series report supports the efficacy of a particular casein-based eHF for the nutritional management of non-IgE mediated CMPA enteropathies.

Food Protein Induced Proctocolitis: A Benign Condition with an Obscure Immunologic Mechanism.

Food protein-induced proctocolitis (FPIP) is the most common colonic manifestation of food allergy in infants, accounting for up to 60% of exclusively breast-fed children. The causative foods derived from the mother's diet, which are then excreted in her milk. The suggested risk factors for the development of FPIP are an immature immune system, altered intestinal permeability and other factors that activate local immune function, such as genetic susceptibility in combination with particularly sensitizing foods. FPIP is an enhanced immune responsiveness of some infants to very small amounts of food antigens, inducing an inflammatory mucosal response, mediated by T cells.

Melanoma of the gallbladder: appropriate surgical management and review of the literature.

KEY CLINICAL MESSAGE: Melanomas of the gallbladder (GB) are extremely rare with a very poor prognosis. They feature in the literature as a few case reports and the method of their management is not clear. We report a case of patient with metastatic cutaneous melanoma to the GB, and our treatment suggestion.

Peanut allergy: effect of environmental peanut exposure in children with filaggrin loss-of-function mutations.

BACKGROUND: Filaggrin (FLG) loss-of-function mutations lead to an impaired skin barrier associated with peanut allergy. Household peanut consumption is associated with peanut allergy, and peanut allergen in household dust correlates with household peanut consumption. OBJECTIVE: We sought to determine whether environmental peanut exposure increases the odds of peanut allergy and whether FLG mutations modulate these odds. METHODS: Exposure to peanut antigen in dust within the first year of life was measured in a population-based birth cohort. Peanut sensitization and peanut allergy (defined by using oral food challenges or component-resolved diagnostics [CRD]) were assessed at 8 and 11 years. Genotyping was performed for 6 FLG mutations. RESULTS: After adjustment for infantile atopic dermatitis and preceding egg skin prick test (SPT) sensitization, we found a strong and significant interaction between natural log (ln [loge]) peanut dust levels and FLG mutations on peanut sensitization and peanut allergy. Among children with FLG mutations, for each ln unit increase in the house dust peanut protein level, there was a more than 6-fold increased odds of peanut SPT sensitization, CRD sensitization, or both in children at ages 8 years, 11 years, or both and a greater than 3-fold increased odds of peanut allergy compared with odds seen in children with wild-type FLG. There was no significant effect of exposure in children without FLG mutations. In children carrying an FLG mutation, the threshold level for peanut SPT sensitization was 0.92 µg of peanut protein per gram (95% CI, 0.70-1.22 µg/g), that for CRD sensitization was 1.03 µg/g (95% CI, 0.90-1.82 µg/g), and that for peanut allergy was 1.17 µg/g (95% CI, 0.01-163.83 µg/g). CONCLUSION: Early-life environmental peanut exposure is associated with an increased risk of peanut sensitization and allergy in children who carry an FLG mutation. These data support the hypothesis that peanut allergy develops through transcutaneous sensitization in children with an impaired skin barrier.

Reintroduction of cow's milk in milk-allergic children.

Even though cow's milk protein allergy (CMPA) is one of the most common food allergies in childhood, its prognosis is generally good and cow's milk (CM) is usually reintroduced in the patient's diet. The natural history of CMPA shows heterogeneity and is closely related to the immunological and clinical phenotype by which CMPA presents. Children with non-IgE-mediated CMPA tend to develop tolerance at an earlier age and at a higher percentage compared to those with IgE-mediated disease. In subjects with severe symptoms CMPA may persist for longer or ever. Although, the majority of children will outgrow their allergy, the individual timing of tolerance acquisition is largely unknown. Most of the current guidelines on the diagnosis and management of CMPA suggest reevaluation of milk-allergic children every 6-12 months, and reintroduction of CM after a negative Oral Food Challenge (OFC). However, OFC procedure is time consuming, expensive and not without risk. Clinical variables and the measurements of sIgE levels and SPT wheal sizes to crude (whole) CM protein and individual milk protein components may provide some useful prognostic information in the course of CMPA. However, no clear-cut clinical or laboratory criteria exist to predict which children and at what age are more likely to pass a repeat (reintroduction) OFC. The identification of factors that could accurately predict the outcome of reintroduction OFC and the timing of tolerance development would be extremely useful in daily clinical practice. Until recently, reintroduction of CM was commonly attempted when children with CMPA were more likely to have become tolerant. Over the last years, a different approach in the management of milk and egg allergy has emerged with specific oral tolerance induction (SOTI) as a promising method for the treatment of food allergies. Furthermore, a number of studies have shown evidence that introduction of heated milk and egg protein into the diet of allergic patients may induce the acquisition of tolerance. Still, the question of when and how to reintroduce cow's milk in milk-allergic children remains challenging and further research in this important field is necessary to provide both clinicians and anxious parents with the desirable answer.

Molecular diagnosis of peanut and legume allergy.

PURPOSE OF REVIEW: To review and discuss recent studies on molecular diagnosis of peanut and other legume allergy. RECENT FINDINGS: Studies from the UK and France suggest that quantification of Ara h 2-specific IgE may accurately discriminate peanut allergy from tolerance. However, the pattern of allergenic component recognition in peanut-sensitized patients from different populations or geographical areas varies, reflecting different pollen and dietary exposures. In the USA, peanut-allergic patients are commonly sensitized to Ara h 1-3, in Spain to Ara h 9 and in Sweden to Ara h 8. Patients with soybean allergy sensitized to Gly m 5 or Gly m 6 allergens may be at greater risk of experiencing severe allergic reactions. SUMMARY: Accurate diagnosis of peanut and legume allergy is challenging and essential. Measurement of IgE response to specific allergenic molecules may be more useful in predicting the presence and severity of clinical allergy than currently used skin or blood tests based on whole extracts. However, given the heterogeneity in component recognition patterns observed in different geographical areas, further studies are essential to identify and confirm potentially useful molecular diagnostic and prognostic markers. Until such markers are confirmed and replicated in different age groups, oral food challenge (OFC) remains the gold standard for accurate diagnosis.

Effect of day care attendance on sensitization and atopic wheezing differs by Toll-like receptor 2 genotype in 2 population-based birth cohort studies.

BACKGROUND: Variation in the Toll-like receptor 2 gene (TLR2/-16934) is associated with allergic diseases among farmers' children but not among children not living on farms. OBJECTIVE: To test the hypothesis that the same genetic variant conferring protection in the farming environment is associated with reduced risk of developing allergic phenotypes among urban children attending day care in early life. METHODS: In 2 population-based birth cohorts (Manchester, United Kingdom, Manchester Asthma and Allergy Study [MAAS]; Tucson, Ariz, Tucson Infant Immune Study [IIS]), participants were recruited prenatally and followed prospectively (MAAS: 3, 5, 8 and 11 years; IIS: 1, 2, 3 and 5 years). We assessed allergic sensitization and atopic wheezing at each follow-up. RESULTS: A total of 727 children participated in Manchester and 263 in Tucson. We found no significant associations between TLR2/-16934 and sensitization and atopic wheeze in either cohort. However, a different pattern emerged when we explored the interaction between TLR2/-16934 and day care attendance on these outcomes. We found a significant interaction between day care and TLR2/-16934 on the development of sensitization in the longitudinal model in MAAS in that children carrying the T allele who attended day care were less likely to be sensitized than those who did not attend day care, whereas among AA homozygotes, the association tended to be in the opposite direction. In a longitudinal model in IIS, we found a significant interaction between day care attendance and TLR2/-16934 on the development atopic wheezing. Significant interactions between TLR2/-16934 and day care were maintained when adjusting for socioeconomic status. CONCLUSION: The effect of day care on sensitization and atopic wheezing may differ among children with different variants of the TLR2 gene.

Residential exposure to motor vehicle emissions and the risk of wheezing among 7-8 year-old schoolchildren: a city-wide cross-sectional study in Nicosia, Cyprus.

BACKGROUND: Several studies have reported associations between respiratory outcomes in children and a range of self-reported, administrative or geographical indicators of traffic pollution. First-time investigation into the frequency of asthmatic symptoms among 7-8 year-old Cypriot children in 1999-2000 showed increased prevalence in the capital Nicosia compared to other areas. Geographical differences on an island the size of Cyprus may reflect environmental and/or lifestyle factors. This study investigates the relationship between self-reported symptoms and residential exposure to motor vehicle emissions among Nicosia schoolchildren. METHODS: The addresses of children in the metropolitan area of Nicosia who participated in the original survey (N = 1,735) were geo-coded and the level of exposure of each child was assessed using distance- and emission-based indicators (i.e. estimated levels of particulate matter and nitrogen oxides emissions due to motor vehicles on main roads around the residence). Odds ratios of wheezing and asthma diagnosis in relation to levels of exposure were estimated in logistic regression models adjusting for person-based factors, co-morbidity and intra-school clustering. RESULTS: We found an increased risk of wheezing at distances less than 50 m from a main road and/or only among those experiencing the highest levels of exposure. The strongest effect estimates were observed when exposure was defined in terms of the cumulative burden at all roads around the residence. Adjusted odds ratios for current wheezing were 2.33 (95% CI 1.27, 4.30) amongst the quartile of participants exposed to the highest levels of PM at all roads 50 m of their residence and 2.14 (95% CI 1.05, 4.35) for NOx, with no effect at intermediate levels of exposure. While the direction of effect was apparent at longer distances, differences were generally not statistically significant. CONCLUSIONS: Children experiencing the highest burden of emissions in Nicosia seem to be at a higher risk of reporting asthmatic symptoms. Due to the small number of children residing at close proximity to main roads and lack of evidence of risk at intermediate levels of exposure or longer distances, the observed pattern alone does not explain the generally higher prevalence observed in urban Nicosia compared to other areas.

Allergy or tolerance in children sensitized to peanut: prevalence and differentiation using component-resolved diagnostics.

BACKGROUND: Not all peanut-sensitized children develop allergic reactions on exposure. OBJECTIVE: To establish by oral food challenge the proportion of children with clinical peanut allergy among those considered peanut-sensitized by using skin prick tests and/or IgE measurement, and to investigate whether component-resolved diagnostics using microarray could differentiate peanut allergy from tolerance. METHODS: Within a population-based birth cohort, we ascertained peanut sensitization by skin tests and IgE measurement at age 8 years. Among sensitized children, we determined peanut allergy versus tolerance by oral food challenges. We used open challenge among children consuming peanuts (n = 45); others underwent double-blind placebo-controlled challenge (n = 34). We compared sensitization profiles between children with peanut allergy and peanut-tolerant children by using a microarray with 12 pure components (major peanut and potentially cross-reactive components, including grass allergens). RESULTS: Of 933 children, 110 (11.8%) were peanut-sensitized. Nineteen were not challenged (17 no consent). Twelve with a convincing history of reactions on exposure, IgE > or =15 kUa/L and/or skin test > or =8mm were considered allergic without challenge. Of the remaining 79 children who underwent challenge, 7 had > or =2 objective signs and were designated as having peanut allergy. We estimated the prevalence of clinical peanut allergy among sensitized subjects as 22.4% (95% CI, 14.8% to 32.3%). By using component-resolved diagnostics, we detected marked differences in the pattern of component recognition between children with peanut allergy (n = 29; group enriched with 12 children with allergy) and peanut-tolerant children (n = 52). The peanut component Ara h 2 was the most important predictor of clinical allergy. CONCLUSION: The majority of children considered peanut-sensitized on the basis of standard tests do not have peanut allergy. Component-resolved diagnostics may facilitate the diagnosis of peanut allergy.

Day-care attendance, position in sibship, and early childhood wheezing: a population-based birth cohort study.

BACKGROUND: There are conflicting data on the effect of day-care attendance and position in sibship on the development of wheezing. OBJECTIVE: To investigate the relationship between day-care attendance and position in sibship with early childhood wheeze. METHODS: Prospective population-based birth cohort. At age 5 years, we collected information on parentally reported symptoms (n = 922); lung function was ascertained using plethysmography (n = 745) and allergic sensitization by skin testing (n = 815). Participants were assigned into categories according to the age of entry to day-care (0-6, 6-12, >12 mo) and number of older siblings (0, 1, 2, >2). RESULTS: Current wheeze was reported by 203 participants (22%); 224 (28%) were sensitized. In the multivariate model, sensitization (odds ratio, 2.47; 95% CI, 1.66-3.67), male sex (1.49, 1.01-2.20), maternal asthma (1.72, 1.10-2.68), and maternal smoking during pregnancy (2.15, 1.26-3.66) significantly increased the risk of wheezing. Entering day-care between 6 and 12 or after 12 months of age was significantly and inversely associated with current wheeze (0.25, 0.11-0.60; and 0.65, 0.44-0.98, respectively). Entry into nursery between 6 and 12 months reduced the risk of persistent wheezing (P = .04). We found no association between day-care attendance and lung function. Entering nursery in the first 6 months of life was associated with increased risk of atopy (2.47, 1.23-4.95). Having older siblings was associated only with rhinoconjunctivitis (0.72, 0.54-0.97). CONCLUSION: Day-care attendance was associated with a reduced risk of current wheezing in 5-year-old children. The protective effect appeared strongest for children who entered day-care between the ages of 6 and 12 months.

Exhaled breath condensate pH and childhood asthma: unselected birth cohort study.

RATIONALE: Exhaled breath condensate pH (EBC-pH) may be useful noninvasive marker for evaluation of patients with asthma. OBJECTIVES: To investigate the relationship between EBC-pH and symptoms suggestive of childhood asthma in an epidemiologic setting and examine its relation to lung function, airway hyperresponsiveness (AHR), and airway inflammation. METHODS: Within the context of a prospective population-based birth cohort, EBC was collected from 630 children at age 8 yr using the RTube (pH measured after deaeration with argon). Lung function was measured by spirometry (FEV1; n = 521) and plethysmography (sRaw; n = 567), and AHR by methacholine challenge (n = 498). Airway inflammation was assessed using exhaled nitric oxide (eNO; n = 305). RESULTS: EBC-pH values ranged widely (4.40-8.29), and did not differ between 54 children with parentally reported asthma and 562 nonasthmatic subjects (median [interquartile range]: 7.75 [7.45-7.85] vs. 7.77 [7.59-7.87]; p = 0.35). There was a trend for lower EBC-pH among current wheezers (n = 98; 7.72 [7.50-7.83]) compared with nonwheezers (n = 532; 7.77 [7.60-7.87]; p = 0.07). Wheeze frequency, severity, and use of antiasthma medication were not associated with EBC-pH. There was no consistent association between EBC-pH and lung function, airway reactivity, and airway inflammation (FEV1, sRaw, PD20 methacholine, or eNO). There was no significant difference in EBC-pH between current wheezers receiving asthma medication who had positive methacholine challenge compared with children without any of these features. CONCLUSIONS: In the epidemiologic setting, EBC-pH does not differ between children with and without parentally reported symptoms suggestive of asthma. We found no consistent association between EBC-pH and lung function, AHR, and airway inflammation in this sample from the general population.

Domestic allergen and endotoxin exposure and allergic sensitization in Cyprus.

We investigated the relationship between domestic allergen and endotoxin exposure and allergic sensitization among children in Cyprus. We skin prick tested 128 children aged 15-16 yr (random samples of 85 children with self-reported asthma and 43 healthy controls) and measured their domestic exposure to endotoxin and allergens (mite, cat, and dog). We analyzed the data using multivariate logistic regression (adjusting for gender, area of residence and parental history) and presented the outcomes as odds ratios (OR) and 95% confidence intervals (CI). Among this selected population, 19% of children were sensitized to mite, 15% to cat and 7% to dog. Male gender (OR 2.74, 95% CI 1.18-6.38, p = 0.02), maternal history of allergic disease (OR 3.53, 95% CI 1.13-11.00, p = 0.03), increasing endotoxin (OR 1.58, 95% CI 1.00-2.49, p = 0.05) and residence in the district of Nicosia (OR 2.48, 95% CI 1.01-6.08, p = 0.05) were independent associates of allergic sensitization. Factors associated with mite sensitization were increasing Der p 1 and endotoxin exposure (OR 1.28, 95% CI 1.01-1.62, p = 0.04 and OR 1.76, 95% CI 1.01-3.08, p = 0.05, respectively) and living in an urban area (OR 6.80, 95% CI 1.37-33.67, p = 0.02). Sensitization to domestic pets was associated only with paternal allergic disease (cat: OR 5.68, 95% CI 1.57-23.56, p = 0.02; dog: OR 13.5, 95% CI 1.79-101.73, p = 0.01), but not with pet ownership or specific allergen or endotoxin exposure. In conclusion, mite allergen exposure was associated with sensitization to mite, but there was no association between cat and dog allergen exposure and specific sensitizations. Surprisingly, in this area, increasing endotoxin exposure was associated with an increased risk of sensitization.