Background: Inhibition of IL-4/IL-13-driven inflammation by dupilumab has shown significant clinical benefits in treatment of Objective: Our aim was to assess longitudinal protein and metabolite composition in AD skin during dupilumab treatment. Methods: Skin tape strips (STSs) were collected from lesional/ nonlesional skin of 20 patients with AD during a 16-week dupilumab treatment course and from 20 healthy volunteers (HVs) followed for 16 weeks. STS extracts were examined by liquid chromatography-mass spectrometry proteomic analysis and targeted metabolomics. Results: Approximately 2500 individual proteins were identified in the STS extracts. Of those proteins, 490 were present in at least 80% of the AD and HV skin samples and differentially expressed in the AD skin; the levels of 249 proteins were significantly reduced (cluster 1), and the levels of 136 were significantly increased (cluster 2) in the AD skin versus in the HV skin (both P < .0001). Functionally, cluster 1 included proteins involved in epidermal barrier formation, lysosomal enzymes required for lamellae assembly, and oxidative response. Cluster 2 was enriched for markers of epidermal hyperplasia, glycolytic enzymes, and actin filament proteins. A significant increase in cluster 1 and a significant inhibition of cluster 2 proteins expression were achieved in AD skin by 16 weeks of dupilumab treatment (P <.0001 for both vs baseline), approaching the levels in HV skin. These improvements were also revealed in differential metabolite changes in the STS extracts, including amino acids, nucleotide breakdown products, and antioxidants. Conclusion: Longitudinal integrated assessment of the skin proteome and metabolome in patients with AD who were treated with dupilumab established significant inhibition of epidermal hyperplasia and improvement in epidermal differentiation. The identified changes were linked to improvements in clinical AD skin assessments, including improvements in transepidermal water loss and disease severity.

Introduction Le dupilumab, un anticorps monoclonal, qui inhibe la synthèse des interleukines IL-4 et IL-13, est utilisé pour le traitement de la dermatite atopique (DA) modérée à sévère chez les sujets âgés de 6 mois et plus. Matériel et méthodes Nous avons examiné la peau des patients atteints de DA âgés de 6 à 11 ans (étude PELISTAD [NCT04718870], n=23) et de 12 à 65 ans (étude BALISTAD [NCT04447417], n=26) pendant 16 semaines de traitement par dupilumab. Le score total de l’indice de sévérité et de l’étendue de l’eczéma (EASI) et les lipides de la couche cornée de la peau prélevés par stripping ont été évalués de manière longitudinale. Résultats Il n’y avait pas de différence dans les scores EASI entre les tranches d’âge 6–11 ans et 12–65 ans à l’inclusion (moyenne±erreur type : 34,85±2,55 vs 31,18±3,25, respectivement). Le traitement par dupilumab a entraîné une amélioration plus rapide des scores EASI dans la tranche d’âge des 6–11 ans que dans celle des 12–65 ans (moyenne±erreur type pour ces tranches d’âge respectif : à la semaine 3, 11,97±2,14 vs 19,52±2,96 [p=0,027], à la semaine 8 : 9,76±2,26 vs 13,48±2,16 [p=0,022]). L’analyse des lipides collectés par stripping des lésions cutanées à la semaine 8 a montré que dans la tranche d’âge 6–11 ans, les céramides (CER) estérifiés en position oméga de l’acide gras avec la base sphingosine (EOS) ont presque atteint les niveaux de CER EOS dans la peau saine (91 % d’amélioration), tandis que dans la tranche d’âge 12–65 ans l’amélioration était de 45 %. Les niveaux de N(C22)S-céramides dans les lésions cutanées de la DA se sont améliorés de 52,9 % dans la tranche d’âge 6–11 ans vs 32 % dans la tranche d’âge 12–65 ans entre l’inclusion et la semaine 8. Discussion Nos données montrent que dans la tranche d’âge 6–11 ans, l’intervention du dupilumab entraîne une amélioration significative et plus rapide de la sévérité clinique par rapport à la tranche d’âge 12–65 ans, ainsi qu’une normalisation des lipides de la barrière cutanée. Ainsi, une intervention précoce pourrait offrir de meilleurs résultats thérapeutiques tant pour les lipides de la barrière cutanée que pour la sévérité de la maladie dans la DA. Conclusion Les enfants atteints de dermatite atopique répondent plus rapidement au traitement par dupilumab que les adolescents et les adultes.

Highly cross-linked insoluble proteins and the extracellular lipids form skin cornified envelop (CE). Keratins (KRTs) are anchored to the CE through conjugation with desmosomal proteins. Type 2-driven inflammation in atopic dermatitis (AD) inhibits CE formation. The role of dupilumab, a humanized monoclonal antibody against IL-4 receptor, in the regulation of CE assembly in AD has not been established. Skin tape strips (STS) were collected from 20 AD patients over the 16-week course of dupilumab treatment and 20 healthy volunteers followed for 16 weeks. Lesional and non-lesional STS extracts were examined at baseline, weeks 8 and 16 of treatment by liquid chromatography mass spectrometry for proteomic analysis. Desmoglein 1 (DSG1) and desmocollin 1 (DSC1) were decreased in AD lesional skin (p<0.01) at baseline and were normalized with dupilumab treatment (p<0.01). Other desmosomal structures, including plakophillins, envoplakin and periplakin, were increased in AD skin (p<0.0001), and were inhibited by dupilumab treatment (p<0.001). KRT1 and KRT10 form bundles with filaggrin in differentiated epidermis, were significantly inhibited in AD lesional skin (p<0.0001) at baseline and were normalized with dupilumab treatment (p<0.0001). KRT16, KRT6A and KRT6B that compete with the assembly of KRT1 andKRT10 filaments were significantly elevated in AD skin at baseline(p<0.0001), and were significantly reduced in AD skin after dupilumab treatment (p<0.0001). The study revealed abnormal expression of KRT filaments required for the CE formation in AD skin. As a result, to compensate for the mechanical stress, the expression of desmosomal proteins was altered in AD skin. Dupilumab-mediated IL-4/IL-13 inhibition resulted in redistribution of KRTs and desmosomal components required for proper CE assembly, suggesting an underlying mechanism of action of Dupilumab in improving skin barrier function in AD. These data provide supportive evidence for the restoration of the appropriate equilibrium of KRTs and desmosomal components in CE following treatment with dupilumab.

Dupilumab, a monoclonal antibody that targets IL-4Ra, has shown significant clinical benefits in modulation of type 2(T2) inflammation in atopic dermatitis(AD). To date, there are no detailed proteomic studies of longitudinal changes in AD skin proteins in patients treated with dupilumab. Assessments of lesional and non-lesional skin of 20 AD patients during 16 weeks of dupilumab treatment were done by skin tape stripping(STS) and compared to STS of 20 healthy volunteers (HV). STS protein extracts were examined at baseline, weeks 8 and 16 of treatment by liquid chromatography mass spectrometry proteomic analysis. 490 proteins were detected in ≥80% of AD and HV skin samples. 139 proteins were differentially expressed between AD and HV at baseline and significantly changed during dupilumab treatment (p<0.05). At baseline, expression of 49 proteins, including proteins involved in epidermal barrier formation (KRT2, KRT77, TGM3, CDSN, DSC3, KLK7, BLMH), lysosomal enzymes required for lamellae assembly (ASAH1, GLB1, HEXA, HEXB, GAA, FUCA1, ALOXE3), oxidative response enzymes (CAT, GSS, GGCT) and proteasome subunits, was significantly lower in AD skin compared to HV (p<0.0001). Expression of these proteins in AD skin significantly increased at weeks 8 and 16 of dupilumab treatment (both p<001) and approached levels observed in HV. Longitudinal assessment of the skin proteome in AD patients treated with dupilumab demonstrated significant improvement in the expression of proteins involved in epidermal differentiation, normalization of lamellae assembly and antioxidant defense by 16 weeks of treatment, suggesting that these proteins are under control of T2 cytokines in AD skin.

Atopic dermatitis (AD) is characterized by skin barrier dysfunction, for which the abnormal stratum corneum (sc) ceramide composition serves as one of the driving factors. Here, we assess effect of dupilumab on the regulation of skin barrier function and sc ceramide composition in patients with AD. BALISTAD (NCT04447417) was an open-label trial, in which Trans-epidermal Water Loss (TEWL) was assessed via skin tape strip (STS) samples from lesional skin of 26 patients with AD treated with dupilumab and from normal skin of 26 healthy volunteers over 16 weeks. Quantitative lipidomic analysis of STS samples was conducted by liquid chromatography tandem mass spectrometry. Wilcoxon signed rank tests were performed to assess the change in TEWL from baseline in response to dupilumab treatment. The median TEWL area under curve up to 10 STS in AD lesions was significantly reduced from baseline starting at Week 2 (P < 0.0001) and sustained through Week 16 (P < 0.0001). Dupilumab treatment significantly increased levels of total C18 esterified omega-hydroxy fatty acid sphingosine ceramides (EOS-CER, long-chain lipids; P < 0.0001), and decreased levels of total non-hydroxy fatty acid sphingosine ceramides (NS-CER, short-chain lipids; P < 0.0001) in AD lesional skin at Week 16. In conclusion, dupilumab treatment significantly improved skin barrier and normalized composition of skin barrier lipids in adults and adolescents with AD.

Epidermal hyperplasia is a characteristic histological feature in psoriasis, a chronic inflammatory skin disease of IL-23/IL-17A axis. Metabolically, proliferating cells require glucose uptake through glucose transporters. Indeed, keratinocytes in psoriasis lesions exhibit increased expression of Glut1, one of the most widely expressed glucose transporters. In addition, Glut1-deficient mice show impaired epidermal hyperplasia in mouse psoriasis models, suggesting that glucose metabolism through Glut1 is essential for epidermal hyperplasia in psoriasis. However, it remains unclear whether increased glucose metabolism in keratinocytes in psoriasis lesions returns to physiologically normal levels after treatments, such as biologics. To examine this, we performed an immunohistochemical analysis of Glut1 expression in psoriasis lesions before and four weeks after the treatment with secukinumab, an anti-IL-17A antibody (n = 6). Epidermal hyperplasia with increased Ki67 expression observed before treatment was markedly diminished after treatment. Before treatment, the expression levels of Glut1 were significantly upregulated in psoriasis lesions compared with the healthy skin in normal controls (n = 8), consistent with the previous reports. However, the expression levels were significantly decreased after treatment and were similar to those in healthy skin and non-lesional skin of psoriasis patients. The mRNA expression analysis using a public database set revealed that the expression of GLUT1 in psoriasis lesions was significantly down-regulated after ixekizumab treatment, another IL-17A antibody (n = 5). These results suggest that blockade of IL-17A pathyways reverses the increased glucose metabolism in keratinocytes and may reduce epidermal hyperplasia in psoriasis.

Collection of skin very top layer, called stratum corneum, by tape stripping and the analysis of stratum corneum components by mass spectrometry provides multiple advantages for clinical studies that aim to understand the origins of allergic skin diseases and food allergy. However, such a methodology has multiple challenges on the way of complex stratum corneum analysis when molecules of different polarity are needed to be analyzed from minimal amount of skin tape strips. This review provides an overview of current knowledge about lipid and polar molecules in the skin, discusses challenging aspects of sample processing when dealing with skin tape strips, and provides some guidance towards approaches that generate complex, quantitative, normalized to total sample protein data that fit best the purpose of analysis of stratum corneum components for the purpose of clinical trials.

its low cost and noninvasive nature, STS allows for the collection of samples from large patient cohorts and can be useful for disease endotyping, as collected STS can be readily used for lipid, protein, and RNA analysis. (5) STS procedure can be useful for skin sampling in various age groups, including infants (Kim et al., 2016; McAleer et al., 2019). In conclusion, STS could be a good standard procedure to evaluate epidermal differentiation markers from the SC and the upper granular layer of the epidermis. Moreover, this method is a safe and reliable strategy to evaluate cellular and molecular characteristics of the skin barrier in both children and adults.

BACKGROUND:The role of CD8 T lymphocytes in the pathogenesis of asthma is not well understood. We investigated whether a subset of IL-13-producing BLT1-positive CD8 T lymphocytes are present in asthmatic airways and are associated with impaired lung function.METHODS:Bronchoalveolar lavage (BAL) cells were obtained from asthmatic (n = 39) and healthy control (n = 28) subjects. Cells were stimulated with phorbol ester and ionomycin in the presence of brefeldin A and stained for CD8, BLT1, and intracellular IL-13. The frequency of IL-13-producing BLT1-positive CD8 T lymphocytes was compared between the two groups and related to lung function, serum IgE levels, and reticular basement membrane (RBM) thickness.RESULTS:A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 were detected in the airways of all asthmatic subjects. The frequency of this subset among recovered lymphocytes was significantly higher in the airways of asthmatic subjects compared with controls (mean ± SEM: 16.2 ± 1.4 vs 5.3 ± 0.5, respectively, P < 0.001) and correlated positively with serum IgE levels and RBM thickness. More importantly, the frequency of CD8 T lymphocytes co-expressing BLT1 and IL-13 was inversely related to FEV1 and FEF[25-75] percent predicted values (P < 0.001).CONCLUSIONS:A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 is present in the airways of asthmatics. The accumulation of these cells is associated with airway obstruction, suggesting that they may play a significant pathogenic role in bronchial asthma.

It is estimated that 1 billion people around the world are vitamin D deficient. Vitamin D deficiency has been linked to various inflammatory diseases. However, the mechanism by which vitamin D reduces inflammation remains poorly understood. In this study, we investigated the inhibitory effects of physiologic levels of vitamin D on LPS-stimulated inflammatory response in human blood monocytes and explored potential mechanisms of vitamin D action. We observed that two forms of the vitamin D, 1,25(OH)2D3, and 25(OH)D3, dose dependently inhibited LPS-induced p38 phosphorylation at physiologic concentrations, IL-6 and TNF-α production by human monocytes. Upon vitamin D treatment, the expression of MAPK phosphatase-1 (MKP-1) was significantly upregulated in human monocytes and murine bone marrow-derived macrophages (BMM). Increased binding of the vitamin D receptor and increased histone H4 acetylation at the identified vitamin D response element of the murine and human MKP-1 promoters were demonstrated. Moreover, in BMM from MKP1−/− mice, the inhibition of LPS-induced p38 phosphorylation by vitamin D was completely abolished. Vitamin D inhibition of LPS-induced IL-6 and TNF-α production by BMM from MKP-1−/− mice was significantly reduced as compared with wild-type mice. In conclusion, this study identified the upregulation of MKP-1 by vitamin D as a novel pathway by which vitamin D inhibits LPS-induced p38 activation and cytokine production in monocytes/macrophages.

The factor(s) leading to increase propensity of disseminated herpes simplex virus (HSV) and vaccinia virus (VV) infections in atopic dermatitis (AD) are poorly understood. A history of eczema herpeticum is frequently associated with Staphylococcus aureus infection. This study investigated whether S. aureus toxins augment viral load in normal human keratinocytes (NHK). NHK were pretreated with commercially purified staphylococcal enterotoxin (SE) B, toxic shock syndrome toxin 1(TSST1), lipoteichoic acid (LTA), peptidoglycan (PGN), and α-toxin; then incubated with VV or HSV-1 for up to 24 hours. Viral replication was evaluated by real-time PCR and viral plaque assay. SEB, TSST1, and sublytic concentrations of α-toxin significantly increased viral load of VV and HSV-1 in NHK. In contrast, LTA and PGN had no effect on viral load in NHK. Using mass spectrometry and western-blot, we found that commercial SEB and TSST1 prepared from S. aureus cultures were contaminated with α-toxin. Recombinant SEB and TSST1, free of α-toxin, did not enhance viral load in NHK, suggesting that viral enhancement in commercial SEB/TSST1 was from α-toxin contamination. Purified α-toxin (40ng/ml) treated NHK, however, showed increased VV (mean: 0.72±0.13 ng VV/ng 18S compared with mock treatment 0.07±0.02; p<0.01) and HSV-1 loads (mean: 241.4±11.4 ng HSV-1/ng 18S compared to mock treatment 91.1±22.7; p<0.01). The mechanism by which sublytic α-toxin increases viral load was related to increased virus uptake into NHK. S. aureus α-toxin augments viral load in keratinocytes. This novel observation suggests that cutaneous S. aureus on AD skin may increase susceptibility to disseminated viral infections.

RationaleTo determine if body mass index (BMI) affects the corticosteroid (CS) response in children with asthma, we studied PBMC and bronchoalveolar lavage (BAL) cells from asthmatic children who underwent clinically indicated bronchoscopies.MethodsChildren were characterized by spirometry (age ≥ 5 y), BMI percentile (%) to adjust for age, plasma leptin (ELISA), daily inhaled CS dose (mcg budesonide equivalent [BE]), and in vitro response to CS. Mitogen-activated protein kinase phosphatase-1 (MKP-1) induction by 10-6 M dexamethasone (DEX) was evaluated by PCR in PBMC and BAL cells. Pearson correlation coefficients (r) were calculated.ResultsSixty-one children with asthma (median [range] FEV1 80% predicted [52-121%], FEV1/FVC 80% [55-99%], FEV1 change by bronchodilator 8% [-11-47%]), age 2 to 18 y (median 11 y) were studied. Fifty-six % (34/61) of children had normal weight (BMI < 85%), 21% (13/61) were overweight (BMI 85-94%), and 23% (14/61) obese (BMI ≥ 95%). Fifty-six children used ICS. Median ICS dose was 1100 mcg BE (range 0 - 4500 mcg). BMI% correlated with plasma leptin (r=0.5, p=0.0001), daily ICS dose (r=0.37; p=0.0035), and DEX-induced MKP-1-fold induction in PBMC (r=-0.45, p=0.0009) and BAL cells (r=-0.42, p=0.0205). MKP-1 induction correlated between PBMC and BAL cells (r=0.46; p=0.0176). A negative correlation was found between MKP-1 induction and daily ICS requirement (r=-0.3960, p=0.0303).ConclusionsChildren with asthma demonstrate a decreased in vitro CS response with increasing BMI% that correlated with a higher daily ICS requirement. Further studies are needed to determine if obese asthmatics will have enhanced CS responses after their BMI is normalized. RationaleTo determine if body mass index (BMI) affects the corticosteroid (CS) response in children with asthma, we studied PBMC and bronchoalveolar lavage (BAL) cells from asthmatic children who underwent clinically indicated bronchoscopies. To determine if body mass index (BMI) affects the corticosteroid (CS) response in children with asthma, we studied PBMC and bronchoalveolar lavage (BAL) cells from asthmatic children who underwent clinically indicated bronchoscopies. MethodsChildren were characterized by spirometry (age ≥ 5 y), BMI percentile (%) to adjust for age, plasma leptin (ELISA), daily inhaled CS dose (mcg budesonide equivalent [BE]), and in vitro response to CS. Mitogen-activated protein kinase phosphatase-1 (MKP-1) induction by 10-6 M dexamethasone (DEX) was evaluated by PCR in PBMC and BAL cells. Pearson correlation coefficients (r) were calculated. Children were characterized by spirometry (age ≥ 5 y), BMI percentile (%) to adjust for age, plasma leptin (ELISA), daily inhaled CS dose (mcg budesonide equivalent [BE]), and in vitro response to CS. Mitogen-activated protein kinase phosphatase-1 (MKP-1) induction by 10-6 M dexamethasone (DEX) was evaluated by PCR in PBMC and BAL cells. Pearson correlation coefficients (r) were calculated. ResultsSixty-one children with asthma (median [range] FEV1 80% predicted [52-121%], FEV1/FVC 80% [55-99%], FEV1 change by bronchodilator 8% [-11-47%]), age 2 to 18 y (median 11 y) were studied. Fifty-six % (34/61) of children had normal weight (BMI < 85%), 21% (13/61) were overweight (BMI 85-94%), and 23% (14/61) obese (BMI ≥ 95%). Fifty-six children used ICS. Median ICS dose was 1100 mcg BE (range 0 - 4500 mcg). BMI% correlated with plasma leptin (r=0.5, p=0.0001), daily ICS dose (r=0.37; p=0.0035), and DEX-induced MKP-1-fold induction in PBMC (r=-0.45, p=0.0009) and BAL cells (r=-0.42, p=0.0205). MKP-1 induction correlated between PBMC and BAL cells (r=0.46; p=0.0176). A negative correlation was found between MKP-1 induction and daily ICS requirement (r=-0.3960, p=0.0303). Sixty-one children with asthma (median [range] FEV1 80% predicted [52-121%], FEV1/FVC 80% [55-99%], FEV1 change by bronchodilator 8% [-11-47%]), age 2 to 18 y (median 11 y) were studied. Fifty-six % (34/61) of children had normal weight (BMI < 85%), 21% (13/61) were overweight (BMI 85-94%), and 23% (14/61) obese (BMI ≥ 95%). Fifty-six children used ICS. Median ICS dose was 1100 mcg BE (range 0 - 4500 mcg). BMI% correlated with plasma leptin (r=0.5, p=0.0001), daily ICS dose (r=0.37; p=0.0035), and DEX-induced MKP-1-fold induction in PBMC (r=-0.45, p=0.0009) and BAL cells (r=-0.42, p=0.0205). MKP-1 induction correlated between PBMC and BAL cells (r=0.46; p=0.0176). A negative correlation was found between MKP-1 induction and daily ICS requirement (r=-0.3960, p=0.0303). ConclusionsChildren with asthma demonstrate a decreased in vitro CS response with increasing BMI% that correlated with a higher daily ICS requirement. Further studies are needed to determine if obese asthmatics will have enhanced CS responses after their BMI is normalized. Children with asthma demonstrate a decreased in vitro CS response with increasing BMI% that correlated with a higher daily ICS requirement. Further studies are needed to determine if obese asthmatics will have enhanced CS responses after their BMI is normalized.

the role of lower airway bacterial communities in asthma is poorly understood. BAL samples were obtained from 23 adults with chronic asthma (FEV1 73.6±13.1%, PC20 1.4±2.3 mg/ml) and 20 normal control subjects. Bacterial DNA was extracted using Qiagen EZ1 system and sequenced using 454 FLX chemistry. Taxonomic identification was performed with RDP classifier and BLAST. Asthma control was measured by asthma control questionnaires (ACQ). Based on microbiome data, asthmatics were subdivided into three groups: group A with commensal flora only (n=6); group B with expansions (>10% of 16s RNA sequences) in organisms also seen in normal controls (n=9); group C with expansions (>10%) of unique organisms not seen in the normal microbiome (n=8) (Comamonas testosteroni (n=1), Simonsiella muelleri (n=1), Asticcacaulis (n=4), Streptococcus pseudopneumoniae (n=2)). A progressive reduction in the number of sequences for Genera Prevotella (Rsq=0.65, p<0.0001) and Veillonella (Rsq=0.53, p=0.0006), considered to be major airway commensal flora, was observed in groups B and C. The lowest number of commensal sequences was observed in group C. There was progressively lower asthma control from groups A to C (ACQ test score 0.86±0.28, 1.63±0.23, 2.02±0.24, for groups A-C, respectively, p=0.0175, ANOVA). Alpha diversity metrics significantly increased for bacterial diversity/richness from groups A to C (Sequences(observed) p=0.0011, ANOVA; richness(Ho) p=0.0017, ANOVA). Expansion of selected groups of bacteria in the lower airways of asthmatics was associated with reduced numbers of commensal flora organisms and increased richness/bacterial diversity. The loss of asthma control was the greatest with expansion of unique organisms.

Background: The effects of serum vitamin D status on atopy, steroid requirement, and functional responsiveness to corticosteroids in children versus adults with asthma have not been studied systematically.Objective: We sought to explore the age-specific effects of vitamin D in asthmatic patients.Methods: Serum vitamin D levels were examined in a prospective study of adults and children (102 healthy control subjects and 103 asthmatic patients). PBMCs were cultured for 3 hours with or without 100 nmol/L dexamethasone, and the expression of corticosteroid-regulated genes was detected by using real-time PCR. Serum IgE levels were measured, and information about asthmatic patients' steroid requirements was collected.Results: Deficient serum vitamin D levels (<20 ng/mL) were found in 47.6% of asthmatic patients and 56.8% of healthy control subjects, with means +/- SDs of 20.7 +/- 9.8 and 19.2 +/- 7.7 ng/mL, respectively. In multivariate regression models a significant positive correlation between serum vitamin D levels and the expression of vitamin D-regulated targets, cytochrome P450, family 24, subfamily a (cyp24a) expression by PBMCs (P = .0084, pediatric asthma group only) and serum LL-37 levels (P = .0006 in the pediatric group but P = .0067 in the adult asthma group), was found. An inverse association between vitamin D and serum IgE levels was observed in the pediatric (P = .0006) asthma group. Serum vitamin D level (P = .05), as well as PBMC cyp24a expression (P = .0312), demonstrated a significant inverse relationship with daily inhaled corticosteroid dose in the pediatric asthma group only. Cyp24a expression in PBMCs correlated positively with in vitro suppression of TNF-alpha by dexamethasone (P = .05) and IL-13 (P = .0094) in PBMCs in the pediatric asthma group only.Conclusions: This study demonstrated significant associations between serum vitamin D status and steroid requirement and in vitro responsiveness to corticosteroids in the pediatric but not the adult asthma group. Vitamin D was also related to IgE levels in children but not in adults. (J Allergy Clin Immunol 2012;129:1243-51.)

SummaryBackgroundGlucocorticoid‐induced TNF receptor‐related protein ligand (GITRL), a ligand for the T cell co‐stimulatory molecule GITR, is expressed by keratinocytes and involved in chemokine production. The expression of GITRL in skin inflammation remains unknown.ObjectivesThis study investigated cytokine regulation of keratinocyte GITRL expression.MethodsGlucocorticoid‐induced TNF receptor expression was evaluated in cytokine‐treated human epidermal keratinocytes (HEK)s, murine PAM 212 cell line, murine and human skin explants by real time PCR, flow cytometry and immunostaining. Functional responses to GITR fusion protein were examined by real time PCR and ELISA. GITRL expression in AD and psoriasis was studied by immunohistochemistry.ResultsSkin biopsies from STAT6VT transgenic mice, which develop spontaneous atopic skin inflammation, were found by immunofluoresence, to have increased keratinocyte GITRL expression. Exposure to Th2 cytokines augmented GITRL mRNA expression in the murine PAM 212 keratinocytic cell line and murine skin explants. In contrast, GITRL mRNA and protein expression was only increased in HEKs and human skin explants in the presence of the combination of TNF‐α and Th2 cytokines. A synergistic effect of Th2 cytokines and GITR fusion protein on production of CCL17, the Th2 chemokine, by murine keratinocytes was demonstrated. Immunohistochemical staining showed that acute AD lesions have increased expression of GITRL compared with normal skin, chronic AD lesions and psoriatic plaques.Conclusions and Clinical RelevanceOur studies demonstrate that GITRL expression is augmented by Th2 cytokines and TNF‐α in keratinocytes. Increased GITRL expression in acute AD skin lesions is shown. This observation suggests a link between cytokine‐regulated keratinocyte GITRL expression and its role in inflammatory responses in AD.

To characterize the effects of bacterial lipopolysaccharide (LPS) on airway inflammation in childhood asthma, we studied bronchoalveolar lavage (BAL) cells and fluid from asthmatic children who underwent clinically indicated bronchoscopies. BAL samples from 33 children, ages 1 to 16 y (median 10 y), with well-characterized asthma, were analyzed based on presence (Group A: 18/33) or absence (Group B: 15/33) of Gram-negative bacteria determined by cultures. Cell counts and differentials, concentrations of LPS (chromogenic limulus lysate assay; [pg/mg total protein]), cytokines and chemokines (Multiplex ELISA; [pg/mg total protein]) in BAL were compared between group A and B using non-parametric tests. Pearson correlation coefficients (r) were calculated. Clinical characteristics including atopic sensitization, BAL cell counts and differentials did not differ between groups. Group A had higher LPS concentrations (825 ± 2246; mean ± SD) than B (159 ± 501; p=0.0166). IL-1 ra and MIP-1 beta were higher in Group A (IL-1 ra: 19 ± 26; MIP-1 beta: 15 ± 23) than in B (IL-1 ra: 7 ± 11; p=0.0119; MIP-1 beta: 1 ± 3; p=0.0558). In Group A, but not B, LPS correlated with TNF-alpha (r=0.9889, p=<0.0001). IL-1 ra correlated with IL-1 alpha, IL-1 beta, and IL-6 (p<0.05). MIP-1 beta correlated with IL-6, IL-8, and G-CSF (p<0.05). Our data suggest that increased LPS exposure through Gram-negative bacteria in lower airways of asthmatic children leads to a distinct pattern of airway inflammation. Future studies are needed to determine the effects of bacterial colonization in the airways of children with difficult to manage asthma.