Ivone Jakasa

Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency

Background Filaggrin (FLG) mutations result in reduced stratum corneum (SC) natural moisturizing factor (NMF) components and consequent increased SC pH. Because higher pH activates SC protease activity, we hypothesized an enhanced release of proinflammatory IL-1 cytokines from corneocytes in patients with atopic dermatitis (AD) with FLG mutations (ADFLG) compared with that seen in patients with AD without these mutations (ADNON-FLG). Objectives We sought to investigate SC IL-1 cytokine profiles in the uninvolved skin of controls and patients with ADFLG versus patients with ADNON-FLG. We also sought to examine the same profiles in a murine model of filaggrin deficiency (Flgft/Flgft [FlgdelAPfal] mice). Methods One hundred thirty-seven patients were studied. NMF levels were ascertained using confocal Raman spectroscopy; transepidermal water loss and skin surface pH were measured. IL-1α, IL-1β, IL-18, IL-1 receptor antagonist (IL-1RA), and IL-8 levels were determined in SC tape strips from 93 patients. All subjects were screened for 9 FLG mutations. Flgft/Flgft (FlgdelAPfal) mice, separated from maFlgft/maFlgft (flaky tail) mice, were used for the preparation and culture of primary murine keratinocytes and as a source of murine skin. RT-PCR was performed using primers specific for murine IL-1α, IL-1β, and IL-1RA. Results SC IL-1 levels were increased in patients with ADFLG; these levels were inversely correlated with NMF levels. NMF values were also inversely correlated with skin surface pH. Skin and keratinocytes from Flgft/Flgft mice had upregulated expression of IL-1β and IL-1RA mRNA. Conclusions ADFLG is associated with an increased SC IL-1 cytokine profile; this profile is also seen in a murine homologue of filaggrin deficiency. These findings might have importance in understanding the influence of FLG mutations on the inflammasome in the pathogenesis of AD and help individualize therapeutic approaches.

South African amaXhosa patients with atopic dermatitis have decreased levels of filaggrin breakdown products but no loss-of-function mutations in filaggrin

To the Editor: n nLoss-of-function (LOF) mutations in the filaggrin gene (FLG) are the strongest known genetic risk factors for atopic dermatitis (AD). The genetic architecture of FLG mutations is well established in European, Japanese, and selected Chinese populations, but their contribution to AD in African populations is not well understood. The only data on FLG mutations in Africans come from a recent study conducted in Ethiopia1 that studied 103 patients with AD, 7 patients with ichthyosis vulgaris (IV), and 103 healthy controls. This study identified only a single novel mutation (a 2-bp deletion, 632del2), by direct sequencing of FLG in a patient with AD. n nTo investigate the role of filaggrin in the etiology of AD in South Africa, we studied 69 children with AD from the amaXhosa community along with 81 age-, ethnic- and sex-matched controls, with no history of AD. The patients (nxa0= 69) and controls (nxa0= 81) were recruited from tertiary referral AD clinics in Cape Town. Clinical and demographic characteristics of control subjects and patients with AD are outlined in Table I. The study was conducted in accordance with the Helsinki Declaration and was approved by the Human Research Ethics Committee of the Faculty of Health Sciences of the University of Cape Town. Written consent/ascent in the amaXhosa language was obtained from the patients or their parents. n n n nTable I n nClinical and demographic characteristics of control subjects and patients with AD n n n nThe entire coding sequence of the FLG gene was directly sequenced (as described previously2) in 31 patients with AD with prominent features of IV, that is, those who should most likely have FLG mutations. Sequencing of PCR products was performed by a core facility (DNA Sequencing and Services, University of Dundee, Dundee, United Kingdom) according to our standard operating procedures. The entire collection was additionally typed for the previously known FLG mutations R501X, 2282del4, R2447X, and S3247X by using custom-made Taqman allelic discrimination assays.2 Although the primers used for the amplification of the FLG gene were originally optimized for the sequencing of European populations,2 in the 31 amaXhosa patients with AD who were sequenced, we identified 124 mutations (synonymous and nonsynonymous) throughout exon 3 of the FLG gene by using these methods. Identification of these silent and nonpathogenic missense mutations in the FLG gene indicates minimal allele dropout using these primer sets. None of these identified mutations was predicted to lead to loss of filaggrin at the protein level. Screening of the entire collection of patients for the FLG mutations R501X, 2282del4, R2447X, and S3247X showed that all samples were wild type for these mutations. n nIn addition to gene sequencing, in all patients with AD and controls, we determined the concentrations of filaggrin breakdown products in the stratum corneum (SC). Filaggrin is degraded in the later stages of epidermal differentiation into free amino acids (FAA) and their derivatives; a major proportion of the total SC FAA (70% to 100%) is derived from filaggrin.3 The most common amino acid residues in filaggrin repeats are basic amino acids such as histidine (413 of 4061 residues; 10.17%) and arginine (440 of 4061 residues; 10.83%) and the polar residue glutamine (367 of 4061; 9.04%) (see Fig E1 in this articles Online Repository at www.jacionline.org). Histidine is enzymatically deaminated to trans-urocanic acid (trans-UCA).3 Trans-UCA, which is converted to cis-UCA on ultraviolet irradiation, functions as a major chromophore and exerts immunomodulatory effects in the skin.3,4 UCA maintains an acidic pH in the skin, which is crucial for the optimal function of several enzymes in the SC and antimicrobial defence.3,4 Another abundant amino acid glutamine is further converted into pyrrolidone-5-carboxylic acid (PCA). PCA is highly hygroscopic and is one of the major components of the natural moisturizing factor, thus providing a humectant effect by retaining water in the SC.3,4 Filaggrin degradation products thus have multiple functions. n nFLG mutations lead to reduced levels of filaggrin degradation products in the SC in a dose-dependent fashion.5 It has been shown that moderate-to-severe AD also has an effect on SC filaggrin expression6 as well as on the levels of filaggrin degradation products5 possibly due to the systemic TH2 immune response.6 n nIn the present study, the levels of both PCA and UCA and their sum were significantly decreased in the SC of patients with AD than in control subjects (Fig 1; see Table E1 in this articles Online Repository at www.jacionline.org). The magnitude of reduction between cases and controls in this study was approximately 20%. Similar results were obtained when comparing total FAA content as well as total FAA including their derivatives, PCA, UCA, citrulline, and ornithine (Fig 1; Table E1). While FLG mutations are the major determinants of the level of filaggrin breakdown products in the SC, it has been previously shown that their levels are significantly reduced in European populations both in nonlesional skin of patients with AD with FLG mutations and in patients without FLG mutations.5 In this study, we have replicated these filaggrin breakdown product findings in patients with AD without FLG mutations in an African population. This is consistent with the inxa0vitro findings of Howell etxa0al6 and Pellerin etxa0al7 and highlights the fact that there is an interplay between thexa0skin barrier and a systemic immunologic process, with systemic TH2 inflammation causing a decrease in SC filaggrin expression. n n n nTable E1 n nThe levels of filaggrin degradation products and differences between healthy controls and patients with AD with corresponding 95% CIs n n n n n nFig 1 n nLevels of filaggrin degradation products in the SC of healthy controls (Ctrl, nxa0= 81) and patients withxa0AD (nxa0= 69) (median with interquartiles). FAAxa0+ derivatives, FAA including UCA, PCA, citrulline, and ornithine. *** ... n n n nThe SC profiles of filaggrin breakdown products are highly informative in this African population because they provide a second look, in addition to direct sequencing, for FLG mutations. In our study, we demonstrate that the diminution of filaggrin breakdown products is consistent with what we have seen in European patients with AD who are wild type for FLG mutations. The magnitude of this reduction is much less than that seen in patients with AD with FLG mutations, even in African patients with clinical findings consistent with IV who could be expected to have FLG mutations. In Europeans with a single FLG LOF mutation, expression of these products is reduced by approximately 50%.5 We here provide strong and complementary evidence for an absence of FLG mutations in this African population through 2 analytical methodologies. Given that FLG mutations are well established as the strongest and most important risk factor for AD in European, Japanese, and Chinese populations, this work clearly implies significant genetic heterogeneity between AD in the African population and AD in these populations. The genetics of AD is not well studied in African populations; in addition to the Ethiopian study referenced earlier, 2 studies in African American populations have found low frequencies of European-derived FLG mutations,8,9 presumably due to European population admixture.10 While it is tempting to speculate, on the basis of this work, that there could be a major (non-FLG) African-specific gene for IV and/or AD, African populations will require specific studies and dedicated collections to disclose African population-specific major genetic risks for AD. n nIn conclusion, FLG LOF mutations are not a significant contributor to AD in the amaXhosa population. When combined with previous findings in the Ethiopian population, the contribution of these mutations to AD in Africans seems to be at great variance with their major role in European and Asian populations. Further work is required in African populations to better understand the genetic basis of AD in these populations.

Evaluation of an HPLC Method for the Determination of Natural Moisturizing Factors in the Human Stratum Corneum

The levels of natural moisturizing factors in the skin can be used as a biomarker of hydration, for studying the effect of skin irritants, or as a biomarker of loss-of-function mutations in the filaggrin gene which are the main risk factor for atopic dermatitis. In this study the chromatographic performance and recovery of natural moisturizing factors and proteins from the skin were investigated using different extraction solvents and adhesive tapes. The uppermost layer of the skin stratum corneum, collected by using commercially available D-squame and corneofix adhesive tapes, was extracted by ammonia or potassium hydroxide. Protein levels used to correct for a variable stratum corneum amount on a tape were assessed by measuring optical density of a tape or indirectly by measuring proteins by a spectrophotometric assay. The measured natural moisturizing factors, pyrrolidone-5-carboxylic acid, histidine, tyrosine, trans-urocanic acid, and cis-urocanic acid were determined by ion pair reverse phase HPLC. Sample preparation and chromatographic performance were favorable when ammonia was used as an extraction solvent. Extraction of the natural moisturizing factors with ammonia avoids a time consuming neutralization step as required with extraction procedures using strong base or acid. The only drawback of the ammonia method is incomplete extraction of proteins from the tapes; however this can be avoided by measuring the optical density of stratum corneum-loaded tapes. The sensitivity of the method was sufficiently high to quantify the analytes even in homozygous filaggrin gene carriers. Reduced natural moisturizing factors levels found in the individuals with filaggrin gene mutation or after exposure to a skin irritant sodium lauryl sulfate were consistent with the previously reported studies.

Skin absorption through atopic dermatitis skin : a systematic review

Patients with atopic dermatitis (AD) have skin barrier impairment in both lesional and nonlesional skin. They are typically exposed daily to emollients and intermittently to topical anti‐inflammatory medicaments, thereby increasing the risk of developing contact allergy and systemic exposure to chemical ingredients found in these topical preparations. We systematically searched for studies that investigated skin absorption of various penetrants, including medicaments, in patients with AD, but also in animals with experimentally‐induced dermatitis. We identified 40 articles: 11 human studies examining model penetrants, 26 human studies examining AD drugs, and three animal studies. We conclude that patients with AD have almost twofold increased skin absorption compared with healthy controls. There is a need for well‐designed epidemiological and dermatopharmacokinetic studies that examine to what extent AD causes patients to be systemically exposed to chemicals compared with nonatopic dermatitis.

Current knowledge on biomarkers for contact sensitization and allergic contact dermatitis

Contact sensitization is common and affects up to 20% of the general population. The clinical manifestation of contact sensitization is allergic contact dermatitis. This is a clinical expression that is sometimes difficult to distinguish from other types of dermatitis, for example irritant and atopic dermatitis. Several studies have examined the pathogenesis and severity of allergic contact dermatitis by measuring the absence or presence of various biomarkers. In this review, we provide a non‐systematic overview of biomarkers that have been studied in allergic contact dermatitis. These include genetic variations and mutations, inflammatory mediators, alarmins, proteases, immunoproteomics, lipids, natural moisturizing factors, tight junctions, and antimicrobial peptides. We conclude that, despite the enormous amount of data, convincing specific biomarkers for allergic contact dermatitis are yet to be described.

Identification and quantification of lignans in wheat bran by gas chromatography-electron capture detection.

Whole grain cereals are an important source of bioavailable lignans, the group of compounds with potential anti-cancerogenic, antioxidant, anti-proliferative, pro-apoptotic, and antiangiogenic properties. The aim of this work was to develop a sensitive method for determination of wheat bran lignans. The analysis of lignans secoisolariciresinol, hydroxymatairesinol, lariciresinol, matairesinol, pinoresinol, syringaresinol is based on derivatization with pentafluoropropionic anhydride (PFPA) and gas chromatography-electron capture detection (GC-ECD), using styrene glycol as internal standard. To our knowledge, this is the first time that EC detection has been used for lignan analysis. The results show that the technique is reproducible and sensitive enough for detecting lignans in wheat at parts-per-billion (ppb) levels, except for hydroxymatairesinol. The method developed showed good recovery (85-105%) and precision (4-20%) for five types of lignans and thus represents a simpler and more affordable alternative to state-of-the-art wheat lignan liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.

Dermal uptake of petroleum substances.

Petroleum products are complex substances comprising varying amounts of linear and branched alkanes, alkenes, cycloalkanes, and aromatics which may penetrate the skin at different rates. For proper interpretation of toxic hazard data, understanding their percutaneous absorption is of paramount importance. The extent and significance of dermal absorption of eight petroleum substances, representing different classes of hydrocarbons, was evaluated. Literature data on the steady-state flux and permeability coefficient of these substances were evaluated and compared to those predicted by mathematical models. Reported results spanned over 5-6 orders of magnitude and were largely dependent on experimental conditions in particular on the type of the vehicle used. In general, aromatic hydrocarbons showed higher dermal absorption than more lipophilic aliphatics with similar molecular weight. The results showed high variation and were largely influenced by experimental conditions emphasizing the need of performing the experiments under in use scenario. The predictive models overestimated experimental absorption. The overall conclusion is that, based on the observed percutaneous penetration data, dermal exposure to petroleum hydrocarbons, even of aromatics with highest dermal absorption is limited and highly unlikely to be associated with health risks under real use scenarios.

Percutaneous penetration of silver from a silver containing garment in healthy volunteers and patients with atopic dermatitis.

Human data on dermal absorption of silver under in use scenario are scarce which hampers health risk assessment. The main objective of the present study was to determine percutaneous penetration of silver after dermal exposure to silver containing garment in healthy individuals and atopic dermatitis (AD) patients. Next to assess pro-inflammatory effect of silver in the skin. Healthy subjects (n=15) and patients with AD (n=15) wore a sleeve containing 3.6% (w/w) silver on their lower arms for 8h during 5 consecutive days. The percutaneous penetration parameters were deduced from the silver concentration-depth profiles in the stratum corneum (SC) collected by adhesive tapes. Furthermore, silver was measured in urine samples collected before and after exposure. Inflammatory response was assessed by measuring IL-1α and IL-1RA in the exposed and non-exposed skin sites. Dermal flux of silver in healthy subjects and AD patients was respectively 0.23 and 0.20 ng/cm(2)/h. The urine silver concentrations showed no increase after exposure. Furthermore, exposure to silver did not lead to the changes in the profiles of IL-1α and IL-1RA. Dermal absorption of silver under real life scenario was lower than the current reference dose. Furthermore, dermal exposure did not lead to altered expression of inflammatory IL-1 cytokines in the skin.

Barrier function and natural moisturizing factor levels after cumulative exposure to a fruit-derived organic acid and a detergent: different outcomes in atopic and healthy skin and relevance for occupational contact dermatitis in the food industry

Fruit‐derived organic compounds and detergents are relevant exposure factors for occupational contact dermatitis in the food industry. Although individuals with atopic dermatitis (AD) are at risk for development of occupational contact dermatitis, there have been no controlled studies on the effects of repeated exposure to multiple irritants, relevant for the food industry, in atopic skin.

Changes in filaggrin degradation products and corneocyte surface texture by season

During the winter in northern countries, the risk of dermatitis is increased due to low temperature and humidity. Dermatitis is particularly common on weather‐exposed skin such as the cheeks and hands. Recently, increased numbers of unidentified nanosized protrusions on the corneocyte surface were associated with dermatitis and deficiency of natural moisturizing factor (NMF).