Nonatopic Research Articles

Signaling sphingolipids are biomarkers for atopic dermatitis prone to disseminated viral infections

Life-threatening viral diseases such as eczema herpeticum (EH) and eczema vaccinatum (EV) occur in <5% of individuals with atopic dermatitis (AD). The diagnosis of AD, however, excludes all individuals with AD from smallpox vaccination. We sought to identify circulatory and skin lipid biomarkers associated with EH and EV. Stratum corneum and plasma samples from 15 subjects with AD and a history of EH, 13 age- and gender-matched subjects with AD and without EH history, and 13 healthy nonatopic (NA) controls were analyzed by liquid chromatography tandem mass spectrometry for sphingolipid content. Sphingosine-1-phosphate (S1P) and ceramide levels were validated in plasma samples from the Atopic Dermatitis Vaccinia Network/Atopic Dermatitis Research Network repository (12 NA, 12 AD, 23 EH) and plasma from 7 subjects with EV and 7 matched subjects with AD. S1P lyase was downregulated in human primary keratinocytes to evaluate its effect on herpes simplex virus 1 (HSV-1) replication invitro. The stratum corneum of patients with EH demonstrated significantly higher levels of free sphingoid bases than those in patients who were NA, indicating enhanced sphingolipid turnover in keratinocytes (P< .05). Plasma from 2 independent cohorts of patients with EH had a significantly increased S1P/ceramide ratio in subjects with EH versus those with AD and or who were NA (P< .01). The S1P level in plasma from subjects with EV was twice the level in plasma from subjects with AD (mean= 1,533 vs 732 pmol/mL; P< .001). Downregulation of S1P lyase expression with silencing RNA led to an increased S1P level and doubled HSV-1 titer in keratinocytes. Our data point to long-term abnormalities in the S1P signaling system as a biomarker for previous disseminated viral diseases and a potential treatment target in recurring infections.

Are skin microbes with lantibiotic biosynthesis genes reduced in atopic dermatitis subjects who are culture positive for Staphylococcus aureus?

Genomic DNA isolated from nonlesional and lesional Atopic Dermatitis (AD) and nonatopic (NA) skin swabs was used to test whether lantibiotics produced by cutaneous microbes and known to inhibit Staphylococcus aureus (SA) growth, may impact SA culturability.

189 Microbial expression of lantibiotics may explain discrepancies between S. aureus culturability and metagenomics in atopic dermatitis subjects and healthy controls

Atopic Dermatitis (AD) is an inflammatory skin disease exacerbated by Staphyloccocus aureus (S. aureus) skin colonization. The Atopic Dermatitis Research Network performed taxonomic analysis of skin swabs from 20 disease severity-matched AD S. aureus culture+ (n=10) and AD culture- (n=10) and 9 culture-, nonatopic (NA) subjects. Metagenomic analysis revealed discrepancies between molecular and culture evidence of S. aureus, suggesting that S. aureus skin burden may not be fully reflected by culture results. We hypothesized that production of lantibiotics by cutaneous microbes, which inhibit S. aureus growth, may explain high genomic S. aureus abundance in culture- samples and vice versa. Since each lantibiotic class (I, II, III & IV) has specific biosynthesis genes (lanB/C, lanM, labKC & lanL, respectively), Hidden Markov Models, previously used to assess oral and stool samples, were used to search metagenomes for these genes. We observed 190 total hits (AD [nonlesional & lesional]: 122, median 1/sample; NA: 68, median 7/sample). Significantly fewer lantibiotic hits were found in S. aureus culture+ AD nonlesional than their metagenomic counterparts (P<0.01) but no difference in lesional samples. BLAST search of peptide sequences revealed 17.0% of lantibiotic hits were from Malassezia restricta, 8.9% from coagulase-negative Staph, 8.9% from Cutibacterium acnes and 7.9% from Malassezia globosa. Our findings suggest that expression of lantibiotics may reduce S. aureus viability and highlights the potential importance of bacterial-fungal dynamics in the skin ecosystem.

Unique skin abnormality in patients with peanut allergy but no atopic dermatitis

The nonlesional skin of children with atopic dermatitis (AD) with peanut allergy (PA) is associated with increased transepidermal water loss; low urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), both of which are filaggrin breakdown products; and a reduced ratio of esterified ω-hydroxy fatty acid sphingosine ceramides (EOS-CERs) to nonhydroxy fatty acid sphingosine ceramides (NS-CERs) in the skin. The skin barrier of subjects with PA without AD (AD-PA+) has not been studied. Our aim was to explore whether AD-PA+ is associated with skin barrier abnormalities. A total of 33 participants were enrolled, including 13 AD-PA+, 9 AD+PA+, and 11 nonatopic (NA) participants. The PCA content in the stratum corneum of AD-PA+ subjects was significantly reduced versus that in NA subjects (median level, 67 vs 97 μg/mg protein [P= .028]). The ratio between cis- and trans-UCA decreased significantly from being highest in the NA group (1.62) to lowest in AD+PA+ group (0.07 [P< .001 vs in the NA group; P= .006 vs in the AD-PA+ group]), with the AD-PA+ group having an intermediate cis/trans-UCA ratio (1.17 [P= .024 vs in the NA group]). The TEWL in AD-PA+ subjects did not differ from that in the group with NA skin. Interestingly, AD-PA+ subjects had an increased EOS/NS-CER ratio versus that in the group of subjects with NA skin (1.9 vs 1.3 [P= .008]), whereas the AD+PA+ group had a decreased proportion of EOS-CERs (0.8 [P= .001] vs in the AD-PA+ group). Our data demonstrate that irrespective of AD, PA is associated with decreased skin cis-UCA and PCA content. An increase in skin EOS-CER/NS-CER ratio separates the AD-PA+ group from the AD+PA+ and NA groups.

344 Longitudinal changes in skin microbial populations as a function of atopic dermatitis severity

Atopic dermatitis (AD) is an inflammatory skin disease associated with increased Staphylococcus aureus (SA) colonization. We and others have found an inverse relationship between levels of SA and a common skin commensal, Cutibacterium acnes (CA). As intra-individual changes in the quantity of CA and SA from the skin of human subjects have not been well studied, we initiated a single-center, pragmatic, longitudinal study to address this, the design of which we present here. We hypothesize that CA, which produces antimicrobial peptides including cutimycin, limits SA growth in nonatopic (NA) and psoriasis (PS) subjects with low SA levels.

The Nonlesional Skin Surface Distinguishes Atopic Dermatitis With Food Allergy as a Unique Endotype

To determine if children with atopic dermatitis and food allergy (AD FA+) to peanut have skin features that distinguish them from children with atopic dermatitis without food allergy (AD FA−) and nonatopic (NA) controls.Sixty-two children with atopic dermatitis (AD) of any severity between the ages of 4 to 17 years were recruited to National Jewish Health. The 3 groups consisted of 21 children with food allergy (AD FA+) to at least peanut, 19 without food allergy (AD FA−), and 22 NA controls. Groups were balanced for age, sex, and race and were reported to have similar AD severity.This was a prospective, clinical mechanistic study to evaluate for differences among the AD FA+, AD FA−, and NA groups. They used skin tape stripping (STS) of lesional and nonlesional skin to examine transepidermal water loss (TEWL) and stratum corneum composition. The primary outcome was TEWL area under the curve measured before STS and after 5, 10, 15, and 20 skin tape tests. This was combined with the use of proteomics, lipidomics, electron microscopy, and transcriptomics to evaluate STS samples. In addition, the microbiome was assessed by skin swabs.TEWL area under the curve assessed on nonlesional skin demonstrated that subjects with AD FA+ had a significant increase in TEWL when compared with subjects with AD FA− and NA controls. There was noted to be significantly higher amounts of filaggrin (FLG) breakdown products at skin tape tests 15 and 16 in the NA group when compared with subjects with AD FA− and AD FA+, with the AD FA+ group demonstrating the lowest amount of FLG breakdown products. They mapped shotgun metagenomics microbial sequence reads and found an increased relative abundance of Staphylococcus and Micrococcus in subjects with AD FA+ compared with NAs on nonlesional skin. In addition KRT5, KRT14 (basal markers), and KRT16 (marker of hyperproliferation) at skin tape tests 15 and 16 were all increased in the AD FA+ group compared with subjects with AD FA− and NA, suggesting poor terminal differentiation of proliferating keratinocytes. Through transcriptome analysis of STS, they found that patients with AD FA+ had more dendritic cell and Th2 immune activation signatures in nonlesional skin. These differences were found to be independent of AD severity because there were no differences in AD severity among the 2 groups with AD, measured by Scoring Atopic Dermatitis, Eczema Area and Severity Index, and Nottingham Eczema Severity Score. In addition, network analysis demonstrated that these keratins (KRT5, KRT 14, and KRT 16) were positively correlated with AD FA+ and that FLG breakdown products were negatively correlated with AD FA+.Increased TEWL, low amounts of FLG breakdown products, and increased keratin proteins in the nonlesional skin of the AD FA+ group all suggest that in children with AD FA+, skin exhibits abnormal barrier function and lack of epidermal terminal differentiation.The authors demonstrate that both AD and food allergy affect the structure of nonlesional skin at the stratum corneum. This highlights that nonlesional skin abnormalities not visible to the eye may exist in our patients with both disorders. Further insights into these differences could help to improve treatment of AD and perhaps even prevent food allergy.

Skin Lipid Abnormalities in Patients with a History of Eczema Herpeticum Indicate the Involvement of the Sphingosine‐1‐Phosphate Signaling System in HSV Viral Replication

RationaleAtopic dermatitis (AD) with eczema herpeticum (EH) can result in systemic infections and can be life‐threatening. The purpose of this study was to identify differences in non‐lesional skin between AD with a history of recurrent EH (ADEH+), AD without EH (ADEH−), and non‐atopic (NA) controls to gain insight into mechanisms involved in EH pathogenesis using a novel minimally invasive skin tape strip (STS) methodology and mass spectrometric approaches.MethodsSTS layers 5–6 from 13 NA, 13 ADEH−, and 15 ADEH+ adult subjects were collected from non‐lesional skin. AD subjects were balanced by gender, age and disease severity; STS were subjected to lipidomic and filaggrin breakdown products analyses by liquid chromatography tandem mass spectrometry (LC‐MS/MS). For the in vitro experiments, differentiated primary human keratinocytes were transfected with control or sphingosine‐1‐phosphate (S1P) lyase (SGPL), sphingosine kinase‐1 (SPHK1), or sphingosine kinase‐2 (SPHK2) siRNA for 48h and infected with HSV‐1 (0.1 MOI). The effect of enzyme silencing on sphingolipidome was evaluated via the LC‐MS/MS. Viral replication was assessed in 24h with qPCR and plaque assay.ResultsADEH+, as compared to ADEH− and NA, subjects had a significantly increased proportion of short‐chain over long‐chain NS‐ceramides (sphingosine N‐acylated with non‐hydroxy fatty acids) (p=0.002), and increased levels of sphingomyelins (p=0.006). In contrast to ADEH−, ADEH+ subjects demonstrated increased level of free C18‐ and C17‐sphingosines (p=0.007 and 0.052, correspondingly) that suggests upregulated skin sphingosine‐1‐phosphate (S1P) metabolism and signaling. A comparison of the effect of silencing of major enzymes that directly regulate S1P levels (SPHK1, SPHK2, SGPL) in primary human keratinocytes in vitro revealed that only the silencing of SGPL resulted in increased viral replication after keratinocyte inoculation with HSV‐1 (Mean±SE, 0.55±0.09 vs. 0.25±0.05 for S1PL vs. control siRNA transfected keratinocytes, p&lt;0.01, as detected by qPCR).ConclusionsSTS from ADEH+ subjects and in vitro studies indicate involvement of the S1P signaling system in EH and suggest novel potential therapeutic targets to treat EH.Support or Funding InformationThis study was funded by the Atopic Dermatitis Research Network supported by NIH/NIAID grant U19 AI117673.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The nonlesional skin surface distinguishes atopic dermatitis with food allergy as a unique endotype.

Skin barrier dysfunction has been reported in both atopic dermatitis (AD) and food allergy (FA). However, only one-third of patients with AD have FA. The purpose of this study was to use a minimally invasive skin tape strip sampling method and a multiomics approach to determine whether children with AD and FA (AD FA+) have stratum corneum (SC) abnormalities that distinguish them from AD without FA (AD FA-) and nonatopic (NA) controls. Transepidermal water loss was found to be increased in AD FA+. Filaggrin and the proportion of ω-hydroxy fatty acid sphingosine ceramide content in nonlesional skin of children with AD FA+ were substantially lower than in AD FA- and NA skin. These abnormalities correlated with morphologic changes in epidermal lamellar bilayer architecture responsible for barrier homeostasis. Shotgun metagenomic studies revealed that the nonlesional skin of AD FA+ had increased abundance of Staphylococcus aureus compared to NA. Increased expression of keratins 5, 14, and 16 indicative of hyperproliferative keratinocytes was observed in the SC of AD FA+. The skin transcriptome of AD FA+ had increased gene expression for dendritic cells and type 2 immune pathways. A network analysis revealed keratins 5, 14, and 16 were positively correlated with AD FA+, whereas filaggrin breakdown products were negatively correlated with AD FA+. These data suggest that the most superficial compartment of nonlesional skin in AD FA+ has unique properties associated with an immature skin barrier and type 2 immune activation.

Ankyrin repeat domain 1 regulates innate immune responses against herpes simplex virus 1: A potential role in eczema herpeticum

Atopic dermatitis (AD) is a common inflammatory skin disease. Asubset of patients with AD are susceptible to disseminated herpes simplex virus (HSV) infection, a complication termed eczema herpeticum (ADEH+). The immune mechanisms causing ADEH+remain elusive. Using RNA sequencing, we recently found that ankyrin repeat domain 1 (ANKRD1) was significantly induced in human PBMCs upon HSV-1 stimulation, and its induction in patients with ADEH+ was significantly reduced compared with that seen in AD patients without a history of eczema herpeticum (ADEH-). We sought to validate ANKRD1 gene expression in nonatopic (NA) subjects, patients with ADEH-, and patients with ADEH+ and to delineate the biological function of ANKRD1 and the signaling pathway or pathways involved. Purification of human PBMCs, monocytes, B cells, dendritic cells, T cells, and natural killer cells; RNA extraction and quantitative RT-PCR; small interfering RNA technique; co-immunoprecipitation; and Western blot assays were used. ANKRD1 expression was significantly reduced in PBMCs from patients with ADEH+after HSV-1 stimulation compared with PBMCs from patients with ADEH-. We found that the induction of ANKRD1 by HSV-1 and multiple pattern recognition receptor agonists are mediated by inflammatory cytokines. Silencing ANKRD1 gene expression in antigen-presenting cells led to increased viral load and reduced IFNB1 and IL29 production. Using co-immunoprecipitation methods, we demonstrated that ANKRD1 formed protein complexes with interferon regulatory factor (IRF) 3 and IRF7, which are important transcription factors regulating signaling transduction of pattern recognition receptors. Overexpression of ANKRD1 enhanced the IRF3-mediated signaling pathways. ANKRD1 is involved in IRF3-mediated antiviral innate immune signaling pathways. Its reduced expression in patients with ADEH+might contribute to the pathogenesis of ADEH+.

Atopic Dermatitis Associated with Eczema Herpeticum is Associated with Abnormal Expression of Keratin Genes in Skin Biopsies

RATIONALE: The mechanisms underlying the increased propensity of disseminated viral infections, such as eczema herpeticum (ADEH+), in atopic dermatitis (AD) are poorly understood. We hypothesized that there would be differences in transcriptional response to vaccinia virus (VV) between ADEH+ vs ADEH- or healthy non-atopic (NA) individuals.

T cell responses to major peanut allergens in children with and without peanut allergy

T cell responses involved in peanut allergy are poorly understood. To investigate T cell responses towards major peanut allergens in peanut-allergic (PA) subjects compared with peanut-sensitized (PS) non-allergic children and non-atopic (NA) controls. Eighteen PA children, seven non-allergic PS children and 11 NA adults were included. Peripheral blood mononuclear cells were stimulated with a crude peanut extract (CPE). Short-term T cell lines were generated and subsequently stimulated with CPE and purified Ara h 1, Ara h 2, Ara h 3 and Ara h 6. The proliferation and production of IL-13, IFN-gamma, IL-10 and TNF-alpha were analysed. Proliferation to CPE and major allergens was enhanced in PA subjects. The primary response to CPE was comparable with PS subjects, with increased production of IL-13 and IFN-gamma compared with NA. Production of IL-10 was not observed. In short-term T cell lines, the response to CPE was stronger in PA than in PS and NA subjects. Only PA children had a detectable response to major peanut allergens, characterized by IL-13 production. The response was the highest after Ara h 3 stimulation, and the lowest after Ara h 2 stimulation. No significant correlation was observed between peanut-specific IgE levels and T cell responses to CPE. T cell responses to CPE in PA and PS children were characterized by Th1 and Th2 cytokines. Only PA children showed enhanced Th2 responses to Ara h 1, Ara h 3 and Ara h 6.

Infiltration of cells in the bronchial mucosa in atopic and non-atopic patients with bronchial asthma

Numbers of T lymphocytes, eosinophils, neutrophils, and mast cells in samples of bronchial mucosa were measured with monoclonal antibodies. Samples were obtained by biopsy from atopic (A) and non-atopic (NA) patients with bronchial asthma, and from healthy control subjects (C). The numbers of T lymphocytes, eosinophils, and neutrophils did not differ significantly between the A group and the NA group, but the A group had significantly more mast cells than the NA group (p < 0.025). The difference was greatest in the mucosal epithelium (p < 0.01). Electron microscopy revealed many degranulating mast cells in the A group. These results suggest that in atopic patients with bronchial asthma, mast cells infiltrate into bronchial mucosal epithelium and contribute to inflammation.

Eosinophilic Gastrointestinal Disease and Peanut Allergy are Alternatively Associated With IL-5+ and IL-5- Th2 Responses

RATIONALE: Both anaphylactic food allergy and eosinophil associated gastrointestinal disorders (EGIDs) are associated with Th2 responses and food specific IgE, yet are clinically different. We determined if the clinical differences between these diseases are reflected in different Th2 responses to foods. METHODS: Peanut allergic (PA), allergic eosinophilic gastroenteritis (AEG) and nonatopic (NA) subjects were enrolled. Antigen specific IL-4, IL-5, IFN-γ and TNF T cell responses to peanut, soy and shrimp were measured using intracellular cytokine staining and polychromatic flow cytometry. RESULTS: Two distinct subpopulations of Th2 cells were found: IL-5+ Th2 (IL-4+, IL-5+) and IL-5- Th2 (IL-4+, IL-5-) cells. Peanut specific IL-5+ Th2 cells were present at a 20-fold greater frequency in AEG vs. PA (81 vs. 4 per 106 CD4 cells, p = 0.05), whereas similar frequencies of IL-5- Th2 cells were found (67 vs. 41 per 106). IL-5+ Th2 cells accounted for a significantly greater fraction of the food antigen specific cells in AEG relative to PA (29% vs. 4%, p < 0.0001). In PA, but not AEG, IL-5- Th2 responses to peanut were highly correlated with peanut specific IgE (r= 0.87 vs. 0.55, respectively). All subject groups elicited similar very low magnitude Th1 responses to food Ags. CONCLUSION: Th2 responses are composed of two subpopulations: IL-5+ Th2 and IL-5- Th2 cells. IL-5+ Th2 food allergen specific T cells are singularly associated with AEG, whereas PA is associated with a dominant IL-5- Th2 response. These results suggest heterogeneity within the Th2 cytokine response, with different Th2 responses alternatively favoring IgE mediated or eosinophil dominant immunopathology.

Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells. However, a relationship between the AS status and FcepsilonRI has not been investigated. We aimed to characterize basophils from AS by looking at histamine release (HR) (sensitivity and reactivity) and the FcepsilonRI molecule, and compare it with nonatopic (NA) or allergic (A) persons. Blood was obtained from NA (n = 14), grass and/or birch A persons (n = 17) and mono-sensitized grass or birch pollen AS (n = 12). The basophil sensitivity and reactivity were examined by anti-IgE triggered HR. Surface expression of FcepsilonRI and IgE were measured by flow cytometry, FcepsilonRIalpha protein was identified using a radioimmunoassay and Western blot. mRNA coding for the classic FcepsilonRIbeta-chain and the truncated form (FcepsilonRIbetaT) were determined by real-time PCR. The AS group was less reactive than NA or A persons when triggered by anti-IgE and had a significant higher number of nonresponders. However, there was no difference in sensitivity among the three groups and furthermore; the groups did not vary in FcepsilonRI- and IgE-surface expression, FcepsilonRIalpha-protein level or beta/betaT ratio. Basophils from AS persons are less reactive and include more nonresponders than basophils from NA and A persons, but do not differ regarding the FcepsilonRI molecule.

The relative effect of the truncated isoform of the FcεRIβ chain (βT) on the FcεRI expression on human basophils

RATIONALE: The βT chain is described to prevent FcεRI surface expression in vitro. We aimed at analysing the effect of βT levels on the FcεRI expression on basophils from non-atopic (NA), IgE-mono-sensitized asymptomatic (AS) and allergic patients (A).

Is the Atopy Patch Test with House Dust Mites Specific for Atopic Dermatitis?

Background: The atopy patch test (APT), namely the patch test with aeroallergens, is regarded as specific for patients with atopic dermatitis (AD), but small numbers of positive APT were reported in the past also in atopic subjects without dermatitis and in healthy persons. Objective: The aim of this study was to evaluate the response to the APT with house dust mites (HDM) in subjects nonaffected by AD and to compare the outcomes observed in these cases with those pointed out in AD patients, evaluating also the differences between two allergen extracts manufactured at different purifications and concentrations. Methods: Forty-seven atopic subjects without eczema (AWE), 33 nonatopic (NA) subjects and 77 adult AD patients were patch tested with an extract of purified bodies of HDM at 20% and with another extract of whole bodies of HDM at 30%, the latter corresponding to 300 µg/g of Der p 1. The reproducibility of APT was also tested in 8 AD patients, in 37 AWE subjects and in 19 NA subjects. Results: Positive responses with extract at 20% were observed in 29 (37.7%) AD, in 5 (10.6%) AWE and in 4 (12.1%) NA subjects. The APT with HDM at 30% was positive in 32 (41.6%) AD, 9 (19.1%) AWE and 4 (12.1%) NA persons. The rates of positivity and the intensity scores of responses were significantly different between AD and non-AD subjects (p < 0.01). The reproducibility of the APT in the three groups was satisfactory. Conclusion: These observations lead to conclude that the APT with HDM is positive also in non-AD subjects but it is probably more specific for AD.

Specific Vβ T Cell Subsets Are Associated with Cat and Birch Pollen Allergy in Humans

AbstractCognate interaction between TCRs and MHC class II molecules plays an important role in initiating the allergen-specific immune response. Therefore, we analyzed the TCR distribution of human PBLs of 56 atopic and nonatopic (NA) individuals, including 4 monozygotic twin pairs, from two extended and four nuclear families. The expression of 23 Vβ and 3 Vα elements was analyzed. The blood samples of symptomatic birch pollen-sensitized individuals that were taken ≤6 wk after the birch pollen season (n = 8) showed a significantly higher frequency of Vβ16.1+ and Vβ20.1+ T cells compared with the blood samples of birch pollen-sensitized individuals that were obtained out of allergen season (n = 10) or from NA individuals (p &amp;lt; 0.0005 and p &amp;lt; 0.0001, respectively). Allergen-specific lymphocyte proliferation was detected in the allergic individuals, and the distribution of Vβ16.1+ and Vβ20.1+ T cells returned to normal levels after the pollen season. The frequency of these Vβ-expressing T cells correlated with the levels of allergen-specific IgE Abs. In addition, cat-sensitized individuals (n = 8) showed a significantly higher frequency of Vβ17.1-expressing T cells than did NA individuals (p &amp;lt; 0.005). Our results indicate restricted TCR-Vβ gene usage in cat and birch pollen allergies; we suggest that both genetic and environmental factors contribute to TCR-Vβ gene expression and to the development of a specific T cell response.

Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4: detection by flow cytometry.

IgE-stimulated cultured basophils from atopic subjects are capable of secreting interleukin-4 (IL-4). We describe a flow-cytometric technique which identified intracellular IL-4 in unstimulated basophils unseparated from peripheral blood mononuclear cells (PBMC) in both atopic (AT) and nonatopic (NC) volunteers. Freshly isolated PBMC were fixed in 4% paraformaldehyde (PFA). Surface staining with 22E7, a noncompetitive anti-FcepsilonRI-alpha antibody, allowed identification of basophils. Permeabilization by 0.1% saponin allowed staining of intracellular cytokines with specific monoclonal antibodies (mAbs). Two series of experiments utilizing different protocols and anticytokine mAbs were performed. The first protocol required a two-stage fluorochrome staining technique. The availability of fluorochrome-conjugated mAbs allowed a simpler, one-stage labelling procedure for the second protocol. With the first protocol, IL-4 (but not IFN-gamma), immunoreactivity was detectable in a majority (median 77%) of peripheral blood basophils from both AT and NC subjects (n=8). Basophil IL-4 immunoreactivity was again evident in experiment 2 but did not differ significantly between AT and NC subjects--either evaluated as percentage of IL-4+ basophils (AT median=66%, NC median=38.4%, P=0.41) or IL-4-specific mean fluorescence (AT median=0.85, NC median=0.3, P=0.07). This simple technique allowed the study of intracellular cytokine expression in unstimulated blood basophils. It demonstrated constitutive basophil expression of IL-4 (but not IFN-gamma) in all subjects, with no significant increases in atopics.

Plasma histamine and clinical tolerance to infused histamine in normal, atopic and urticarial subjects

Five subjects with a recent history of urticaria (U), five atopic (A) subjects and a non-atopic (NA) control group were given intravenous infusions of histamine starting at 0.05 micrograms/kg/min, increasing by 0.05 micrograms/kg/min every 30 minutes to a maximum of 0.35 micrograms/kg/min. Plasma histamine levels were monitored every 15 minutes. The infusion was stopped when an objective clinical endpoint was reached, involving either evidence of peripheral vasodilatation (rise of skin temperature by at least 1 degree C) or a 20% fall of peak expiratory flow rate. There were no significant differences in resting plasma histamine in the three groups. Those with urticaria reached the clinical endpoint at a lower infusion rate than non-atopic subjects (U 0.22 +/- 0.02 micrograms/kg/min; A 0.26 +/- 0.02 micrograms/kg/min; NA 0.32 +/- 0.2 micrograms/kg/min. p less than 0.008) they also received a lower total histamine dose (U 1.12 +/- 0.33 mg; A 1.42 +/- 0.38 mg, NA 2.2 +/- 0.51 mg, p less than 0.008). Atopic subjects with a history of asthma, eczema or rhinitis also tolerated histamine poorly, some subjects reaching a clinical endpoint while the plasma histamine level was still relatively low (U 1.52 +/- 0.4 ng/ml, A 0.85 +/- 0.19 ng/ml, NA 1.4 +/- 0.44 ng/ml, p = 0.05). After the histamine infusion was stopped, the fall in the blood level of histamine was slower in urticarial subjects than in the other two groups, with a half-life of 6.2 +/- 1.3 min (A 3.0 +/- 1.2 min, NA 4.0 +/- 0.7 min, p less than 0.02). There were thus differences in the metabolism of histamine in our non-atopic urticarial subjects and increased histamine sensitivity in atopic subjects which require further study.